URINE

FIELD OF THE INVENTION

[0001] The invention is directed systems and methods to isolate stem cells from urine. BACKGROUND OF THE INVENTION

[0002] Mesenchymal stem cells (MSCs) line all blood vessels in the human body, hence the term "pericytes". Any vascular tissue would be expected to be rich in MSCs, hence their observed abundance in adipose tissue. The kidneys are the most heavily vascularized area of the human body as the kidneys are responsible for the filtering of blood; one would expect that MSCs would be abundant in the kidneys. In the scientific literature, multiple published papers exist which have isolated urine-derived MSCs from rodents as well as humans; however, the processes for isolation and expansion frequently involve the use of multiple media, all containing fetal bovine serum (FBS). Such approaches could not be expected to be suitable for human therapeutics due to the use of non-human products, which have the potential to cause immunoreactivity issues upon administration.

[0003] What is needed is a non-invasive, animal-free process for successfully isolating and expanding MSCs from urine.

BRIEF SUMMARY OF THE INVENTION

[0004] In a first embodiment, the invention is directed to a process to isolate stem cells from urine.

[0005] In another embodiment, the invention is directed to a method for collection, processing and therapeutic use of stem cells. [0006] In another embodiment, the invention is directed to a business method for the collection, transport and use of urine derived mesenchymal stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Fig 1A is a diagram of the renal system; and

[0008] Fig IB illustrates stem cell originating site in the kidney.

DETAILED DESCRIPTION OF THE INVENTION

[0009] Certain terminology is used herein for convenience only and is not to be taken as a limitation on the present invention. The terminology includes the words specifically mentioned, derivatives thereof and words of similar import. The embodiments discussed herein are not intended to be exhaustive or to limit the invention to the precise form disclosed. These embodiments are chosen and described to best explain the principle of the invention and its application and practical use and to enable others skilled in the art to best utilize the invention.

[0010] Referring to Fig. 1A, IB and Appendix A (incorporated herein in its entirety), in a first embodiment, the invention is directed to a process to isolate stem cells from urine. The process includes the steps as provided in steps 1-9.

1. Urine collected using standard urine collection techniques

2. Urine is centrifuged at 500-550g for 10 minutes to pellet all cells

3. Supernatant is aspirated and discarded

4. Cells are re-suspended in animal-free cell culture growth medium supplemented with l(Vg/ml Gentamicin sulfate

5. Cell are centrifuged at 500-550g for 10 minutes

6. Supernatant is aspirated and discarded 7. Cells are resuspended in animal-free cell culture growth medium supplemented with l(Vg/ml Gentamicin sulfate and seeded onto fibronectin-coated cultureware

8. Cell culture growth medium is replaced the following day with fresh culture growth medium supplemented with K^g/ml Gentamicin sulfate; thereafter, growth medium is replaced every 2-3 days

9. Cells are passaged upon achieving >90 confluency.

[0011] The process requires that 1) the urine samples are obtained by non-invasive methods 2) is limited to animal-free cell culture growth medium and 3) does not include bovine based serum. As the process uses animal free serum most regulatory concerns are avoided in addition to a further reduction of cost. The ability to obtain urine naturally or collection via an implemented catheter avoids the necessity to the cells by intrusive methods such as surgical or extraction by suction. Further, having the ability to obtain the cells by non-invasive methods substantially reduces costs and trauma to the patient.

[0012] The cells derived from this process would be characterized by flow-based immunophenotyping against multiple positive and negative antigenic markers as well as differentiation assays to demonstrate the classic multipotency seen in MSCs. These cells would then be expanded under optimal culture conditions in an animal-free medium to provide commercialized production and application of urine-derived MSCs for various therapies, most specifically, autologous therapies within days or a couple of weeks of obtaining a sample. The process would not require isolation of MSCs from an encapsulating tissue matrix, digestive enzymes, or any of the things that the FDA regards as exceeding "minimal manipulation". As urine-derived mesenchymal stem cells (herein termed "UDMSCs") require no liberation from a pre-existing tissue matrix, this would be expected to be the case. Furthermore, it is believed that, because of the relative ease of harvest and expansion, that UDMSCs could conceivably be obtained in large numbers continuously from catheterized patients during periods of hospitalization and used without the need for excessive expansion.

[0013] In another embodiment, the invention is directed to a method for collection, processing and therapeutic use of stem cells including the steps of: a. obtaining a urine sample from a patient;

b. processing the urine sample to isolate stem cells contained in the sample; c. expanding the cells in a growth medium; and

d. providing a therapeutic amount of the expanded cells as required to the patient from which the urine sample was obtained.

[0014] The urine sample is obtained by a non-invasive method and uses only animal free serum. The expanded cells are stored at ambient temperatures. It is appreciated within the scope of the present invention, that it would be possible to provide therapeutic uses of stem cells which are obtained from a patient for use in another patient. However, those skilled in the art will appreciate the benefits of autologuous use.

[0015] In another embodiment, the invention is directed to a business method for the collection, transport and use of urine derived mesenchymal stem cells. Appendix A provides illustration and explanation regarding the process and characterization of the isolated stem cells and, in addition, the related steps regarding commercialization aspects for use of the cells. The business method includes the steps of: a. establishing a physician network with patients interested in therapeutic stem cell uses;

b. obtaining a urine sample from a patient;

c. implementation of a method to ensure sample is referenced to patient from which is was obtained.

d. providing a temperature controlled box for transportation of urine sample; e. diluting the urine sample in a growth medium;

f. decontaminating the urine samples during shipment in a nutrient-rich environment;

g. processing the urine sample to isolate stem cells contained in the sample; h. expanding the cells in a growth medium; and providing a therapeutic amount of the expanded cells as required to the patient from which the urine sample was obtained.

[0016] It will be recognized that expansion, cryopreservation and distribution of the cells are within scope of the invention. Methods of cryopreservation and distribution of the cells for use is defined in PCT/2011/039260 (American Cryostem Corporation) incorporated herein in relevant part.

EXAMPLES

[0017] The following are examples of the cells of the present invention providing specific identification to define the cells.

[0018] Whereas MSCs derived from bone marrow, umbilical cord, placenta, and adipose tissue generally illustrate expression of the cell surface markers CD73, CD90, and CD105, cells derived from urine illustrate very low or absent expression of CD73 and no detectable expression of CD105. These cells may illustrate positive or negative expression of CD90, suggesting that two cellular populations may be present.

CD 105 Positive Positive Positive Positive Negative

CD146 Positive Positive Positive Positive Positive

[0019] Whereas MSCs derived from bone marrow, umbilical cord, placenta, and adipose tissue are capable of differentiating into adipocytes, chondrocytes, and osteocytes, UDMSCs near the end of their lifespans appear to differentiate into an epithelial-like phenotype with broad, flattened cells.

[0020] Without being limited to any specific theory, evidence provided herein suggests UDMSCs are likely derived by shedding from kidney tubules suggesting that they have potential therapeutic application in kidney repair.

[0021] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.