METHODS FOR INCREASING CELL CULTURE TRANSFECTION EFFICIENCY

AND CELLULAR REPROGRAMMING

CROSS REFERENCE

[0001] This Application claims the benefit of United States Provisional Application No.

62/405,725, filed October 7, 2016; United States Provisional Application No. 62/362,214, filed July 14, 2016; and United States Provisional Application No. 62/353,435, filed June 22, 2016, each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Transfection methods can be used to introduce nucleic acids into cultured cells.

Transfection methods have become a mainstay of studies related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy. Transfection efficiency can vary based on cell culture conditions, cell type, cell viability and health, cell confluency, cell culture media, serum, and type of nucleic acid used for transfection. A method for increasing cell culture transfection efficiency could lead to improvements in genetic manipulation of cells and, in turn, future therapeutic studies.

[0003] Stem cell reprogramming is a cell culture technique that can be used in the field of regenerative medicine. Induced pluripotent stem cells (iPSCs) can be used to replace those cells lost due to damage or disease in afflicted patients. Current methods of stem cell reprogramming can be inefficient and time-consuming. Thus, a method for increasing stem cell reprogramming efficiency could lead to improvements in future therapeutic studies.

SUMMARY OF THE INVENTION

[0004] In some embodiments, the invention provides a method for increasing transfection efficiency of a nucleic acid that is introduced into a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases expression of a polypeptide encoded by the nucleic acid that is introduced into the cell as compared to expression of the polypeptide encoded by a nucleic acid that is introduced into a cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

[0005] In some embodiments, the invention provides a method for reprogramming a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein the cell exhibits a rate of reprogramming that is higher than the rate of reprogramming of a cell cultured in the absence of the hypoxic condition and the positive pressure condition. INCORPORATION BY REFERENCE

[0006] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually.

BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGURE 1 depicts an illustrative transfection workflow of the invention

[0008] FIGURE 2 depicts an experimental procedure for comparison of electroporation versus a method described herein.

[0009] FIGURE 3 depicts the results of transfection of cells using a method described herein.

[0010] FIGURE 4 depicts the results of transfection of cells using a method described herein.

[0011] FIGURE 5 depicts the results of transfection of PBMCs from two different donors using a method described herein.

[0012] FIGURE 6 is an illustrative computer system to be used with a method described herein.

[0013] FIGURE 7 depicts a workflow that can be used for the reprogramming of stem cells.

[0014] FIGURE 8 illustrates the average fold increase in stem cell colony number using a method described herein.

[0015] FIGURE 9 illustrates the distribution of stem cell colony area using a method described herein.

[0016] FIGURE 10 depicts the cell morphology of cells cultured using a method described herein.

[0017] FIGURE 11 provides the reprogramming kinetics of stem cells cultured using a method described herein.

[0018] FIGURE 12 depicts cardiomyocyte differentiation using a method described herein.

[0019] FIGURE 13 depicts the effect of conditions described herein on stem cell pluripotency and differentiation.

[0020] FIGURE 14 depicts the effect of conditions described herein on stem cell differentiation markers.

[0021] FIGURE 15 depicts immunofluorescence of stem cell markers using a method described herein.

[0022] FIGURE 16 illustrates the average colony area size of differentiated stem cells using a method described herein.

[0023] FIGURE 17 shows the gene expression profile of a population of cells as a function of oxygen concentration and pressure as compared to a standard cell culture incubator.

[0024] FIGURE 18 depicts the change in transfection efficiency with changes in oxygen and pressure levels.

[0025] FIGURE 19 depicts the change in transfection efficiency with changes in oxygen and pressure levels.

[0026] FIGURE 20 provides a workflow for measuring transfection efficiency using a method disclosed herein.

[0027] FIGURE 21 shows the change in transfection efficiency via GFP expression with changes in oxygen and pressure levels.

[0028] FIGURE 22 shows the quantification of transfection efficiency via GFP expression of FIGURE 21 with changes in oxygen and pressure levels.

[0029] FIGURE 23 shows a comparison between the transfection of CD8+ cells enriched from

PBMCs and PBMCs with a GFP plasmid using a method described herein.

[0030] FIGURE 24 shows the quantification of the results of FIGURE 23.

[0031] FIGURE 25 shows that the GFP-transfected CD8+ cells cultured under hypoxic and high pressure conditions developed more multicellular clusters than did cells grown at standard incubator conditions.

[0032] FIGURE 26 shows the percent GFP in the multicellular clusters in cells grown under hypoxic and high pressure conditions compared to cells grown under standard incubator conditions.

[0033] FIGURE 27 shows the quantification of the results of FIGURE 26.

[0034] FIGURE 28 depicts that when a CRISPR/Cas9 system was used to knockout CTLA4, and knock-in GFP using homology-directed repair, the transfection efficiency of the

CRISPR/Cas9 was higher in the cells grown under hypoxic and high pressure conditions than in standard incubator conditions.

[0035] FIGURE 29 shows that the cells grown under hypoxic and high pressure conditions developed a higher percentage of GFP-positive multicellular clusters than the cells grown at standard culture conditions.

[0036] FIGURE 30 shows that the proliferation of the CD8+ cells grown under hypoxic and high pressure conditions was enriched over the cells grown under standard incubator conditions.

[0037] FIGURE 31 depicts a limited dilution assay workflow to assess GFP-positive colonies using the CRISPR/Cas9 system.

[0038] FIGURE 32 shows genome editing of the CD8-positive T-cells as indicated by the GFP signal. [0039] FIGURE 33 shows that a combination of low oxygen and high pressure enhances ectoderm commitment in defined medium, while causing changes in colony morphology to more mesoderm-like morphology.

[0040] FIGURE 34 shows the change in various stem cell markers upon incubation of cells using a method disclosed herein.

[0041] FIGURE 35 show that different combinations of tumor (disease) extracellular matrix

(ECM), low oxygen, and high pressure can alter the gene expression of EGFR and other metabolic regulators in DU145 (prostate cancer) and PanclO (pancreatic cell lines).

[0042] FIGURE 36 shows that PDL1 expression increased in ARV7-positive, 22RV1 prostate cancer cells during low oxygen and high pressure culturing conditions.

[0043] FIGURE 37 (top panel) provides a western blot showing increased PDL1 protein expression under various conditions of high pressure and hypoxia in both DU145 and 22Rvl prostate cancer cells. The bottom panel of FIGURE 37 provides a quantification of the western blot results normalized to actin.

[0044] FIGURE 38 shows identification of pressure and oxygen sensitive gene expression signatures in various cell lines.

[0045] FIGURE 39 shows a workflow of taking a biopsy culture taken from a patient having prostate cancer.

[0046] FIGURE 40 shows thst prostate cancer cells were able to form an organoid after two weeks of culture under high pressure and low oxygen conditions.

[0047] FIGURE 41 shows a workflow of taking an apheresis culture taken from a patient having prostate cancer.

[0048] FIGURE 42 shows the mutations found using the COSMIC database from pancreatic ductal adenocarcinoma (PDAC) and circulating tumor cells (CTC) using whole exome sequencing (top panels).

[0049] FIGURE 43 shows that there was increased ex vivo expansion of primary cells under low oxygen and high pressure.

[0050] FIGURE 44 shows that there was increased ex vivo expansion of primary cells under low oxygen and high pressure.

[0051] FIGURE 45 shows the effect that various oxygen and pressure conditions had on the gene expression of immunotherapeutic targets in donor PBMCs.

[0052] FIGURE 46 shows the results of the ex vivo culture and expansion of tumor- infiltrating lymphocytes (TILs) enriched from renal cell carcinoma tumors using high pressure and low conditions. [0053] FIGURE 47 shows that hypoxic and high pressure conditions can lead to greater enrichment of CD8+ cells from fresh blood samples than culture under standard incubator conditions.

[0054] FIGURE 48 shows an expanded culture time, which indicated that the culture under hypoxic and high pressure conditions generates more CD8+ cells from whole blood than culture under standard conditions.

[0055] FIGURE 49 shows induction of neural precursor markers, PAX6 and NESTIN, in iPSCs after two weeks in culture under 5% 0 ₂ and 2 PSI in stem cell maintenance media.

[0056] FIGURES 50 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

[0057] FIGURES 51 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

[0058] FIGURES 52 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

[0059] FIGURE 53 shows the transfection of human dermal fibroblasts using electroporation of a GFP plasmid.

[0060] FIGURE 54 shows the transfection of PBMCs using electroporation of a GFP plasmid.

[0061] FIGURE 55 shows the transfection of activated CD8+ T-cells using electroporation of a GFP plasmid.

[0062] FIGURE 56 shows the post-transfection effects of CD8+ T-cells using low oxygen and high pressure conditions.

[0063] FIGURES 57 shows a heatmap of the effect on pressure-sensitive genes under various experimental conditions.

[0064] FIGURE 58 shows a heatmap of the effect on oxygen- sensitive genes under various experimental conditions.

[0065] FIGURE 59 is a molecular confirmation of a genome editing experiment.

[0066] FIGURE 60 is a molecular confirmation of a genome editing experiment.

DETAILED DESCRIPTION

Transfection.

[0067] A method described herein can be used to increase, for example, transfection and transduction efficiency in cells. Transduction can be used, for example, to introduce a viral vector in a cell. Viral nucleic acid delivery systems can use recombinant viruses to deliver nucleic acids for gene therapy. Non- limiting examples of viruses that can be used to deliver nucleic acids include retrovirus, adenovirus, herpes simplex virus, adeno-associated virus, vesicular stomatitis virus, reovirus, vaccinia, pox virus, lentivirus, and measles virus.

[0068] Transfection methods that can be used with methods of the invention include, for example, lipofection, electroporation, calcium phosphate transfection, chemical transfection, polymer transfection, gene gun, magnetofection, or sonoporation. FIGURE 1 depicts an illustrative transfection workflow of the invention. FIGURE 1 shows the transfection of, for example, DU145 (human prostate cancer), LnCaP (androgen- sensitive human prostate adenocarcinoma), U87 (human primary glioblastoma), PANCIO (pancreatic adenocarcinoma), or PBMCs (peripheral blood mononuclear cells) with a GFP (green fluorescent protein) plasmid. The cells can be cultured in hypoxic conditions, for example, at 1% or 5% oxygen, and at conditions that are about 2 PSI greater or less than normal pressure conditions. The transfection allows introduction of the GFP-expressing plasmid into the cell.

[0069] Viral nucleic acid delivery methods can use recombinant viruses for nucleic acid transfer. Non-viral nucleic acid delivery can comprise injecting naked DNA or RNA, use of carriers including lipid carriers, polymer carriers, chemical carriers and biological carriers such as biologic membranes, bacteria, and virus-like particles, and physical/mechanical approaches. A combination of viral and non- viral nucleic acid delivery methods can be used for efficient gene therapy.

[0070] Non- viral nucleic acid transfer can include injection of naked nucleic acid, for example, nucleic acid that is not protected or devoid of a carrier. Hydrodynamic injection methods can increase the targeting ability of naked nucleic acids.

[0071] Non-viral nucleic acid delivery systems can include chemical carriers. These systems can include lipoplexes, polyplexes, dendrimers, and inorganic nanoparticles. A lipoplex is a complex of a lipid and a nucleic-acid that protects the nucleic acid from degradation and facilitates entry into cells, and can be prepared from neutral, anionic, or cationic lipids. Lipoplexes can enter cells by endocytosis, and release the nucleic acid contents into the cytoplasm. A polyplex is a complex of a polymer and a nucleic acid, and are prepared from cationic polymers that facilitate assembly by ionic interactions between nucleic acids and polymers. Uptake of polyplexes into cells can occur by endocytosis. Inside the cells, polyplexes require co-transfected endosomal rupture agents such as inactivated adenovirus, for the release of the polyplex particle from the endocytic vesicle. Examples of polymeric carriers include polyethyleneimine, chitosan, poly(beta-amino esters) and polyphosphoramidate. Dendrimers can be constructed to have a positively-charged surface and/or carry functional groups that aid temporary association of the dendrimer with nucleic acids. These dendrimer-nucleic acid complexes can be used for gene therapy. The dendrimer-nucleic acid complex can enter the cell by endocytosis. Nanoparticles prepared from inorganic material can be used for nucleic acid delivery. Examples of inorganic material can include gold, silica/silicate, silver, iron oxide, and calcium phosphate. Inorganic nanoparticles with a size of less than 100 nm can be used to encapsulate nucleic acids efficiently. The nanoparticles can be taken up by the cell via endocytosis, and the nucleic acid can be released from the endosome without degradation. Nanoparticles based on quantum dots can be prepared and offers the use of a stable fluorescence marker coupled with gene therapy. Organically modified silica or silicate can be used to target nucleic acids to specific cells in an organism.

