Stem cell cryopreservation system and method

Abstract

The present application relates to the technical field of stem cell cryopreservation, in particular, the present application relates to a stem cell cryopreservation system and a cryopreservation method.The present application provides a stem cell cryopreservation system, comprising: a cryoprotective solution and a solid support for the attachment of stem cell film suspended in the cryoprotective solution, wherein the solid support is a star-shaped-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane.The stem cell cryopreservation system contains a special solid support for the attachment of stem cell film, which can ensure the integrity of the stem cell film during the cryopreservation process and after recovery, and at the same time can ensure the survival rate and good dryness of the cells in the stem cell film.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stem cell cryopreservation, in particular, the present application relates to a stem cell cryopreservation system and a cryopreservation method. BACKGROUND

[0002] Stem cells are a kind of self-replicating multipotent cells. Under certain conditions, it can differentiate into a variety of functional cells. It is a kind of primitive undifferentiated cells with multi-directional differentiation potential and self-replication ability, which is the original cell of forming various tissues and organs of mammals. Stem cells have commonality in morphology, usually round or round, small cell volume, relatively large nucleus, cell nucleus is mostly euchromatin, and has high telomerase activity.

[0003] With the development of biology and tissue engineering technology, mesenchymal stem cells are concerned due to their potential repair function. However, the current method of infusing cell suspension often causes low cell regeneration efficiency and utilization rate after reinfusion due to the destruction of extracellular matrix (ECM) by hydrolase. To solve the above problems, cell sheet as a kind of cell material without exogenous material has attracted widespread attention in cell transplantation application. Cell sheet refers to the isolated cells cultured on a specific material under in vitro culture conditions, and the complete monolayer cell sheet obtained by separating the cells from the culture substrate without damaging the cell connection. Compared with cell injection and polymer-cell composite implant, cell sheet has the following advantages in clinical application: (1) cell sheet can be directly transplanted to the required site during transplantation surgery; (2) cell sheet can be directly applied to these sites to play a key therapeutic role; (3) cell sheet retains the extracellular matrix, which is beneficial to cell regeneration and in vivo utilization. Therefore, the new type of biological dressing based on stem cell membrane is an ideal means for treating skin damage.

[0004] However, the wide application of stem cell membrane is still limited by many factors, and establishing a safe and effective standardized cryopreservation scheme is a major challenge. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art. To this end, one object of the present application is to provide a stem cell cryopreservation system and a mesenchymal stem cell membrane cryopreservation method, wherein the stem cell cryopreservation system contains a special solid support for the attachment of stem cell membrane, which can ensure the integrity of stem cell membrane during cryopreservation and after recovery, and at the same time can ensure the survival rate and good dryness of cells in stem cell membrane.

[0006] To this end the present invention provides in a first aspect a stem cell cryopreservation system. According to embodiments of the present invention the stem cell cryopreservation system comprises:

[0007] a cryoprotective solution and a solid support suspended in the cryoprotective solution for attachment of a stem cell pellicle,

[0008] wherein the solid support is a star-polydimethylaminoethylacrylate hydrophilically modified PVDF membrane.

[0009] Mesenchymal stem cells can exert their immunomodulatory effects through direct cell-to-cell contact, as well as by secreting bioactive factors such as growth factors, cytokines and chemokines. For example, MSCs interact with macrophages and upregulate the expression of molecules associated with phagocytic function in macrophages, resulting in enhanced phagocytosis of microorganisms by macrophages and reducing further stimulation of immune cells by invading microorganisms. In addition, mesenchymal stem cells can effectively inhibit the overactivation of T cells. They can inhibit the formation of dendritic cells from PBMCs (peripheral blood mononuclear cells), inhibit lymphocyte proliferation, and reduce the number of T cells. They can also directly regulate the content of activated subpopulations of T cells, thereby inhibiting the overactivation of T cells. Infusion of mesenchymal stem cell suspension into patients undergoing allogeneic liver transplantation mainly distributes mesenchymal stem cells in the liver and lung tissues, making it difficult to achieve specific targeting of the transplant. However, using mesenchymal stem cell pellicle, stem cell pellicle can be transplanted in the required organ tissue during the operation process, which can better achieve cell targeting.

