Cell sheet protection solution

Abstract

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

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application bearing application number CN201910550572.X and entitled "Cell Sheet Protective Solution" filed on June 24, 2019. The disclosures of said Chinese patent application are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to the fields of tissue engineering and regenerative medicine, and in particular to a cell sheet protective solution and its use in the storage and transport of cell sheets. [Background technology]

[0003] Currently, the incidence of tissue and organ damage due to degenerative diseases and trauma (e.g., heart failure, Parkinson's disease, Alzheimer's disease) is increasing year by year, and it is difficult to completely treat these diseases using conventional medical methods. Stem cells include pluripotent stem cells and adult stem cells, which have the potential to differentiate into a variety of functional cells and are also able to secrete various cytokines that help repair tissues and organs, making them ideal cells for repairing damaged tissues and organs.

[0004] Currently, numerous basic and clinical studies are being conducted on stem cells to repair damaged tissues and organs and treat diseases. Most of these studies have used direct cell injection or combined cell and biological material transplantation, but both methods have certain limitations. Direct cell injection results in the loss of many stem cells, which not only affects the efficacy of stem cell therapy but also causes side effects such as pulmonary embolism. Simultaneous transplantation of combined cells and biological materials solves the problem of cell loss, but the degradation of biological materials in the body can cause varying degrees of inflammation, and the degradation process and degradation products can cause local tissue lesions.

[0005] Cell sheet technology offers a new technique for stem cell transplantation and application. During the culture process, stem cells establish connections between cells and between the cells and the culture dish through the extracellular matrix. By changing the temperature and adjusting the temperature-sensitive intelligent culture dish to alter the contact conditions between the cells and the culture dish, cells and the extracellular matrix can be separated from the bottom of the temperature-sensitive culture dish without enzymatic digestion, resulting in a complete cell sheet. The cell sheet obtained using this method has high cell density, uniform thickness, and a complete structure. Because the cell sheet produced using this method does not lose its extracellular matrix, extracellular matrix components such as fibronectin can adhere the cell sheet to the surface of the target tissue or organ without relying on additional sutures or adhesives.

[0006] From the time cell sheets are produced in the laboratory until they are used by patients in hospitals, they must undergo a storage and transportation process. If the cell sheet preservation solution is inappropriate during this process, the activity and secreted factors of the cells in the cell sheet cannot be guaranteed, and the integrity of the cell sheet and the connections between the cells within the membrane may be destroyed, severely impacting the clinical application of cell sheets.

[0007] Therefore, there is a great need to develop a cell sheet transport protection solution to ensure that the activity of cells and the amount of secreted factors from cells are not affected during the storage and transportation of cell sheets, and that the integrity of cell sheets and intramembrane cell connections are not affected. Summary of the Invention

[0008] The present disclosure provides a cell sheet protection solution that can maintain the activity and cytokine secretion ability of cells in a cell sheet during storage and transportation of the cell sheet, maintain the integrity of the cell sheet and cell connectivity in the sheet, and protect cells from apoptosis.

[0009] Accordingly, in one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a. Buffered salt solutions; b. Extracellular matrix components; c. Reducing agent; d. Substances necessary for cellular metabolism; and e. A cell sheet protection solution containing a hormone substance.

[0010] In one embodiment, non-limiting examples of the buffered salt solution include phosphate buffered saline (PBS), Hanks' buffer, HEPES buffer, Dulbecco's phosphate buffered saline (DPBS), and MOPS buffer. Other buffered salt solutions known in the art, particularly those used in cell and tissue-related experiments, can also be used.

[0011] Non-limiting examples of extracellular matrix components include collagen, gelatin, fibronectin, laminin, and vitronectin. In some embodiments of the cell sheet protective solution of the present disclosure, the extracellular matrix components include one or more selected from collagen, gelatin, fibronectin, laminin, and vitronectin, for example, at least one, at least two, at least three, at least four, or all five extracellular matrix components selected from collagen, gelatin, fibronectin, laminin, and vitronectin.