[0072] Non-viral nucleic acid delivery systems can include biological methods including bactofection, biological liposomes, and virus-like particles (VLPs). The bactofection method comprises using attenuated bacteria to deliver nucleic acids to a cell. Biological liposomes, such as erythrocyte ghosts and secretion exosomes, are derived from the subject receiving gene therapy to avoid an immune response. Virus-like particles (VLP) or empty viral particles are produced by transfecting cells with only the structural genes of a virus and harvesting the empty particles. The empty particles are loaded with nucleic acids to be transfected for gene therapy.

[0073] Examples of physical methods of transfection include electroporation, gene gun, sonoporation, and magnetofection. The electroporation method uses short high-voltage pulses to transfer nucleic acid across the cell membrane. These pulses can lead to formation of temporary pores in the cell membrane, thereby allowing nucleic acid to enter the cell. Electroporation can be efficient for a broad range of cells. Electron-avalanche transfection is a type of electroporation method that uses very short, for example, microsecond, pulses of high- voltage plasma discharge for increasing efficiency of nucleic acid delivery. The gene gun method utilizes nucleic acid- coated gold particles that are shot into the cell using high-pressure gas. Force generated by the gene gun allows penetration of nucleic acid into the cells, while the gold is left behind on a stopping disk. The sonoporation method uses ultrasonic frequencies to modify permeability of cell membrane. Change in permeability allows uptake of nucleic acid into cells. The

magnetofection method uses a magnetic field to enhance nucleic acid uptake. In this method, nucleic acid is complexed with magnetic particles. A magnetic field is used to concentrate the nucleic acid complex and bring them in contact with cells.

[0074] Non- limiting examples of viruses that can be used to deliver nucleic acids include retrovirus, adenovirus, herpes simplex virus, adeno-associated virus, vesicular stomatitis virus, reovirus, vaccinia, pox virus, and measles virus.

[0075] Non- limiting examples of retroviral vectors include Moloney murine leukemia viral (MMLV) vectors, HIV-based viral vectors, gammaretroviral vectors, C-type retroviral vectors, and lentiviral vectors. Lentivirus is a subclass of retrovirus. While some retroviruses can infect only dividing cells, lentiviruses can infect and integrate into the genome of actively dividing cells and non-dividing cells.

[0076] An adenovirus is a non-enveloped virus with a linear double- stranded genome.

Adenoviruses can enter host cells using interactions between viral surface proteins and host cell receptors that lead to endocytosis of the adenovirus particle. Once inside the host cell cytoplasm, the adenovirus particle is released by the degradation of the endosome. Using cellular

microtubules, the adenovirus particle gains entry into the host cell nucleus, where adenoviral DNA is released. Inside the host cell nucleus, the adenoviral DNA is transcribed and translated, without integrating into the host cell genome.

[0077] Herpes simplex virus (HSV)-based vectors can be used in the disclosure. The HSV is an enveloped virus with a linear double- stranded DNA genome. Interactions between surface proteins on the host cell and HSV lead to pore formation in the host cell membrane. These pores allow HSV to enter the host cell cytoplasm, and once inside the host cell, the HSV uses the nuclear entry pore to enter the host cell nucleus where HSV DNA is released. HSV can persist in host cells in a state of latency. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known as human herpes virus 1 and 2 (HHV-1 and HHV-2), are members of the herpes virus family.

[0078] Alphavirus-based vectors can be used to deliver nucleic acids. Examples of alphavirus- based vectors include vectors derived from semliki forest virus and sindbis virus.

[0079] Pox/vaccinia-based vectors such as orthopox or avipox vectors can be used in the present invention. Pox virus is a double stranded DNA virus that can infect diving and non-dividing cells. Pox viral genome can accommodate up to 25kb transgenic sequence. Multiple genes can be delivered using a single vaccinia viral vector.

[0080] Adeno-associated virus (AAV) is a small, non-enveloped virus that belongs to the

Parvoviridae family. The AAV genome is a linear single- stranded DNA molecule of about 4,800 nucleotides. The AAV DNA comprises two inverted terminal repeats (ITRs) at both ends of the genome and two sets of open reading frames. The ITRs serve as origins of replication for the viral DNA and as integration elements. The open reading frames encode for the Rep (nonstructural replication) and Cap (structural capsid) proteins. AAV can infect dividing cells and quiescent cells. AAV can be engineered for use as a gene therapy vector by substituting the coding sequence for both AAV genes with a transgene (transferred nucleic acid) to be delivered to a cell. The substitution eliminates immunologic or toxic side effects due to expression of viral genes. The transgene can be placed between the two ITRs (145 bp) on the AAV DNA molecule.

[0081] A pseudotyped virus can be used for the delivery of nucleic acids. Pseudotyping involves substitution of endogenous envelope proteins of the virus by envelope proteins from other viruses or chimeric proteins. The foreign envelope proteins can confer a change in host tropism or alter stability of the virus. An example of a pseudotyped virus useful for gene therapy includes vesicular stomatitis virus G-pseudotyped lentivirus (VSV G-pseudotyped lentivirus) that is produced by coating the lentivirus with the envelope G-protein from Vesicular stomatitis virus. VSV G-pseudotyped lentivirus can transduce almost all mammalian cell types.

[0082] A hybrid vector having properties of two or more vectors can be used for nucleic acid delivery to a host cell. Hybrid vectors can be engineered to reduce toxicity or improve

therapeutic transgene expression in target cells. Non- limiting examples of hybrid vectors include AAV/adeno virus hybrid vectors, AAV/phage hybrid vectors, and retrovirus/adenovirus hybrid vectors.

[0083] A viral vector can be replication-competent. A replication-competent vector contains all the genes necessary for replication, making the genome lengthier than replication-defective viral vectors. A viral vector can be replication-defective, wherein the coding region for the genes essential for replication and packaging are deleted or replaced with other genes. Replication- defective viruses can transduce host cells and transfer the genetic material, but do not replicate. A helper virus can be supplied to help a replication-defective virus replicate.

[0084] A viral vector can be derived from any source, for example, humans, non-human primates, dogs, fowl, mouse, cat, sheep, and pig.

[0085] The nucleic acid of the disclosure can be generated using any method. The nucleic acid can be synthetic, recombinant, isolated, and/or purified.

[0086] A vector of the present disclosure can comprise one or more types of nucleic acids. The nucleic acids can include DNA or RNA. RNA nucleic acids can include a transcript of a gene of interest. DNA nucleic acids can include the gene of interest, promoter sequences, untranslated regions, and termination sequences. A combination of DNA and RNA can be used. The nucleic acids can be double- stranded or single-stranded. The nucleic acid can include non-natural or altered nucleotides.

[0087] A vector of the disclosure can comprise nucleic acids encoding a selectable marker. The selectable marker can be positive, negative or bifunctional. The selectable marker can be an antibiotic-resistance gene. Examples of antibiotic resistance genes include markers conferring resistance to kanamycin, gentamicin, ampicillin, chloramphenicol, tetracycline, doxycycline, hygromycin, puromycin, zeomycin, or blasticidin. The selectable marker can allow imaging of the host cells, for example, a fluorescent protein. Examples of imaging marker genes include GFP, eGFP, RFP, CFP, YFP, dsRed, Venus, mCherry, mTomato, and mOrange. [0088] The transfection can be a stable or transient transfection. The transfection can be used to transfect DNA plasmids, RNA, siRNA, shRNA, or any nucleic acid. The plasmids can encode, for example, green fluorescent protein (GFP), selectable markers, and other proteins of interest. The selectable markers can provide resistance to, for example, G418, hygromycin B, puromycin, and blasticidin.

[0089] A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)— CRISPR associated (Cas) (CRISPR-Cas) system can be used to modify a target or deliver a nucleic acid of the disclosure. The CRIPSR-Cas system is a targeted genome-editing system comprising a Cas nuclease that is guided to specific DNA sequences, for example, a genomic locus in a subject, by a guide RNA molecule. The Cas nuclease can modify the genomic locus, for example, by cleaving the genomic locus, thus generating mutations that result in loss of function of the target sequence. The Cas nuclease can also modify the genomic locus, for example, by cleaving the genomic locus, and adding a transgene, for example, a therapeutic nucleic acid of the disclosure. The CRIPSR/Cas system can be used in conjunction with other nucleic acid delivery methods such as viral vectors and non- viral methods as described herein.

[0090] A CRISPR interference (CRISPRi) system can be used to modify the expression of a target of the disclosure. The CRISPRi system is a targeted gene regulatory system comprising a nuclease deficient Cas enzyme fused to a transcriptional regulatory domain that is guided to specific DNA sequences, for example, a genomic locus in a subject, by a guide RNA molecule. The Cas/regulator fusion protein can occupy the genomic locus and induce, for example, transcriptional repression of the target gene through the function of a negative regulatory domain fused to the Cas protein. The CRISPRi system can be used in conjunction with other nucleic acid delivery methods such as viral vectors and non- viral methods as described herein.

[0091] A method of the invention can increase the transfection or transduction efficiency by, for example, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold.

[0092] In some embodiments, a hypoxic or positive pressure condition is applied to a cell prior to transfection. In some embodiments, a hypoxic or positive pressure condition is applied to a cell after transfection. A method described herein can comprise a conditioning step, where the conditioning step is for 24-48 hours and comprises culturing the cell to be transfected in a hypoxic or high pressure condition prior to the transfection. A method described herein can comprise a recovery period, where the recovery period comprises culturing a cell post- transfection in a hypoxic or positive pressure condition. In some embodiments, a transfection method described herein comprises a conditioning step, where the conditioning step comprises culturing the cell prior to transfection in a hypoxic or positive pressure condition for 24-48 hours. In some embodiments, a transfection method described herein comprises a recovery period, where the recovery period comprises culturing the cell after transfection in a hypoxic or positive pressure condition. In some embodiments, a transfection method described herein comprises both a conditioning step and a recovery period.

[0093] In some embodiments, a conditioning step prior to transfection can use moderate oxygen and moderate pressure levels to efficiently propagate cells while maintaining, for example, pluripotency. The oxygen levels can vary from about 5% to about 15%. Pressure levels can vary from about 0.1 PSI to about 2 PSI.

[0094] In some embodiments, a recovery phase after a transfection can use low oxygen and high pressure levels to increase transfection and recovery of cells by increasing cell viability. The oxygen levels can vary from about 0.1% to about 2%. Pressure levels can vary from about 2 PSI to about 5 PSI.

[0095] In some embodiments, positive pressure is used to increase transfection efficiency. In some embodiments, hypoxia is used to increase transfection efficiency. In some embodiments, hypoxia and positive pressure are used to increase transfection efficiency.

Stem cells.

[0096] A method disclosed herein can be used to reprogram, for example, fibroblasts to pluripotent stem cells. A method disclosed herein can, for example, increase the efficiency and increase the rate of cell reprogramming. A method disclosed herein can further increase, for example, the number and size of stem cell colonies that form as a result of the reprogramming protocol. The cells can be reprogrammed into, for example, totipotent, pluripotent, multipotent, oligopotent, or unipotent stem cells.

[0097] Reprogramming of cells into pluripotent stem cells can be enhanced by, for example, culturing the cells under hypoxic and positive pressure conditions. The cells can be

reprogrammed by transfecting cells with, for example, an RNA replicon vector encoding several stem cell transformation factors. The stem cell transformation factors can include, for example, Oct4, Sox2, KLF-4, GLIS l, and c-MYC. Additional stem cell transformation factors include, for example, Nanog and Lin28. After transfection of the cells with the reprograming factors, the cells can be maintained in media designed to differentiate and maintain stem cell populations. The cells can be grown under hypoxic and high pressure conditions as disclosed herein to induce differentiation of the cells. [0098] Adult stem cells can be found in many organs and tissues including, for example, brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. The stem cells can reside in stem cell niches within the various areas of the body. In many tissues, some types of stem cells are pericytes, which are cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent non- dividing for long periods of time until they are activated by a normal need for more cells to maintain tissues, or by disease or tissue injury.

[0099] Markers that can be used to identify iPSCs include, for example, SSEA-3, SSEA-4, TRA- 1-60, TRA-1-81, TRA-2-49/6E, Nanog, Oct3/4, Sox2, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.

[00100] The iPSCs can be induced to differentiate into, for example, neuronal cells, hippocampal progenitors, dentate granule cell neurons, MGE progenitors, cortical interneurons, dorsal cortical progenitors, excitatory cortical neurons, glial progenitors, astrocytes, neural crest stem cells, dopaminergic neurons, oligodendrocytes, dopaminergic neurons, hematopoietic cells, B-cells, T- cells, NK cells, granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, platelets, cardiomyocytes, hepatocytes, skeletal muscle cells, adipocytes, pancreatic beta-cells, or cells from the ectoderm, mesoderm, or endoderm.

[00101] The stem cells obtained using a method disclosed herein can be cultured on, for example, a gelatin-coated culture dish. The cells can be in cultured in medium containing inactivated mouse embryonic fibroblast (MEF) medium, basic FGF solution, pluripotent culture medium, leukemia inhibitory factor, and a collagenase solution. The stem cells can additionally be grown over a layer of feeder cells, which can be, for example, MEFs, JK1 cells, or SNL 76/7 cells.