[0010] Mesenchymal stem cell pellicle, because the cells retain the complete extracellular matrix and intercellular connections, its paracrine function is not greatly affected. Its main pathway for inducing immune tolerance is through the secretion of exosomes and cytokines, including interleukin-6 (IL-6), transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, etc., to regulate inflammation and tissue homeostasis. However, because it is not injected into the blood, it cannot effectively come into direct contact with T lymphocytes and inhibit T cell activation. The use of mesenchymal stem cell pellicle can effectively avoid the risks that may be caused by intravenous injection of mesenchymal stem cell suspension, such as thrombosis.

[0011] The existing stem cell cryopreservation system cannot guarantee the integrity of the stem cell sheet after recovery, and the cell survival rate after recovery is low. The inventors found that the use of a special solid support for the attachment of the stem cell sheet can guarantee the integrity of the stem cell sheet during the cryopreservation process and after recovery, and can also guarantee the survival rate of the cells in the stem cell sheet and good dryness.

[0012] The stem cell cryopreservation system according to the above embodiments of the present application can also have at least one of the following additional technical features:

[0013] According to an embodiment of the present application, the cryoprotective solution comprises:

[0014] 2-10% v / v DMSO, 5-15% v / v glycol, 40-60% v / v FBS, 1-10% BSA, 1-3 mmol / L glutamine.

[0015] The cryoprotective solution provided by the present application provides nutrients for cells while preventing bacterial contamination of the stem cell cryopreservation system. The DMSO used is dimethyl sulfoxide, a sulfur-containing compound. DMSO is a permeability protective agent that enters the cell interior after passing through the cell membrane to form a certain concentration, preventing excessive water exosmosis in the cell and avoiding excessive dehydration of the cell. It also reduces the electrolyte concentration in the non-frozen solution in the cell, thereby protecting the cell from damage caused by high concentrations of electrolytes. When it permeates into the cell, DMSO can combine with the active water molecules in the cell, slowing down the crystallization of water and preventing low-temperature cryopreservation damage to the cell. However, DMSO can also bind to proteins in the cell, leading to cell damage and death, and thus has a certain cytotoxicity. To minimize the cytotoxicity of DMSO while ensuring that DMSO fully protects cells at low temperatures, the concentration of DMSO is adjusted to 5%. To compensate for the reduced cell protection caused by the reduced concentration of DMSO, glycol, another permeability protective agent, is added. Glycol also enters the cell to prevent the formation of ice crystals in the cell that can cause damage to the cell. Glycol is less toxic than DMSO, and 10% glycol is selected to work together with 5% DMSO to protect stem cells at low temperatures while reducing the toxicity of the cryoprotective agent to the cells.

[0016] According to a preferred embodiment of the present application, the cryoprotective solution comprises:

[0017] 5% v / v DMSO, 10% v / v glycol, 50% v / v FBS, 1-10% BSA, 2 mmol / L glutamine.

[0018] According to an embodiment of the present application, the cryoprotective solution further comprises a DMRM medium and an antibiotic selected from at least one of penicillin and streptomycin.

[0019] According to an embodiment of the present application, the concentration of the penicillin is 80-120 U / mL.

[0020] According to an embodiment of the present application, the concentration of the streptomycin is 80-120 U / mL.

[0021] According to a preferred embodiment of the present application, the concentration of the penicillin is 100 U / mL and the concentration of the streptomycin is 100 U / mL.

[0022] According to an embodiment of the present application, the cryoprotective solution further comprises trehalose.

[0023] According to an embodiment of the present application, the concentration of the trehalose is 0.1-0.2 mmol / L.

[0024] According to an embodiment of the present application, the stem cell sheet is a single layer or a composite mesenchymal stem cell sheet.

[0025] According to an embodiment of the present application, the composite mesenchymal stem cell sheet comprises 2-5 single layer mesenchymal stem cell sheets. Thereby, the toughness and firmness of the cell sheet can be further improved.

[0026] According to an embodiment of the present application, the single layer or composite mesenchymal stem cell sheet is formed of at least one selected from umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and fat-derived mesenchymal stem cells.

[0027] According to an embodiment of the present application, the single layer mesenchymal stem cell sheet is obtained by:

[0028] (1) culturing mesenchymal stem cells in a culture dish using a mesenchymal stem cell medium to form a single layer mesenchymal stem cell sheet;

[0029] (2) removing the mesenchymal stem cell medium and placing the single layer mesenchymal stem cell sheet in a stripping solution to obtain a free single layer mesenchymal stem cell sheet.

