CELL SHEET PROTECTION SOLUTION

Abstract

The present disclosure relates to a cell sheet protection solution, such as a stem cell sheet protection solution, such as a mesenchymal stem cell sheet protection solution. The present disclosure also relates to use of the cell sheet protection solution in storage and transportation of the cell sheet.

Claims

1. A cell sheet protection solution comprising the following components: a. a buffered salt solution; b. an extracellular matrix component; c. a reducing agent; d. a substance required for cell metabolism; and e. a hormone.

2. The cell sheet protection solution according to claim 1, wherein the buffered salt solution is selected from phosphate buffered saline (PBS), Hanks buffer, HEPES buffer, Dulbecco's phosphate buffered saline (DPBS) and MOPS buffer.

3. The cell sheet protection solution according to claim 1, wherein the extracellular matrix component includes one or more selected from collagen, gelatin, fibronectin, laminin, and vitronectin.

4. The cell sheet protection solution according to claim 1, wherein the reducing agent includes one or more selected from vitamin C, sodium selenate, sodium pyruvate and glutathione.

5. The cell sheet protection solution according to claim 1, wherein the substance required for cell metabolism includes one or more selected from transferrin, human serum albumin, putrescine, ethanolamine, carnitine, linoleic acid, linolenic acid and an amino acid.

6. The cell sheet protection solution according to claim 5, wherein the amino acid is selected from one or more of alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine, taurine, cysteine, arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine.

7. The cell sheet protection solution according to claim 1, wherein the hormone includes one or more selected from hydrocortisone, insulin and progesterone.

8. The cell sheet protection solution according to claim 1, wherein (a) the buffered salt solution is PBS; the extracellular matrix component includes gelatin; the reducing agent includes vitamin C and glutathione; the substance required for cell metabolism includes transferrin, linoleic acid and linolenic acid; and the hormone includes insulin; (b) the buffered salt solution is Hanks buffer; the extracellular matrix component includes fibronectin, the reducing agent includes vitamin C and sodium selenate, the substance required for cell metabolism includes transferrin, putrescine, human serum albumin and carnitine; and the hormone includes insulin and progesterone, (c) the buffered salt solution is HEPES buffer; the extracellular matrix component includes vitronectin, the reducing agent includes sodium pyruvate, glutathione and sodium selenate, the substance required for cell metabolism includes linoleic acid, linolenic acid and ethanolamine, and the hormone includes insulin and progesterone; (d) the buffered salt solution is HEPES buffer; the extracellular matrix component includes vitronectin, the reducing agent includes sodium pyruvate, glutathione and sodium selenate; the substance required for cell metabolism includes alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine and taurine, and the hormone includes insulin and progesterone; or (e) the buffered salt solution Is MOPS buffer, the extracellular matrix component includes fibronectin, and the reducing agent includes glutathione and vitamin C; the substance required for cell metabolism includes alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine and taurine; and the hormone includes insulin.

9-12. (canceled)

13. The cell sheet protection solution according to claim 3, wherein the concentration of collagen is 0.5-5 mg/L; the concentration of gelatin is 0.5-5 mg/L; the concentration of fibronectin is 5-30 mg/L; the concentration of laminin is 2-10 mg/L; and the concentration of vitronectin is 5-30 mg/L.

14. The cell sheet protection solution according to claim 4, wherein the concentration of vitamin C is 100-1000 mg/L; the concentration of sodium selenate is 0.01-0.05 mg/L; the concentration of sodium pyruvate is 10-100 mg/L; and the concentration of glutathione is 5-30 mg/L.

15. The cell sheet protection solution according to claim 5, wherein the concentration of transferrin is 5-30 mg/L; the concentration of human serum albumin is 1000-5000 mg/L; the concentration of putrescine is 5-30 mg/L; the concentration of ethanolamine is 0.5-5 mg/L; the concentration of carnitine is 0.5-5 mg/L; the concentration of linoleic acid is 0.5-5 mg/L; and the concentration of linoleic acid is 0.5-5 mg/L.

16. The cell sheet protection solution according to claim 6, wherein the concentration of alanine is 0.025-0.4 mM; the concentration of aspartic acid is 0.025-0.4 mM; the concentration of asparagine is 0.025-0.4 mM; the concentration of glutamic acid is 0.025-0.4 mM; the concentration of alanylglutamine is 0.1-2 mM; the concentration of glycine is 0.025-0.4 mM; the concentration of proline is 0.025-0.4 mM; the concentration of serine is 0.025-0.4 mM; the concentration of taurine is 0.025-0.4 mM, the concentration of cysteine is 0.1-2 mM; the concentration of arginine is 0.15-2.4 mM; the concentration of cystine is 0.025-0.4 mM; the concentration of histidine is 0.05-0.8 mM; the concentration of isoleucine is 0.1-1.6 mM, the concentration of leucine is 0.1-1.6 mM, the concentration of lysine is 0.1-1.6 mM; the concentration of methionine is 0.025-0.4 mM; the concentration of phenylalanine is 0.05-0.8 mM; the concentration of threonine is 0.1-1.6 mM; the concentration of tryptophan is 0.1-2 mM; the concentration of tyrosine is 0.05-0.8 mM; and the concentration of valine is 0.1-1.6 mM.

