Cell sheet cryopreservation liquid and cell sheet cryopreservation method

Abstract

The invention provides a cell sheet cryopreservation solution. The cell sheet cryopreservation solution comprises a cryoprotectant, a buffer solution, a reducing agent, a calcium ion channel inhibitor and an apoptosis inhibitor. The invention also provides a method for cryopreserving the cell sheet by using the cell sheet cryopreservation liquid. According to the cell sheet cryopreservation liquid and the method for cryopreserving the cell sheet, the structural integrity and the cell activity of the cell sheet can be kept.

Description

Cell sheet freezing solution and cell sheet freezing method Technical Field

[0001] The present invention belongs to the field of cell biology and specifically relates to a cell membrane cryopreservation solution and a cell membrane cryopreservation method. Background Art

[0002] Cell sheets are composed of multiple layers of cells and the extracellular matrix they secrete. They offer a more efficient method for stem cell transplantation. Compared to previous methods involving stem cell suspension injection, cell sheets effectively prevent stem cell loss during transplantation, improving the efficiency of stem cell transplantation. Furthermore, cell sheets are prepared without the use of enzymes or similar agents for cell digestion, effectively avoiding the damage to the extracellular matrix and reduced cell function that can occur with enzymatic digestion. Cell sheet transplantation allows stem cells to better function in vivo.

[0003] Currently, cell membranes are typically prepared and used immediately, requiring patients to schedule an appointment at least one week in advance. Furthermore, the cell membranes have a limited shelf life in the preservation solution, typically no longer than 24 hours, and must be used on patients as soon as possible after preparation. Furthermore, due to the short window of use, some classic safety tests, such as sterility and mycoplasma testing, cannot obtain results before use, necessitating the use of rapid testing methods or risk-based release, increasing the risks and testing costs of using cell membranes.

[0004] Therefore, developing cell sheet cryopreservation technology to transform cell sheets from a ready-to-use product into a shelf-ready product is crucial. Unlike traditional single-cell suspension cryopreservation, cell sheet cryopreservation requires overcoming two major challenges: 1) ensuring cell viability at high cell densities per unit volume, and 2) preserving cell-to-cell connectivity. Traditional and commercially available cryopreservation solutions are unable to successfully cryopreserve cell suspensions, resulting in damage to the sheet structure and low cell viability after thawing.

[0005] Therefore, it is necessary to develop a cell membrane freezing solution and a method for freezing cell membranes that can ensure the structural integrity of cell membranes and cell activity.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. One aspect of the present disclosure provides a cell membrane cryopreservation solution comprising: a cryoprotectant, a buffer, a reducing agent, a calcium ion channel inhibitor, and an apoptosis inhibitor.

[0008] For example, the apoptosis inhibitor is selected from one or more of a p53 inhibitor and a caspase inhibitor. Optionally, the concentration of the apoptosis inhibitor is 1-50 μM based on the whole cell sheet cryopreservation solution. In one embodiment, the apoptosis inhibitor is 1-50 μM of a p53 inhibitor or 1-50 μM of a caspase inhibitor based on the whole cell sheet cryopreservation solution.

[0009] For example, the calcium channel inhibitor includes Anipamil, Falipamil, Devapamil, Gallopamil, Tiapamil or Verapamil. Optionally, the concentration of the calcium channel inhibitor is 5-500 μM based on the whole cell membrane cryopreservation solution. In one embodiment, the calcium channel inhibitor is Verapamil 5-500 μM based on the whole cell membrane cryopreservation solution.

[0010] For example, based on the whole cell membrane cryopreservation solution, the reducing agent includes one or more of VC 1-100 μg / mL, reduced glutathione 0.3-30 mM, allopurinol 0.1-10 mM, and adenylate 0.5-50 mM.

[0011] For example, based on the whole cell membrane freezing solution, the cryoprotectant includes one or more of DMSO 2 volume%-10 volume%, sucrose 2g / 100mL-10g / 100mL, ethylene glycol 1 volume%-5 volume%, cross-linked polyvinyl pyrrolidone 2g / 100mL-10g / 100mL, propylene glycol 2 volume%-10 volume%, trehalose 2g / 100mL-10g / 100mL, methylcellulose 0.05g / 100mL-0.5g / 100mL, and glycerol 5 volume%-15 volume%.

[0012] For example, the buffer comprises one of a HEPES buffer system, a phosphate buffer system, a MOPS buffer system, and a PIPES buffer system.