[0030] According to an embodiment of the present application, the method of obtaining the composite mesenchymal stem cell sheet further comprises:

[0031] collecting the free single layer mesenchymal stem cell sheets from the stripping solution and stacking a plurality of the free single layer mesenchymal stem cell sheets to obtain a composite mesenchymal stem cell sheet.

[0032] According to an embodiment of the present application, in step (1), the mesenchymal stem cells are subcultured no more than 7 times.

[0033] According to an embodiment of the present application, the inner surface of the bottom of the culture dish is coated with a coating layer, and the surface free energy of the coating layer is no more than 90 mJ / m 2 .

[0034] According to an embodiment of the present application, in step (1), the mesenchymal stem cells are subcultured no more than 7 times. With the mesenchymal stem cells subcultured no more than 7 times, the integrity of the mesenchymal stem cell film obtained can be further improved, and the state of the cell film can be improved. However, when cells subcultured 8 times or more are used to prepare the cell film, the cell film may, occasionally, be broken, the cell film may, occasionally, be automatically detached, and it may be difficult to form a complete cell film. According to an embodiment of the present application, the inner surface of the bottom of the culture dish is coated with a coating layer, and the surface free energy of the coating layer is no more than 90 mJ / m 2 . In this way, the cells are more likely to adhere and grow, and the cell film is more likely to be detached from the culture dish under the stimulation of specific external conditions.

[0035] According to an embodiment of the present application, the surface free energy of the coating layer is no more than 60 mJ / m 2 . In this way, the cell film is more likely to be detached from the culture dish.

[0036] The surface free energy of the culture dish affects the hydrophilicity / hydrophobicity of the surface of the culture dish. If the hydrophilicity is too strong, the adhesion proteins on the surface of the cells are too strong to the culture dish, which may result in that the cells cannot be detached into a single-layer cell film, or the cells are partially detached to cause the cell film to be broken; and if the hydrophobicity is too strong, the cells cannot adhere and grow.

[0037] According to an embodiment of the present application, the thickness of the coating layer is 2-100 nm, preferably 2-50 nm, and more preferably 2-30 nm. In this way, the cell film is more likely to be detached from the culture dish. In addition, the inventors have found that if the thickness of the coating layer is too thick, the growth of the cells will be adversely affected.

[0038] According to an embodiment of the present application, the coating layer comprises a high polymer and / or a block copolymer.

[0039] According to an embodiment of the present application, the coating layer is formed of a poly(2-vinylpyridine-co-styrene) block copolymer. The coating layer can effectively adjust the surface free energy of the culture dish, so that the cells adhere and are easily detached under specific external conditions.

[0040] According to an embodiment of the present application, the coating is formed by solid phase deposition of 2-vinylpyridine and styrene. Thereby, the effect of detaching the cell sheet from the culture dish is better.

[0041] According to an embodiment of the present application, the stripping solution is DPBS buffer.

[0042] The second aspect of the present application provides a cryopreservation method of mesenchymal stem cell sheet, according to an embodiment of the present application, the cryopreservation method comprises:

[0043] The stem cell cryopreservation system, the monolayer or the composite mesenchymal stem cell sheet to be cryopreserved in the first aspect are sealed in a container and cryopreserved.

[0044] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0045] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0046] Figure 1 A nuclear magnetic resonance hydrogen spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to one embodiment of the present application is shown;

[0047] Figure 2 A SEM spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to one embodiment of the present application is shown;

[0048] Figure 3 A state of a cell sheet obtained after resuscitation of a stem cell sheet cryopreserved by the method of Example 1 using a PVDF support film specially modified according to the present application and a commercially available PVDF support film respectively in Example 2 of the present application is shown, wherein, A is a stem cell sheet obtained after resuscitation of a stem cell sheet cryopreserved using the modified PVDF support film according to the present application, and B is a stem cell sheet obtained after resuscitation of a stem cell sheet cryopreserved using the commercially available PVDF support film;

[0049] Figure 4 A live and dead cell activity fluorescence staining result of a human umbilical cord mesenchymal stem cell sheet after resuscitation according to Example 3 of the present application is shown, wherein, the white area in the left figure shows the fluorescence of living cells, and the middle figure shows the fluorescence of dead cells;

[0050] Figure 5 A survival rate of a human umbilical cord mesenchymal stem cell obtained after resuscitation of a stem cell sheet cryopreserved by the method of Example 1 using a PVDF support film specially modified according to the present application and a commercially available PVDF support film respectively according to Example 3 of the present application is shown.