[0012] Non-limiting examples of reducing agents include vitamin C, sodium selenate, sodium pyruvate, and glutathione. In one embodiment of the cell sheet protective solution of the present disclosure, the reducing agent comprises one or more selected from vitamin C, sodium selenate, sodium pyruvate, and glutathione, for example, at least one, at least two, at least three, or all four reducing agents selected from vitamin C, sodium selenate, sodium pyruvate, and glutathione.

[0013] Non-limiting examples of substances necessary for cell metabolism include transferrin, human serum albumin, putrescine, ethanolamine, carnitine, linoleic acid, and linolenic acid. In one embodiment of the cell sheet protective solution of the present disclosure, the substances necessary for cell metabolism include one or more selected from transferrin, human serum albumin, putrescine, ethanolamine, carnitine, linoleic acid, linolenic acid, and amino acids, for example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight substances necessary for cell metabolism selected from transferrin, human serum albumin, putrescine, ethanolamine, carnitine, linoleic acid, linolenic acid, and amino acids.

[0014] In one embodiment, the amino acids may include one or more selected from 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.

[0015] Non-limiting examples of hormones include hydrocortisone, insulin, and progesterone. In some embodiments of the cell sheet protective solution of the present disclosure, the hormone substance includes one or more selected from hydrocortisone, insulin, and progesterone, for example, at least one, at least two, or all three hormone substances selected from hydrocortisone, insulin, and progesterone.

[0016] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solutions: PBS, Hanks buffer or HEPES buffer; b. Extracellular matrix components: gelatin, fibronectin, vitronectin, or any combination thereof; c. Reducing agents: Vitamin C, glutathione, sodium selenate, sodium pyruvate, or any combination thereof; d. Substances necessary for cellular metabolism: transferrin, linoleic acid, linolenic acid, putrescine, human serum albumin, carnitine, ethanolamine, or any combination thereof; and e. Hormonal substances: insulin, progesterone, or a combination thereof.

[0017] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solution: PBS; b. Extracellular matrix components: gelatin; c. Reducing agents: Vitamin C and glutathione; d. Substances necessary for cellular metabolism: transferrin, linoleic acid, and linolenic acid; and e. Hormonal substance: insulin.

[0018] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solutions: Hanks buffer solution; b. Extracellular matrix components: fibronectin; c. Reducing agents: Vitamin C and sodium selenate; d. Substances necessary for cellular metabolism: transferrin, putrescine, human serum albumin, and carnitine; and e. Hormonal substances: insulin and progesterone.

[0019] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solution: HEPES buffer; b. Extracellular matrix components: vitronectin; c. Reducing agents: sodium pyruvate, glutathione and sodium selenate; d. Substances necessary for cellular metabolism: linoleic acid, linolenic acid, and ethanolamine; and e. Hormonal substances: insulin and progesterone.

[0020] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solution: HEPES buffer; b. Extracellular matrix components: vitronectin; c. Reducing agents: sodium pyruvate, glutathione and sodium selenate; d. Substances necessary for cellular metabolism: alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine, and taurine; and e. Hormonal substances: insulin and progesterone.

[0021] In certain embodiments, the cell sheet protective solution of the present disclosure comprises the following components: a. Buffered salt solution: MOPS buffer; b. Extracellular matrix components: fibronectin; c. Reducing agents: glutathione and vitamin C; d. Substances necessary for cellular metabolism: alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine, taurine; and e. Hormonal substance: insulin.

[0022] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of collagen as an extracellular matrix component may be, for example, 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0023] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of gelatin as an extracellular matrix component may be, for example, 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0024] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of fibronectin as an extracellular matrix component may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0025] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of laminin as an extracellular matrix component may be 1-50 mg / L, for example, 2-20 mg / L, for example, 2-10 mg / L, for example, about 5 mg / L.