[0013] Optionally, the cell membrane cryopreservation solution further comprises nutritional additives, including transferrin, amino acids, and glucose. For example, the amino acids are selected from one or more of the following: glycine 0.4-1.2 mM, alanine 0.2-0.6 mM, arginine 0.3-2.4 mM, aspartic acid 0.1-0.7 mM, aspartic acid 0.05-0.5 mM, cysteine ​​0.3-0.9 mM, cystine 0.05-0.3 mM, glutamic acid 0.3-0.9 mM, glutamine 1-3 mM, histidine 0. 0.1-0.8, isoleucine 0.2-1.0mM, leucine 0.2-1.0mM, lysine 0.2-1.0mM, methionine 0.05-0.5mM, phenylalanine 1.1-0.6mM, proline 0.1-0.6mM, serine 0.1-0.6mM, threonine 0.2-1.0mM, tryptophan 0.01-0.15mM, tyrosine 0.1-0.6mM, valine 0.2-1.0mM. Optionally, the nutritional additives further include one or more of fetal bovine serum, bovine serum albumin, human serum albumin, and platelet lysate.

[0014] In one embodiment of the cell sheet freezing solution, the cryoprotectant is DMSO based on the entire cell sheet freezing solution. 5 volume % and methylcellulose 0.1g / 100mL; the buffer system is HEPES buffer system; the nutritional additives are human serum albumin 1g / 100mL, glucose 1000μg / mL, transferrin 1μg / mL, glycine 0.7mM, alanine 0.3mM, arginine 0.6mM, aspartic acid 0.3mM, aspartic acid 0.2mM, cysteine ​​0.6mM, cystine 0.1mM, glutamic acid 0.5mM, glutamine 2mM, histidine 0.2mM, isoleucine 0.4mM, leucine 0.4mM, lysine 0.4mM, methionine 0.1mM, phenylalanine 0.2mM, proline 0.3mM, serine 0.2mM, threonine 0.4mM, tryptophan 0.05mM, tyrosine 0.2mM and valine 0.4mM; the reducing agent is VC 10μg / mL; the calcium channel inhibitor is verapamil 30μM; the apoptosis inhibitor is Caspase inhibitor Q-VD-OPh 10μM.

[0015] In another embodiment of the cell sheet freezing solution, the cryoprotectant is DMSO based on the entire cell sheet freezing solution. 10% by volume; the buffer system is a phosphate buffer system; the nutritional additives are: bovine serum albumin 1g / 100mL, glucose 1000μg / mL, transferrin 1μg / mL, glycine 0.7mM, alanine 0.3mM, arginine 0.6mM, aspartic acid 0.3mM, aspartic acid 0.2mM, cysteine ​​0.6mM, cystine 0.1mM, glutamic acid 0.5mM, glutamine 2mM, histidine 0.2mM, isoleucine 0.4mM, leucine 0.4mM, lysine 0.4mM, methionine 0.1mM, phenylalanine 0.2mM, proline 0.3mM, serine 0.2mM, threonine 0.4mM, tryptophan 0.05mM, tyrosine 0.2mM and valine 0.4mM; the reducing agent is VC 5μg / mL and reduced glutathione 3mM; the calcium channel inhibitor is verapamil 20μM; the apoptosis inhibitor is Caspase inhibitor Q-VD-OPh 10μM.

[0016] In another embodiment of the cell sheet freezing solution, the cryoprotectant is DMSO based on the entire cell sheet freezing solution. 10% by volume; the buffer system is a MOPS buffer system; the nutritional additives are 1% by volume of platelet lysate, 1200 μg / mL of glucose, 5 μg / mL of transferrin, 0.7 mM of glycine, 0.3 mM of alanine, 0.6 mM of arginine, 0.3 mM of aspartic acid, 0.2 mM of aspartic acid, 0.6 mM of cysteine, 0.1 mM of cystine, 0.5 mM of glutamic acid, 2 mM of glutamine, 0.2 mM of histidine, 0.4 mM of isoleucine, 0.4 mM of leucine, 0.4 mM of lysine, 0.1 mM of methionine, 0.2 mM of phenylalanine, 0.3 mM of proline, 0.2 mM of serine, 0.4 mM of threonine, 0.05 mM of tryptophan, 0.2 mM of tyrosine, and 0.4 mM of valine; the reducing agent is VC 5μg / mL, reduced glutathione 3mM and allopurinol 1mM; the calcium channel inhibitor is verapamil 25μM; the apoptosis inhibitor is P53 inhibitor Pifithrin-α 10μM.

[0017] Another aspect of the present disclosure provides a method for freezing a cell membrane sheet, comprising: contacting the cell membrane sheet with the cell membrane sheet freezing solution; and freezing the cell membrane sheet and the cell membrane sheet freezing solution together. Optionally, the cell membrane sheet freezing solution is pre-cooled to 2-8°C before contacting the cell membrane sheet.

[0018] Another aspect of the present disclosure provides a composition comprising the above-mentioned cell membrane sheet freezing solution and a cell membrane sheet.

[0019] The cell membrane cryopreservation solution and the method for cryopreserving cell membranes disclosed herein can better ensure the structural integrity and cell activity of the cell membranes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, and are not intended to limit the present invention.