17. The cell sheet protection solution according to claim 7, wherein the concentration of hydrocortisone is 5-30 mg/L; the concentration of insulin is 5-30 mg/L; and the concentration of progesterone is 0.005-0.05 mg/L.

18. The cell sheet protection solution according to claim 1, wherein the pH of the cell sheet protection solution is 7.0-7.4.

19. The cell sheet protection solution according to claim 1, wherein the osmotic pressure of the cell sheet protection solution is 240-340 mOSM/L.

20. The cell sheet protection solution according to claim 1, wherein the cell sheet protection solution is free of serum.

21. The cell sheet protection solution according to claim 1, which is a stem cell sheet protection solution.

22. The cell sheet protection solution according to claim 21, which is a mesenchymal stern cell sheet protection solution.

23. Use of the cell sheet protection solution according to claim 1 in storage of a cell sheet.

24. Use of the cell sheet protection solution according to claim 1 in transportation of a cell sheet.

25-26. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0100] FIG. 1 shows exemplary photos of mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation B).

[0101] FIG. 2 shows a histogram of the levels of cytokines HGF, IL-6 and VEGF secreted by mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation A, Formulation B, and Formulation C).

[0102] FIG. 3 shows a histogram of the levels of cytokines HGF, IL-6, and VEGF secreted by mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation D and Formulation E) and the control buffer.

[0103] FIG. 4 shows the graphs of results of fluorescent staining of fibronectin and integrin-β1 in the mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation B).

[0104] FIG. 5 shows a histogram of the results of the apoptosis level (percentage of living cells) in the mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation A, Formulation B, and Formulation C).

[0105] FIG. 6 shows a histogram of the results of the apoptosis level (percentage of viable cells) in the mesenchymal stem cell sheets before and after transportation in the cell sheet protection solution of the present disclosure (Formulation D and Formulation E) and the control buffer.

SPECIFIC EMBODIMENTS

EXAMPLE 1

Preparation of a Mesenchymal Stem Cell Sheet

[0106] Before preparing the umbilical cord mesenchymal stem cell sheet, the surface of the thermo-sensitive petri dish was first coated with a matrix that facilitates the adherence of mesenchymal stem cells. The matrix used was fibronectin at a concentration of 10 mg/L. The thermo-sensitive petri dish was coated for 8 hours. Subsequently, the coating solution was discarded, and the single-cell suspension of mesenchymal stem cells was added to the smart petri dish. After the cells were adhered and proliferated for 12 hours, the temperature was decreased to 25° C. The cells were detached from the bottom of the thermo-sensitive smart petri dish in a sheet form, and become a cell sheet with complete connection of extracellular matrixes. The sheet is off-white, of dense structure, and of a smooth and flat surface.

EXAMPLE 2

Preparation of Cell Sheet Protection Solutions

[0107] Exemplary cell sheet protection solutions with the following Formulation A, Formulation B, and Formulation C were prepared for further experiments.

[0108] Formulation A:

[0109] Buffered salt solution: PBS

[0110] Extracellular matrix components: gelatin (1 mg/L)

[0111] Reducing agents: vitamin C (500 mg/L); glutathione (10 mg/L)

[0112] Substances required for cell metabolism: transferrin (10 mg/L); linoleic acid (1.0 mg/L); linolenic acid (1.0 mg/L)

[0113] Hormones: insulin (10 mg/L)

[0114] Formulation B:

[0115] Buffered salt solution: Hanks buffer

[0116] Extracellular matrix components: fibronectin (10 mg/L)

[0117] Reducing agents: vitamin C (500 mg/L); sodium selenate (0.02 mg/L)

[0118] Substances required for cell metabolism: transferrin (10 mg/L); putrescine (10 mg/L); human serum albumin (2000 mg/L); carnitine (2 mg/L)

[0119] Hormones: insulin (10 mg/L); progesterone (0.01 mg/L)

[0120] Formulation C:

[0121] Buffered salt solution: HEPES buffer

[0122] Extracellular matrix components: vitronectin (10 mg/L)

[0123] Reducing agents: sodium pyruvate (25 mg/L); glutathione (10 mg/L); sodium selenate (0.02 mg/L)

[0124] Substances required for cell metabolism: linoleic acid (1 mg/L); linolenic acid (1 mg/L); ethanolamine (1 mg/L)

[0125] Hormones: insulin (10 mg/L); progesterone (0.01 mg/L).

