A protective liquid and its preparation method and application

Abstract

The present application discloses a protective solution and its preparation method and application. The protective solution of the present application includes a cryopreservation agent and a complete culture medium; the cryopreservation agent is composed of a permeable protective agent and a non-permeable protective agent, and the non-permeable protective agent contains β-1,3 glucan; the complete culture medium is composed of a serum-free basal culture medium, a serum substitute and L-glutamine; the protective solution does not contain fetal bovine serum, autologous umbilical cord blood serum and human albumin. The protective solution of the present application can provide a good cryopreservation environment for tissue cells, and β-1,3 glucan can increase the stability of the protective solution, improve the protective effect of frozen tissues and the cell viability after recovery; the complete culture medium provides nutrition for umbilical cord tissue and cells, and is consistent with the culture medium for stem cell recovery and culture, which increases the convenience of preparation and application of the protective solution. The protective solution of the present application has the advantages of easy use, short preparation cycle of cryopreservation preparations, low cost, and safe and reliable clinical application.

Description

Technical Field

[0001] The present application relates to the technical field of tissue cell cryopreservation, and in particular to a protective solution for tissue cell cryopreservation, and a preparation method and application thereof. Background Art

[0002] The umbilical cord is a tubular structure connecting the placenta and the fetus. It has an outer layer of amniotic membrane, two umbilical arteries and one umbilical vein. The gelatinous connective tissue between the inner wall of the umbilical cord and the umbilical blood vessels is Wharton's jelly, that is, human umbilical cord Wharton's jelly tissue. Through culture, about 10 6 Primary mesenchymal stem cells.

[0003] Umbilical cord mesenchymal stem cells are pluripotent stem cells with the ability of self-renewal and multidirectional differentiation. They can differentiate into a variety of cells and tissues, have low immunogenicity, can secrete a variety of cytokines, regulate the immune system, and promote the repair of tissue and organ damage. They can be used to treat osteoarthritis, graft-versus-host disease, diabetes and other diseases; therefore, umbilical cord Wharton's jelly tissue and umbilical cord mesenchymal stem cells have broad clinical application prospects. Moreover, compared with other tissue sources, choosing newborn umbilical cord as a source of mesenchymal stem cells has many advantages, such as rich content, simple collection process, no damage to donors, and no ethical issues.

[0004] At present, stem cell transplantation and treatment generally use pre-stored stem cells, that is, the Wharton's jelly tissue is first separated and primary cultured to obtain umbilical cord mesenchymal stem cells, which are then frozen for subsequent use. This freezing method has a long preparation cycle and high cost. The cell culture and expansion process will increase the number of cell generations, and the freezing process will also have a certain adverse effect on cell activity, reducing the stability of cell culture and clinical application after recovery. Therefore, there are also studies on the direct freezing of Wharton's jelly tissue. Whether the Wharton's jelly tissue is directly frozen or the cells are separated and frozen, the composition of the cryopreservation protection solution will directly affect the freezing effect and affect subsequent use.

[0005] The existing Wharton's jelly tissue cryopreservation protective solution mainly includes protective solutions containing fetal bovine serum or autologous umbilical cord blood serum and protective solutions containing human serum albumin. For protective solutions containing fetal bovine serum or autologous umbilical cord blood serum, fetal bovine serum is derived from a foreign animal and may contain dangerous components such as untested proteins or viruses, which will greatly increase the risk of clinical application of stem cells; while the source of autologous umbilical cord blood serum cannot be guaranteed, the collection volume is limited, and the serum quality will be affected by multiple factors such as current physical conditions, and the quality cannot be guaranteed to be stable and reliable. In addition, there are batch differences between fetal bovine serum and autologous umbilical cord blood serum, and the uniformity of quality cannot be guaranteed. Therefore, the quality and stability of the protective solution cannot be guaranteed. For protective solutions containing human serum albumin, human serum albumin is a human blood product and an allogeneic component; when using cryopreservation protective solutions containing human serum albumin to store tissues and cells, there is a potential risk of transmitting certain pathogens. In addition, human serum albumin also has batch differences, and the uniformity of quality cannot be guaranteed. Moreover, the cost of human serum albumin is high, which is not conducive to production cost control.