[0021] FIG1 is an appearance diagram of a cell sheet before and after freezing and thawing using the cell sheet freezing solution of Example 1, with the left side showing before freezing and the right side showing after thawing.

[0022] FIG2 is an appearance diagram of the cell sheets after being revived using the cell sheet freezing solutions of Examples 2, 3, and 4, respectively.

[0023] FIG3 is an appearance diagram of the cell membrane after thawing using the cell membrane cryopreservation solutions of Comparative Example 1 and Comparative Example 2, with Comparative Example 1 on the left and Comparative Example 2 on the right.

[0024] FIG4 shows the cell survival rates of the cell sheets after freezing and thawing using the cell sheet freezing solutions of Examples 1, 2, 3 and Comparative Examples 1 and 2.

[0025] FIG5 shows the cell survival rates of the cell sheets after freezing and thawing using the cell sheet freezing solutions of Examples 1 and 4 and Comparative Examples 5, 6, and 7. DETAILED DESCRIPTION

[0026] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The present invention may be implemented in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0028] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0029] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0031] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0032] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0033] Cell sheets are multilayered structures composed of multiple cells, connected by a secreted extracellular matrix (ECM). They are milky white and translucent, with a smooth surface and neat edges. Cell sheets can be harvested using a variety of methods, such as by stimulating extracellular matrix (ECM) secretion with Vitamin C, or by coating culture dishes with temperature-sensitive, light-sensitive, electrosensitive, or polymerized human fibrin. For a review, see "Application of Cell Sheet Technology in Tissue Engineering," published in the December 2017 issue of the Chinese Journal of Cell and Stem Cells, Volume 7, Issue 6.

[0034] The cell membrane of the present application is an animal cell membrane, preferably a mammalian cell membrane, and more preferably a human cell membrane. Human cell membranes include, for example, human umbilical cord mesenchymal stem cell membranes, but other membrane types are also possible. The cell membranes can be of various specifications and sizes, for example, a thickness of 20-300 μm.

[0035] The cell membrane cryopreservation solution disclosed herein comprises: a cryoprotectant, a buffer, a reducing agent, a calcium ion channel inhibitor, and an apoptosis inhibitor. The cell membrane cryopreservation solution disclosed herein can be obtained by mixing the above components.

[0036] Cryoprotectants are divided into non-permeable cryoprotectants and permeable cryoprotectants according to whether they penetrate the cell membrane. Non-permeable cryoprotectants such as sucrose, cross-linked polyvinyl pyrrolidone, trehalose, methylcellulose, polyethylene glycol, dextran, hydroxyethyl starch etc., can not penetrate the cell membrane, but can improve the extracellular fluid osmotic pressure, so that intracellular water enters the extracellular fluid, reduces the formation of ice crystals in the cell. Permeable cryoprotectants can penetrate the cell membrane to enter the cell and play a role, and their direct effect on the cell is better than non-permeable cryoprotectants, such as dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, glycerol etc., are small molecules, inhibit ice crystal formation in the cell by hydrogen bonding with water molecules, reduce the cryoprotectant crystallization temperature, and form a vitrified state of semi-solid-solid state at low temperatures.

[0037] In one embodiment, the cryoprotectant may include one or more of DMSO 2% to 10% by volume, sucrose 2g / 100mL to 10g / 100mL, ethylene glycol 1% to 5% by volume, cross-linked polyvinyl pyrrolidone 2g / 100mL to 10g / 100mL, propylene glycol 2% to 10% by volume, trehalose 2g / 100mL to 10g / 100mL, methylcellulose 0.05g / 100mL to 0.5g / 100mL, and glycerol 5% to 15% by volume, based on the whole cell sheet freezing solution. For example, based on the whole cell sheet freezing solution, the cryoprotectant is DMSO 5% by volume and methylcellulose 0.1g / 100mL. For another example, based on the whole cell sheet freezing solution, the cryoprotectant is DMSO 10% by volume.

[0038] The buffer may be a biological buffer conventionally used for cell culture, for example, a HEPES buffer system, a phosphate buffer system, a MOPS buffer system, or a PIPES buffer system.

[0039] Reducing agents are used to protect cells from damage caused by reactive oxygen species, such as reduced glutathione, VC, allopurinol, and adenylate.

[0040] In one embodiment, the reducing agent comprises one or more of VC 1-100 μg / mL, reduced glutathione 0.3-30 mM, allopurinol 0.1-10 mM, and adenylate 0.5-50 mM, based on the whole cell sheet cryopreservation solution. For example, based on the whole cell sheet cryopreservation solution, the reducing agent is VC 10 μg / mL. For another example, based on the whole cell sheet cryopreservation solution, the reducing agent comprises VC 5 μg / mL and reduced glutathione 3 mM. For another example, based on the whole cell sheet cryopreservation solution, the reducing agent comprises VC 5 μg / mL, reduced glutathione 3 mM, and allopurinol 1 mM.

