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Title:
AN EXPLANT CULTURE TECHNIQUE FOR ISOLATION OF MESENCHYMAL STEM CELLS FROM ADIPOSE TISSUE
Document Type and Number:
WIPO Patent Application WO/2012/070001
Kind Code:
A1
Abstract:
The present disclosure relates to a method for isolating and culturing adipose tissue- derived mesenchymal stromal/stem cells (ASC) by explant culture method. The adipose tissue obtained by surgical or other known method is processed to isolate a enriched population of MSC more economically. The multipotent ASC so obtained by explant culture method are capable of self renewal, and are immunosuppressive and hypoimmunogenic. The explant method induces minimal or no stress on the stem cells unlike the conventional methods.

Inventors:
RAJ, Swathi, Sundar (9th Floor Manipal Hospital, 98, Rustom,Bagh, Airport Road, Bangalore Karnataka 7, 560 01, IN)
PRIYA, Nancy (9th Floor Manipal Hospital, 98, Rustom,Bagh, Airport Road, Bangalore Karnataka 7, 560 01, IN)
Application Number:
IB2011/055252
Publication Date:
May 31, 2012
Filing Date:
November 23, 2011
Export Citation:
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Assignee:
STEMPEUTICS RESEARCH PVT. LTD. (9th Floor Manipal Hospital, 98,Rustom Bagh, Airport Road, Bangalore Karnataka 7, 560 01, IN)
RAJ, Swathi, Sundar (9th Floor Manipal Hospital, 98, Rustom,Bagh, Airport Road, Bangalore Karnataka 7, 560 01, IN)
PRIYA, Nancy (9th Floor Manipal Hospital, 98, Rustom,Bagh, Airport Road, Bangalore Karnataka 7, 560 01, IN)
International Classes:
C12N5/077
Other References:
JING WEI ET AL: "Explant culture: an efficient method to isolate adipose-derived stromal cells for tissue engineering.", ARTIFICIAL ORGANS FEB 2011 LNKD- DOI:10.1111/J.1525-1594.2010.01054.X PUBMED:20946305, vol. 35, no. 2, 14 October 2010 (2010-10-14), pages 105-112, XP55021341, ISSN: 1525-1594 cited in the application
HE XIAOLI ET AL: "Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.", JOURNAL OF MOLECULAR CELL BIOLOGY AUG 2010 LNKD- PUBMED:20584786, vol. 2, no. 4, August 2010 (2010-08), pages 199-208, XP55021461, ISSN: 1759-4685
"Retraction: Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.", JOURNAL OF MOLECULAR CELL BIOLOGY DEC 2011 LNKD- PUBMED:22128322, vol. 3, no. 6, December 2011 (2011-12), page 382, XP55021470, ISSN: 1759-4685
"Technical Resources - Media Formulations 10569 - DMEM, high glucose, GlutaMAX(TM), pyruvate", , 9 March 2012 (2012-03-09), XP55021491, Retrieved from the Internet: URL:http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation.46.html [retrieved on 2012-03-09]
Attorney, Agent or Firm:
VIJAYAKRISHNAN, Sindhu et al. (K&S Partners, 4121/B, 6th Cross, 19th A Main, HAL II,Stage, Bangalore Karnataka 8, 56003, IN)
Download PDF:
Claims:
We claim:

1. A method of isolating mesenchymal stem cells from adipose tissue non- enzymatically, said method comprising acts of:

a. harvesting adipose tissue;

b. washing the harvested adipose tissue with buffer solution and separating fat tissue from the buffer solution;

c. mincing the separated fat tissue to obtain tissue explants; and

d. adding pre -warmed culture media over the tissue explants and incubating to isolate the mesenchymal stem cells non-enzymatically.

2. A media for isolating mesenchymal stem cells from adipose tissue non- enzymatically, said media comprising basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a-MEM), supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.

3. The method as claimed in claim 1 , wherein the harvesting is carried out using liposuction, lipectomy, lipoaspiration and resection or any combination thereof.

4. The method as claimed in claim 1, wherein the buffer solution is selected from group comprising normal saline, Phosphate Buffered Saline and basal medium; preferably Phosphate Buffered Saline.

5. The method as claimed in claim 1, wherein the media comprises of basal medium selected from group comprising DMEM or a-MEM, supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.

6. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the Serum is selected from group comprising Human serum (HS), Fetal Bovine Serum (FBS), Human Plasma (HP) and Human Platelet Lysate (HPL).

7. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the DMEM is selected from group comprising DMEM-KO, DMEM-LG, DMEM- HG and DMEM-F12, each having concentration ranging from about 80% to about 99%.

8. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the a-MEM is having concentration ranging from about 80% to about 99%.

9. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the Serum is having concentration ranging from about 1% to about 20%, preferably about 10%.

10. The method and the media as claimed in claim 6, wherein the (HP) is having concentration ranging from about 1 to about 20% and the (HPL) is having concentration ranging from about 1% to about 20%.

11. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the growth factor is selected from group comprising basic Fibroblast Growth Factor (bFGF) and Platelet Derived Growth Factor (PDGF) or combination thereof, each having concentration ranging from about 1 ng/ml to about 20 ng/ml.

12. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the Sphingosine- 1 -Phosphate is having concentration ranging from about 0.01 μΜ to about 5 μΜ.

13. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the Glutamine or Glutamine-dipeptide is having concentration ranging from about 50 mM to about 500 mM, preferably about 200 mM.