[00102] Expression markers that can be measured to assess the differentiation or gene expression profile of an initial cell culture to iPSCS can include, for example, IGF1, CTNNB 1, AXIN1, KAT2A, CD4, CXCL12, FZD9, CD44, ACTC1, JAG1, BMP1, FZD2, IL6ST, FZD7, LIFR, SMAD4, DVLl, CTNNAl, FGFRl, WNTl, PPARG, COLlAl, FGFl, GLL, DNMT3B, PSENl, ALDH1A1, JUND, SDAD1, NCSTN, FZD6, TCF7, NOTCH1, APC, RB I, NUMB, CREBBP, GATA6, PSEN2, HDAC2, CCND1, CCNE1, EP300, Notch2, MME, GLI2, BTRC, STAT3, PPARD, Notch3, Notch4, GLI3, CDC42, CCNA2, ISL1, BMP2, PAX6, S 100B, CD3D, FZD5, Nanog, CDH1, Soxl, DLL1, CCND2, SMO, COL2AI, LIFR, or COX2.

Plant cells.

[00103] A method disclosed herein can be used to genetically engineer or to reprogram plant cells. A method disclosed herein can be used to create plant cells with a particular genotype that alters the cell's ability to produce a specific molecule or that results in a specific phenotype. Some embodiments of the invention comprise modulating local pressure and oxygen conditions during transformation of plant cells.

[00104] A method disclosed herein can be applied to any type of plant cell or tissue. Plant cells or tissues used in the invention can include roots, leaves, monocotyledons such as cotton, soybean, Brassica, and peanut, dicotyledons such as asparagus, barley, maize, oat, rice, sugarcane, tall fescue, and wheat, hypocotyl tissue, callus tissue, nodal explants, shoot meristem, cell cultures, immature embryos, scutellar tissue, and immature inflorescence.

[00105] In addition to or in conjunction with the methods described herein, the invention can include the use of Agrobacterium tumor- inducing (Ti) plasmid genes, which can contain a transfer DNA region (T-DNA), for engineering a plant cell's DNA. Agrobacterium can be used in the invention to produce Ti plasmid genes, and Agrobacterium strains used in the invention can include Agrobacterium tumefaciens strain C58, nopaline strains, octopine strains such as LBA4404, and agropine strains such as EHA101, EHA105, and EHA 109.

[00106] The invention can also include the use of promoters such as nopaline synthase (NOS) promoter, octopine synthase (OCS) promoter, caulimo virus promoters such as cauliflower mosaic virus (CaMV) 19S and 35S promoters, enhanced CaMV 35S promoter (e35S), figwort mosaic virus (FMV) 35S promoter, and promoters from the ribulose bisphosphate carboxylase (Rubisco) family such as Rubisco small subunit and Rubisco activase promoters in engineered plant cells.

Conditions used in methods disclosed herein.

[00107] The present invention can use a substrate to culture the cells during transfection. The cells can be applied to, for example, a culture dish coated with a substrate that can promote growth and enrichment of the cells. Cells that do not adhere to the substrate can be washed away with media. Once adhered, the cells can spread and begin dividing on the substrate.

[00108] The substrate can comprise, for example, 1, 2, 3, 4, or 5 layers. The distance between two substrates layers may range from about 0.1 to about 20 mm, about 1 to about 10 mm, or about 1 to about 5 mm and each layer can be about 0.1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, or about 20 mm.

[00109] The cells can be plated on a material made of, for example, plastic, glass, gelatin, polyacrylamide, or any combination thereof. The dishes used to the plate the cells can be, for example, microscope slides, culture plates, culture dishes, Petri dishes, microscope coverslips, an enclosed environmental chamber, a sealed culture dish, or multi-well culture dishes. [00110] The binding surface layer of the substrate can be the portion of the substrate that is in contact with the cells. In some instances, the binding surface layer is the only layer, adjacent to the base layer, or separated from the base layer by one or more middle layers.

[00111] The binding surface layer of the substrate can comprise, for example, cell monolayers, cell lysates, biological materials associated with the extracellular matrix (ECM), gelatin, or any combination thereof.

[00112] Biological materials associated with the ECM can include, for example, collagen type I, collagen type IV, laminin, fibronectin, elastin, reticulin, hygroscopic molecules,

glycosaminoglycanse, roteoglycans, glycocalyx, bovine serum albumin, Poly-L-lysine, Poly-D- lysine, or Poly-L-ornithine. The gelatin can be from an animal source, for example, the gelatin can porcine or bovine.

[00113] The monolayer of cells used in the substrate can be, for example, mammalian cells, endothelial cells, vascular cells, venous cells, capillary cells, human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HLMVEC). The cell lines can be obtained from a primary source or from an immortalized cell line. The monolayer of cells can be irradiated by ultraviolet light or X-ray sources to cause senescence of cells. The monolayer can also contain a mixture of one or more different cell types. The different cell types may be co- cultured together. One non- limiting example of co-culture is a combination of primary human endothelial cells co-cultured with transgenic mouse embryonic fibroblasts mixed to form a monolayer.

[00114] The binding surface layer of the substrate can contain, for example, a mixture of intracellular components. One method that can be used to obtain a mixture of intracellular components is lysis of the cells and collection of the cytosolic components. The lysed cells can be primary or immortalized. The lysed cells can be from either mono- or co-cultures.

[00115] The binding surface layer of the substrate can contain biological materials associated with the extracellular matrix (ECM) or binding moieties. For example, gelatin can be mixed directly with cells, binding moieties, biological materials associated with the ECM, or any combination thereof, to make a binding surface layer for the substrate. For example, the binding surface layer can be comprised of a gelatin mixed with a collagen.

[00116] The substrate can have one or more middle layers. The middle layer of the substrate can be one or more monolayers of cells. The cells of the monolayer can be of varying origin. For example, the middle layer of the substrate can be made by growing a confluent monolayer of mouse embryonic fibroblasts on the base layer and then growing another layer of cells, for example, the binding surface layer, on top of the confluent mouse embryonic fibroblasts. [00117] A feeder layer can be used in the substrate for growth or reprogramming of the cells. A feeder layer can sit adjacent to a base layer and can be separated from the binding surface layer of the substrate. The feeder layer can be a monolayer of feeder cells. The cells of the monolayer can be of varying origin. For example, the feeder layer can be made by growing a monolayer of human endothelial cells or mouse embryonic fibroblasts on a base layer.

[00118] Conjugation of layers of the substrate can be done by allowing cells to grow in a monolayer on top of the base layer or middle layer. Conjugation of layers can also be done by pre-treating the surface with a surface of either net positive, net negative, or net neutral charge. The conjugation procedure can be aided by chemical moieties, linkers, protein fragments, nucleotide fragments, or any combination thereof.

[00119] The media used for growing the cells can be supplemented or made with culture media that has been collected from cell cultures, blood plasma, or any combination thereof. The enrichment media can be, for example, Plating Culture Medium, Type R Long Term Growth Medium, Type DF Long Term Growth Medium, Type D Long Term Growth Medium, and MEF - Enrichment Medium, or any combination thereof. The enrichment medium can contain, for example, a primary nutrient source, animal serum, ions, elements, calcium, glutamate, magnesium, zinc, iron, potassium, sodium, amino acids, vitamins, glucose, growth factors, hormones, tissue extracts, proteins, small molecules, or any combination thereof. In some embodiments, the culture media used for transfection does not contain serum.

[00120] Non-limiting examples of amino acids include essential amino acids, phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, and histidine, arginine, cysteine, glycine, glutamine, proline, serine, tyrosine, alanine, asparagine, aspartic acid, glutamic acid, or any combination thereof.

[00121] Non-limiting examples of growth factors include Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Fibroblast Growth Factor (FGF), Stem Cell Factor (SCF), Insulin-like Growth Factor (IGF), Transforming Growth Factor-beta (TGF-β), or any combination thereof.

[00122] Non- limiting examples of hormones include peptide hormones, insulin, steroidal hormones, hydrocortisone, progesterone, testosterone, estrogen, dihydro testosterone, or any combination thereof.

[00123] Non-limiting examples of tissue extracts include pituitary extract. Non-limiting examples of small molecule additives include sodium pyruvate, endothelin-1, transferrin, cholesterol, or any combination thereof.

[00124] The culturing conditions in a method of the invention can be adjusted to simulate oxygen and pressure levels found, for example, in pathological conditions. The oxygen level used during culturing conditions can be, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% oxygen in the incubator. In some embodiments, the cells can be grown under hypoxic conditions during transfection.

[00125] The culturing condition in a method of the invention can be adjusted to simulate the pressure found, for example, in pathological conditions. The pressure used during culturing conditions can be about 0 PSI, about 0.1 PSI, about 0.15 PSI about 0.2 PSI, about 0.25 PSI, about 0.3 PSI, about 0.35 PSI, about 0.4 PSI, about 0.45 PSI, 0.5 PSI, about 0.55 PSI, about 0.6 PSI, about 0.65 PSI, about 0.7 PSI, about 0.75 PSI, about 0.8 PSI, about 0.85 PSI, about 0.9 PSI, about 0.95 PSI, about 1 PSI, about 1.1 PSI, about 1.2 PSI, about 1.3 PSI, about 1.4 PSI, about 1.5 PSI, about 1.6 PSI, about 1.7 PSI, about 1.8 PSIG, about 1.9 PSI, about 2 PSI, about 2.1 PSI, about 2.2 PSI, about 2.3 PSI, about 2.4 PSI, about 2.5 PSI, about 2.6 PSI, about 2.7 PSI, about 2.8 PSI, about 2.9 PSI, about 3 PSI, about 3.5 PSI, about 4 PSI, about 4.5 PSI, about 5 PSI, about 6 PSI, about 7 PSI, about 8 PSI, about 9 PSI, or about 10 PSI. A pressure used in a method disclosed herein can be an above atmospheric pressure value. A pressure used in a method disclosed herein can be positive pressure.

[00126] The culturing condition in a method of the invention can be adjusted to simulate the pressure found, for example, in pathological conditions. The pressure used during culturing conditions can be a PSI gauge (PSIG) reading of, for example, about 0.5 PSIG, about 0.6 PSIG, about 0.7 PSIG, about 0.8 PSIG, about 0.9 PSIG, about 1 PSIG, about 1.1 PSIG, about 1.2 PSIG, about 1.3 PSIG, about 1.4 PSIG, about 1.5 PSIG, about 1.6 PSIG, about 1.7 PSIG, about 1.8 PSIG, about 1.9 PSIG, about 2 PSIG, about 2.5 PSIG, about 3 PSIG, about 3.5 PSIG, about 4 PSIG, about 4.5 PSIG, about 5 PSIG, about 6 PSIG, about 7 PSIG, about 8 PSIG, about 9 PSIG, about 10 PSIG, about 15 PSIG, about 20 PSIG, about 25 PSIG, about 30 PSIG, about 35 PSIG, about 40 PSIG, about 45 PSIG, about 50 PSIG, or about 55 PSIG.

[00127] The pressure used during culturing conditions can be, for example, about 3.45 kPa, about 4.14 kPa, about 4.83 kPa, about 5.52 kPa, about 6.21 kPa, about 6.89 kPa, about 7.58 kPa, about 8.27 kPa, about 8.96 kPa, about 9.65 kPa, about 10.3 kPa, about 11 kPa, about 11.7 kPa, about 12.4 kPa, about 13.1 kPa, about 13.8 kPa, about 17.2 kPa, about 20.7 kPa, about 24.1 kPa, about 27.6 kPa, about 31 kPa, about 34.4 kPa, about 41.4 kPa, about 48.3 kPa, about 55.2 kPa, about 62.1 kPa, about 68.9 kPa, about 103 kPa, about 138 kPa, about 172 kPa, about 207 kPa, about 241 kPa, about 276 kPa, about 310 kPa, about 345 kPa, or about 379 kPa.

[00128] The pressure used in a method of the invention can be delivered continuously or via pulses of pressure produced by repeated depressurizations and pressurizations of an incubator used in the method. The pulses of pressure can be separated by, for example, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours.

[00129] The pH of the media used in a method of the invention can be, for example, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.55, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.5, about 6, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, or about 11 pH units.

[00130] The viscosity of the media can be adjusted by, for example, at least 0.001 Pascal-second (Pa.s), at least 0.001 Pa.s, at least 0.0009 Pa.s, at least 0.0008 Pa.s, at least 0.0007 Pa.s, at least 0.0006 Pa.s, at least 0.0005 Pa.s, at least 0.0004 Pa.s, at least 0.0003 Pa.s, at least 0.0002 Pa.s, at least 0.0001 Pa.s, at least 0.00005 Pa.s, or at least 0.00001 Pa.s depending on the cell types being cultured.