[0026] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of vitronectin as an extracellular matrix component may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0027] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of vitamin C as a reducing agent may be 50-2000 mg / L, for example, 100-1000 mg / L, for example, 200-1000 mg / L, for example, about 500 mg / L.

[0028] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of sodium selenate as a reducing agent may be 0.001-0.1 mg / L, for example, 0.005-0.05 mg / L, for example, 0.01-0.05 mg / L, for example, about 0.02 mg / L.

[0029] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of sodium pyruvate as a reducing agent may be 5-200 mg / L, for example, 10-100 mg / L, for example, 10-50 mg / L, for example, about 25 mg / L.

[0030] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of glutathione as a reducing agent may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0031] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of transferrin, a substance necessary for cell metabolism, may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0032] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of human serum albumin, a substance necessary for cell metabolism, may be 500-5000 mg / L, for example, 1000-5000 mg / L, for example, about 2000 mg / L.

[0033] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of putrescine, a substance necessary for cell metabolism, may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0034] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of ethanolamine, a substance necessary for cell metabolism, may be, for example, 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0035] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of carnitine, a substance necessary for cell metabolism, may be 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0036] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of linoleic acid, a substance necessary for cell metabolism, may be 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0037] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of linolenic acid, a substance necessary for cell metabolism, may be 0.1-10 mg / L, for example, 0.5-5 mg / L, for example, 0.5-2 mg / L, for example, about 1 mg / L.

[0038] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of alanine, a substance necessary for cell metabolism, may be 0.025-0.4 mM, for example, 0.05-0.2 mM, for example, about 0.1 mM; the concentration of aspartic acid may be 0.025-0.4 mM, for example, 0.05-0.2 mM, for example, about 0.1 mM; the concentration of asparagine may be 0.025-0.4 mM, for example, 0.05-0.2 mM, for example, about 0.1 mM; and the concentration of glutamic acid may be 0.025-0.4 mM, for example, 0.05-0.2 mM, for example, about 0. the concentration of alanylglutamine may be 0.1-2 mM, for example 0.2-1 mM, for example about 0.5 mM; the concentration of glycine may be 0.025-0.4 mM, for example 0.05-0.2 mM, for example about 0.1 mM; the concentration of proline may be 0.025-0.4 mM, for example 0.05-0.2 mM, for example about 0.1 mM; the concentration of serine may be 0.025-0.4 mM, for example 0.05-0.2 mM, for example about 0.1 mM; the concentration of taurine may be 0.025-0.4 mM, for example 0.05-0.2 the concentration of cysteine may be 0.1-2 mM, such as 0.2-1 mM, for example about 0.5 mM; the concentration of arginine may be 0.15-2.4 mM, for example 0.3-1.2 mM, for example about 0.6 mM; the concentration of cystine may be 0.025-0.4 mM, for example 0.05-0.2 mM, for example about 0.1 mM; the concentration of histamic acid may be 0.05-0.8 mM, for example 0.1-0.4 mM, for example about 0.2 mM; the concentration of isoleucine may be 0.1-1.6 mM, for example The concentration of leucine may be 0.1-1.6 mM, for example, 0.2-0.8 mM, for example, about 0.4 mM; the concentration of lysine may be 0.1-1.6 mM, for example, 0.2-0.8 mM, for example, about 0.4 mM; the concentration of methionine may be 0.025-0.4 mM, for example, 0.05-0.2 mM, for example, about 0.1 mM; the concentration of phenylalanine may be 0.05-0.8 mM, for example, 0.1-0.4 mM, for example, 0.2 mM; and the concentration of threonine may be 0.1-1.The concentration of tryptophan may be 0.1-2 mM, for example, 0.2-1 mM, for example, about 0.5 mM; the concentration of tyrosine may be 0.05-0.8 mM, for example, 0.1-0.4 mM, for example, about 0.2 mM; and the concentration of valine may be 0.1-1.6 mM, for example, 0.2-0.8 mM, for example, about 0.4 mM.