[0006] In general, the existing technologies have many problems that are not conducive to the future clinical application of stem cells, such as the cryopreservation protective fluids generally contain animal serum or human albumin, the preparation cycle of cryopreservation preparations is long, the preparation cost is high, and the cryopreservation and resuscitation process damages cell activity. Currently, the industry has an urgent need for safer, more reliable and low-cost cell and tissue cryopreservation technologies. Summary of the invention

[0007] The purpose of the present application is to provide an improved protective solution for tissue cryopreservation and a preparation method and application thereof.

[0008] This application specifically adopts the following technical solutions:

[0009] One aspect of the present application discloses a protective solution, comprising a cryopreservation agent and a complete culture medium; the cryopreservation agent is composed of a permeable protective agent and a non-permeable protective agent, and the non-permeable protective agent contains β-1,3 glucan; the complete culture medium is composed of a serum-free basal culture medium, a serum substitute and L-glutamine; and the protective solution does not contain fetal bovine serum, autologous umbilical cord blood serum and human serum albumin.

[0010] It should be noted that the protective solution of the present application, in which the permeable protective agent is a small molecule substance, can penetrate into the interior of tissue cells through the cell membrane. For example, in one implementation of the present application, it can penetrate into the interior of the umbilical cord Wharton's jelly tissue cells, reduce the electrolyte concentration inside and outside the cells, protect the cells from damage by high concentration electrolytes, and avoid excessive dehydration of the cells. Non-permeable protective agents are macromolecules that can combine with water molecules in the solution outside the tissue cells, reduce the content of free water, lower the freezing point, reduce the formation of ice crystals and the physical damage of ice crystals to tissue cells, and reduce the electrolyte concentration in the solution, thereby reducing damage to tissues and cells. β-1,3 glucan is a naturally occurring non-starch polysaccharide that is widely present in the cell walls of microorganisms and plants such as yeast, seaweed, and oats. β-1,3 glucan is a polysaccharide with different branch lengths formed by D-glucose monomers connected by β-1,3 glycosidic bonds. This connection method can form a spiral molecular structure, making β-1,3 glucan easily accepted by the immune system. β-1,3 glucan is a safe compound that can replace a portion of whole blood during blood transfusion. It can be used as a plasma volume expander and can also be used in food, medicine, skin care products, feed, etc. β-1,3 glucan has the functions of enhancing the immune system, anti-bacterial, anti-viral, anti-fungal, anti-parasitic, lowering cholesterol and blood lipids, and promoting wound healing. It is a good biological effect regulator. The present application creatively adds β-1,3 glucan to the protective solution. Since β-1,3 glucan has high viscosity and water absorption, it can increase the stability of the cryopreservation protective solution, improve the protective effect of frozen tissues and the viability of cells after recovery. In addition, the protective solution of the present application also contains a complete culture medium, which can provide nutrition for tissues and cells, and is consistent with the culture medium used for stem cell recovery and culture, which increases the convenience of preparation and application of the cryopreservation protective solution.

[0011] It should also be noted that the protective solution of the present application does not contain fetal bovine serum, which avoids the risk of introducing components from xenobiotic animals and improves the safety of cryopreserved preparations and future clinical applications of mesenchymal stem cells; it does not contain human serum albumin, which avoids the potential risk of transmitting certain pathogens and reduces production costs; it does not contain components with batch differences such as autologous umbilical cord blood serum, which can effectively ensure the quality uniformity of the protective solution.

[0012] Preferably, in the protective solution of the present application, the concentration of β-1,3 glucan in the protective solution is 0.1 mg / mL-9 mg / mL.

[0013] It should be noted that one of the key points of this application is the use of β-1,3 glucan in the protective solution, which has high viscosity and water absorption, can increase the stability of the cryopreservation protective solution, improve the protective effect on the frozen tissue and the viability of the cells after recovery; it can be understood that as long as β-1,3 glucan is added to the protective solution, its effect can be exerted to varying degrees. The concentration specified in this application is only a specific concentration that meets the experimental design and requirements in one implementation of this application. According to different usage requirements or experimental requirements, it can be adjusted within or outside this concentration range. In principle, the effect on the stability of the protective solution is reduced below the concentration specified in this application; and if the concentration of β-1,3 glucan is too high, on the one hand, its effect is not significantly increased, and on the other hand, it will affect the content of other components in the protective solution, which is not conducive to cryopreservation, and too high a concentration of β-1,3 glucan will also increase the cost of the protective solution.