[00131] The oxygen solubility of the media can be, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[00132] In some embodiments, a culture media used for a method described herein can contain, for example, an L-alanine-L-glutamine dipeptide, B27 TM supplement, human bFGF, human EGF, human HGH, 1 mg/mL human insulin, 0.55 mg/mL human transferrin, 0.5 μg/mL sodium selenite, beta-mercaptoethanol, non-essential amina acids solution, and high glucose media.

[00133] In some embodiments, for PBMC or CD8+ cell culture, a culture media for a method described herein can contain, for example, PHA-P (10 μg/mL), IL-2 (100 U/mL), IL-4 (20 ng/mL), IL-15 (100 ng/mL), GM-CSF (20 ng/mL), and LPS (100 ng/mL).

[00134] The oxygen concentration used in a method disclosed herein can be used to mimic oxygen concentration found in, for example, solid tumors (about 1.1%), muscle (about 3.8%), prostate (about 3.9%), brain (about 4.4%), peripheral tissues (about 5.3%), venous blood (about 5.3%), lung (about 5.6%), bone marrow (about 6.4%), intestinal tissue (about 7.6%), kidney (about 9.5%), and arterial blood (about 13.2 %).

[00135] The pressure conditions used in a method disclosed herein can be used to mimic the interstitial fluid pressure found in, for example, normal breast (about 0.02 PSI), normal skin (about 0.04 PSI), lymphoma (about 0.14 PSI), brain tumors (about 0.15 PSI), sarcoma (about 0.17 PSI), lung carcinoma (about 0.25 PSI), rectal carcinoma (about 0.33 PSI), breast carcinoma (about 0.37 PSI), head and neck carcinoma (about 0.41 PSI), metastatic melanoma (about 0.43 PSI), colorectal carcinoma liver metastases (about 0.43 PSI), cervical carcinoma (about 0.44 PSI), ovarian carcinoma (about 0.48 PSI), and renal cell carcinoma (about 0.72 PSI).

Therapeutic Uses.

[00136] Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants. Subjects can be non-human animals, for example, a subject can be a mouse, rat, cow, horse, donkey, pig, sheep, dog, cat, or goat. A subject can be a patient.

[00137] A method disclosed herein can be used to identify a therapeutic, a biomarker, a genetic mutation, or a therapeutic target for, for example, stem cell differentiation or differentiation of various cell types.

[00138] Genomic, proteomic, and metabolic analysis can be conducted on the transfected cells to, for example, identify biomarkers that can be used for development of cancer therapies, drug development, cancer vaccines, cancer screening, diagnostics, personalized antibody development, hematopoietic stem cell transplantation, organ transplantation, or cardiovascular disease treatment. A method described herein can be used to induce phenotypic and genotypic changes in cells to determine the effect of cancer therapies. The cancer therapies can include, for example, chemo therapeutics or gene therapy.

[00139] Non-limiting examples of cancers that can be analyzed in a method disclosed herein include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bo ne/osteo sarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer,

osteo sarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,

Waldenstrom macroglobulinemia, and Wilms tumor.

[00140] Methods that can be used to determine the presence of, for example, biological markers or transfection of desired genes can include, for example, qPCR, RT-PCR, immunofluorescence, immunohistochemistry, western blotting, high-throughput sequencing, or mRNA sequencing. Computer Systems.

[00141] A method of the invention can be used to, for example, sequence, image, or characterize the transfected cells. Further methods can be found in PCT/US 14/13048, the entirety of which is incorporated herein by reference.

[00142] The invention provides a computer system that is configured to implement the methods of the disclosure. The system can include a computer server ("server") that is programmed to implement the methods described herein. FIGURE 6 depicts a system 600 adapted to enable a user to detect, analyze, and process images of cells and sequence cells. The system 600 includes a central computer server 601 that is programmed to implement exemplary methods described herein. The server 601 includes a central processing unit (CPU, also "processor") 605 which can be a single core processor, a multi core processor, or plurality of processors for parallel processing. The server 601 also includes memory 610 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 615 (e.g. hard disk); communications interface 620 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 625 which may include cache, other memory, data storage, and/or electronic display adaptors. The memory 610, storage unit 615, interface 620, and peripheral devices 625 are in communication with the processor 605 through a communications bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit for storing data. The server 601 is operatively coupled to a computer network ("network") 630 with the aid of the communications interface 620. The network 630 can be the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a telecommunication or data network. The network 630 in some cases, with the aid of the server 601, can implement a peer-to-peer network, which may enable devices coupled to the server 601 to behave as a client or a server. The microscope and micromanipulator can be peripheral devices 625 or remote computer systems 640.

[00143] The storage unit 615 can store files, such as individual images, time lapse images, data about individual cells, cell colonies, or any aspect of data associated with the invention. The data storage unit 615 may be coupled with data relating to locations of cells in a virtual grid.

[00144] The server can communicate with one or more remote computer systems through the network 630. The one or more remote computer systems may be, for example, personal computers, laptops, tablets, telephones, Smart phones, or personal digital assistants. [00145] In some situations the system 600 includes a single server 601. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the Internet.

[00146] The server 601 can be adapted to store cell profile information, such as, for example, cell size, morphology, shape, migratory ability, proliferative capacity, kinetic properties, and/or other information of potential relevance. Such information can be stored on the storage unit 615 or the server 601 and such data can be transmitted through a network.

[00147] Methods as described herein can be implemented by way of machine (e.g., computer processor) computer readable medium (or software) stored on an electronic storage location of the server 601, such as, for example, on the memory 610, or electronic storage unit 615. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610. Alternatively, the code can be executed on a second computer system 640.

[00148] Aspects of the systems and methods provided herein, such as the server 601, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium (e.g., computer readable medium). Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless likes, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[00149] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, tangible storage medium, a carrier wave medium, or physical transmission medium. Non-volatile storage media can include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such may be used to implement the system. Tangible transmission media can include: coaxial cables, copper wires, and fiber optics (including the wires that comprise a bus within a computer system). Carrier- wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH- EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables, or links transporting such carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

EXAMPLES

EXAMPLE 1: Transfection efficiency using a method disclosed herein.

[00150] FIGURE 2 depicts an experimental procedure for comparison of electroporation versus a method of the invention, which includes hypoxic and high pressure conditions. The cells were cultured in either a standard C0 ₂ incubator or under hypoxic and positive pressure conditions. The cells were further cultured in media and a substrate that contained FBS or serum-free media and a serum-free substrate.

[00151] FIGURE 3 depicts the results of the transfection of DU145 (human prostate cancer) cells. 5 x 10 ^A 6 cells/mL were transfected with 0.5 μg GFP at 1260 V for 40 ms with 2 pulses. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIGURE 3 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum- free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom-right panel.

[00152] FIGURE 4 depicts the results of the transfection of human dermal fibroblast cells. 5 x 10 ^Λ 6 cells/mL were transfected with 0.5 μg GFP at 1000 V for 30 ms with 1 pulse. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIGURE 4 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum- free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom-right panel.

[00153] FIGURE 5 depicts the results of the transfection of healthy donor peripheral blood mononuclear cells (PBMCs). 2 x 10 ^A 7 cells/mL were transfected with 1 μg GFP at 1500 V for 10 ms with 1 pulse. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIGURE 5 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum- free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom panels.

[00154] TABLE 1 below provides a quantitative analysis of the change in fold expression of the GFP plasmid using different methods.

EXAMPLE 2: Transfection of human dermal fibroblasts.

[00155] FIGURE 18 depicts the transfection of human dermal fibroblasts under 21% 0 ₂ and 0 PSI, 5% 0 ₂ and 0, 2, or 5 PSI, and 1% 0 ₂ , and 0, 2, or 5 PSI. The cells were transfected with a GFP plasmid and imaged 48 hours post-transfection. The experiments were repeated in triplicate. FIGURE 18 shows that transfection efficiency increased at lower oxygen and higher pressure conditions as indicated by brighter GFP expression.

[00156] FIGURE 19 provides a quantitative analysis of GFP transfection of human dermal fibroblasts grown under various hypoxic and high pressure conditions in FIGURE 18. The results indicate that cells grown under the hypoxic and positive pressure conditions provided higher GFP transfection efficiency than cells grown under 21% 0 ₂ and 0 PSI.

[00157] FIGURE 53 shows the transfection of human dermal fibroblasts using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 2 PSI pressure provided the greatest proportion of transfected cells (52.8% GFP+ cells versus 9.4% GFP+ under standard culture conditions).

[00158] FIGURE 54 shows the transfection of PBMCs using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 5 PSI pressure provided the greatest proportion of transfected cells (7.8% GFP+ cells versus 3.7% GFP+ under standard culture conditions).

[00159] FIGURE 55 shows the transfection of activated CD8+ T-cells using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 5 PSI pressure provided the greatest proportion of transfected cells (55.6% GFP+ cells versus 3.7% GFP+ under standard culture conditions).

[00160] FIGURE 56 shows the post-transfection effects of CD8+ T-cells using low oxygen and high pressure conditions. The results indicated that lower oxygen (10% (¾ and positive pressure (5 PSI) promotes cell proliferation post-transfection (1.1 X 10 ⁵ cells under higher oxygen and positive pressure versus 3 X 10 ⁴ cells under standard conditions 2 days after transfection).

EXAMPLE 3: Transfection of immune cells.

[00161] FIGURE 20 depicts a sample workflow for transfection of immune cells using a method disclosed herein. FIGURE 20 shows that after a sample is obtained from a donor, the sample can be enriched for CD8+ cells. A DNA plasmid is transfected using electroporation. The transfected cells are then subjected to decreasing oxygen levels and either 0 PSI or high pressure conditions. Then, the percent GFP-positive cells, percent viable cells, and relative expression of GFP in the cells are assessed.

[00162] FIGURE 21 shows the transfection of peripheral blood mononuclear cells (PBMC) with a GFP plasmid using electroporation of cells at passage zero. The 2-5 PSI pulsed pressure condition was performed at a frequency of 30 minute pulses. The experiments were repeated four times. FIGURE 22 depicts a quantification of the results from FIGURE 21 indicating that there was an almost 2.5-fold increase in GFP-positive cells using the hypoxic and positive pressure conditions compared to standard incubator conditions.

[00163] FIGURE 23 shows a comparison between the transfection of CD8+ cells enriched from PBMC and PBMC with a GFP plasmid using electroporation of cells at passage zero. The experiment was performed in triplicate. FIGURE 24 depicts a quantification of the results from FIGURE 23 indicating that enriched CD8+ cells have higher transfection efficiency than the PBMC. "ST" in FIGURE 24 denotes standard culture conditions. [00164] FIGURE 25 shows that the GFP-transfected CD8+ cells cultured under hypoxic and positive pressure conditions developed more multicellular clusters than did cells grown at standard incubator conditions. In each panel, the first number indicates the oxygen level; the second number represents the pressure level in PSI. The top left panel is a control without transfection. FIGURE 26 shows the percent GFP in the multicellular clusters in cells grown under hypoxic and positive pressure conditions compared to cells grown under standard incubator conditions. FIGURE 27 is a quantification of the results of FIGURE 26. "ST" in FIGURE 27 denotes standard culture conditions.

[00165] FIGURE 47 shows that hypoxic and positive pressure conditions can lead to greater enrichment of CD8+ cells from fresh blood samples than culture under standard incubator conditions. For example, FIGURE 47 shows that 8.3 million CD8+ cells were obtained after one week under hypoxic and positive pressure conditions versus 3 million CD8+ cells obtained under standard conditions. An expanded culture time up to 11 days is shown in FIGURE 48 indicating that the culture under hypoxic and positive pressure conditions generates more CD8+ cells from whole blood than culture under standard conditions.

EXAMPLE 4: Immune Cell Genome Editing.

[00166] A method disclosed herein can be used to introduce the CRISPR/Cas9 system into immune cells, for example, CD8+ T-cells, as shown in FIGURES 28-30. PBMCs were freshly isolated from healthy donors, and post-cell counting PBMCs were enriched for CD8+ cells. The CD8+ cells were cultured at 2 million cells/mL in IL-2 containing media in a standard incubator, or with PBMC media under low oxygen and positive pressure conditions. Once the cells demonstrated doubling times of 36-48 hours, the cells were transfected at 20 million cells/ml with 1 μg/μl CTLA4 CRISPR Knockout and a GFP HDR DNA plasmid. The cells were expanded under low oxygen and positive pressure conditions or under conventional culture conditions. Images were taken 1, 3, and 5 days post-transfection prior to cell splitting and counting. Quantification was performed through cell counting across entire wells and assessed as GFP+ multicellular clusters/total multiceullar clusters at 5 days post-transfection as well as total cell count at 5 days post-transfection.

[00167] FIGURE 28 shows that when a CRISPR/Cas9 system was used to knockout CTLA4, and knock-in GFP using homology-directed repair, the transfection efficiency of the

CRISPR/Cas9 system was higher in the cells grown under hypoxic and high pressure conditions than in standard incubator conditions. This is indicated by the bright GFP signal seen in the top panels of FIGURE 28. The results also indicate that the GFP expression persisted through subsequent expansion of the CD8+ cells for at least five days. The experiment was repeated four times, and the cells were transfected at passage 1+ while in the exponential growth phase.