[0051] Figure 6 Fig. 4 shows the results of the identification of cell surface sternness determinants of mesenchymal stem cells recovered after cryopreservation according to Example 4 of the present application using a PVDF support membrane specially modified according to the present application as a support membrane.

[0052] Figure 7 Fig. 5 shows the results of the identification of cell surface sternness determinants of mesenchymal stem cells recovered after cryopreservation according to Example 4 of the present application using a commercially available PVDF support membrane as a support membrane. DETAILED DESCRIPTION

[0053] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein the same or like reference numerals and characters throughout the figures denote the same or like elements or features. The embodiments described below are illustrative of the present application and are not intended to be limiting thereof. In the description of the embodiments, unless otherwise specified, the technical or conditional terms used are those conventionally used in the art or those described in the product manual. Where the manufacturer of the reagent or instrument is not indicated, it is a conventional product available from the market.

[0054] In the description of the present application, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0055] In addition, the terms "first", "second", and the like are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second", and the like can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "plurality" is at least two, such as two, three, and the like, unless otherwise specifically limited.

[0056] In the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature can be "above", "over" and "on" the second feature, which can be that the first feature is directly above or obliquely above the second feature, or only means that the first feature is horizontally higher than the second feature. The first feature can be "below", "under" and "under" the second feature, which can be that the first feature is directly below or obliquely below the second feature, or only means that the first feature is horizontally lower than the second feature.

[0057] According to one specific embodiment of the present application, the present application provides a stem cell cryopreservation system, comprising:

[0058] A cryoprotective solution and a solid support for the stem cell film to adhere in the cryoprotective solution,

[0059] The solid support is a star-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane.

[0060] According to an embodiment of the present application, the cryoprotective solution comprises:

[0061] DMEM medium, 2-10% v / v DMSO, 5-15% v / v ethylene glycol, 40-60% v / v FBS, 1-10% BSA, 1-3 mmol / L glutamine, 80-120 U / mL penicillin, 80-120 U / mL streptomycin, 0.1-0.2 mmol / L trehalose.

[0062] The cryoprotective solution in the stem cell cryopreservation system provided by the present application has the effect of low cytotoxicity. DMSO and ethylene glycol are used as cryoprotective agents to reduce the cytotoxicity of DMSO. Secondly, FBS is used in combination with DMSO and ethylene glycol to simulate the internal environment in which the stem cells are located in vivo, thereby improving the viability of the stem cells and their resistance to DMSO, further reducing the damage of DMSO to the stem cells. Thirdly, while reducing the concentration of DMSO, non-permeable protective agent trehalose is used to protect the stem cells outside the stem cells to ensure the recovery rate of the stem cells and reduce the cytotoxicity of the cryoprotective agent. Fourthly, the microcarrier technology in cell culture is applied to the stem cell cryopreservation system, which has the advantages of high recovery rate and large amount of cryopreservation, and achieves the effects of low cytotoxicity, high recovery rate, large amount of cryopreservation and low cryopreservation cost.

[0063] According to an embodiment of the present application, the stem cell film is a single layer or a composite mesenchymal stem cell film. The composite mesenchymal stem cell film comprises 2-5 single layer mesenchymal stem cell films. In this way, the toughness and firmness of the cell film can be further improved.

[0064] According to an embodiment of the present application, the single-layer mesenchymal stem cell membrane is obtained by:

[0065] (1) culturing mesenchymal stem cells in a culture dish with a mesenchymal stem cell culture medium to form a single-layer mesenchymal stem cell membrane;

[0066] (2) removing the mesenchymal stem cell culture medium and placing the single-layer mesenchymal stem cell membrane in a stripping solution to obtain a free single-layer mesenchymal stem cell membrane.

[0067] According to an embodiment of the present application, the method for obtaining a composite mesenchymal stem cell membrane further comprises:

[0068] collecting the free single-layer mesenchymal stem cell membranes from the stripping solution and stacking a plurality of the free single-layer mesenchymal stem cell membranes to obtain a composite mesenchymal stem cell membrane.

[0069] According to an embodiment of the present application, in step (1), the number of passages of the mesenchymal stem cells is not more than 7. The number of passages of the mesenchymal stem cells is not particularly limited, but preferably the number of passages of the mesenchymal stem cells is not more than 7, because the mesenchymal stem cells meeting this requirement can further improve the integrity of the obtained mesenchymal stem cell membrane and improve the state of the cell membrane, which is not prone to early shedding.