14. The method as claimed in claim 5 and the media as claimed in claim 2, wherein the antibiotic is selected from group comprising Penicillin having concentration ranging from about 50 to about 200 U/ml, preferably about 100 U/ml; Streptomycin having concentration ranging from about 50 to about 200 μg/ml, preferably about 100 μg/ml; and Gentamycin having concentration ranging from about 10 μg/ml to about 200 μg/ml, preferably about 25 μg/ml.

15. The method as claimed in claim 1, wherein the incubation is carried out in humidified 5% C02 incubator at about 37°C, for time duration ranging from about 4 days to about 20 days and sub-cultured for duration ranging from about 7 days to about 10 days.

16. The method as claimed in claim 1 and the media as claimed in claim 2, wherein the media is used in amount ranging from about 1ml to about 5ml, preferably about 2.5 ml.

17. A kit comprising media components as claimed in claim 2 along with instructions for preparation of the media.

18. A method of assembling a kit as claimed in claim 17, said method comprising act of combining components as claimed in claim 2 to arrive at the kit.

Description:
AN EXPLANT CULTURE TECHNIQUE FOR ISOLATION OF

MESENCHYMAL STEM CELLS FROM ADIPOSE TISSUE"

TECHNICAL FIELD

The present disclosure relates to the field of stem cells in general, while in particular it relates to the isolation of mesenchymal stem/stromal cells (MSC) from adipose tissue by explant culture method. The method provides stress-free, cost effective isolation of enriched mesenchymal stem cells for further expansion and use in clinical/therapeutic applications.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE

Mesenchymal stem/stromal cells (MSC) are multipotent adult stem cell populations that reside in the connective tissue stroma of almost all organs. Since their isolation and characterization from the bone marrow, MSC have been under the spotlight owing to their potential in tissue engineering and regenerative medicine. They have a unique capacity for self renewal and multilineage differentiation into mesodermal, endodermal, and ectodermal lineages. In addition to their plasticity, they have the capacity to home towards injured tissue and have been shown to secrete a wide variety of trophic factors capable of tissue repair and regeneration. MSC are also immune privileged and are well documented to have potent anti-inflammatory and immunosuppressive activity.

Tremendous progress has been made in preclinical studies using MSC, including the ability to use the cells in allogeneic and xenogenic environments. The safety and therapeutic efficacy of human MSC in preclinical models has driven their application toward the clinical setting. While MSC are conventionally obtained from the bone marrow, they can also be isolated from adipose tissue, umbilical cord matrix or Wharton's jelly, umbilical cord blood, synovium, skeletal muscle, dental pulp, limbal tissue, amniotic fluid, placenta and menstrual blood. Subcutaneous adipose tissue is a particularly advantageous source as it can be obtained in much larger quantity when compared to other tissues, has a very high MSC content, and can be easily harvested by liposuction. Adipose tissue-derived MSC (ASC) are in fact being evaluated in clinical trials for several pathologies including ischemic heart disease, Crohn's disease, liver cirrhosis, limb ischemia and diabetes, as well as in a variety of cosmetic and reconstructive procedures. Adipose tissue is composed of several cell types including mature adipocytes, preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells and their progenitors, resident monocytes/macrophages, lymphocytes, and the multipotent stem/stromal cells..

The Conventional method for isolation of MSC from adipose tissue universally involves dissociation of the adipose tissue to release the different cell populations by chemical (enzymatic, collagenase treatment) or physical (ultrasonic) methods, or a combination of both. Research Articles titled "Isolation of Stromal Stem Cells from Human Adipose Tissue" by collaslab and "Systems and methods for isolating stromal cells from adipose tissue and uses thereof by, HIGGINS JOEL C et al, refer to the isolation of stromal stem cells from adipose tissue. The PCT Application WO/2003/053346 refers to a closed device for processing lipoaspirate for obtaining stem cells and a method of treating patients with processed cells. Here, "tissue dissociation" is defined as the process of obtaining single cell suspensions from primary tissue by breaking down the tissue extracellular matrix. Such methods typically place considerable stress on the cells due to enzyme treatment and time spent (3-4 hours) outside the physiological environment (37°C, 5% C0 2 ).

Enzymatic process although widely followed, has several disadvantages. Collagenase and other enzymes are extremely costly, and are rarely pure homogeneous preparations. Commonly available commercial collagenase products are crude or enriched, and may contain pigments, endotoxin [Jahr, H et al (1999). Journal of Molecular Medicine, 77: 118-120 and Vargas, F.et al (1997). Lancet, 350:641], xenoantigens, [http://www.celltherapysociety.org/files/PDF/Resources/Risk_ BSE_in_Collagenase_Enz ymes.pdf] and undesirable proteases. Their overall composition is also highly variable from lot-to-lot, and manufacturer to manufacturer [Yamamoto, T et al (2007). Transplantation, 84:997-1002, Barnett, M.J et al., (2005). Transplantation, 80:723-728 and Cavanagh, T.Jet al (1997). Transplantation Proceedings, 29: 1942-1944]. Inconsistency in protease profiles and activity could contribute to variability in the cell populations isolated, and hence the differences in ASC surface phenotype and function that have been reported from different laboratories [Zuk, P. A., et al. (2002). Molecular Biology of the Cell, 13:4279-4295, Planat-Benard, et al (2004). Circulation, 109:656- 663, Mitchell, et al. (2006). Stem Cells, 24:376-385 and Astori, G., et al. (2007). Journal of Translational Medicine, 5:55.]. ASC have recently been shown to be composed of several subpopulations that differ in surface marker expression and exhibit distinct differentiation potentials [Rada, T., et al (201 1). Stem Cell Reviews, 7:64-76 and Paredes, B et al. (2010). Journal of Tissue Engineering and Regenerative Medicine, 10.1002/term.351]. Enzymatic digestion followed by centrifugation is also a time consuming and stressful process. Cell lysis and decreased viability due to enzyme activity and centrifugal stress have been reported [Ishige, I et al. (2009). International Journal of Hematology, 90:261-269 andlgura, K., et al (2004). Cytotherapy, 6:543-553.]. Mechanical dissociation techniques have also been employed to a limited extent to disaggregate fat tissue [Baptista, L.S et al (2009). Cytotherapy, 6:706-715]. Physical forces such as centrifugation, ultrasonic force as well as shear stress used are also stressful to the cells, and have been demonstrated to affect the proliferation and undifferentiated state of ASC. The following two publications clearly show the stress that is placed on the cell using the conventional method: Oedayrajsingh-Varma, M.J et al. (2006). Cytotherapy, 8: 166-177 and Fischer, L.J et al. (2009). Journal of Surgical Research, 152: 157-166.