[00168] FIGURES 29 and 30 provide a quantification of the results of from FIGURE 28.

FIGURE 29 shows that the cells grown under hypoxic and positive pressure conditions developed a higher percentage of GFP-positive multicellular clusters than the cells grown at standard culture conditions. FIGURE 30 shows that the proliferation of the CD8+ cells grown under hypoxic and positive pressure conditions was enriched over the cells grown under standard incubator conditions. The error bars represent standard deviation.

[00169] FIGURE 31 depicts a limited dilution assay workflow to assess GFP-positive colonies using the CRISPR/Cas9 system. A sample is taken from a donor and then subjected to

enrichment for CD8+ cells. Then, the cells are split to be cultured under either positive pressure and low oxygen conditions or under standard incubator conditions. The cells are then transfected using electroporation using a guide RNA and a homo logy-directed repair plasmid to effect the genomic GFP insertion. The cells are then expanded under either positive pressure and low oxygen conditions or under standard incubator conditions. After one week, the GFP-positive cells are assessed.

[00170] FIGURE 32 shows that less input of T-cells still allowed for successful genome editing of the CD8-positive T-cells as indicated by the GFP signal visible in the cells even at the lowest concentration of 5000 cells. TABLE 2 below provides a quantification of the results of the

FIGURE 32. The results indicate that a use of hypoxic and positive pressure conditions to culture cells provided 45-times increased transfection efficiency over standard culture conditions.

The p-value of the experiment was 1.68 xlO ^" 18.

[00171] FIGURES 59-60 provide molecular confirmation of the genome editing experiments performed above. FIGURE 59 provides data relating to the PD1 knockout, and FIGURE 60 provides data relating to the CTLA4 knockout. Both figures show that the highlighted bands in the DNA gels (upper panels) were extracted and used as a template for sequencing. For FIGURE 59, genome editing was confirmed for Donors 76, 78, 82, and MOLT4. The PDl-pos- HDR was a positive control from previous MOLT4 knockout cells. For FIGURE 60, genome editing was confirmed for Donors 74, 75, and 76.

EXAMPLE 5: Reprogramming of cells using a method of the invention.

[00172] FIGURE 7 depicts a workflow of a stem cell reprogramming experiment using a method of the invention. Prior to initiation of the reprograming process, human fibroblasts were plated in fibroblast medium until the cells reached a desired confluency. The cells were cultured either under standard conditions or under hypoxic and positive pressure conditions. The fibroblasts were then transfected with a RNA vector encoding Oct4, KLF-4, SOX2, GLIS 1, and c-MYC, and a puromycin resistance gene. After 5 days of puromycin selection post-transfection, the cells were cultured in reprogramming media for the remainder of the reprogramming induction phase until the induced pluripotent stem cell (iPSC) colonies emerged. Recombinant B 18R Protein was also added during the first 2 weeks after transfection to inhibit the interferon response and increase cell viability. After about 20 days, the iPSC colonies were isolated and propagated in maintenance media. The maintenance media was a complete, serum-free media designed for the feeder-free maintenance and expansion of stem cells. The maintenance media contained recombinant human basic fibroblast growth factor and recombinant human

transforming growth factor β. The reprogramming media was a complete, xeno-free defined reprogramming media designed for generating iPSCs under feeder-free conditions.

[00173] The colonies formed from standard or hypoxic and high pressure conditions were assessed for various markers. FIGURE 8 shows that by day 19, the average fold increase in colony number was higher in cells grown under hypoxic and either standard or high pressure conditions as compared to standard conditions (18% oxygen and 0 PSI). For example, cells grown under 5% oxygen and 0 PSI had about a 13-fold increase in colony number as compared to cells grown in 18% oxygen and 0 PSI, which increase was statistically significant. Further, cells grown under 5% oxygen and 2 PSI had about a 10-fold increase in colony number as compared to cells grown in 18% oxygen and 0 PSI, which increase was statistically significant.

[00174] FIGURE 9 depicts the frequency distribution of colony area between cells cultured in 18% oxygen; 0 PSI, 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The graph illustrates that the colony area (measured in μιη ), was greater in cells grown under hypoxic and either standard or positive pressure conditions.

[00175] FIGURE 10 shows microscopy images of the morphology of cells grown at 18% oxygen; 0 PSI, 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The images show that cells grown under hypoxic and either standard or high pressure conditions had a greater cell area than those cells grown under standard conditions.

[00176] FIGURE 11 shows the reprogramming kinetics of cells grown under 18% oxygen; 0 PSI (standard), 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The graph shows that cells grown under hypoxic and either standard or positive pressure conditions had a higher rate of reprogramming, as indicated by the increase in stem cell colony count per days post-transfection, as compared to cells grown under standard conditions.

[00177] FIGURE 12 shows the effect of hypoxia and positive pressure conditions on pre- cardiomyocyte differentiation and morphology. H9c2 pre-cardiomyocytes were cultured under 20% oxygen; 0 PSI (standard conditions), 5% oxygen; 2 PSI, and 1% oxygen; 5 PSI. The cells were then stained with DAPI to identify the nuclei of the cells, F-actin to visualize the individual cells, and cardiac troponin, a marker of cardiomyocytes. The results indicated that with decreasing oxygen levels and increasing pressure levels, the cells expressed increasing levels of cardiac troponin as shown by the more intense staining in the cytoplasm of the cells.

[00178] FIGURE 13 shows staining of fibroblasts with DAPI to identify the nuclei of the cells and Sox2, a stem cell marker. After removal of pluripotency supporting mouse embryonic fibroblasts (MEFs), the cells grown under standard conditions (20% oxygen and 0 PSI) differentiated, while the cells grown under 1% oxygen and 2 PSI conditions maintained their dedifferentiated state. The differentiation was indicated by the Sox2 staining and the morphology of the cells after culture in the varying oxygen and pressure conditions. The cells were assessed for other stem cells markers by qPCR as shown in FIGURE 14.

[00179] FIGURE 14 shows that the cells grown under 1% oxygen and 2 PSI had higher levels of Nanog, Oct4, and Sox2 as compared to cells grown under standard conditions.

[00180] FIGURE 15 shows staining of fibroblasts 23 days post-transfection of the

reprogramming RNA vector as described above under 20% oxygen; 0 PSI (standard conditions); 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI. The cells were stained with DAPI, Sox2, and SSEA4, the latter two of which are stem cells markers. Cells of the same size from each condition were analyzed for expression of SSEA4, a human embryonic stem cell marker. The results indicated that the cells grown under hypoxic and either standard or high pressure conditions showed greater staining for SSEA4, as indicated by the more intense staining around the periphery of the cells in FIGURE 15. FIGURE 16 shows the average colony area over the experimental period for the aforementioned conditions.

[00181] FIGURE 49 shows induction of neural precursor markers, PAX6 and NESTIN, in iPSCs after two weeks in culture under 5% 0 ₂ and 2 PSI in stem cell maintenance media. [00182] FIGURE 33 shows that a combination of low oxygen and positive pressure enhances ectoderm commitment in defined medium, while causing changes in colony morphology to more mesoderm-like morphology. The directed differentiation of iPSCs to all three germ layers was performed using defined medium under the indicated cell culture conditions. Each experiment was performed in triplicate using independent iPSC reprogrammed cells lines at passage 5. The results indicate that the brachyury (mesoderm indicator) was more prominent along the edges of the cells under culture at hypoxic and high pressure conditions. FOXA2 (endoderm indicator) was more prominent in cells under culture at hypoxic and high pressure conditions. Finally, PAX6 (ectoderm indicator) was more highly expressed in the cells under grown under low oxygen and high pressure conditions.

[00183] FIGURE 34 shows that induction of PAX6 in iPSCs was accompanied by a loss of E- cadherin (indicated by CDHl staining) under conditions of low oxygen and positive pressure (A; left panel is PAX6 staining; right panel is CDHl staining). Additionally, SOX2, SSEA4, and NANOG staining decreased at low oxygen and positive pressure conditions, while OCT4 remained fairly constant throughout the three experimental conditions (B; left panel is SOX2 staining, right panel is SSEA4 staining. C; left panel is OCT4 staining, right panel is NANOG staining).

[00184] FIGURE 17 shows the gene expression profiles as a function of oxygen concentration and pressure as compared to standard incubation conditions. The results indicated that oxygen concentration and pressure had an effect on the gene expression profile of several iPSC marker genes of interest. FIGURE 45 shows the effect that various oxygen and pressure conditions had on the gene expression of immunotherapeutic targets in donor PBMCs. The cell culture conditions were created to mimic the vasculature and tumor microenvironments. The sample size for each condition was 12. The results of FIGURE 45 are quantified (on a logarithmic scale) in TABLE 3 below.

CD8A 10.58403 10.74872 11.73939 11.03479 11.5482 11.26316

IL1B 12.26734 12.36899 13.85733 13.13851 17.2512 16.41079

CTLA4 7.218212 7.548015 7.96451 8.191502 8.123371 8.438327

ICOS 8.459099 8.514474 9.260944 9.310994 9.568987 9.989088

PDCD1 5.298568 5.887669 5.870134 5.689587 6.187069 6.070108

CX3CR1 6.848331 6.521741 6.770097 6.755467 7.244198 7.098837

BTLA 6.550678 6.491376 7.731419 7.463986 8.060028 7.759103

IL12A 5.238696 4.780255 5.055161 4.893 4.910049 5.020782

CXCL9 4.88878 4.39225 4.078183 6.442779 5.653539 4.950788

CXCL10 6.477897 6.717857 5.181365 7.381816 4.957034 4.986273

CXCL13 8.705631 8.945058 8.734548 9.69251 9.49176 10.32852

IL13 3.67695 4.39225 4.078183 4.453252 3.883496 4.668556

IL4 4.262466 4.885367 4.356257 5.157029 3.736401 4.668556

HAVCR2 9.170135 9.146919 9.582496 9.055441 8.495221 8.383747

MICA 9.5119 9.485452 8.457327 8.581001 8.713503 8.310856

VEGFA 10.82009 10.66857 11.57516 10.85657 12.58646 12.20046

IL17A 4.394877 4.019712 3.658276 4.555729 5.5696 4.950788

IL6 10.67563 10.36244 9.83626 11.30245 13.4968 12.44336

FOXP3 7.649995 7.533283 8.282547 8.390411 8.306643 8.195861

CD40LG 6.142305 6.637095 6.475081 7.027289 6.392924 7.242464

IDOl 10.16081 10.41552 9.648315 11.00966 9.463818 10.17725

IL7 5.298568 5.509989 5.591429 5.876287 6.829225 6.680652

CD274 9.902727 9.830315 8.97929 9.246269 10.94272 10.59646

PDCD1LG2 9.004413 8.817339 6.071134 7.61544 6.963616 6.700756

IL33 7.31835 7.105141 3.066 10.27267 3.883496 3.066

IL15 7.450272 7.687581 7.290701 7.714615 7.036545 7.434076

PRF1 9.608044 9.799925 10.05587 10.23814 9.88916 10.17636

CD27 7.499824 7.739823 8.341071 7.842887 7.983301 8.152978

LAG3 7.703818 7.605478 7.701923 8.135379 8.251017 8.432363

CD4 9.963468 10.03466 10.39339 10.6011 10.25358 10.45002

IFNG 3.066 4.019712 4.356257 4.339943 4.701601 5.020782

CXCL1 13.46389 13.51905 14.50338 14.0498 15.36751 14.96332

CCL7 10.178 10.03205 11.89334 9.941749 6.974265 7.373004 CCL5 11.94975 12.08067 12.31532 12.19939 11.9531 12.05798

CD40 8.757388 8.690513 9.051137 8.895786 8.983975 8.414324

CD70 5.700041 5.446335 5.546533 6.159697 5.988991 5.736881

ICAM1 10.56529 10.50489 9.923909 10.34862 10.86652 10.23831

TGFB 1 11.26375 11.17533 11.36419 11.25554 11.26814 11.0061

CD33 7.582629 7.291983 9.093345 7.724167 7.278922 6.414203

[00185] TABLE 4 below shows the effect that high atmospheric pressure had on iPSCs grown under hypoxic conditions as assessed by digital PCR. The results indicate that that was a change in gene expression of various neuronal, bone, and cardiomyocyte factors. TABLE 4 shows relative gene expression changes with 1 being no change, and values above 1 indicating greater expression, and values below 1 indicating lower expression.