[0014] Preferably, the concentration of β-1,3 glucan in the protective solution is 0.45 mg / mL, 2.25 mg / mL or 9 mg / mL.

[0015] Preferably, in the protective solution of the present application, the volume ratio of serum-free basal culture medium, serum substitute and L-glutamine is 97:2:1; or, the volume ratio of serum-free basal culture medium, serum substitute and L-glutamine is 95:4:1.

[0016] It should be noted that in the protective solution of the present application, serum-free basal medium, serum substitute and L-glutamine constitute a complete medium. The role of the complete medium is to provide nutrition for tissues and cells on the one hand, and to be consistent with the medium used for stem cell resuscitation and culture on the other hand. Therefore, the distribution ratio of each group can be adjusted as needed while ensuring the supply of nutrition so that it is consistent with the medium for stem cell resuscitation and culture. The volume ratio of serum-free basal medium, serum substitute and L-glutamine is 97:2:1 or 95:4:1, which is only a specific ratio used in one implementation of the present application, and it is not ruled out that other ratio schemes can also be used.

[0017] Preferably, in the protective solution of the present application, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservative is 1:1, and the volume ratio of the complete culture medium to the cryopreservative is 9:1; or, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservative is 3:1, and the volume ratio of the complete culture medium to the cryopreservative is 4:1; or, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservative is 4:1, and the volume ratio of the complete culture medium to the cryopreservative is 3:1; or, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservative is 9:1, and the volume ratio of the complete culture medium to the cryopreservative is 1:1.

[0018] It should be noted that these four ratios are all feasible, but the preservation effects may vary, and the specific ones can be determined according to the needs. It can be understood that these four ratios are only one implementation of the present application, and adaptive adjustments can be made on this basis, which is not specifically limited here.

[0019] Preferably, the protective solution of the present application consists of 5-20 volumes of an osmotic protective agent, 2-50 volumes of an impermeable protective agent, 0.2-1 volumes of L-glutamine, 1-10 volumes of a serum replacement, and 45-90 volumes of a serum-free basal culture medium.

[0020] Preferably, the protective solution of the present application consists of 5 volumes of an osmotic protective agent, 45 volumes of an impermeable protective agent, 0.5 volumes of L-glutamine, 1 volume of a serum replacement, and 48.5 volumes of a serum-free basal culture medium.

[0021] Preferably, in the protective solution of the present application, the permeable protective agent includes at least one of dimethyl sulfoxide, glycerol, ethylene glycol and propylene glycol.

[0022] Preferably, the osmotic protectant is dimethyl sulfoxide.

[0023] Preferably, the non-permeable protective agent consists of at least one of hydroxyethyl starch, sucrose, polyethylene glycol and trehalose, and β-1,3 glucan.

[0024] Preferably, the non-permeable protective agent consists of hydroxyethyl starch and β-1,3 glucan.

[0025] Preferably, the L-glutamine is clinical grade L-glutamine, the serum substitute is clinical grade serum substitute, and the serum-free basal medium is clinical grade basal medium.

[0026] Another aspect of the present application discloses a method for preparing the protective solution of the present application, comprising the following steps:

[0027] A. Mix serum-free basal medium, serum replacement and L-glutamine evenly to prepare complete medium and store at 2-8℃;

[0028] B. Add the permeable protective agent to the non-permeable protective agent containing β-1,3 glucan, mix well, prepare a cryopreservative, and store at 2-8°C;

[0029] C. Evenly mix the complete culture medium prepared in step A and step B with the cryopreservation agent to obtain the protective solution of the present application, and store it at 2-8°C.

[0030] Preferably, the non-permeable protective agent containing β-1,3 glucan is prepared using physiological saline.

[0031] Another aspect of the present application discloses the use of the protective solution of the present application in the cryopreservation of tissues or cells.