The PCT application WO/2008/129563 relates to a process of isolation of MSC mainly from bone marrow and culture of the stem cells. This application does not specifically disclose the method of isolation of MSC from Adipose tissue but makes a statement that MSC can be isolated from various other sources including adipose tissue.

Derivation of primary cell cultures from tissue explants has been recently used to obtain MSC from umbilical cord, dental pulp, synovium and placenta. Explant culture has been reported for isolation of murine ASC from mouse fat pads [Jing, W et al. (201 1). Artificial Organs, 35: 105-112]. The above report specifically describes explanting from resected fat tissue from mouse. However, it is not known if the explanting technique disclosed in the report can be applied to human adipose tissue, particularly lipoaspirated fat tissue. Surgical resection for obtaining fat tissue is an invasive procedure which is associated with complications of post surgical morbidity, healing and recovery. Liposuction used in present disclosure on the other hand is a minimally invasive procedure, and hence the preferred procedure for fat harvesting for stem cell isolation. The immunogenicity and immunomodulatory nature of cells derived from fat tissue explants has also not been determined in the report by Jing et.al. As adipose tissue is composed of several cell types described earlier, it is not known if the cells obtained by explant culture would have immune privilege, which is absolutely essential for clinical utility of the ASC.

Accordingly what is needed is a reproducible, economical, simple method for isolating and culturing Adipose tissue-derived Mesenchymal Stem Cells (ASC), in which ASC can be obtained without subjecting the cells to the stress-strain as well as all the above mentioned disadvantages associated with the normal conventional method of isolation. Present disclosure discloses the utility of the explant culture system for derivation of ASC from adipose tissue. The method was evaluated not only with adipose tissue obtained by surgical resection, but also with tissue obtained by liposuction, which is minimally invasive and hence the preferred procedure for fat harvesting.

STATEMENT OF THE DISCLOSURE Accordingly, the present disclosure relates to a method of isolating mesenchymal stem cells from adipose tissue non-enzymatically, said method comprising acts of a) harvesting adipose tissue, b) washing the harvested adipose tissue with buffer solution and separating fat tissue from the buffer solution, c) mincing the separated fat tissue to obtain tissue explants and d) adding pre -warmed culture media over the tissue explants and incubating to isolate the mesenchymal stem cells non-enzymatically; a media for isolating mesenchymal stem cells from adipose tissue non-enzymatically, said media comprising basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a-MEM), supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof; a kit comprising media components as above, along with instructions for preparation of the media; and a method of assembling a kit as above, said method comprising act of combining components as above to arrive at the kit.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Fig. 1 Shows isolation of ASC by explant culture: A, adipose tissue explants; B, C, migration of fibroblastic cells from the tissue explants on day 3. D, E, explant derived cells on day 6; F, G, explant derived cells on day 9. B, D and F represent lipoaspirate tissue explants; C, E and G represent resected tissue explants. Scale bars represent a magnification of 100 microns.

Fig. 2 Shows comparison graphs and photos of Growth kinetics of ASC derived by explant culture and collagenase digestion methods. A, fold expansion from P0 to P10 for explant (lipoaspirate and resected tissue) and collagenase-derived ASC; B, cumulative population doubling at each passage for explant (lipoaspirate and resected tissue) and collagenase-derived ASC from P0 to P10; C, mean doubling time for explant (lipoaspirate and resected tissue) and collagenase-derived ASC over 10 passages. Ex-L, lipoaspirate explant-derived ASC; Ex-T, resected tissue explant-derived ASC; Coll., collagenase-derived ASC. Data is represented as mean ± standard deviation for at least 4 biological replicates for each condition. D, senescence associated β-galactosidase staining for ASC derived by explant and enzymatic techniques at P20. Micrographs show a representative set of images from 4 biological replicates for each condition, scale bars represent a magnification of 50 microns.

Fig. 3 Shows comparison of detection of cell surface markers expressed by explant and collagenase-derived ASC at P4 by immunophenotyping and flow cytometric analysis. Open histograms represent staining with isotype control antibodies, shaded histograms represent staining with the specific antibody. The percentage positive cells for each marker is calculated after subtraction of the non-specific fluorescence obtained with the isotype control antibodies. Data shows a representative set of histograms from 4 biological replicates for each condition. Coll- ASC, collagenase derived ASC.