[0041] Calcium channel inhibitors are agents that block the transmembrane transport of calcium ions through calcium channels on the cell membrane. In one embodiment, the calcium channel inhibitors include one or more of Anipamil, Falipamil, Devapamil, Gallopamil, Tiapamil, and Verapamil. The concentration of the calcium channel inhibitor is typically 5-500 μM based on the entire cell membrane cryopreservation solution. For example, the calcium channel inhibitor is 30 μM, 20 μM, or 25 μM of Verapamil based on the entire cell membrane cryopreservation solution. It is believed that the calcium channel inhibitors in the cell membrane cryopreservation solution may prevent apoptosis caused by changes in calcium ion concentration by hindering changes in calcium ion concentration in the cell.

[0042] Apoptosis inhibitors are agents that can inhibit cell apoptosis. The inventors have found that adding specific apoptosis inhibitors (such as P53 inhibitors, Caspase inhibitors) to cell membrane cryopreservation solution can improve the survival rate of frozen cells after recovery. In one embodiment, the apoptosis inhibitor in the cell membrane cryopreservation solution includes one or more of P53 inhibitors and Caspase inhibitors. Caspase inhibitors are, for example, Q-VD-Oph (CAS No.: 1135695-98-5, purchased from Abcam). P53 inhibitors are, for example, Pifithrin-α (purchased from stemgent, molecular formula C 16 H 18 N2OS·HBr).

[0043] Preferably, the concentration of the apoptosis inhibitor is 1-50 μM based on the whole cell membrane cryopreservation solution. For example, the apoptosis inhibitor is 1-50 μM of a P53 inhibitor or 1-50 μM of a caspase inhibitor based on the whole cell membrane cryopreservation solution.

[0044] In one embodiment, the apoptosis inhibitor in the cell sheet cryopreservation solution is 10 μM of a p53 inhibitor based on the whole cell sheet cryopreservation solution. In one embodiment, the apoptosis inhibitor in the cell sheet cryopreservation solution is 10 μM of a caspase inhibitor based on the whole cell sheet cryopreservation solution.

[0045] The cell membrane cryopreservation solution of the present disclosure may further optionally contain nutritional additives, including transferrin, amino acids, and glucose. In one embodiment, the amino acids are selected from one or more of the following: glycine 0.4-1.2 mM, alanine 0.2-0.6 mM, arginine 0.3-2.4 mM, aspartic acid 0.1-0.7 mM, aspartic acid 0.05-0.5 mM, cysteine ​​0.3-0.9 mM, cystine 0.05-0.3 mM, glutamic acid 0.3-0.9 mM, glutamine 1-3 mM, histidine, based on the entire cell membrane cryopreservation solution. 0.1-0.8mM, isoleucine 0.2-1.0mM, leucine 0.2-1.0mM, lysine 0.2-1.0mM, methionine 0.05-0.5mM, phenylalanine 1.1-0.6mM, proline 0.1-0.6mM, serine 0.1-0.6mM, threonine 0.2-1.0mM, tryptophan 0.01-0.15mM, tyrosine 0.1-0.6mM, valine 0.2-1.0mM.

[0046] Optionally, the nutritional additives further include one or more of fetal bovine serum, bovine serum albumin, human serum albumin, and platelet lysate. In one embodiment, the cell sheet freezing solution does not contain bovine serum, bovine serum albumin, human serum albumin, or platelet lysate.

[0047] The present disclosure also provides a method for freezing a cell membrane sheet, comprising: contacting the cell membrane sheet with the cell membrane sheet freezing solution; and freezing the cell membrane sheet and the cell membrane sheet freezing solution together.

[0048] Contacting the cell sheet with the cell sheet freezing solution may include, for example, immersing the cell sheet in the freezing solution.

[0049] Preferably, the cell membrane freezing solution is pre-cooled at 2-8° C. before contacting the cell membrane.

[0050] The present disclosure also provides a method for reviving a cell sheet, comprising:

[0051] The frozen cell membrane containing the cell membrane and the cell membrane freezing solution is placed in a constant temperature device at 35-39°C for thawing, and then the thawing cell membrane is transferred to a culture dish pre-added with PBS or saline, the liquid in the culture dish is aspirated, and then PBS or saline is added.

[0052] The cell membrane cryopreservation solution and the method for cryopreserving cell membranes disclosed in the present invention can better maintain the structural integrity and cell activity of the cell membranes, wherein the cell activity rate of the cell membranes after thawing is above 80%.

[0053] The present invention is further described below with reference to the following examples, but the present invention is not limited to the following examples. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents and instruments used, for which the manufacturer is not specified, are commercially available conventional products.

[0054] (1) Preparation of cell membrane cryopreservation solution

[0055] Cell membrane cryopreservation solution was obtained by mixing the components of the following formula. As an exception, the commercial cryopreservation solution of Comparative Example 2 was purchased from outside.