[0070] According to an embodiment of the present application, the inoculation density of the mesenchymal stem cells inoculated into the culture medium contained in the culture dish for culturing the mesenchymal stem cells is not particularly limited and can be controlled according to the length of time required for harvesting the cell membrane, for example, if a cell membrane is required to be obtained quickly, the inoculation density of the mesenchymal stem cells is increased, for example, the density can be maintained at 24,000-30,000 / cm

[0071] According to an embodiment of the present application, the mesenchymal stem cell culture medium can be a conventional culture medium suitable for culturing mesenchymal stem cells in the art. In addition, additional ingredients can be added to the culture medium, for example, ascorbic acid of a suitable concentration is added to facilitate the synthesis of extracellular matrix by cells and to enhance the strength of the cell membrane.

[0072] According to an embodiment of the present application, the inner surface of the bottom of the culture dish is coated with a coating layer, and the surface free energy of the coating layer is not more than 90 mJ / m 2 , preferably not more than 60 mJ / m 2 . Thus, the cell membrane is better detached from the culture dish.

[0073] According to an embodiment of the present application, the thickness of the coating is 2-100 nm, preferably 2-50 nm, and more preferably 2-30 nm. Thereby, the effect of detaching the cell sheet from the culture dish is better.

[0074] According to an embodiment of the present application, the coating comprises a high polymer and / or a block copolymer. The kind of the high polymer and / or the block copolymer in the material of the coating is not particularly limited, as long as the surface free energy of the coating is not more than 90 mJ / m 2 For example, the coating is formed of a poly(2-vinylpyridine-co-styrene) block copolymer, or the coating is formed by solid-phase deposition of 2-vinylpyridine and styrene.

[0075] According to an embodiment of the present application, after the mesenchymal stem cells are cultured to near 100% confluence in the culture dish in step (1), the cell sheet should be treated within 48 hours, otherwise the cell sheet will be detached or broken.

[0076] According to an embodiment of the present application, the stripping solution is DPBS buffer. The stripping solution has a good effect of reducing the binding force between the cell sheet and the culture dish.

[0077] According to an embodiment of the present application, the present application provides a freezing method of mesenchymal stem cell sheet, comprising:

[0078] 1) resuscitating human umbilical cord mesenchymal stem cells and inoculating them into a low surface free energy culture dish and continuously culturing them to form a cell sheet;

[0079] 2) adding DPBS and incubating the support sheet at room temperature, so that the mesenchymal stem cell sheet is detached from the low surface free energy culture dish;

[0080] 3) preparing a cryoprotective solution, the cryoprotective solution comprising: DMEM medium, 5% v / v DMSO, 10% v / v ethylene glycol, 50% v / v FBS, 1%-10% BSA, 2 mmol / L glutamine, 100 U / mL penicillin, 100 U / mL streptomycin, and 0.1 mmol / L trehalose;

[0081] 4) immersing the mesenchymal stem cell sheet together with the support sheet in the cryopreservation solution and sealing it in a cryopreservation bag;

[0082] 5) placing the cryopreservation bag in a programmed cooling box and placing the cell cryopreservation tube in liquid nitrogen for long-term storage.

[0083] For the convenience of understanding, the above-mentioned star-polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane is described in detail.

[0084] Star-shaped PDMAEA has a structure as shown in Formula I

[0085]

[0086] In Formula I, R is n is a positive integer of 15-105. The star-shaped PDMAEA has a polymerization degree of 100-400 and a molecular weight of 10,000-60,000 Dalton. The inventors have found that the long alkyl chain in the star-shaped PDMAEA has hydrophobicity and good compatibility with PVDF, and the PDMAEA chain segment has high hydrophilicity, which improves the hydrophilicity and increases the stability of the modified PVDF membrane. Thus, the PVDF membrane modified by the star-shaped PDMAEA has higher hydrophilicity, permeability and antifouling property, and has high recovery performance after multiple and long-time uses.