[0126] Formulation D:

[0127] Buffered salt solution: HEPES buffer

[0128] Extracellular matrix components: vitronectin (10 mg/L)

[0129] Reducing agents: sodium pyruvate (25 mg/L); glutathione (10 mg/L); sodium selenate (0.02 mg/L)

[0130] Substances required for cell metabolism: alanine (0.1 mM); aspartic acid (0.1 mM); asparagine (0.1 mM); glutamic acid (0.1 mM); alanylglutamine (0.5 mM); glycine (0.1 mM); proline (0.1 mM); serine (0.1 mM); taurine (0.1 mM)

[0131] Hormones: insulin (10 mg/L); progesterone (0.01 mg/L).

[0132] Formulation E:

[0133] Buffered salt solution: MOPS buffer

[0134] Extracellular matrix components: fibronectin (10 mg/L)

[0135] Reducing agents: glutathione (10 mg/L); vitamin C (500 mg/L)

[0136] Substances required for cell metabolism: alanine (0.1 mM); aspartic acid (0.1 mM); asparagine (0.1 mM); glutamic acid (0.1 mM); alanylglutamine (0.5 mM); glycine (0.1 mM); proline (0.1 mM); serine (0.1 mM); taurine (0.1 mM)

[0137] Hormones: insulin (10 mg/L).

EXAMPLE 3

Test of Cell Secreted Factors Before and After Transportation

[0138] The mesenchymal stem cell sheets prepared as described in Example 1 were placed in the cell sheet protection solution with Formulation A, Formulation B, Formulation C, Formulation D or Formulation E, respectively, and transported at 4° C. for 48 hours. Exemplary photos of the cell sheet before and after transportation in the cell sheet protection solution of Formulation B are shown in FIG. 1. It can be seen from the photos that the integrity of cell sheets were maintained before and after transportation, with no significant change in their morphology, and with features of smooth edges, flat surfaces, and no looseness or folds.

[0139] The therapeutic effects of mesenchymal stem cells on self-tissue repair and immune-related diseases are mainly achieved through the secretion and expression of immunosuppressive factors, cytokines, growth factors and the like to regulate inflammation and tissue homeostasis. In this example, the levels of secreted cytokines HGF (R&D, cat. No. DHG00B), IL-6 (R&D, cat. No. VAL102) and VEGF (Life Technologies, cat. No. KHG0112) in the supernatant of mesenchymal stem cell sheets were tested before and after transportation using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. Specifically, the cell sheet was placed in a fresh medium. After 16 hours, the supernatant was collected and placed in a 15 mL centrifuge tube, centrifuged at 400 g for 5 minutes to remove dead cells. The supernatant was taken directly for ELISA assay or frozen at −80° C. for subsequent ELISA assay.

[0140] The results are shown in FIG. 2 and FIG. 3. From these results, it can be seen that the levels of cytokines secreted by mesenchymal stem cells did not change significantly before and after transportation in the cell sheet protection solution of the present disclosure, indicating that the viability of mesenchymal stem cells was maintained. On the contrary, the mesenchymal stem cell sheets transported in the control PBS buffer has a significantly reduced ability to secrete the cytokines HGF, IL-6 and VEGF.

EXAMPLE 4

Test of Adhesion Proteins in Cell Sheets

[0141] The mesenchymal stem cell sheets obtained above were fixed with paraformaldehyde or formalin fixative, and the cell sheets were then made into tissue sections with a thickness of 4-10 μm by paraffin section or frozen section method for staining to observe the profile of proteins contained in the extracellular matrix in the cell sheets, while the DAPI dye was used to stain the nuclei to assist in positioning.

[0142] FIG. 4 shows the results of staining mesenchymal stem cell sheets with fluorescein-labelled anti-fibronectin and integrin-β1 antibodies before and after transportation in the cell sheet protection solution of Formulation B. These results indicate that the mesenchymal stem cell sheets contain a large amount of fibronectin and integrin-β1, which serve as the extracellular matrix in the sheets to connect mesenchymal stem cells. In addition, the amounts of adhesion proteins, fibronectin and integrin-β1 in the cell sheets before and after transportation did not change significantly, indicating that the intercellular connections in the cell sheets transported in the cell sheet protection solution of the present disclosure were maintained well.

EXAMPLE 5

Test of Apoptosis in Cell Sheets

[0143] In addition, the apoptosis of mesenchymal stem cells in the cell sheets before and after transportation was tested. The specific testing steps were performed using Annexin V-FITC Apoptosis Detection Kit (Life Technologies, cat. No. BMS500FI) according to the manufacturer's instructions.

[0144] The results are shown in FIG. 5 and FIG. 6. These results show that the survival rate of the mesenchymal stem cells in the cell sheet did not change significantly before and after transportation in the cell sheet protection solution with Formulation A, Formulation B, Formulation C, Formulation D or Formulation E. In contrast, the percentage of viable cells in the cell sheet transported in the control PBS buffer decreased significantly. The above results indicate that the cell sheet protection solution of the present disclosure protects mesenchymal stem cells from apoptosis.

[0145] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should all fall within the scope of the present invention.