[0032] Preferably, in the application of the present application, the tissue is human umbilical cord Wharton's jelly tissue.

[0033] It should be noted that the protective solution of the present application is developed for the cryopreservation of human umbilical cord Wharton's jelly tissue; it can be understood that it can be used not only for the cryopreservation of human umbilical cord Wharton's jelly tissue, but also for the cryopreservation of other tissues or cells; other tissues include various tissues from humans or other animals, such as skin, muscle, internal organs, bones and other tissues; other cells also include various cells from humans or other animals.

[0034] Another aspect of the present application discloses a method for cryopreservation of human umbilical cord Wharton's jelly tissue, comprising the following steps:

[0035] A. Clean and disinfect the fresh umbilical cord sample, peel off the Wharton's jelly tissue, and cut the Wharton's jelly tissue into small pieces;

[0036] B. adding the Wharton's jelly tissue block obtained in step A to the protective solution of the present application, mixing well and dispensing into cryopreservation tubes to prepare an umbilical cord tissue cryopreservation preparation;

[0037] C. The umbilical cord tissue cryopreservation preparation obtained in step B is subjected to programmed cooling and stored in a liquid nitrogen tank.

[0038] Preferably, in the umbilical cord tissue cryopreservation preparation, the volume ratio of Wharton's jelly tissue to protective solution is 1:2.

[0039] It should be noted that the volume ratio of Wharton's jelly tissue to protective solution is mainly to ensure that the Wharton's jelly tissue is effectively protected with as little protective solution as possible. In principle, the volume of protective solution is twice the volume of Wharton's jelly tissue to meet the demand. Of course, more protective solution can also be used.

[0040] Preferably, the Wharton's jelly tissue is cut into small pieces with a size of 1-3 mm. 3 The specification of the cryotube is 2mL.

[0041] It should be noted that the purpose of cutting the Wharton's jelly tissue into pieces is to allow it to better contact the protective solution and mix evenly with the protective solution. The specific size of the pieces can be determined according to actual conditions. As for the specifications of the cryopreservation tubes, 2 mL centrifuge tubes are generally used, which can be convenient for storage and use. Of course, according to different needs, larger centrifuge tubes or 1.5 mL centrifuge tubes can also be used, which are not specifically limited here.

[0042] The beneficial effects of this application are:

[0043] The protective solution of the present application can provide a good freezing environment for tissue cells, among which β-1,3 glucan can increase the stability of the cryopreservation protective solution, improve the protective effect on frozen tissues and the viability of cells after recovery; the complete culture medium can not only provide nutrition for umbilical cord tissue and cells, but also keep consistent with the culture medium used for stem cell recovery and culture, which increases the convenience of preparation and application of the cryopreservation protective solution. The protective solution of the present application does not contain fetal bovine serum, autologous umbilical cord blood serum and human albumin, which not only improves the safety and reliability of clinical application, but also avoids the quality uniformity problems caused by these components. The protective solution of the present application has the advantages of easy use, short preparation cycle of cryopreservation preparations, low cost, safe and reliable clinical application, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is an inverted microscope observation result of P0 generation cells of umbilical cord mesenchymal stem cells of different umbilical cord tissue cryopreservation preparations and fresh umbilical cord tissue in the examples of the present application;

[0045] Figure 2 It is a growth curve diagram of P0 generation cells of umbilical cord mesenchymal stem cells from different umbilical cord tissue cryopreservation preparations and fresh umbilical cord tissue in the examples of the present application. DETAILED DESCRIPTION

[0046] The existing cryopreservation protection fluids generally contain animal serum or human albumin, the preparation cycle of cryopreservation preparations is long, the preparation cost is high, and the cryopreservation and resuscitation process damages cell activity, which are not conducive to the future clinical application of stem cells. Therefore, the present application has developed an improved tissue cell cryopreservation protection fluid, which solves the above unmet needs at the same time, is easy to use, shortens the preparation cycle of cryopreservation preparations, reduces the cost of cryopreservation preparations, and improves the safety and reliability of the clinical application of umbilical cord stem cells.