Fig. 4 Shows comparison of multipotentiality of explant and collagenase-derived ASC. A, B, oil-red O staining of adipogenically induced ASC isolated by explant culture (A) and the corresponding collagenase control (B). C, the stain from both uninduced and induced cultures was extracted using chloroform and methanol (2: 1) and quantified by spectrophotometry. D, E, alizarin red S staining of osteogenically induced ASC isolated by explant culture (D) and the corresponding collagenase control (E). F, the stain from both uninduced and induced cultures was extracted using acetic acid and ammonium hydroxide and colorimetrically estimated. G, H, alcian blue staining of induced chondrocyte pellets of ASC isolated by explant culture (G) and the corresponding collagenase control (H). I, semi-quantitative RT-PCR analysis for expression of chondrogenic genes in induced pellets of explant-derived ASC (Ex.) and the corresponding collagenase control (Coll.). 18s ribosomal R A expression was used to normalize cDNA concentration for each sample set. Data depicts gene expression in two pairs of samples out of 4 pairs of biological replicates analyzed. Micrographs show a representative set of images from 4 biological replicates for each condition, scale bars represent a magnification of 50 microns. Graphs represent mean ± standard deviation from 4 biological replicates for each condition. Fig. 5 shows graphical representation of Immunogenicity and immunosuppressive capacity of explant-derived ASC. A, mitomycin-C arrested ASC fail to stimulate proliferation of allogeneic PBMC. Mismatched PBMC (allo-PBMC) were used as positive controls for PBMC stimulation at the corresponding stimulator to responder ratios. B, inhibition of mixed lymphocyte reaction by mitomycin-C arrested ASC. Data is represented as mean ± standard deviation for 4 biological replicates for each condition.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a method of isolating mesenchymal stem cells from adipose tissue non-enzymatically, said method comprising acts of:

a. harvesting adipose tissue;

b. washing the harvested adipose tissue with buffer solution and separating fat tissue from the buffer solution;

c. mincing the separated fat tissue to obtain tissue explants; and

d. adding pre -warmed culture media over the tissue explants and incubating to isolate the mesenchymal stem cells non-enzymatically.

The present disclosure also relates to a media for isolating mesenchymal stem cells from adipose tissue non-enzymatically, said media comprising basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a- MEM), supplemented with Serum, growth factor, Sphingosine-1- Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.

In an embodiment of the present disclosure, the harvesting is carried out using liposuction, lipectomy, lipoaspiration and resection or any combination thereof. In another embodiment of the present disclosure, the buffer solution is selected from group comprising normal saline, Phosphate Buffered Saline and basal medium; preferably Phosphate Buffered Saline.

In yet another embodiment of the present disclosure, the media comprises of basal medium selected from group comprising DMEM or a-MEM, supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.

In still another embodiment of the present disclosure, the Serum is selected from group comprising Human serum (HS), Fetal Bovine Serum (FBS), Human Plasma (HP) and Human Platelet Lysate (HPL).

In still another embodiment of the present disclosure, the DMEM is selected from group comprising DMEM-KO, DMEM-LG, DMEM-HG and DMEM-F12, each having concentration ranging from about 80% to about 99%.

In still another embodiment of the present disclosure, the a-MEM is having concentration ranging from about 80% to about 99%.

In still another embodiment of the present disclosure, the Serum is having concentration ranging from about 1% to about 20%, preferably about 10%.

In still another embodiment of the present disclosure, the (HP) is having concentration ranging from about 1 to about 20% and the (HPL) is having concentration ranging from about 1% to about 20%.

In still another embodiment of the present disclosure, the growth factor is selected from group comprising basic Fibroblast Growth Factor (bFGF) and Platelet Derived Growth Factor (PDGF) or combination thereof, each having concentration ranging from about 1 ng/ml to about 20 ng/ml.

In still another embodiment of the present disclosure, the Sphingosine-1 -Phosphate is having concentration ranging from about 0.01 μΜ to about 5 μΜ.

In still another embodiment of the present disclosure, the Glutamine or Glutamine- dipeptide is having concentration ranging from about 50 mM to about 500 mM, preferably about 200 mM. In still another embodiment of the present disclosure, the antibiotic is selected from group comprising Penicillin having concentration ranging from about 50 to about 200 U/ml, preferably about 100 U/ml; Streptomycin having concentration ranging from about 50 to about 200 μg/ml, preferably about 100 μg/ml; and Gentamycin having concentration ranging from about 10 μg/ml to about 200 μg/ml, preferably about 25 μg/ml.

In still another embodiment of the present disclosure, the incubation is carried out in humidified 5% C0 2 incubator at about 37°C, for time duration ranging from about 4 days to about 20 days and sub-cultured for duration ranging from about 7 days to about 10 days.

In still another embodiment of the present disclosure, the media is used in amount ranging from about 1ml to about 5ml, preferably about 2.5 ml. The present disclosure relates to a kit comprising media components as above along with instructions for preparation of the media.

The present disclosure also relates to a method of assembling a kit as above, said method comprising act of combining components as above to arrive at the kit.