[0087] According to one specific embodiment of the present application, the preparation method of the star-shaped PDMAEA is as follows:

[0088] A proper amount of initiator azobisisobutyronitrile, dimethylaminoethyl acrylate and star-shaped chain transfer reagent four-branched 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (its structure is as shown in Formula II) with a molar ratio of (60-420): 1 are dissolved in 50 mL 2-butanone, the oxygen in the reaction container is removed by freeze-degassing-thawing method, and nitrogen or argon is injected as protective gas, and the mixture is stirred in a metal bath at 70-120℃ for 4-20 h; the obtained polymer solution is dropped into hexane, and the product is collected by precipitation method, and the obtained product is purified and dried by rotary evaporation, vacuum drying and other steps to constant weight to obtain the star-shaped PDMAEA, whose 1H NMR spectrum is as shown in Figure 1 , and the SEM spectrum is as shown in Figure 2 .

[0089] wherein R' is

[0090] Subsequently, the prepared star-poly (dimethylaminoethyl acrylate) polymer is added as an additive to a PVDF casting solution, and a star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modified PVDF membrane is prepared by using a doctor blade method. The specific method comprises: taking 50 g of PVDF, 2 g of PVP, 2.7 g of star-poly (dimethylaminoethyl acrylate) polymer and an appropriate amount of DMF into a reaction container, stirring the reaction at 70°C in a metal bath for 10-15 h, and then deaerating in a vacuum oven at 60°C for 8 h to obtain a casting solution. A PVDF flat membrane is prepared by using a solvent-induced phase separation method, water is used as a coagulation bath, the temperature is room temperature, the casting solution is poured onto a clean glass plate, and an automatic film doctor is used for film coating, with a doctor blade thickness of 150 μm. After the coated film is exposed to air for 30 s, it is placed in the coagulation bath until it falls off the glass plate, and the prepared membrane (M-0, M-1, M-2, M-3 and M-4) is immersed in distilled water, which is changed every 12 h to remove residual solvents and pore-forming agents in the membrane, to obtain a star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modified PVDF membrane product.

[0091] The following describes the embodiments of the present application in detail. The embodiments described below are exemplary and are only used to explain the present application and cannot be understood as a limitation of the present application.

[0092] The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by purchase.

[0093] Example 1: Cryopreservation and recovery of human umbilical cord mesenchymal stem cell thin film

[0094] 1. Preparation of human umbilical cord mesenchymal stem cell thin film

[0095] (1) Culture dish treatment

[0096] The bottom of the culture dish used for preparing the human umbilical cord mesenchymal stem cell thin film has a poly (2-vinylpyridine-co-styrene) block copolymer coating layer with a thickness of 8 nm. After the culture dish is sterilized by irradiation sterilization, it is used to culture human umbilical cord mesenchymal stem cells to prepare a human umbilical cord mesenchymal stem cell thin film. The surface free energy of the bottom surface of the coating coated culture dish is 49±2.5 mJ / m 2 .

[0097] (2) Preparation of human umbilical cord mesenchymal stem cell thin film

[0098] Cell culture medium for culturing human umbilical cord mesenchymal stem cells: basal medium (DMEM), 10% serum serum substitute (Takara Cellartis (R) MSC Xeno-Free Supplement, JP), 5% Glutamax additive (ThermoFisher, US).

[0099] 1) Resuscitate human umbilical cord mesenchymal stem cells (purchased from Zhongyuan Union) and inoculate in the treated culture dish in (1) for continuous culture to form a cell film: obtain human umbilical cord-derived mesenchymal stem cells (fourth generation) and resuscitate, resuspend in cell culture medium, centrifuge at 300x g for five minutes, add cell culture medium and inoculate cells at 30,000 / cm2 in a 35mm diameter treated culture dish in (1), and place the culture dish in a 37°C, 5% carbon dioxide, humidity-saturated cell culture incubator. Replace the medium every two days, observe the cell growth state, and when the cells grow to a supersaturated state (100% confluence), obtain an intact human umbilical cord mesenchymal stem cell film.

[0100] 2) After completely removing the culture medium in the culture dish, add DPBS to completely cover the cell film, and place it at room temperature until the cell film spontaneously falls off. After washing the cell film with DPBS three times, the cell film completely separates from the cell culture dish and floats in the form of an intact monolayer film in the DPBS solution. The star-shaped-polyacrylamide dimethylaminoethyl ester polymer hydrophilic modified PVDF membrane product (support film) obtained by the method described above is used to collect the prepared monolayer cell film, and about 2ml of DPBS is added for incubation at room temperature for 20 minutes. After incubation, the three layers of human umbilical cord mesenchymal stem cell films are stacked with tweezers, and the stacked cell film is separated from the culture dish together with the support film to obtain the human umbilical cord mesenchymal stem cell film to be frozen.