[0039] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of hydrocortisone as a hormone substance may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0040] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of insulin as a hormone substance may be 1-50 mg / L, for example, 5-30 mg / L, for example, 5-20 mg / L, for example, about 10 mg / L.

[0041] In any embodiment of the cell sheet protective solution of the present disclosure, the concentration of progesterone as a hormone substance may be 0.001-0.1 mg / L, for example, 0.005-0.05 mg / L, for example, 0.005-0.02 mg / L, for example, about 0.01 mg / L.

[0042] As used herein, the term "about" is meant to include the specified value and up to + / - 20%, preferably up to + / - 10%, and more preferably up to + / - 5% of the specified value, where such variations are appropriate to make in the disclosed invention. It should be understood that a value referred to by the modifier "about" is itself specifically and preferably disclosed.

[0043] In one embodiment, the cell sheet protective solution has a pH close to neutral or close to a physiological environment. For example, in one embodiment, the pH of the cell sheet protective solution is 6.0-8.0. In a preferred embodiment, the pH of the cell sheet protective solution is 7.0-7.4.

[0044] In one embodiment, the cell sheet protective solution is isotonic or nearly isotonic with the cells. For example, in one embodiment, the osmotic pressure of the cell sheet protective solution is 240-340 mOSM / L. In a preferred embodiment, the osmotic pressure of the cell sheet protective solution is approximately 280 mOSM / L.

[0045] In one embodiment, the cell sheet protection solution may further contain serum, for example, the serum may be selected from fetal bovine serum, newborn bovine serum, calf serum, and human serum. In one embodiment, the serum content in the cell sheet protection solution is 1-20%, for example, 5-10%.

[0046] In another embodiment, the cell sheet protective solution is serum-free.

[0047] Serum-free cell sheet protection solutions offer an additional advantage. This is because serum derived from different organisms or species poses potential immune risks when cell sheets are administered to subjects such as patients and may be carriers of pathogenic microorganisms. While using serum derived from the cell itself avoids immune risks, its supply is limited, and it is difficult to control the quality between cells, which is detrimental to the stability of the cell sheet protection solution system.

[0048] In one embodiment, the cell sheet protective solution is a stem cell sheet protective solution.

[0049] In one embodiment, the cell sheet protection solution is a mesenchymal stem cell sheet protection solution. The mesenchymal stem cells may have different sources, for example, may be derived from tissues selected from amniotic fluid, amniotic membrane, chorion, chorionic villi, decidua, placenta, umbilical cord blood, Wharton's jelly, umbilical cord, adult bone marrow, adult peripheral blood, and adult adipose tissue.

[0050] In one embodiment, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0051] In one embodiment, the cell sheet protection solution is used for cell sheet preservation, while in another embodiment, the cell sheet protection solution is used for cell sheet transportation.

[0052] In one aspect, the present disclosure relates to the use of the cell sheet protective solution of the present disclosure in preserving a cell sheet.

[0053] In another aspect, the present disclosure relates to the use of the cell sheet protective solution of the present disclosure in transporting a cell sheet.

[0054] In one embodiment of the above use, the cell sheet is a stem cell sheet, such as a mesenchymal stem cell sheet.

[0055] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a. maintaining the cell sheet in the cell sheet protective solution of the present disclosure; and b. A method for preserving a cell sheet, comprising preserving the cell sheet.

[0056] In one embodiment, the cell sheet is stored at a temperature of 0-37°C, such as about 4°C or room temperature (about 25°C).

[0057] In one embodiment, the cell sheet can be stored for a period of 12 hours to 2 weeks, such as about 24 hours, about 48 hours, about 72 hours, about 48 hours, about 5 days, about 6 days, about 1 week, or about 2 weeks.

[0058] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a. maintaining the cell sheet in the cell sheet protective solution of the present disclosure; and b. A method for transporting a cell sheet, comprising transporting the cell sheet.