As 'explanting' refers to incubation of undissociated tissue in a suitable culture medium, derivation of MSC from the explants would rely on the mesenchymal migratory capacity of the cells. Human adipose tissue samples were obtained with informed consent from patients undergoing elective cosmetic surgery, following approval by the Institutional committee for Stem Cell Research and Therapy (ICSCRT) and the Institutional Ethics Committee (IEC) at the Manipal Hospital, Bangalore, India. Lipoaspirate tissue (n=5) was collected from abdominal liposuction procedures and processed by both explant culture and enzymatic method for comparison. Resected adipose tissue was obtained from abdominoplasty (n=4) procedures and was processed by explant culture alone. In an embodiment of the present disclosure, the buffer solution is selected from a group comprising normal saline, phosphate buffered saline (PBS) and basal medium in order to remove blood.

In yet another embodiment of the present disclosure, the completed culture media comprises basal medium and supplements to support cell growth. Basal medium is selected from DMEM or a-MEM, supplemented with bovine or human serum, or plasma or human platelet lysate, growth factors; Sphingosine-1 -Phosphate, Glutamine or glutamine-dipeptide and antibiotics, and the explant tissues are cultured in complete culture media.

In an embodiment the instant disclosure presents a media composition used for culturing the MSC isolated by explants method/non-enzymatic method. In the explants method the MSC are isolated without the use of any enzymes for tissue dissociation unlike the conventional method wherein enzymes like collagenase are used, hence it is also referred to as non-enzymatic method.

The instant disclosure describes a novel, gentle, much simplified and cost-effective technique for isolation of adipose tissue-derived MSC (ASC) by explant culture, without using conventionally known tissue dissociation methods. The present disclosure demonstrates that MSC are isolated from undissociated adipose tissue. 'Explant culture' refers to transferring intact tissue obtained by lipectomy/liposuction into a suitable culture medium. The tissue here is not dissociated or processed using chemical or physical means, and thus places minimal stress on the cells. The processing time is also very rapid, as the surgically removed tissue is transferred to a physiological environment (37°C, 5% C0 2 , isotonic culture medium containing relevant growth factors) with minimal time delay (30-60 min). This instant method also eliminates use of enzymes such as collagenase, hence also referred to as non-enzymatic method, which would dramatically reduce cost of processing, as well as negate problems surrounding use of xeno-derived or bacterial-derived agents for isolating cells intended for clinical use in humans. Another advantage is that the amount of tissue/lipoaspirate required for explant culture is also much lesser than the amount of tissue/lipoaspirate required for isolating cells by enzymatic means. This technique yields a population of stromal cells that is highly enriched for MSC.

ASC isolated and cultured as described by explant culture method of present disclosure showed typical spindle shaped morphology characteristic of MSC. The explant-derived ASC show potent immunosuppressive capacity in a dose-dependent manner and efficiently differentiate towards adipogenic, osteogenic and chondrogenic cell lineages.

In an embodiment of the instant disclosure, the tissue used in the instant disclosure is Adipose tissue obtained by any known medical procedure in art but not limiting to methods such as liposuction, lipoaspiration and lipectomy. In an embodiment of the instant disclosure, the composition of the culture medium comprises of Basal medium selected from Dulbecco's Modified Eagle Medium (DMEM) or Alpha-Minimum essential medium (a-MEM). Further DMEM is selected from a group comprising DMEM-KO, DMEM-LG, DMEM-HG, DMEM-F12 (1 : 1); basal medium is supplemented with 1 to 20% Fetal Bovine Serum (FBS) or 1 to 20% Human Serum (HS) or 1 to 20% Human Plasma or 1 to 20% Human Platelet Lysate (HPL); growth factors such as Fibroblast Growth Factor (bFGF) or Platelet Derived Growth Factor (PDGF), Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotics. In another embodiment of the instant disclosure, as the composition of fat tissue in humans and large animals such as dogs and horses is very similar, explant culture technique can also be used for isolation of ASC from animal fat tissue, for applications in veterinary regenerative medicine. The present disclosure is further elaborated by the following examples and figures. However, these examples should not be construed to limit the scope of the disclosure. EXAMPLE:

Example 1: Method of isolation of Mesenchymal Stem Cells by Explant culture

In one of the embodiment of the present disclosure for explants culture all steps are carried out in the sterile environment of a biosafety cabinet. The adipose tissue obtained by Lipoaspirate (-10 ml) or resected tissue (~5g) is washed with Saline or Phosphate Buffered Saline (PBS) to remove blood. Lipoaspirated tissue is preferably used as liposuction is a less invasive method of fat harvest compared to the surgical method, and less tedious for ASC isolation when compared to resected tissue. Fat tissues can be lipoaspirated from mammal for explants culture. The tissue is washed by mixing it with an equal a m o u n t o f buffer in a sterile tube and resting it for 2-5 min, t h u s allowing the aqueous buffer fraction containing blood to separate below the fat fraction. The buffer is then removed by aspiration. A small quantity of the fat tissue (~ 1 g for a 100 mm culture dish) is then transferred to a sterile culture dish and minced into smaller fragments of about 2-10 mm 3 ; preferably 5mm 3 with the help of sterile forceps.