[0101] (3) Cryopreservation and resuscitation of human umbilical cord mesenchymal stem cell film

[0102] 1) Preparation of cryoprotective solution

[0103] Commercially available DMEM medium is purchased and the following ingredients are added: 5% v / v DMSO, 10% v / v ethylene glycol, 50% FBS by volume, 1%-10% BSA, and 2mmol / L glutamine.

[0104] 2) Cryopreservation of human umbilical cord mesenchymal stem cell film

[0105] The human umbilical cord mesenchymal stem cell film prepared in (2) above is immersed in the cryopreservation solution together with the support film, sealed in a cryopreservation bag;

[0106] The cryopreservation bag is placed in a programmed cooling box and stored in a refrigerator at -80°C for 12 hours, and then the cell cryopreservation bag is placed in liquid nitrogen for long-term storage. When the human umbilical cord mesenchymal stem cell membrane is needed, the cell cryopreservation bag is taken out from the liquid nitrogen for resuscitation of the human umbilical cord mesenchymal stem cell membrane.

[0107] 3) Resuscitation of the human umbilical cord mesenchymal stem cell membrane

[0108] The cell cryopreservation bag taken out from the liquid nitrogen is placed in a water bath at 37°C for rapid thawing, and the cell membrane is transferred into the cell culture medium in (2) above together with the support, and the stem cell freezing system is washed thoroughly. Then the human umbilical cord mesenchymal stem cell membrane is placed in physiological saline for use.

[0109] Example 2: Integrity evaluation of the human umbilical cord mesenchymal stem cell membrane after resuscitation

[0110] After the cell membranes are cryopreserved by the special modified PVDF support membrane in the present application and the commercially available PVDF support membrane in Example 1, the cell membranes are resuscitated and washed with DPBS, so that the human umbilical cord mesenchymal stem cell membrane is peeled off, and the integrity of the cell membrane is observed, and the results are shown in Figure 3 , wherein Figure A is the stem cell membrane obtained by resuscitation of the cryopreservation using the modified PVDF support membrane in the present application, and Figure B is the stem cell membrane obtained by resuscitation of the cryopreservation using the commercially available PVDF support membrane. It can be found by comparison that the human umbilical cord mesenchymal stem cell membrane obtained by resuscitation of the cryopreservation using the modified PVDF support membrane has a more complete shape and a clearer boundary than the commercially available PVDF support membrane.

[0111] Example 3: Determination of cell survival rate after resuscitation of the stem cell membrane

[0112] The resuscitated human umbilical cord mesenchymal stem cell membrane obtained by the method in Example 1 is dyed and identified by a live and dead cell activity fluorescent kit (ABCAM, item number ab115347, US), and the living cells are dyed green fluorescent Figure 4 in the white area on the leftmost side of the figure), and the dead cells are red Figure 4 in the middle figure, and there is basically no dead cell). After dyeing, the cell activity and survival percentage are calculated by ImageJ and statistical software.

[0113] To ensure the accuracy of the results, another fluorescence staining combined with a cell counter method was used to determine the activity of the cell thin film obtained by using the specially modified PVDF support film and the commercially available PVDF support film as the material for freezing and resuscitation. After the resuscitated cell thin film was washed with PDBS, the cells were digested using trypsin and then stained using a cell activity fluorescence kit. Then, the live and dead cells were counted using a cell counter, and the survival rate was calculated. The results are shown in Figure 5 As can be seen from the results, the cell survival rate after freezing and resuscitation of human umbilical cord mesenchymal stem cell thin film using the specially modified PVDF support film in the present application is high, and is maintained at more than 90%.

[0114] Example 4: Dryness determination of cell thin film

[0115] The dryness of human umbilical cord mesenchymal stem cells was identified by flow cytometry. The mesenchymal stem cell thin film obtained by freezing and resuscitation of mesenchymal stem cells using the specially modified PVDF support film and the commercially available PVDF support film as the support film in Example 1 was digested with trypsin, and then fluorescently stained with fluorescently labeled antibodies specific to CD105, CD90, CD73, CD45, and CD34. Then, the cell surface dryness determinants of the mesenchymal stem cells were identified by flow cytometry. The results are shown in Figure 6 and Figure 7 The data results show that for the mesenchymal stem cell thin film obtained by freezing and resuscitation using the specially modified PVDF support film and the commercially available PVDF support film as the material in the present application, the mesenchymal stem cell thin film has high expression of stem cell specific determinants such as CD105, CD90, and CD73, and low expression of non-stem cell specific determinants such as CD45 and CD34. By comparing the dryness of the mesenchymal stem cells using the two types of support films, it can be seen that the stem cell dryness of the stem cell film obtained by using the freezing and resuscitation technology of the present application is well maintained.