[0047] The protective solution of the present application includes a cryopreservation agent and a complete culture medium; the cryopreservation agent is composed of a permeable protective agent and a non-permeable protective agent, and the non-permeable protective agent contains β-1,3 glucan; the complete culture medium is composed of a serum-free basal culture medium, a serum substitute and L-glutamine; and the protective solution does not contain fetal bovine serum, autologous umbilical cord blood serum and human serum albumin.

[0048] The protective liquid of this application has the following advantages:

[0049] (1) The protective solution of the present application contains β-1,3 glucan, which is a good biological effect regulator with high viscosity and water absorption. It can increase the stability of the cryopreservation protective solution, improve the protective effect on frozen tissues and the viability of cells after recovery.

[0050] (2) The protective solution of the present application does not contain fetal bovine serum, thus avoiding the risk of introducing components from xenobiotic animals and improving the safety of cryopreserved preparations and future clinical applications of mesenchymal stem cells.

[0051] (3) The protective liquid of the present application does not contain human serum albumin, thereby avoiding the potential risk of transmitting certain pathogens and reducing production costs.

[0052] (4) The protective solution of the present application contains a complete culture medium, which can provide nutrition for umbilical cord tissue and cells, and is consistent with the culture medium used for stem cell recovery and culture, thereby increasing the convenience of preparation and application of the cryopreservation protective solution.

[0053] (5) The protective solution of the present application, in one implementation of the present application, all raw materials used are clinical-grade reagents with defined ingredients, which have stable and reliable quality and can meet the quality requirements of drug production.

[0054] (6) The protective solution of the present application can be used for direct cryopreservation of human umbilical cord Wharton's jelly tissue, which reduces the preparation cost of the cryopreservation preparation, shortens the preparation cycle, reduces the potential toxicity of the cryopreservation protective solution to cells, improves the quality of umbilical cord mesenchymal stem cells obtained by resuscitation culture, and ensures the reliability of the cryopreservation preparation.

[0055] Compared with the traditional cryopreservation protective solution and cryopreservation method of umbilical cord mesenchymal stem cells, the present invention can save the time of preparing cryopreservation preparations, reduce the cost of preparing cryopreservation preparations, and improve the cryopreservation effect of umbilical cord Wharton's jelly tissue. Compared with the cryopreservation of umbilical cord mesenchymal stem cells after resuscitation using traditional methods, the cell morphology and cell activity of umbilical cord mesenchymal stem cells separated from the cryopreservation of umbilical cord Wharton's jelly tissue after resuscitation in the present invention are better.

[0056] In the protective solution of the present application, L-glutamine is clinical-grade L-glutamine, such as products from Sigma-Aldrich. Serum substitutes are clinical-grade serum substitutes, such as products from PALL, Thermo Fisher or Helios, such as Ultroser G or UltraGRO Cell Culture Supplement. Serum-free basal medium is clinical-grade basal medium, such as products from Lonza, Thermo Fisher or Hyclone, such as UltraCULTURE Serum-free Medium, DMEM, DMEM / F12, α-MEM, etc.

[0057] The protective solution of the present application uses a clinical-grade serum substitute with defined ingredients, which can avoid the risk of introducing animal-derived proteins such as fetal bovine serum, while ensuring the uniformity of different batches and improving the safety and stability of the cryopreservation protective solution. In one implementation of the present application, the protective solution uses UltraCULTURE Serum-free Medium serum-free culture medium, Ultroser G serum substitute and Gibco L-glutamine, the permeable protective agent uses dimethyl sulfoxide, and a non-permeable protective agent formed by dissolving β-1,3 glucan and hydroxyethyl starch in physiological saline.

[0058] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0059] Example

[0060] In this case, high-concentration β-1,3-glucan, medium-concentration β-1,3-glucan, and low-concentration β-1,3-glucan protective solutions were prepared for human umbilical cord Wharton's jelly tissue cryopreservation experiments, and protective solutions without β-1,3-glucan and traditional cryopreservation protective solutions were used as controls; the cell morphology, cell activity, proliferation ability, and surface marker expression of umbilical cord mesenchymal stem cells after 30 days of cryopreservation using the five protective solutions were tested; at the same time, fresh human umbilical cord Wharton's jelly tissue that had not been cryopreserved was used as a control for the same tests, as detailed below:

[0061] 1. Preparation of protective solution

[0062] 1. High concentration β-1,3 glucan protective solution

[0063] Mix 97 mL of UltraCULTURE Serum-free Medium, 2 mL of pre-dissolved and filtered Ultroser G serum substitute, and 1 mL of 200 mM L-glutamine to prepare 100 mL of complete culture medium and store in a refrigerator at 4°C.