[0056] Example 1 (Formula 1):

[0057] Cryoprotectant: DMSO 5% by volume, methylcellulose 0.1 g / 100 mL.

[0058] Buffer system: HEPES buffer system.

[0059] Nutritional additives: human serum albumin 1 g / 100 ml (human serum albumin at a concentration of 20 g / 100 ml by adding 5% of the total formula volume), glucose 1000 μg / mL, transferrin 1 μg / mL, and amino acids: glycine 0.7 mM, alanine 0.3 mM, arginine 0.6 mM, aspartic acid 0.3 mM, aspartic acid 0.2 mM, cysteine ​​0.6 mM, cystine 0.1 mM, glutamic acid 0.5 mM, glutamine 2 mM, histidine 0.2 mM, isoleucine 0.4 mM, leucine 0.4 mM, lysine 0.4 mM, methionine 0.1 mM, phenylalanine 0.2 mM, proline 0.3 mM, serine 0.2 mM, threonine 0.4 mM, tryptophan 0.05 mM, tyrosine 0.2 mM, and valine 0.4 mM. The amino acids were prepared with water for injection to prepare a 100-fold concentrated stock solution of the amino acid combination, and the volume added during preparation was 1% of the total formula.

[0060] Reducing agent: VC 10 μg / mL. VC is prepared into a 1000-fold concentrated stock solution (10 mg / mL) with water for injection. The volume added during preparation is 0.1% of the total formulation.

[0061] Calcium channel inhibitor: Verapamil 30 μM. Verapamil was prepared into a 1000-fold concentrated stock solution (30 mM) using water for injection. The volume added during preparation was 0.1% of the total formulation.

[0062] Apoptosis inhibitor: Caspase inhibitor Q-VD-Oph (CAS No. 1135695-98-5, purchased from Abcam), 10 μM. Caspase inhibitor Q-VD-Oph was prepared in DMSO to a 1000-fold concentrated stock solution, i.e., 10 mM. The volume added during preparation was 0.1% of the total formulation.

[0063] Example 2 (Formula 2):

[0064] The cryoprotectant DMSO was 10% of the total formulation volume.

[0065] Buffer system: phosphate buffer system.

[0066] Nutritional additives: bovine serum albumin 1g / 100ml (bovine serum albumin is prepared into a concentrated stock solution of 20g / 100ml with water for injection, and the volume added during preparation is 5% of the total formula), glucose 1000μg / mL (prepared into a 100-fold concentrated stock solution with water for injection, and the volume added during preparation is 1% of the total formula), transferrin 1μg / mL (prepared into a 1000-fold concentrated stock solution with water for injection, and the volume added during preparation is 0.1% of the total formula) and amino acids. The amino acids are glycine 0.7mM, alanine 0.3mM, arginine 0.6mM, aspartic acid 0.3mM, aspartic acid 0.2mM, cysteine ​​0.6mM, cystine 0.1mM, glutamic acid 0.5mM, glutamine 2mM, histidine 0.2mM, isoleucine 0.4mM, leucine 0.4mM, lysine 0.4mM, methionine 0.1mM, phenylalanine 0.2mM, proline 0.3mM, serine 0.2mM, threonine 0.4mM, tryptophan 0.05mM, tyrosine 0.2mM, and valine 0.4mM (prepared with water for injection into a 100-fold concentrated stock solution of the amino acid combination, the volume added during preparation being 1% of the total formula).

[0067] Reducing agent: VC 5 μg / mL (prepared into a 1000-fold concentrated stock solution with water for injection, the volume added during preparation is 0.1% of the total formula), reduced glutathione 3 mM (prepared into a 1000-fold concentrated stock solution with water for injection, the volume added during preparation is 0.1% of the total formula).

[0068] Calcium channel inhibitor: Verapamil 20 μM (prepared into a 1000-fold concentrated stock solution with water for injection, the volume added during preparation is 0.1% of the total formulation).

[0069] Apoptosis inhibitor: Caspase inhibitor Q-VD-Oph (CAS No. 1135695-98-5, purchased from Abcam), 10 μM. Caspase inhibitor Q-VD-Oph was prepared in DMSO to a 1000-fold concentrated stock solution, i.e., 10 mM. The volume added during preparation was 0.1% of the total formulation.

[0070] Example 3 (Formula 3):

[0071] The cryoprotectant DMSO was 10% of the total formulation volume.

[0072] Buffer system: MOPS buffer system.

[0073] Nutritional supplements: platelet lysate (1% of the total formula volume), glucose 1200 μg / mL, transferrin 5 μg / mL, and amino acids. The amino acids are glycine 0.7 mM, alanine 0.3 mM, arginine 0.6 mM, aspartic acid 0.3 mM, aspartic acid 0.2 mM, cysteine ​​0.6 mM, cystine 0.1 mM, glutamate 0.5 mM, glutamine 2 mM, histidine 0.2 mM, isoleucine 0.4 mM, leucine 0.4 mM, lysine 0.4 mM, methionine 0.1 mM, phenylalanine 0.2 mM, proline 0.3 mM, serine 0.2 mM, threonine 0.4 mM, tryptophan 0.05 mM, tyrosine 0.2 mM, and valine 0.4 mM.