REST 1.323 1.651

HSPA9 1.276 1.060

GPI 1.274 1.253

GABRB3 1.264 0.821

CCNA2 1.259 1.173

MYBL2 1.258 1.165

CDK1 1.229 1.233

LEFTY1 1.225 0.360

TCF3 1.218 1.439

ALPL 1.209 1.119

DPPA3 1.208 0.531

SFRP2 1.197 1.921

GRB7 1.189 0.533

PSMB4 1.167 0.997

NAT1 1.151 0.792

APC 1.145 1.361

GDF3 1.138 0.443

REEP5 1.133 0.996

LIN28A 1.125 1.065

BRIX1 1.111 0.957

PSMB2 1.104 1.016

NUMB 1.090 1.090

HPRT1 1.089 0.774

AFP 1.082 1.135

CCNE1 1.073 0.977

PODXL 1.068 0.737

GJA1 1.067 1.177

PARD6A 1.060 0.766

KAT7 1.052 1.037

KAT8 1.019 0.843

POU5F1 0.982 0.548

RAB7A 0.973 1.062

CDH1 0.954 0.365

KAT2A 0.946 0.996

DNMT3B 0.935 0.589

CD9 0.894 0.398

FOXD3 0.880 0.343

DPPA2 0.876 0.425

MYCN 0.867 1.018

KLF4 0.806 1.032

MYC 0.759 0.822

TDGF1 0.757 0.291

TBX3 0.750 1.250 HDAC2 0.739 0.773

ALDH1A1 0.708 1.542

RUNX2 0.684 0.947

EMX2 0.667 44.000

ZFP42 0.606 0.464

NODAL 0.583 0.313

GAL 0.485 0.175

NANOG 0.485 0.182

NR5A2 0.218 0.113

FGF5 1.500

EXAMPLE 6: Change in gene expression in cancer cells.

[00186] FIGURE 35 show that different combinations of tumor (disease) extracellular matrix (ECM), low oxygen, and high pressure can alter the gene expression of EGFR and other metabolic regulators in DU145 (prostate cancer) and PanclO (pancreatic cell lines). The markers measured included EGFR, ErbB2, LDHA, SLC1A5, and TRX1. The results indicate that, generally, gene expression increased with low oxygen, high pressure, and tumor ECM conditions. However, for ErbB2 in DU145 cells, the ErbB2 expression decreased compared to standard incubator conditions when the cells were cultured under hypoxic and high pressure conditions.

[00187] FIGURE 36 shows that PDLl expression increased in ARV7-positive, 22RV1 prostate cancer cells during low oxygen and positive pressure culturing conditions. The top panel of

FIGURE 37 provides a western blot showing increased PDLl protein expression under various conditions of high pressure and hypoxia in both DU145 and 22Rvl prostate cancer cells. The bottom panel of FIGURE 37 provides a quantification of the western blot results normalized to actin. The results indicated that the change in PDLl expression was more pronounced in the 22Rvl prostate cancer cells, rather than the DU145 prostate cancer cells.

[00188] TABLES 5-10 below show the effect of varying oxygen and pressure on the gene expression profiles of various cancer targets when compared to traditional culturing approaches.

EGFR (higher) SPG7 (lower) PARP2 (lower)

HDAC3 (higher) CDK5 (lower) SSSCA1 (higher)

HPRT 1 (higher) RHOB (lower) ABCC 1 (lower) GP1 (higher) PI3KC2a (lower) P992CB (higher)

HK2 (higher) CDC25A (lower) CDK7 (lower)

PARP2 (lower) PLK4 (lower) HDAC3 (lower)

PLK1 (lower) PRPF40A (lower) ERBB2 (higher)

SSSCA1 (lower) MAN2B 1 (higher) PEA15 (higher)

AURKA (lower) HSP90B 1 (higher) HK2 (lower)

HRAS (lower) COL12A1 (lower) ENOl (lower)

NAA10 (lower) TXNRD1 (higher) ITGAV (lower)

[00189] FIGURE 38 shows identification of pressure and oxygen sensitive gene expression signatures in various cell lines. The results indicated that there was an enrichment of metabolic processes involved in cell survival. The results in the top panel of FIGURE 38 were corrected for any false discovery rate. The top panel of FIGURE 38 provides various gene ontology terms that were enriched under low oxygen or positive pressure conditions. The bars to the right of the 0 on the x-axis indicated upregulated genes, and the bars to the left of the 0 on the x-axis indicated downregulated genes. The bottom panel of FIGURE 38 shows that over 130 genes were shown to be co-regulated by low oxygen and positive pressure conditions across various cell lines.

[00190] FIGURES 57 and 58 show the effect of various experimental conditions on various cells lines. FIGURE 57 shows pressure-sensitive genes, while FIGURE 58 depicts oxygen- sensitive genes. The cells lines tested for both figures was (from left to right) PANCIO, LNCaP, PC3, and 22Rvl. Within each group of cell lines, the cells were exposed to high (>18%; leftmost columns of cells) and low (<5%; right-most columns of cells) oxygen, low pressure (0 PSI; left-most columns of cells) and positive pressure (2 PSI; right-most columns of cells). TABLES 11-16 below provide the quantitative data for the heatmaps of FIGURES 57-58.