[0059] In one embodiment, the cell sheet is transported at a temperature of 0-37°C, such as about 4°C or room temperature (about 25°C).

[0060] In one embodiment, the cell sheet can be transported for a period of 12 hours to 2 weeks, such as about 24 hours, about 48 hours, about 72 hours, about 48 hours, about 5 days, about 6 days, about 1 week, or about 2 weeks. [Brief explanation of the drawings]

[0061] [Figure 1] FIG. 1 shows exemplary photographs of a mesenchymal stem cell sheet before and after transportation using the cell sheet protective solution (formulation B) of the present disclosure. [Figure 2] FIG. 2 shows histograms of the secretion levels of cytokines HGF, IL-6, and VEGF from mesenchymal stem cell sheets before and after transportation using the cell sheet protective solutions of the present disclosure (preparation A, preparation B, and preparation C). [Figure 3] FIG. 3 shows histograms of the secretion levels of cytokines HGF, IL-6, and VEGF from mesenchymal stem cell sheets before and after transportation using the cell sheet protective solutions of the present disclosure (Formulations D and E) and a control buffer solution. [Figure 4] FIG. 4 shows the results of fluorescent staining of fibronectin and integrin-β1 in a mesenchymal stem cell sheet before and after transportation using the cell sheet protective solution (formulation B) of the present disclosure. [Figure 5] Figure 5 shows histograms of the detection results of apoptosis levels (percentage of viable cells) in mesenchymal stem cell sheets before and after transportation using the cell sheet protective solutions of the present disclosure (Formulation A, Formulation B, and Formulation C). [Figure 6] FIG. 6 shows a histogram of the detection results of apoptosis levels (percentage of viable cells) in mesenchymal stem cell sheets before and after transportation using the cell sheet protective solutions of the present disclosure (Formulations D and E) and a control buffer solution. DETAILED DESCRIPTION OF THE INVENTION

[0062] Example 1. Preparation of mesenchymal stem cell sheet Before preparing umbilical cord mesenchymal stem cell sheets, the surface of a temperature-sensitive culture dish is first coated with a substrate that promotes the attachment of mesenchymal stem cells. The substrate used is fibronectin at a concentration of 10 mg / L. The temperature-sensitive culture dish is coated for 8 hours. The coating solution is then discarded, and a single-cell suspension of mesenchymal stem cells is added to the intelligent culture dish. After the cells have attached and grown for 12 hours, the temperature is lowered to 25°C. The cells are separated in layers from the bottom of the temperature-sensitive intelligent culture dish, forming a cell sheet with a fully connected extracellular matrix. The sheet is off-white, has a dense structure, and has a smooth, flat surface.

[0063] Example 2. Preparation of cell sheet protective solution Exemplary cell sheet protection solutions having the following formulations A, B, and C were prepared and used in further experiments.

[0064] Formulation A: Buffered salt solution: PBS Extracellular matrix component: gelatin (1 mg / L) Reducing agents: Vitamin C (500 mg / L); Glutathione (10 mg / L) Substances necessary for cellular metabolism: transferrin (10 mg / L); linoleic acid (1.0 mg / L); linolenic acid (1.0 mg / L) Hormonal substances: insulin (10 mg / L)

[0065] Formulation B: Buffered salt solution: Hanks buffer solution Extracellular matrix component: fibronectin (10 mg / L) Reducing agents: Vitamin C (500 mg / L); Sodium selenate (0.02 mg / L) Substances necessary for cellular metabolism: transferrin (10 mg / L); putrescine (10 mg / L); human serum albumin (2000 mg / L); carnitine (2 mg / L) Hormones: insulin (10 mg / L); progesterone (0.01 mg / L)

[0066] Formulation C: Buffered salt solution: HEPES buffer Extracellular matrix component: vitronectin (10 mg / L) Reducing agents: sodium pyruvate (25 mg / L); glutathione (10 mg / L); sodium selenate (0.02 mg / L) Substances necessary for cellular metabolism: linoleic acid (1 mg / L); linolenic acid (1 mg / L); ethanolamine (1 mg / L) Hormonal substances: insulin (10 mg / L); progesterone (0.01 mg / L).