The figure la shows the fragments of adipose tissue, henceforth referred to as tissue explants, evenly distributed over the surface of the culture dish. Approximately 0.5-2 g preferably lg of tissue was plated per 100 mm dish. The explants are cultured in Knock out Dulbecco's medium (DMEM-KO) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2mM glutamine and antibiotic (Invitrogen). Minimal quantity of about 1-5 ml, preferably 2.5 ml of pre-warmed (37°C) culture medium is then added over the tissue explants such that the explants still remain in contact with the surface of the culture dish. The term culture media, complete media, means one and the same. The complete media comprises of a basal medium supplement with serum, and other growth factor which support the cell expansion. Serum supplement FBS when replaced with HS, HPL or HS it is referred to as xeno-free culture condition. 'Explant culture' refers to transferring intact tissue obtained by lipectomy/liposuction/lipoaspiration (fig. lb, d, f) and by surgical resection (fig. lc, e, and g) into a suitable culture medium thus placing minimal stress on the cells. The dish containing the explants is then transferred into a humidified incubator where it is maintained at 37°C, 5% C0 2 for a period of 4-10 days during which it is monitored regularly for outgrowth of MSC from the explants. The explants tissue is then removed from the dish and the outgrown MSC are further cultured for 7-10 days in fresh culture medium until a confluent monolayer is obtained. These initial cells, referred to as passage 0 (P0), were further sub-cultured at a seeding density of 2000 cells /cm 2 and serially passaged until they reached replicative senescence. Figure If and lg show the cell yield obtained at confluence (passage 0), which is in the range of 5-8 xlO 5 cells /g of initial explant tissue plated. Figure lb, d, and f show the explant culture performed using adipose tissue obtained by liposuction and Figure lc, e, g by surgical resection. Figure lb, c shows spindle-shaped cells migrating out of both types of explants (liposuction & surgical resection) onto the culture dish within 3-4 days of culture. Figure Id and figure le depicts that the migrated cells possess typical mesenchymal morphology and continue to proliferate after removal of the explant tissue.

The yield and morphology of cells obtained from lipoaspirate and resected tissue explants were identical and no difference is seen. Explant method of present disclosure yields a pure and enriched population of ASC that are capable of self renewal, multilineage differentiation and immunosuppression. They are also hypoimmunogenic and hence suitable for both autologous and allogenic cell therapy upon further expansion. Alternatively this method can be used to obtain clinical grade ASC from adipose tissue by using a xeno-free culture condition comprising of basal medium supplemented with Human Serum or Human Platelet Lysate(HPL) or serum-free basal medium supplemented with growth factors and sphingosine-1 -phosphate in culture medium instead of using FBS. The Xenofree culture conditions comprises of using basal medium plus human serum or HPL instead of FBS. Similarly the ASC can be cultured in a Serum- free conditions comprising of serum-free media (proprietary) such as Mesencult-XF and Stempro MSC-SFM supplemented with growth factor. Advantage here is that since explant culture does not use collagenase or any other enzyme, culturing in xeno-free or serum free medium will make the complete process xeno/serum-free, as compared to enzymatic digestion where the enzyme is typically of bacterial origin. Present disclosure also shows that the cells isolated by the explant technique (a non- enzymatic method) are similar to cells obtained by enzymatic digestion in terms of their physical and biological characteristics while having several advantages when compared to enzymatic method.

The isolation of ASC by enzymatic method from lipoaspirate tissue is carried out according to the conventional collagenase digestion method disclosed in Yu et al., (2011) Methods in Molecular Biology, 702: 17-27. Growth kinetics and senescence

The ASC obtained by explants method is checked for growth kinetics and senescence against ASC obtained by enzymatic (collagenase) method.

The lipoaspirates taken for explant culture were also simultaneously processed enzymatically by collagenase digestion, and the SVF obtained was seeded at a density of

4 2

10 cells/cm to obtain passage 0 (P0) cells(see Fiure2). The growth characteristics of explant-derived ASC is then validated by comparing against the corresponding ASC samples isolated by collagenase digestion, cultured under identical conditions from P0 onwards. Both explant and collagenase derived ASC showed very similar proliferation kinetics as observed in their rate of expansion (Fig. 2a), cumulative population doublings (Fig. 2b) and doubling time (Fig. 2c). No difference is observed in the growth parameters of cells obtained from lipoaspirate and resected tissue explants. Both explant and collagenase-derived ASC can be maintained in culture for up to 20 passages, corresponding to an expansion range of 10 10 -10 15 fold over 42-52 population doublings, beyond which their proliferation declined.

Cell senescence is detected using the Senescence β-Galactosidase Staining - Characteristics of replicative senescence such as enlarged cells, quiescence, and increase in senescence associated β-galactosidase activity could be observed in cells beyond P20 for both ASC (Fig. 2d). These results demonstrate that ASC obtained by explant culture harbor proliferation potential equivalent to conventionally derived ASC. Immunophenotypic and functional characterization of explant and coUagenase- derived ASC

In order to examine the cell surface antigens of spindle shaped ASC isolated and cultured as described above, immunophenotype analysis of explant-derived ASC cultures was carried out, which showed positive expression of mesenchymal markers and negative expression of immunogenic hematopoietic markers.

Table 1: Immunophenotypic characterization of ASC

Values represent mean percentage positive cells ± standard deviation.

It is confirmed from table 1 and figure 3 that in the case of ASC isolated and cultured in accordance with the present disclosure, HLA-DR, CD80 and CD86 which are characteristic indicators of hematopoietic cells, showed negative expression; and CD34, CD90, CD73, CD 105, CD44, and HLA-ABC which are characteristic indicators of expression, showed positive reaction, This result is interpreted as showing that cells isolated and cultured in accordance with the present disclosure are bonafide MSC.

The flow cytometric analysis of cell surface markers for explant and collagenase-derived ASC is presented in Table 1. The percentage of CD34+ cells for collagenase-derived ASC ranged between 14-16 % (average: 15%) at P0 and 0.5-5.6 % (average: 3%) at PI . Explant-derived ASC however had a lower percentage of CD34+ cells at P0 (range: 0.5- 9.0 %, average: 4%) when compared to collagenase-derived ASC and this difference was found to be statistically significant (p=0.05), and negligible CD34+ cells at PI (range: 0.4-0.8%, average: 0.6%).