[0116] The results of the examples of the present application show that in the freezing method of stem cell thin film, the use of the special support thin film of the present application makes the stem cell thin film preserved by the freezing method have good integrity, high stem cell survival rate, and good stem cell dryness, which has obvious advantages compared to the commercially available PVDF support film.

[0117] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0118] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A stem cell cryopreservation system, characterized by, The method comprises: a cryoprotective solution and a solid support for the attachment of a stem cell sheet suspended in the cryoprotective solution, wherein the solid support is a star-polyacryl-dimethylaminoethyl polymer hydrophilic modified PVDF membrane.

2. The stem cell cryo-preservation system of claim 1, wherein, The cryoprotective solution comprises: 2-10% v / v DMSO, 5-15% v / v glycol, 40-60% v / v FBS, 1-10% BSA, 1-3 mmol / L glutamine.

3. The stem cell cryo-preservation system of claim 1, wherein, The cryoprotective solution further comprises DMEM medium and antibiotics selected from at least one of penicillin and streptomycin; Optionally, the concentration of the penicillin is 80-120 U / mL; Optionally, the concentration of the streptomycin is 80-120 U / mL; Optionally, the cryoprotective solution further comprises trehalose; Optionally, the concentration of the trehalose is 0.1-0.2 mmol / L.

4. The stem cell cryo-preservation system of claim 1, wherein, The stem cell sheet is a single-layer or composite mesenchymal stem cell sheet; Optionally, the composite mesenchymal stem cell sheet comprises 2-5 single-layer mesenchymal stem cell sheets.

5. The stem cell cryo-preservation system of claim 4, wherein, The single-layer or composite mesenchymal stem cell sheet is formed by at least one selected from umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and adipose tissue-derived mesenchymal stem cells.

6. The stem cell cryo-preservation system of claim 4, wherein, The single-layer mesenchymal stem cell sheet is obtained by: (1) culturing mesenchymal stem cells in a culture dish using a mesenchymal stem cell medium to form a single-layer mesenchymal stem cell sheet; (2) removing the mesenchymal stem cell medium and placing the single-layer mesenchymal stem cell sheet in a stripping solution to obtain a free single-layer mesenchymal stem cell sheet.

7. The stem cell cryo-preservation system of claim 6, wherein, The method further comprises: collecting the free single-layer mesenchymal stem cell sheet from the stripping solution and stacking a plurality of the free single-layer mesenchymal stem cell sheets to obtain a composite mesenchymal stem cell sheet.

8. The stem cell cryo-preservation system of claim 6 or 7, wherein the at least one of the plurality of cryo-preservation containers is a single-use container. In step (1), the number of passages of the mesenchymal stem cells is not more than 7 generations; Optionally, the inner surface of the bottom of the culture dish is coated with a coating having a surface free energy of no more than 90 mJ / m 2 ; Optionally, the surface free energy of the coating is no more than 60 mJ / m 2 ; Optionally, the thickness of the coating is 2-100 nm; Optionally, the coating comprises a high polymer and / or a block copolymer; Optionally, the coating is formed by a poly(2-vinylpyridine-co-styrene) block copolymer; Optionally, the coating is formed by solid-phase deposition of 2-vinylpyridine and styrene.

9. The stem cell cryo-preservation system of claim 6 or 7, wherein, The stripping solution is a DPBS buffer.

10. The stem cell cryo-preservation system of claim 8, wherein, The thickness of the coating is 2-50 nm.

11. The stem cell cryo-preservation system of claim 8, wherein, The thickness of the coating is 2-30 nm.

12. A method for cryopreservation of a mesenchymal stem cell sheet, characterized by, The method comprises: sealing the stem cell cryopreservation system of any one of claims 1-11 and the single-layer or composite mesenchymal stem cell sheet to be cryopreserved in a container for cryopreservation.

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