TABLE 13 - Oxy gen-Sensitive Candidates

02 PSI Expt CellLine BNIP3 PPP1R3G KDM3A BHLHE40

LNCaP_25 20 0 A LNCaP 1884.493 0 336.6374 201.3058

22RV1_3 20 0 A 22RV1 377.7168 1.339421 310.7458 2.678843

PC3_3 20 0 A PC3 535.9478 26.82655 506.7885 2151.956

DU145_3 20 0 A DU145 1537.72 43.74737 534.8115 313.8873

PANC10_12 20 0 B PANC10 6.94829 13.89658 821.2879 187.6038

LNCaP_l 20 0 B LNCaP 2310.132 1.1352 353.0473 188.4432

22RV1_12 20 0 B 22RV1 455.3357 1.782136 336.8237 5.346407

DU145_12 20 0 B DU145 2355.062 58.90487 412.3341 404.4046

PANC10_21 20 0 C PANC10 4.41638 14.57405 1080.247 197.4122

LNCaP_10 20 0 C LNCaP 2106.632 0 274.8215 273.8222

22RV1_21 20 0 C 22RV1 384.4058 2.351106 355.0169 8.22887

PC3_25 20 0 C PC3 473.4583 21.48327 481.7211 1764.933

DU145_21 20 0 C DU145 1772.991 56.54185 465.4606 379.6382

PANC10_8 20 0 A PANC10 12.12097 7.272581 898.7699 173.3299

LNCaP_26 20 0 A LNCaP 1737.064 0 346.6829 209.8344

22RV1_6 20 0 A 22RV1 375.8526 0 370.718 1.02692

PC3_6 20 0 A PC3 652.4015 31.44985 544.1555 1584.195

DU145_6 20 0 A DU145 1697.921 55.25438 534.1256 370.6648

PANC10_15 20 0 B PANC10 11.38174 3.414522 1059.64 175.2788

LNCaP_4 20 0 B LNCaP 2423.8 0 315.4445 227.821

22RV1_15 20 0 B 22RV1 447.4213 0.974774 380.1618 5.848644

PC3_15 20 0 B PC3 859.6456 43.93141 709.411 1654.75

DU145_15 20 0 B DU145 2327.797 101.8222 502.054 634.1204

PANC10_24 20 0 C PANC10 9.549055 6.820754 841.681 201.4396

LNCaP_13 20 0 C LNCaP 2220.541 0 262.4169 267.1239

22RV1_24 20 0 C 22RV1 497.1909 4.220636 406.8693 11.81778

PC3_26 20 0 C PC3 486.6086 29.25811 480.449 1790.905

DU145_24 20 0 C DU145 1195.662 46.98399 460.1191 272.1831

PANC10_9 20 0 A PANC10 9.740092 6.493395 986.996 181.8151

LNCaP_27 20 0 A LNCaP 1826.249 0 368.8781 288.249

22RV1_9 20 0 A 22RV1 418.7484 2.052688 336.6409 2.052688

PC3_9 20 0 A PC3 882.2434 50.94172 802.1921 2231.359

DU145_9 20 0 A DU145 3016.435 74.80582 741.4577 861.367

PANC10_18 20 0 B PANC10 8.218557 8.218557 1145.667 261.3501

LNCaP_7 20 0 B LNCaP 2469.786 2.091267 397.3407 248.8608

22RV1_18 20 0 B 22RV1 360.8076 0 382.6358 1.284013

PC3_18 20 0 B PC3 726.6674 42.62574 606.9094 1504.756

DU145_18 20 0 B DU145 1682.782 56.06259 432.2245 348.1306

PANC10_27 20 0 C PANC10 3.428841 15.42979 917.215 231.4468

LNCaP_16 20 0 C LNCaP 2055.264 0 289.3514 295.5606

22RV1_27 20 0 C 22RV1 369.6848 3.038506 383.8645 10.12835

PC3_27 20 0 C PC3 432.4036 14.43532 459.9619 1728.958

DU145_27 20 0 C DU145 996.1885 0 0 110.6876

PC3_PSI_1 20 0 A PC3 1212.009 49.31381 1193.255 2956.051

PC3_PSI_2 20 0 B PC3 1408.695 53.87734 1015.128 3076.922 PC3_PSI_3 20 0 C PC3 1089.705 60.49494 1467.798 3081.262

PANC10_4 10 2 A PANCIO 6.812705 44.28258 1556.703 436.0131

PC3_2 10 2 A PC3 756.8398 47.30249 706.0334 1985.537

DU145_2 10 2 A DU145 1657.301 74.22405 546.156 310.19

PANC10_11 10 5 B PANCIO 7.344738 8.26283 893.3037 181.7823

LNCaP_2 10 5 B LNCaP 3307.754 0 434.7494 406.6103

22RV1_11 10 5 B 22RV1 422.9914 2.862886 388.6368 5.010051

DU145_11 10 5 B DU145 2160.376 45.84041 443.124 266.8147

PANC10_19 20 2 C PANCIO 6.672573 14.67966 898.1284 218.8604

PANC10_20 20 5 C PANCIO 6.763815 15.9872 863.3087 270.5526

LNCaP_ll 20 2 C LNCaP 2048.336 1.482154 385.3599 183.7871

LNCaP_12 20 5 C LNCaP 1867.387 1.741965 341.4252 209.0358

22RV1_19 20 2 C 22RV1 388.0382 6.424474 489.5449 3.854685

PC3_19 20 5 C PC3 448.7851 26.50305 519.8133 1988.082

PC3_22 20 2 C PC3 454.248 28.50929 527.1051 2062.805

DU145_19 20 2 C DU145 1517.187 52.92512 459.7221 214.8137

DU145_20 20 5 C DU145 1555.453 59.33068 324.3411 163.1594

LNCaP_23 10 2 A LNCaP 1740.446 0 395.007 222.5149

22RV1_5 10 2 A 22RV1 405.2257 3.508448 440.3102 0.877112

PC3_5 10 2 A PC3 759.6934 52.39265 621.007 1768.252

DU145_5 10 2 A DU145 2189.969 97.77537 631.5491 451.9617

PANC10_14 10 5 B PANCIO 12.60679 15.9686 1015.267 196.6659

LNCaP_5 10 5 B LNCaP 2971.391 2.617197 423.1135 247.7613

22RV1_14 10 5 B 22RV1 448.52 3.417295 387.0087 2.562971

PC3_14 10 5 B PC3 1152.941 83.42015 827.3535 1848.939

DU145_14 10 5 B DU145 2995.52 122.0707 499.0539 581.6312

PANC10_22 20 2 C PANCIO 8.039135 13.84518 803.5786 181.3272

PANC10_23 20 5 C PANCIO 12.50035 11.41336 705.9978 197.8316

LNCaP_14 20 2 C LNCaP 2168.046 0 295.1601 237.8268

LNCaP_15 20 5 C LNCaP 2407.96 0 291.3245 273.1923

22RV1_22 20 2 C 22RV1 427.6037 2.50794 408.7942 0

22RV1_23 20 5 C 22RV1 399.0337 0 342.8647 7.02112

PC3_20 20 5 C PC3 667.6491 40.60478 574.4578 1722.042

PC3_23 20 2 C PC3 632.2531 49.76066 530.2925 1614.782

DU145_22 20 2 C DU145 1321.005 34.14363 492.1391 250.779

DU145_23 20 5 C DU145 1298.832 57.50957 452.1137 222.0754

PANC10_6 10 2 A PANCIO 15.11898 16.79887 1069.248 212.9256

LNCaP_24 10 2 A LNCaP 2202.072 1.309978 421.8128 273.7853

22RV1_8 10 2 A 22RV1 425.7942 1.517983 412.8914 3.035966

PC3_8 10 2 A PC3 1170.217 52.93647 982.533 2756.707

DU145_8 10 2 A DU145 1988.923 83.53478 681.2063 594.6881

PANC10_17 10 5 B PANCIO 5.104033 15.3121 1093.397 250.0976

LNCaP_8 10 5 B LNCaP 2407.26 0 306.1569 268.2259

22RV1_17 10 5 B 22RV1 427.1449 2.64486 407.3084 3.96729

PC3_17 10 5 B PC3 858.4368 53.15173 736.115 1716.145

DU145_17 10 5 B DU145 1856.556 97.77131 515.2219 582.2337

PANC10_26 20 5 C PANCIO 7.498328 11.75414 959.3807 143.6842 LNCaP_17 20 2 C LNCaP 2044.425 1.269041 280.4581 291.8795

LNCaP_18 20 5 C LNCaP 2672.67 0 345.4538 241.9144

22RV1_25 20 2 C 22RV1 390.7094 3.412309 412.8894 3.412309

22RV1_26 20 5 C 22RV1 420.5327 1.605087 418.9277 3.210174

PC3_21 20 5 C PC3 500.5573 35.04888 484.267 1771.696

PC3_24 20 2 C PC3 491.581 24.89636 471.0781 1757.878

DU145_25 20 2 C DU145 1469.877 60.67242 375.8503 206.1983

DU145_26 20 5 C DU145 964.7252 0 241.1813 0

PC3_PSI_4 20 0.5 A PC3 1100.137 90.7685 1469.531 3095.321

PC3_PSI_7 20 2 A PC3 918.0267 43.90757 1006.738 2725.854

PC3_PSI_10 20 5 A PC3 907.5433 41.77361 820.3236 2921.858

PC3_PSI_5 20 0.5 B PC3 1087.4 70.17042 1380.18 3091.854

PC3_PSI_8 20 2 B PC3 913.2592 46.1511 1042.837 2678.539

PC3_PSI_11 20 5 B PC3 872.1883 46.45264 863.7788 3215.243

PC3_PSI_6 20 0.5 C PC3 1179.131 120.4338 1625.856 2820.392

PC3_PSI_9 20 2 C PC3 888.8183 68.85212 973.8403 2787.989

PC3_PSI_12 20 5 C PC3 916.3415 54.72688 1090.533 2917.877

PANC10_1 1 2 A PANCIO 10.44371 130.5464 2637.037 877.2717

LNCaP_19 1 2 A LNCaP 11168.95 2.913893 1041.717 926.6181

22RV1_1 1 2 A 22RV1 467.7926 1.430558 702.4041 0

PC3_1 1 2 A PC3 1877.161 114.1301 1523.597 3456.626

DU145_1 1 2 A DU145 4441.451 204.9732 1043.5 1381.103

PANC10_10 1 5 B PANCIO 10.04338 92.62231 2322.253 790.0795

LNCaP_3 1 5 B LNCaP 9018.051 6.091631 877.1949 565.3034

22RV1_10 1 5 B 22RV1 2023.365 16.86138 919.5071 21.35775

DU145_10 1 5 B DU145 4712.332 162.5827 842.8631 1135.512

PANC10_2 1 2 A PANCIO 11.57364 82.02187 3135.449 946.5223

22RV1_4 1 2 A 22RV1 1756.816 22.55541 1064.281 11.6954

PC3_4 1 2 A PC3 5423.365 169.4506 1770.238 5201.848

DU145_4 1 2 A DU145 4238.848 313.1314 1129.466 1706.992

PANC10_13 1 5 B PANCIO 6.359951 131.7418 2111.504 914.9243

LNCaP_6 1 5 B LNCaP 8195.015 6.04263 861.679 576.4669

22RV1_13 1 5 B 22RV1 1859.612 20.94158 894.2053 32.45944

PC3_13 1 5 B PC3 4191.345 268.4914 2037.781 5547.57

DU145_13 1 5 B DU145 5865.239 364.627 652.0583 1364.888

PANC10_3 1 2 A PANCIO 5.984618 133.1578 2558.424 951.5543

LNCaP_21 1 2 A LNCaP 8482.947 2.823884 1042.013 883.8756

22RV1_7 1 2 A 22RV1 3168.179 34.55534 1164.879 24.55248

PC3_7 1 2 A PC3 5787.271 200.7905 2149.898 6611.701

DU145_7 1 2 A DU145 4169.23 240.674 1067.794 1692.075

PANC10_16 1 5 B PANCIO 18.07991 142.5122 2173.843 1168.813

LNCaP_9 1 5 B LNCaP 7559.562 6.743587 987.9356 560.8417

22RV1_16 1 5 B 22RV1 1931.307 14.18093 937.2917 31.06298

PC3_16 1 5 B PC3 4419.143 254.2238 1826.518 4920.72

DU145_16 1 5 B DU145 8228.795 590.7299 304.37 633.954

1

TABL] 16 - Pressure-Sensil ive Candidates

02 PSI Expt CellLine CXorf38 PPID HIST1H2AG AC112229.1

LNCaP_25 20 0 A LNCaP 157.323 796.7649 159.0146 16.91645

22RV1_3 20 0 A 22RV1 300.0304 661.6742 45.54033 8.036528

PC3_3 20 0 A PC3 409.9796 470.0478 39.65664 2.915929

DU145_3 20 0 A DU145 150.9284 185.9263 76.55789 12.03053

PANC10_8 20 0 A PANC10 441.2033 994.5255 42.42339 67.87743

LNCaP_26 20 0 A LNCaP 162.3936 915.9727 149.621 12.77253

22RV1_6 20 0 A 22RV1 344.0181 634.6364 44.15755 17.45764

PC3_6 20 0 A PC3 330.5891 599.0099 70.945 1.462784

DU145_6 20 0 A DU145 146.1939 177.2745 65.61457 11.51133

PANC10_9 20 0 A PANC10 370.1235 1038.943 35.71367 81.16744

LNCaP_27 20 0 A LNCaP 183.4312 896.9986 165.2896 18.14155

22RV1_9 20 0 A 22RV1 283.271 607.5958 45.15914 14.36882

PC3_9 20 0 A PC3 358.2714 525.0916 66.61609 2.798996

DU145_9 20 0 A DU145 127.6099 254.1198 111.1086 9.90077

PC3_PSI_1 20 0 A PC3 346.5858 366.7281 66.67783 0

PANC10_12 20 0 B PANC10 362.7008 715.6739 119.5106 31.96214

LNCaP_l 20 0 B LNCaP 121.4664 863.8873 160.0632 18.1632

22RV1_12 20 0 B 22RV1 310.9827 687.9044 60.59261 5.346407

DU145_12 20 0 B DU145 159.7228 258.2752 147.2622 10.19507

PANC10_15 20 0 B PANC10 363.0775 1083.542 50.07966 71.70497

LNCaP_4 20 0 B LNCaP 128.0651 699.6397 163.1145 16.17664

22RV1_15 20 0 B 22RV1 297.3061 620.931 59.46121 9.74774

PC3_15 20 0 B PC3 318.9095 532.0582 42.84669 2.711816

DU145_15 20 0 B DU145 151.2211 228.8479 112.9117 14.11397

PANC10_18 20 0 B PANC10 350.1105 1150.598 93.69155 37.80536

LNCaP_7 20 0 B LNCaP 146.3887 928.5225 170.4383 9.410701

22RV1_18 20 0 B 22RV1 294.0389 608.622 60.34859 10.2721

PC3_18 20 0 B PC3 255.0779 515.5685 71.7195 2.706396

DU145_18 20 0 B DU145 183.5598 332.7586 141.0607 11.75506

PC3_PSI_2 20 0 B PC3 334.4338 421.1631 76.21673 0

PANC10_21 20 0 C PANC10 352.8687 1048.89 70.22044 51.67164

LNCaP_10 20 0 C LNCaP 141.9078 635.5872 101.9338 4.996755

22RV1_21 20 0 C 22RV1 353.8414 587.7764 62.3043 4.702211

PC3_25 20 0 C PC3 279.2825 552.7811 58.66586 1.652559

DU145_21 20 0 C DU145 148.4224 277.6609 101.9773 15.14514

PANC10_24 20 0 C PANC10 404.2433 807.1225 49.56414 69.11697

LNCaP_13 20 0 C LNCaP 131.7968 669.5749 147.0947 9.414058

22RV1_24 20 0 C 22RV1 324.989 683.743 112.2689 10.97365

PC3_26 20 0 C PC3 354.1772 461.9702 37.72757 3.849752

DU145_24 20 0 C DU145 183.0756 371.0115 98.8284 11.34096

PANC10_27 20 0 C PANC10 298.3092 756.0595 30.85957 39.43167

LNCaP_16 20 0 C LNCaP 136.6037 710.339 103.0737 3.725554

22RV1_27 20 0 C 22RV1 294.735 653.2787 100.2707 10.12835

PC3_27 20 0 C PC3 259.1797 490.1449 72.17662 4.593058 DU145_27 20 0 C DU145 110.6876 221.3752 221.3752 0

PC3_PSI_3 20 0 C PC3 331.9262 322.3744 66.86283 0

PANC10_4 10 2 A PANCIO 600.6535 778.9193 39.74078 38.60533

PC3_2 10 2 A PC3 472.4409 450.8336 30.36703 4.087869

DU145_2 10 2 A DU145 275.8476 124.076 42.09722 2.215643

LNCaP_23 10 2 A LNCaP 162.1426 550.2499 81.0713 10.34953

22RV1_5 10 2 A 22RV1 380.6666 448.2042 29.82181 3.508448

PC3_5 10 2 A PC3 372.9124 481.5501 50.85169 0

DU145_5 10 2 A DU145 246.4338 149.6562 30.92894 0.997708

PANC10_6 10 2 A PANCIO 419.9716 833.6437 47.4568 29.81799

LNCaP_24 10 2 A LNCaP 157.1973 672.0185 58.94899 2.619955

22RV1_8 10 2 A 22RV1 375.7008 509.2833 34.91361 6.830923

PC3_8 10 2 A PC3 401.836 542.9999 49.7282 0

DU145_8 10 2 A DU145 201.8757 157.1249 94.47386 1.988923

PC3_PSI_4 20 0.5 A PC3 328.605 267.7096 61.46981 0

PC3_PSI_7 20 2 A PC3 481.1912 353.0527 24.19397 0

PC3_PSI_10 20 5 A PC3 460.4279 359.4367 28.92019 0

PANC10_11 10 5 B PANCIO 454.4556 695.9139 33.05132 43.15033

LNCaP_2 10 5 B LNCaP 182.9043 536.0503 63.31302 14.06956

22RV1_11 10 5 B 22RV1 371.4595 611.9419 46.5219 5.725772

DU145_11 10 5 B DU145 228.0267 211.5711 117.5395 9.403162

PANC10_14 10 5 B PANCIO 415.1836 770.6951 51.26762 39.50128

LNCaP_5 10 5 B LNCaP 153.5422 827.0341 89.85708 5.234393

22RV1_14 10 5 B 22RV1 334.8949 598.0267 33.31863 5.980267

PC3_14 10 5 B PC3 423.3261 421.4585 32.99454 3.112692

DU145_14 10 5 B DU145 209.4351 163.9578 111.2998 9.574176

PANC10_17 10 5 B PANCIO 421.9334 943.679 47.07053 26.08728

LNCaP_8 10 5 B LNCaP 146.3051 621.7965 86.69929 10.83741

22RV1_17 10 5 B 22RV1 363.6682 566 31.73832 6.61215

PC3_17 10 5 B PC3 284.6894 405.555 58.97657 0.728106

DU145_17 10 5 B DU145 147.2062 210.9224 147.2062 6.591324

PC3_PSI_5 20 0.5 B PC3 335.8502 326.6554 55.16847 0

PC3_PSI_8 20 2 B PC3 469.4987 316.845 31.06324 0

PC3_PSI_11 20 5 B PC3 539.0108 343.1889 28.03176 0.400454

PANC10_19 20 2 C PANCIO 483.0943 533.8059 40.03544 20.01772

PANC10_20 20 5 C PANCIO 452.5607 662.2389 43.04246 30.74461

LNCaP_ll 20 2 C LNCaP 240.1089 422.4138 59.28615 4.446461

LNCaP_12 20 5 C LNCaP 158.5188 505.1699 74.9045 5.225895

22RV1_19 20 2 C 22RV1 442.0038 444.5736 50.1109 2.56979

PC3_19 20 5 C PC3 365.3888 425.8157 43.11164 2.826993

PC3_22 20 2 C PC3 442.2107 392.7946 48.14902 3.167699

DU145_19 20 2 C DU145 178.4925 199.2475 75.75556 4.150989

DU145_20 20 5 C DU145 176.0144 205.6797 74.16335 0.988845

PANC10_22 20 2 C PANCIO 467.6097 684.5547 33.94302 30.48172

PANC10_23 20 5 C PANCIO 519.5796 717.4112 30.97912 13.58733

LNCaP_14 20 2 C LNCaP 160.3208 605.1844 91.30852 6.370362

LNCaP_15 20 5 C LNCaP 130.5521 777.2683 126.9256 6.044077 22RV1_22 20 2 C 22RV1 321.0163 484.0324 68.96835 3.76191