In both cases however, CD34 positivity of the cells is lost upon further sub-culturing and

no CD34+ cells were detected from P2 onwards. The observed dilution of CD34

expression and decrease in hematopoietic cell contamination is an indicator of enrichment of ASC in explant culture is.

Both preparations of ASC are positive for the mesenchymal markers CD90, CD44, CD73

and CD 105, although explant-derived ASC are enriched for CD90, CD44 and CD73

when compared to collagenase-derived ASC at P0. The explant technique also yielded a

lower percentage of HLA-DR+ hematopoietic cells at P0 (range: 1.1-1.6%, average:

1.4%) when compared to the enzymatic technique (range: 4.7-10.7%, average: 8.2%), and this difference is found to be significant (p=0.02). Subsequent passages of ASC

obtained by both methods were found to be negative for CD34 and HLA-DR, and

showed comparable levels of expression of CD90, CD44, CD73 and CD 105 with respect to percentage positive cells as well as mean fluorescence intensity, which is found to be

stable up to passage 10. Both explant and collagenase-derived ASC are also found

positive for HLA-ABC, and negative for the immune co-stimulatory molecules CD40,

CD80 and CD86 (Fig. 3). No difference is observed in mesenchymal, hematopoietic or

co-stimulatory marker expression between ASC derived from lipoaspirate and resected

tissue explants (Table 2).

Table 2: Surface phenotype of lipoaspirate-explant and tissue-explant derived ASC

at P4

Marker CD34 CD90 CD44 CD73 CD105 HLA HLA CD80 CD86 CD40

ABC DR

Explant neg. 97±4 98±3 97±5 92±9 93±4 <l- 5 <l- 5 <l- 5 <l- 5 (Lipoaspirate) Explant neg. 98±1 97±2 88±6 93±3 99±1 <l- 5 <l- 5 <l- 5 <l- 5

(Tissue)

Values represent mean percentage positive cells ± standard deviation.

Immunogenicity and immunosuppressive capacity of explant-derived ASC

ASC isolated by explant and collagenase method is mitotically arrested and cultured with naive allogeneic peripheral blood mononuclear cells (PBMC) at different stimulator:

responder ratios. As positive control for lymphocyte stimulation, mismatched PBMC is used as stimulator cells at corresponding stimulator: responder ratios. Both preparations of ASC did not elicit a proliferative response in T cells of the PBMC at all ratios tested

(Fig. 5a), confirming the hypo-immunogenic nature of the ASC. No significant difference is observed between the immunogenicity of explant and collagenase-derived ASC, thus establishing the immune privileged status of ASC isolated by the explant technique.

The immunosuppressive properties of ASC isolated by explant culture and collagenase digestion are compared by testing their ability to suppress a mixed lymphocyte reaction

(MLR). MLRs is established using PBMC from mismatched donors, and cultured either in the presence or absence of growth-arrested ASC at different MSC:MLR ratios.

Lymphocyte proliferation is strongly suppressed in the presence of ASC, and this inhibition is found to be dose dependent (Fig. 5b). Explant-derived ASC is thus found to be equivalent to collagenase-derived ASC in their immunosuppressive potency.

Induction and quantitation of differentiation

The multipotentiality of ASC obtained by explant and enzymatic techniques is evaluated using standard in-vitro differentiation assays. Adipocyte differentiation of both ASC is detected by staining of accumulated lipid vacuoles with oil red O (Fig. 4a, b). The uptake of stain is then quantified from the extracted lipids by spectrophotometry, which showed that adipogenesis in explant-derived ASC is comparable to that of collagenase-derived

ASC (Fig. 4c). Induction of osteogenic differentiation resulted in matrix mineralization, and is confirmed by staining with Alizarin red S (Fig. 4d, e) that is then extracted and colorimetrically estimated (Fig. 4f). Explant-derived ASC is found to be as competent as collagenase-derived ASC for osteogenic differentiation. Chondrocyte pellets obtained from both ASC is stained with alcian blue to visualize proteoglycan deposition (Fig. 4g, h). Gene expression analysis of the differentiated pellets confirmed equivalent expression of the chondrogenesis markers SOX9, collagen 1 and collagen X in both ASC preparations (Fig. 4i).

An ideal source of MSC for clinical application must be abundantly available, easily accessible for harvest using procedures that have minimal invasiveness and morbidity, and a high content of stem/stromal cells. Adipose tissue fits all these criteria and is therefore a preferred tissue for obtaining MSC for cellular therapy and tissue engineering. Extraction of MSC from the source tissue must also ideally be easy, cost-effective, avoid chemical or mechanical stress and use of bacterial and xeno-derived products for clinical utility. Enzymatic digestion, although widely practiced for obtaining MSC from adipose tissue, does not fulfill all the above criteria. Present disclosure highlights the advantages of explant culture over enzymatic methods for isolation of ASC for laboratory and clinical use.

The method disclosed in present disclosure describes ASC is reliably isolated from explants of adipose tissue obtained by two different surgical procedures: resection and liposuction. The expansion, phenotypic and functional properties of ASC obtained using the explant method of present disclosure are comparatively better, and the present disclosure is also far more economically viable than the conventional and widely used enzymatic techniques. The proliferation potential of both explant and enzymatically derived ASC is nearly identical. In fact, explant culture is advantageous for processing very small quantities of adipose tissue, as 5-10 xlO 5 enriched ASC could be derived from just 1 g of explant tissue. Further culturing yields an expansion of 10 4 fold over just 5 passages, which corresponded to 12.5 ± 2 population doublings (Fig. 2).