[0074] Reducing agent: VC 5μg / mL, reduced glutathione 3mM, allopurinol 1mM.

[0075] Calcium channel inhibitor: Verapamil 25 μM.

[0076] Apoptosis inhibitor: P53 inhibitor Pifithrin-α (purchased from stemgent company, molecular formula C 16 H 18 N2OS·HBr), 10 μM.

[0077] Example 4 (Formula 1-human serum albumin):

[0078] The formula of Example 4 is basically the same as that of Example 1, except that human albumin is missing.

[0079] Comparative Example 1 (FBS+DMSO)

[0080] Formula: 90% FBS (purchased from Gibco) + 10% DMSO (purchased from Sigma)

[0081] Comparative Example 2 (commercial cryopreservation solution)

[0082] Name: MesenCult TM -ACF Freezing Medium

[0083] Brand: STEMCELL technologies

[0084] Item No.: 05490

[0085] Comparative Example 3 (Formula 1-reducing agent):

[0086] The formulation of Comparative Example 3 is substantially the same as that of Example 1, except that the reducing agent is omitted.

[0087] Comparative Example 4 (Formula 1-calcium channel inhibitor):

[0088] The formulation of Comparative Example 4 is substantially the same as that of Example 1, except that the calcium channel inhibitor is absent.

[0089] Comparative Example 5 (Formula 1 - apoptosis inhibitor):

[0090] The formulation of Comparative Example 5 is substantially the same as that of Example 1, except that the apoptosis inhibitor is absent.

[0091] (2) Cryopreservation of cell membranes

[0092] After harvesting the cell membrane, wash the cell membrane with PBS, add 2-8°C pre-cooled freezing solution, and transfer the cell membrane and freezing solution to a cryovial. Transfer the cryovial to a gradient cooling box, place it in a -80°C freezer, and then transfer it to liquid nitrogen overnight.

[0093] (3) Cell sheet recovery

[0094] Thaw the cryovial rapidly in a 37°C water bath until the ice crystals disappear. Transfer the cell membrane together with the cryovial to a culture dish pre-filled with PBS or saline. Aspirate the liquid in the culture dish and then add PBS or saline.

[0095] (4) Study on the morphology of cell membranes before and after cryopreservation

[0096] Before freezing the cell membrane, flatten the membrane in a 100mm sterile culture dish and take a photo with a mobile phone to obtain the morphological photos of the cell membrane before freezing.

[0097] After the cell membrane is revived, the cryopreservation tube is quickly thawed in a 37°C water bath until the ice crystals in the cryopreservation tube disappear. The cell membrane and the cryopreservation solution are transferred to a 100 mm culture dish pre-filled with PBS or saline. The liquid in the culture dish is aspirated and the membrane is flattened. A mobile phone photo is taken to obtain the morphological photos of the cell membrane after resuscitation.

[0098] Figure 1 (left) shows the appearance of a cell membrane sheet before cryopreservation using the cell membrane cryopreservation solution of Example 1. Figure 1 (right) shows the appearance of a cell membrane sheet after cryopreservation and thawing using the cell membrane cryopreservation solution of Example 1. As shown in Figure 1, the cell membrane sheet is morphologically intact before and after cryopreservation, indicating that the cell membrane viability after thawing is not significantly decreased compared to before cryopreservation. The experimental results of the cell membrane cryopreservation solutions of Examples 2, 3, and 4 are similar to those of the cell membrane cryopreservation solution of Example 1 (see Figure 2).

[0099] Figure 3 shows the morphology of cell sheets recovered from cryopreservation in Comparative Example 1 (FBS+DMSO) and Comparative Example 2 (commercial cryopreservation solution). As can be seen from Figure 3, the edges of the cell sheets are uneven and the morphology is incomplete.

[0100] (5) Cell counting and viability detection

[0101] After the cell membrane sheet recovers, the liquid in the culture dish containing the cell membrane sheet is aspirated, and the digestive enzyme TrypLE is added. The TrypLE and cell membrane sheet are transferred to a 37°C incubator and digested until the membrane sheet is loose. PBS is added to terminate the digestion, centrifuged, and the supernatant is discarded. The cell pellet is resuspended in PBS and then the cell count and viability test are performed.

[0102] The cell viability test method is as follows:

[0103] 1) Add 3 mL of TrypLE (purchased from Life Technologies) to the human umbilical cord mesenchymal stem cell membrane sheet, transfer the mixture to a sterile 50 mL centrifuge tube (purchased from Corning), and incubate at 37°C in a 5% carbon dioxide-saturated humidity incubator (purchased from ThermoFisher, model 371) for 3 minutes until the cells on the human umbilical cord mesenchymal stem cell membrane sheet are loose.