[0067] Formulation D: Buffered salt solution: HEPES buffer Extracellular matrix component: vitronectin (10 mg / L) Reducing agents: sodium pyruvate (25 mg / L); glutathione (10 mg / L); sodium selenate (0.02 mg / L) Substances necessary for cellular 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) Hormonal substances: insulin (10 mg / L); progesterone (0.01 mg / L).

[0068] Formulation E: Buffered salt solution: MOPS buffer Extracellular matrix component: fibronectin (10 mg / L) Reducing agents: glutathione (10 mg / L); vitamin C (500 mg / L) Substances necessary for cellular 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) Hormonal substances: insulin (10 mg / L).

[0069] Example 3. Detection of cell-secreted factors before and after transport Mesenchymal stem cell sheets prepared as described in Example 1 were placed in cell sheet protection solutions containing formulation A, formulation B, formulation C, formulation D, or formulation E, respectively, and transported at 4°C for 48 hours. Exemplary photographs of the cell sheet before and after transportation in the cell sheet protection solution of formulation B are shown in Figure 1. The photographs reveal that the cell sheet maintained its integrity before and after transportation, its morphology did not change significantly, and it was characterized by having smooth edges, a flat surface, and no looseness or wrinkles.

[0070] The therapeutic effects of mesenchymal stem cells on tissue repair and immune-related diseases are primarily achieved by regulating inflammation and tissue homeostasis through the secretion and expression of immunosuppressants, cytokines, and growth factors. In this example, we used an enzyme-linked immunosorbent assay (ELISA) kit to detect the secreted cytokines HGF (R&D, catalog number DHG00B), IL-6 (R&D, catalog number VAL102), and VEGF (Life Technologies, catalog number KHG0112) in the supernatant of mesenchymal stem cell sheets before and after transportation, according to the manufacturer's instructions. Specific procedure: The cell sheet was placed in fresh medium. After 16 hours, the supernatant was collected and placed in a 15 mL centrifuge tube. It was then centrifuged at 400 g for 5 minutes to remove dead cells. The supernatant was either used directly for ELISA detection or frozen at -80°C for subsequent ELISA detection.

[0071] The results are shown in Figures 2 and 3. These results indicate that the levels of cytokines secreted by mesenchymal stem cells did not change significantly before and after transport using the cell sheet protective solution of the present disclosure, indicating that mesenchymal stem cell activity was maintained. Conversely, mesenchymal stem cell sheets transported in the control PBS buffer solution showed a significantly reduced ability to secrete the cytokines HGF, IL-6, and VEGF.

[0072] Example 4. Detection of connexin in cell sheets The mesenchymal stem cell sheet obtained as described above is fixed with paraformaldehyde or formalin fixative, and then the cell sheet is cut into 4-10 μm thick tissue sections using paraffin sectioning or frozen sectioning, and stained to observe the proteins contained in the extracellular matrix of the cell sheet. At the same time, DAPI dye is used to stain the cell nuclei and assist in positioning.

[0073] Figure 4 shows the results of staining a mesenchymal stem cell sheet with fluorescein-labeled anti-fibronectin and integrin-β1 antibodies before and after transport in the cell sheet protective solution of Formulation B. These results indicate that the mesenchymal stem cell sheet contains large amounts of fibronectin and integrin-β1, which function as an extracellular matrix connecting the mesenchymal stem cells in the sheet. Furthermore, the amounts of connexins fibronectin and integrin-β1 in the cell sheet before and after transport did not change significantly, indicating that the cell sheet transported in the cell sheet protective solution of the present disclosure fully maintains its intercellular connections.