ASC are known to express CD34 in the adipose tissue niche. Freshly isolated SVF therefore contains CD34+ cells in the ASC fraction [Planat-Benard, et al (2004). Circulation, 109:656-663 and Mitchell, et al. (2006). Stem Cells, 24:376-385] and the expression of this marker has been shown to decline upon ex-vivo culture and expansion, accompanied by an increase in expression of the mesenchymal markers [Mitchell, et al. (2006). Stem Cells, 24:376-385]. Enzymatically derived SVF is also reported to contain undesired hematopoietic cells such as monocytes/macrophages and lymphocytes [Mitchell, et al. (2006). Stem Cells, 24:376-385 and Mcintosh, et al. (2006). Stem Cells, 24: 1246-1253]. Dilution of CD34 expression and decrease in hematopoietic cell contamination is therefore an indicator of enrichment of ASC in culture. ASC obtained by present explant technique shows significantly lower percentage of CD34+ and HLA- DR+ cells at P0 when compared to collagenase-derived ASC (Fig. 3) indicating an enriched MSC population. Further expansion of cells derived by both techniques showed nearly identical surface marker profiles validating the mesenchymal stem cell phenotype of explant-derived ASC. ASC isolated by explant technique also possessed trilineage differentiation capacity comparable with collagenase-derived ASC (Fig. 4), thus confirming their phenotypic and functional equivalence. The ASC obtained by explanting therefore fulfill the criteria for defining multipotent mesenchymal stromal cells as proposed by the International Society for Cellular Therapy namely, they are plastic adherent with spindle shaped fibroblastic morphology, express the cell surface antigens CD90, CD73 and CD105, and lack expression of hematopoietic markers. They also demonstrate trilineage differentiation into adipogenic, osteogenic and chondrogenic lineages.

Immune privilege and immunosuppressive capacity are two properties of tremendous therapeutic utility for MSC, which eliminates the requirement for histocompatibility matching. Allogeneic donor-derived MSC can thus be made readily available for acute clinical conditions, closing the lead time from the bench to the bedside. ASC derived by explant culture is found to be negative for HLA-DR and immune co-stimulatory molecules CD80, CD86 and CD40 (Fig. 3), and they did not provoke stimulation of allogeneic lymphocytes confirming their low immunogenicity (Fig. 5). They were also capable of immunosuppression to a similar extent as collagenase-derived ASC.

Advantages: Not only does the present explant culture method yield ASC, this technique also has several advantages over the conventional enzymatic technique from a clinical and regulatory perspective. During explant culture, the tissue is not dissociated or processed using chemical or physical means, and thus places minimal stress on the cells. The processing time is also very rapid, as the surgically removed tissue can be transferred to a physiologic environment (37°C, 5% C0 2 , isotonic culture medium containing relevant growth factors) with minimal time delay thus ensuring that the adipose tissue/MSC spends minimal time outside physiological environment (less than 30 min). Eliminating use of enzymes such as collagenase would dramatically reduce cost of processing, as well as negate problems surrounding use of xeno-derived or bacterial-derived agents for isolating cells intended for clinical use in humans, including endotoxin contamination. Most collagenase preparations are concentrated from bacterial (Eg: Clostridium histolyticum) culture supernatants. These preparations are heterogeneous, containing different enzymes, cellular debris, pigments, and endotoxin. Endotoxin levels (Jahr et al (1999) Journal of Molecular Medicine and Vargas(1997) Lancet) and variability (Yamamoto et al (2007) Transplantation and Barnett et. al., (2005) Transplantation) are the most significant disadvantages associated with collagenase digestion. Moreover, collagenase has been reported to lose tissue dissociation efficacy over time (Cavanagh et.el.,(1997) Transplantation Proceedings) which would compromise the efficiency of cell isolation. Xeno-antigen contamination is also a potential threat while using enzymes. In fact, collagenase preparations have also been shown to activate human complement [Jahr et.al., (1995). Experimental and Clinical Endocrinology and Diabetes] which could induce local inflammatory reactions. Thus, avoiding the use of enzymes for cell therapy products is definitely recommended. Explant-based isolation method of present disclosure also has the potential to be adapted for xeno-free isolation and expansion of ASC for clinical scale, which would further simplify regulatory approvals.

Present disclosure of using explant method demonstrates that ASC can be efficiently isolated not only from resected fat tissue, but also from liposuctioned fat. As liposuction procedures are preferred over other invasive techniques of fat harvest, the combined methods of lipoaspiration and explanting has implications in reducing the cost associated with, and hugely simplifying, the ASC-isolation process. Present disclosure shows explanting adipose tissue results in an enriched ASC population at P0. The migration of plastic adherent cells from the explant tissue served as the criterion for isolation, and resulted in significantly reduced hematopoietic cells and contaminants in ASC preparation using explants method of present disclosure. The examples provide evidence that explant-derived ASC are immune privileged and exhibit immunosuppressive activity critical for MSC-based therapy, particularly for allogeneic cell therapy. Therefore, presently disclosed explant culture method is an efficient, reproducible and economical technique of clinical utility, which may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem cells for tissue engineering and regenerative medicine.




 
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