[0104] 2) Add 7 mL of PBS to dilute the TrypLE cells in a centrifuge tube and pipette into single cells using a 10 mL centrifuge tube or a 1 mL pipette.

[0105] 3) The cell suspension was centrifuged at 300×g for 5 minutes at room temperature (centrifuge purchased from ThermoFisher, model ST16).

[0106] 4) Resuspend the cell pellet in 10 mL of PBS and mix thoroughly. Take 100 μL of the aliquot for cell counting and viability testing.

[0107] 5) Take three 1.5 mL sterile EP tubes and add 20 μL of AOPI (purchased from Countstar) to each centrifuge tube.

[0108] 6) Add 20 μL of cell suspension to each 1.5 mL sterile EP tube, mix thoroughly, and add 20 μL to the cell counting plate.

[0109] 7) Insert the cell counting plate into a fully automatic cell counter (purchased from Countstar, model S2) and read the cell viability, total cell density, and live cell density.

[0110] Figure 4 compares the cell viability of the cell membranes after recovery in Examples 1, 2, and 3 and Comparative Example 1 (FBS+DMSO) and Comparative Example 2 (commercial freezing solution). The cell viability of Examples 1, 2, and 3 is above 80%, which is significantly higher than that of Comparative Example 1 and Comparative Example 2.

[0111] Figure 5 compares the post-resuscitation cell viability of the cell membrane sheets from Examples 1 and 4 and Comparative Examples 3, 4, and 5. As shown in Figure 5, the removal of the reducing agent, calcium channel inhibitor, and human albumin from the formulation of Example 1 all affected post-resuscitation cell viability, but the cell viability was still maintained above 70%. The cell viability of Example 4 (the formulation of Example 1 with human albumin removed) was minimally affected. However, the removal of the apoptosis inhibitor significantly reduced cell viability.

[0112] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cell membrane cryopreservation solution, comprising: a cryoprotectant, a buffer, a reducing agent, a calcium ion channel inhibitor and an apoptosis inhibitor, wherein the apoptosis inhibitor comprises one or more of a P53 inhibitor and a Caspase inhibitor. 2 . The cell membrane cryopreservation solution according to claim 1 , wherein the concentration of the apoptosis inhibitor is 1-50 μM based on the entire cell membrane cryopreservation solution.

3. The cell membrane cryopreservation solution according to claim 1 or 2, wherein the apoptosis inhibitor is a P53 inhibitor 1-50 μM or a Caspase inhibitor 1-50 μM based on the entire cell membrane cryopreservation solution.

4. The cell membrane cryopreservation solution according to any one of claims 1 to 3, wherein the calcium ion channel inhibitor comprises one or more of Anipamil, Falipamil, Devapamil, Gallopamil, Tiapamil, and Verapamil. The cell membrane cryopreservation solution according to claim 4 , wherein the concentration of the calcium ion channel inhibitor is 5-500 μM based on the entire cell membrane cryopreservation solution. The cell membrane cryopreservation solution according to claim 4 , wherein the calcium ion channel inhibitor is 5-500 μM verapamil based on the entire cell membrane cryopreservation solution.

7. The cell membrane cryopreservation solution according to any one of claims 1 to 6, wherein the reducing agent comprises one or more of VC 1-100 μg / mL, reduced glutathione 0.3-30 mM, allopurinol 0.1-10 mM, and adenylic acid 0.5-50 mM, based on the entire cell membrane cryopreservation solution.

8. The cell membrane freezing solution according to any one of claims 1 to 7, wherein the cryoprotectant comprises, based on the entire cell membrane freezing solution, one or more of DMSO 2 volume %-10 volume %, sucrose 2 g / 100 mL-10 g / 100 mL, ethylene glycol 1 volume %-5 volume %, cross-linked polyvinyl pyrrolidone 2 g / 100 mL-10 g / 100 mL, propylene glycol 2 volume %-10 volume %, trehalose 2 g / 100 mL-10 g / 100 mL, methylcellulose 0.05 g / 100 mL-0.5 g / 100 mL, and glycerol 5 volume %-15 volume %.

9. The cell membrane cryopreservation solution according to any one of claims 1 to 8, wherein the buffer comprises one of a HEPES buffer system, a phosphate buffer system, a MOPS buffer system, and a PIPES buffer system. 10 . The cell membrane cryopreservation solution according to claim 1 , wherein the cell membrane cryopreservation solution further comprises nutritional additives, wherein the nutritional additives include transferrin, amino acids and glucose.