[0074] Example 5. Detection of apoptosis in cell sheets Furthermore, apoptosis of mesenchymal stem cells in the cell sheets before and after transportation was detected using the Annexin V-FITC Apoptosis Detection Kit (Life Technologies, catalog number BMS500FI) according to the manufacturer's instructions.

[0075] The detection results are shown in Figures 5 and 6. The results indicate that the activity of mesenchymal stem cells in the cell sheet did not change significantly before or after transport of the cell sheet protective solution of Formulation A, Formulation B, Formulation C, Formulation D, or Formulation E. Conversely, the percentage of viable cells in the cell sheet transported with the control PBS buffer solution was significantly reduced. The above results indicate that the cell sheet protective solution of the present disclosure protects mesenchymal stem cells from apoptosis.

[0076] Although certain embodiments of the present invention have been described in detail above, these are merely examples, and the present invention is not limited to the above specific embodiments. Equivalent modifications and substitutions made by those skilled in the art to the present invention are also within the scope of the present invention. Therefore, all equivalent substitutions and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims

1. a. Buffered salt solutions; b. Extracellular matrix components; c. a reducing agent; d. Substances necessary for cellular metabolism; and e. Contains hormonal substances; Serum-free and The buffered salt solution is PBS, the extracellular matrix component is gelatin, the reducing agent is vitamin C and glutathione, the substances necessary for cellular metabolism are transferrin, linoleic acid, and linolenic acid, and the hormone substance is insulin, or The buffered salt solution is Hanks' buffer solution, the extracellular matrix component is fibronectin, the reducing agent is vitamin C and sodium selenate, the substances necessary for cellular metabolism are transferrin, putrescine, human serum albumin, and carnitine, and the hormone substances are insulin and progesterone, or The buffered salt solution is a HEPES buffer, the extracellular matrix component is vitronectin, and the reducing agent is sodium pyruvate, glutathione, and sodium selenate. The substances necessary for cellular metabolism are linoleic acid, linolenic acid, and ethanolamine, and the hormone substances are insulin and progesterone, or The buffered salt solution is a HEPES buffer, the extracellular matrix component is vitronectin, the reducing agent is sodium pyruvate, glutathione, and sodium selenate, the substances necessary for cellular metabolism are alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine, and taurine, and the hormone substances are insulin and progesterone, or the buffered salt solution is a MOPS buffer solution, the extracellular matrix component is fibronectin, the reducing agent is glutathione and vitamin C, the substances necessary for cellular metabolism are alanine, aspartic acid, asparagine, glutamic acid, alanylglutamine, glycine, proline, serine, and taurine, and the hormone substance is insulin; The concentration of gelatin is 0.5-5 mg / L, the concentration of fibronectin is 5-30 mg / L, and the concentration of vitronectin is 5-30 mg / L; 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; 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 linolenic acid is 0.5-5 mg / L; 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, and the concentration of taurine is 0.025-0.4 mM, Insulin concentration is 5-30 mg / L and progesterone concentration is 0.005-0.05 mg / L. Mesenchymal stem cell sheet protective solution.

2. The cell sheet protective solution according to claim 1, having a pH of 7.0 to 7.

4.

3. The cell sheet protective solution according to claim 1 or 2, having an osmotic pressure of 240-340 mOSM / L.

4. Use of the cell sheet protective solution according to any one of claims 1 to 3 in preserving a mesenchymal stem cell sheet.

5. Use of the cell sheet protective solution according to any one of claims 1 to 3 in transporting a mesenchymal stem cell sheet.

6. a. Maintaining a mesenchymal stem cell sheet in the cell sheet protective solution according to any one of claims 1 to 3; and b. A method for preserving a mesenchymal stem cell sheet, comprising preserving the mesenchymal stem cell sheet.

7. a. Maintaining a mesenchymal stem cell sheet in the cell sheet protective solution according to any one of claims 1 to 3; and b. A method for transporting a mesenchymal stem cell sheet, comprising transporting a mesenchymal stem cell sheet.

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