22RV1_23 20 5 C 22RV1 349.8858 559.3492 65.53046 1.170187

PC3_20 20 5 C PC3 364.1117 422.6891 29.95435 1.331304

PC3_23 20 2 C PC3 389.304 441.5039 35.12517 2.439248

DU145_22 20 2 C DU145 191.9107 206.0391 70.64198 9.418931

DU145_23 20 5 C DU145 167.2201 223.845 98.20866 11.50191

PANC10_26 20 5 C PANCIO 404.7071 856.228 55.93347 14.99666

LNCaP_17 20 2 C LNCaP 148.4778 553.302 60.91398 7.614247

LNCaP_18 20 5 C LNCaP 188.6932 736.3874 87.08918 7.741261

22RV1_25 20 2 C 22RV1 365.117 479.4294 54.59694 3.412309

22RV1_26 20 5 C 22RV1 346.6987 537.7041 64.20347 1.605087

PC3_21 20 5 C PC3 274.961 448.2308 56.76932 0.493646

PC3_24 20 2 C PC3 313.4012 483.7704 50.76904 0.976328

DU145_25 20 2 C DU145 164.7058 204.7694 73.14764 4.451509

DU145_26 20 5 C DU145 0 241.1813 0 0

PC3_PSI_6 20 0.5 C PC3 270.2759 310.8873 47.61337 0

PC3_PSI_9 20 2 C PC3 498.1347 301.4888 28.16678 0

PC3_PSI_12 20 5 C PC3 464.5111 288.9846 37.37445 1.001101

PANC10_1 1 2 A PANCIO 579.626 704.9505 46.9967 62.66227

LNCaP_19 1 2 A LNCaP 166.0919 463.309 75.76123 8.74168

22RV1_1 1 2 A 22RV1 376.2368 414.8619 38.62507 2.861117

PC3_1 1 2 A PC3 580.228 418.2111 35.11696 3.990564

DU145_1 1 2 A DU145 178.6665 160.0325 46.03676 2.192227

PANC10_2 1 2 A PANCIO 587.7395 823.2379 30.1921 19.12166

22RV1_4 1 2 A 22RV1 435.2359 399.3143 36.75697 0.835386

PC3_4 1 2 A PC3 383.3937 441.1394 46.38591 0.946651

DU145_4 1 2 A DU145 218.0954 155.9565 42.64435 3.65523

PANC10_3 1 2 A PANCIO 408.4502 715.1619 91.26543 19.45001

LNCaP_21 1 2 A LNCaP 200.4958 553.4812 70.5971 2.823884

22RV1_7 1 2 A 22RV1 361.0124 435.5792 35.4647 1.818702

PC3_7 1 2 A PC3 437.861 455.3754 41.28404 1.251031

DU145_7 1 2 A DU145 223.8583 146.0859 98.79193 3.152934

PANC10_10 1 5 B PANCIO 398.3875 639.4287 20.08677 49.10099

LNCaP_3 1 5 B LNCaP 170.5657 650.5862 102.3394 13.40159

22RV1_10 1 5 B 22RV1 338.3516 611.506 69.69369 6.744551

DU145_10 1 5 B DU145 158.3042 209.6461 89.84835 8.556986

PANC10_13 1 5 B PANCIO 421.5739 652.3492 69.05089 19.98842

LNCaP_6 1 5 B LNCaP 160.734 729.9497 102.7247 14.50231

22RV1_13 1 5 B 22RV1 362.2893 560.1872 41.88315 3.141237

PC3_13 1 5 B PC3 443.7305 362.9954 26.91172 1.251708

DU145_13 1 5 B DU145 225.0176 197.0957 80.48074 4.927392

PANC10_16 1 5 B PANCIO 424.3461 656.1943 72.31963 22.334

LNCaP_9 1 5 B LNCaP 206.8033 877.7903 107.8974 8.99145

22RV1_16 1 5 B 22RV1 360.6007 596.2742 47.26976 2.701129

PC3_16 1 5 B PC3 352.1344 346.4086 57.25762 2.290305

DU145_16 1 5 B DU145 203.5136 72.04023 102.6573 0 EXAMPLE 7: Primary tumor culture.

[00191] To culture primary tumors using a method disclosed herein, target cells are isolated from a patient tumor. The cells are enriched for, for example, T-cells, dendritic cells, macrophages, B- cells, neutrophils, cancer cells, cancer stem cells, fibroblasts, and endothelial cells. The isolated cells are then co-cultured to re-establish tumor heterogeneity. To replicate the metastatic microenvironment, the cells are grown under low oxygen and high pressure conditions in an ex vivo setting. The cells are then subcutaneously injected into mice and downstream molecular assays are performed to determine gene expression changes.

[00192] FIGURES 50-52 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate. The cells were stained for DAPI. The cancerous cells were further stained for EpCAM (around periphery of cell) and CK7. The cells that did not get labeled with either CK7 or EpCAM represent the stromal cells derived from the biopsy.

[00193] FIGURE 42 shows the mutations found using the COSMIC database from pancreatic ductal adenocarcinoma (PDAC) and circulating tumor cells (CTC) cultured under low oxygen (1% 0 ₂ ) and positive pressure (2 PSI) using whole exome sequencing (top panels). The bottom panels of FIGURE 42 show that the NANOG and Wnt signaling pathways exhibited increased gene expression in both PDAC and CTC colonies as determined by mRNA sequening of the cells.

[00194] FIGURE 43 shows that there was increased ex vivo expansion of primary cells. The individual lines represent patient PBMC populations from cryopreserved blood samples. The viability of individual patient PBMC populations was tracked following transfection and subsequent recovery and expansion under low oxygen and positive pressure conditions.

FIGURE 44 shows that there was increased ex vivo expansion of primary cells. The individual lines represent patient PBMC populations from cryopreserved blood samples. The viability of individual patient PBMC populations was tracked following transfection and subsequent recovery and expansion under low oxygen and positive pressure conditions.

[00195] FIGURE 46 shows the results of the ex vivo culture and expansion of tumor- infiltrating lymphocytes (TILs) enriched from renal cell carcinoma tumors using positive pressure and low oxygen conditions. The experiments were performed in duplicate; RCC1 and RCC2 indicate the two different cell populations analyzed. For each data point, the left bar is RCC1 and the right bar is RCC2. The results indicate that 1% 0 ₂ and 2 PSI maintained the immune cell viability of CD3+, CD4+, CD8+, and CDl lb+ cell types, as indicated by FACS analysis. The two different tumors were additionally cultured in two different culture media formulations: Media A and Media B. Media A was supplemented with 10% fetal calf serum, while Media B was animal- component free, chemically defined, and composed of recombinant human growth factors.

EXAMPLE 8: Three-dimensional cell culturing using a method disclosed herein.

[00196] FIGURE 39 shows a biopsy culture taken from a patient having prostate cancer. The cells were cultured for 48 hours under either 21% oxygen and 0 PSI or 1% oxygen and 2 PSI. The results indicated that the cells had a two-fold increase in viable cell adherence under positive pressure and low oxygen conditions. The bottom panels of FIGURE 39 show that only cells grown under high pressure and low oxygen yielded enough cells for passaging at day 6, and were then able to form organoids by day 10. The tumor cell cultures were then subcutaneously injected into mice.

[00197] FIGURE 40 shows prostate cancer cells obtained from another patient could form organoids after two weeks of culture under high pressure and low oxygen conditions.

[00198] FIGURE 41 shows an apheresis culture taken from a patient having prostate cancer. The cells were cultured for 10 days under high pressure and low oxygen conditions to form organoids FIGURE 41 shows the results of culturing under both 2D and 3D conditions. The left panels of FIGURE 41 show viable and proliferating tumor cells that were positively selected for EpCAM (prostate cancer marker), and the right panels show viable and proliferating tumor cells that are EpCAM negative. After ten days in culture, the cells were subcutaneously injected into mice.

EMBODIMENTS

[00199] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[00200] Embodiment 1. A method for increasing transfection efficiency of a nucleic acid that is introduced into a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases expression of a polypeptide encoded by the nucleic acid that is introduced into the cell as compared to expression of the polypeptide encoded by a nucleic acid that is introduced into a cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

[00201] Embodiment 2. The method of embodiment 1, wherein the cell is cultured in a culture medium that does not contain serum.

[00202] Embodiment 3. The method of any one of embodiments 1-2, wherein the cell is contacted with a substrate.

[00203] Embodiment 4. The method of any one of embodiments 1-3, wherein the substrate does not contain serum.

[00204] Embodiment 5. The method of any one of embodiments 1-4, wherein the hypoxic condition comprises an oxygen level of about 2%.

[00205] Embodiment 6. The method of any one of embodiments 1-4, wherein the hypoxic condition comprises an oxygen level of about 5%.

[00206] Embodiment 7. The method of any one of embodiments 1-6, wherein the positive pressure condition comprises a pressure level from about 2 PSI to about 10 PSI.

[00207] Embodiment 8. The method of any one of embodiments 1-7, wherein the nucleic acid is

DNA.

[00208] Embodiment 9. The method of any one of embodiments 1-7, wherein the nucleic acid is RNA.

[00209] Embodiment 10. The method of any one of embodiments 1-7, wherein the nucleic acid is circular DNA.

[00210] Embodiment 11. The method of any one of embodiments 1-7, wherein the nucleic acid is supercoiled DNA.

[00211] Embodiment 12. The method of any one of embodiments 1-11, wherein the nucleic acid that is introduced into the cell is introduced via electroporation of the cell.

[00212] Embodiment 13. The method of any one of embodiments 1-11, wherein the nucleic acid that is introduced into the cell is introduced via encapsulation of the nucleic acid in a cationic liposome.

[00213] Embodiment 14. The method of any one of embodiments 1-13, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases an entry rate of the nucleic acid into the cell as compared to the entry rate of the nucleic acid that is introduced into the cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

[00214] Embodiment 15. The method of any one of embodiments 1-14, wherein the positive pressure condition is applied continuously to the cell.

[00215] Embodiment 16. The method of any one of embodiments 1-14, wherein the positive pressure condition is applied in pulses of positive pressure to the cell.

[00216] Embodiment 17. The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs after the nucleic acid is introduced into the cell. [00217] Embodiment 18. The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs before the nucleic acid is introduced into the cell.

[00218] Embodiment 19. The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs before the nucleic acid is introduced into the cell and after the nucleic acid is introduced into the cell.

[00219] Embodiment 20. The method of any one of embodiments 1-19, wherein the nucleic acid is introduced into the cell in the absence of the hypoxic condition and the positive pressure condition.

[00220] Embodiment 21. The method of any one of embodiments 1-20, wherein the cell is a mammalian cell.

[00221] Embodiment 22. A method for reprogramming a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein the cell exhibits a rate of reprogramming that is higher than the rate of reprogramming of a cell cultured in the absence of the hypoxic condition and the positive pressure condition.

[00222] Embodiment 23. The method of embodiment 22, wherein the hypoxic condition comprises an oxygen level of about 2%.

[00223] Embodiment 24. The method of embodiment 22, wherein the hypoxic condition comprises an oxygen level of about 5%.

[00224] Embodiment 25. The method of any one of embodiments 22-24, wherein the positive pressure condition comprises a pressure level of about 2 PSI to about 10 PSI.

[00225] Embodiment 26. The method of any one of embodiments 22-25, wherein the rate of reprogramming of the cell cultured in the hypoxic condition and the positive pressure condition is about 10% higher than the rate of reprogramming of the cell cultured in the absence of the hypoxic condition and the positive pressure condition.

[00226] Embodiment 27. The method of any one of embodiments 22-25, wherein the rate of reprogramming of the cell cultured in the hypoxic condition and the positive pressure condition is about 20% higher than the rate of reprogramming of the cell cultured in the absence of the hypoxic condition and the positive pressure condition.

[00227] Embodiment 28. The method of any one of embodiments 22-27, wherein the cell is a somatic cell.

[00228] Embodiment 29. The method of any one of embodiments 22-27, wherein the cell is a fibroblast.

[00229] Embodiment 30. The method of any one of embodiments 22-29, wherein the cell is reprogrammed into a stem cell.

[00230] Embodiment 31. The method of any one of embodiments 22-30, wherein the cell is reprogrammed into a pluripotent stem cell.

[00231] Embodiment 32. The method of any one of embodiments 22-29, wherein the cell is reprogrammed into an immune cell.

[00232] Embodiment 33. The method of any one of embodiments 22-32, wherein the cell cultured in the hypoxic condition and the positive pressure condition exhibits a greater expression level of a stem cell marker as compared to the expression level of the stem cell marker for a cell cultured in the absence of the hypoxic condition and the positive pressure condition.

[00233] Embodiment 34. The method of embodiment 33, wherein the stem cell marker is Oct4.

[00234] Embodiment 35. The method of embodiment 33, wherein the stem cell marker is Nanog.

[00235] Embodiment 36. The method of embodiment 33, wherein the stem cell marker is Sox2.

[00236] Embodiment 37. The method of any one of embodiments 22-36, wherein the cell is contacted with a substrate.

[00237] Embodiment 38. The method of any one of embodiments 22-37, wherein a nucleic acid encoding a reprogramming factor polypeptide is introduced into the cell.