11. The cell membrane cryopreservation solution according to claim 10, wherein the amino acids are selected from one or more of the following based on the entire cell membrane cryopreservation solution: glycine 0.4-1.2 mM, alanine 0.2-0.6 mM, arginine 0.3-2.4 mM, aspartic acid 0.1-0.7 mM, aspartic acid 0.05-0.5 mM, cysteine ​​0.3-0.9 mM, cystine 0.05-0.3 mM, glutamic acid 0.3-0.9 mM, glutamine 1- 3mM, histidine 0.1-0.8mM, isoleucine 0.2-1.0mM, leucine 0.2-1.0mM, lysine 0.2-1.0mM, methionine 0.05-0.5mM, phenylalanine 1.1-0.6mM, proline 0.1-0.6mM, serine 0.1-0.6mM, threonine 0.2-1.0mM, tryptophan 0.01-0.15mM, tyrosine 0.1-0.6mM, valine 0.2-1.0mM.

12. The cell membrane cryopreservation solution according to claim 10 or 11, wherein the nutritional additive further comprises one or more of fetal bovine serum, bovine serum albumin, human serum albumin, and platelet lysate.

13. The cell membrane cryopreservation solution according to claim 10, wherein the cryoprotectant is 5 volume % DMSO and 0.1 g / 100 mL methylcellulose based on the entire cell membrane cryopreservation solution; the buffer system is a HEPES buffer system; the nutritional additives are 1 g / 100 mL human serum albumin, 1000 μg / mL glucose, 1 μg / mL transferrin, 0.7 mM glycine, 0.3 mM alanine, 0.6 mM arginine, 0.3 mM aspartic acid, 0.2 mM aspartic acid, 0.6 mM cysteine, 0.1 mM cystine, 0.5 mM glutamic acid, 2 mM glutamine, and 100 μg / mL glutamate. The ingredients were as follows: amino acid 0.2mM, isoleucine 0.4mM, leucine 0.4mM, lysine 0.4mM, methionine 0.1mM, phenylalanine 0.2mM, proline 0.3mM, serine 0.2mM, threonine 0.4mM, tryptophan 0.05mM, tyrosine 0.2mM and valine 0.4mM; the reducing agent was VC 10μg / mL; the calcium channel inhibitor was verapamil 30μM; the apoptosis inhibitor was Caspase inhibitor Q-VD-OPh 10μM.

14. The cell membrane cryopreservation solution according to claim 10, wherein the cryoprotectant is DMSO based on the entire cell membrane cryopreservation solution. 10 volume %; the buffer system is a phosphate buffer system; nutritional additives: bovine serum albumin 1g / 100mL, glucose 1000μg / mL, transferrin 1μg / mL, glycine 0.7mM, alanine 0.3mM, arginine 0.6mM, aspartic acid 0.3mM, aspartic acid 0.2mM, cysteine ​​0.6mM, cystine 0.1mM, glutamate 0.5mM, glutamine 2mM, histidine 0.2mM, isoleucine 0.4mM, leucine 0.4mM, lysine 0.4mM, methionine 0.1mM, phenylalanine 0.2mM, proline 0.3mM, serine 0.2mM, threonine 0.4mM, tryptophan 0.05mM, tyrosine 0.2mM and valine 0.4mM; the reducing agent is VC 5μg / mL and reduced glutathione 3mM; the calcium channel inhibitor is verapamil 20μM; the apoptosis inhibitor is Caspase inhibitor Q-VD-OPh 10μM.

15. The cell membrane cryopreservation solution according to claim 10, wherein the cryoprotectant is DMSO based on the entire cell membrane cryopreservation solution. 10 volume %; the buffer system is a MOPS buffer system; the nutritional additives are 1 volume % platelet lysate, 1200 μg / mL glucose, 5 μg / mL transferrin, 0.7 mM glycine, 0.3 mM alanine, 0.6 mM arginine, 0.3 mM aspartic acid, 0.2 mM aspartic acid, 0.6 mM cysteine, 0.1 mM cystine, 0.5 mM glutamate, 2 mM glutamine, 0.2 mM histidine, 0.4 mM isoleucine, 0.4 mM leucine, 0.4 mM lysine, 0.1 mM methionine, 0.2 mM phenylalanine, 0.3 mM proline, 0.2 mM serine, 0.4 mM threonine, 0.05 mM tryptophan, 0.2 mM tyrosine and 0.4 mM valine; the reducing agent is VC 5μg / mL, reduced glutathione 3mM and allopurinol 1mM; the calcium channel inhibitor is verapamil 25μM; the apoptosis inhibitor is P53 inhibitor Pifithrin-α 10μM.

16. A method for freezing a cell membrane sheet, comprising: contacting the cell membrane sheet with a cell membrane sheet freezing solution according to any one of claims 1 to 15; and Cryopreservation of the cell sheets together with the cell sheet freezing solution.

17. The method for freezing cell membranes according to claim 16, wherein the cell membrane freezing solution is pre-cooled at 2-8°C before contacting the cell membranes.

18. A composition comprising the cell membrane cryopreservation solution according to any one of claims 1 to 15 and a cell membrane.

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