[0064] Use physiological saline to prepare 9 mL of a solution containing 20 mg / mL β-1,3 glucan and 20% hydroxyethyl starch, then slowly add 1 mL of dimethyl sulfoxide, mix well, prepare 10 mL of cryopreservative, and store in a refrigerator at 4°C.

[0065] 5 mL of complete culture medium and 5 mL of cryopreservative were mixed evenly to prepare 10 mL of human umbilical cord Wharton's jelly tissue cryopreservation protective solution, that is, the high-concentration β-1,3 glucan protective solution in this example, wherein the concentration of β-1,3 glucan in the protective solution was 9 mg / mL, and the solution was refrigerated and stored at 4°C.

[0066] 2. Medium concentration β-1,3 glucan protective solution

[0067] Mix 97 mL of UltraCULTURE Serum-free Medium, 2 mL of pre-dissolved and filtered Ultroser G serum substitute, and 1 mL of 200 mM L-glutamine to prepare 100 mL of complete culture medium and store in a refrigerator at 4°C.

[0068] Use physiological saline to prepare 9 mL of a solution containing 5 mg / mL β-1,3 glucan and 20% hydroxyethyl starch, then slowly add 1 mL of dimethyl sulfoxide, mix well, prepare 10 mL of a cryopreservative, and store at 4°C.

[0069] 5 mL of complete culture medium and 5 mL of cryopreservative were mixed evenly to prepare 10 mL of human umbilical cord Wharton's jelly tissue cryopreservation protective solution, that is, the medium concentration β-1,3 glucan protective solution in this example, wherein the concentration of β-1,3 glucan in the protective solution was 2.25 mg / mL, and the solution was stored in a refrigerator at 4°C.

[0070] 3. Low concentration β-1,3 glucan protective solution

[0071] Mix 97 mL of UltraCULTURE Serum-free Medium, 2 mL of pre-dissolved and filtered Ultroser G serum substitute, and 1 mL of 200 mM L-glutamine to prepare 100 mL of complete culture medium and store in a refrigerator at 4°C.

[0072] Use physiological saline to prepare 9 mL of a solution containing 1 mg / mL β-1,3 glucan and 20% hydroxyethyl starch, then slowly add 1 mL of dimethyl sulfoxide, mix well, prepare 10 mL of cryopreservative, and store in a refrigerator at 4°C.

[0073] 5 mL of complete culture medium and 5 mL of cryopreservative were mixed evenly to prepare 10 mL of human umbilical cord Wharton's jelly tissue cryopreservation protective solution, that is, the low-concentration β-1,3 glucan protective solution in this example, wherein the concentration of β-1,3 glucan in the protective solution was 0.45 mg / mL, and the solution was stored in a refrigerator at 4°C.

[0074] 4. Protective solution without β-1,3 glucan

[0075] Mix 97 mL of UltraCULTURE Serum-free Medium, 2 mL of pre-dissolved and filtered Ultroser G serum substitute, and 1 mL of 200 mM L-glutamine to prepare 100 mL of complete culture medium and store in a refrigerator at 4°C.

[0076] Use physiological saline to prepare 9 mL of a solution containing 20% ​​polyethylene glycol and 20% hydroxyethyl starch, then slowly add 1 mL of dimethyl sulfoxide, mix well, prepare 10 mL of a cryopreservative, and store in a refrigerator at 4°C.

[0077] 5 mL of complete culture medium and 5 mL of cryopreservative were mixed evenly to prepare 10 mL of human umbilical cord Wharton's jelly tissue cryopreservation protective solution, i.e., the protective solution without β-1,3 glucan in this example, and stored in a refrigerator at 4°C.

[0078] 5. Traditional protective fluid

[0079] 6 mL of DMEM culture medium, 2 mL of fetal bovine serum and 2 mL of dimethyl sulfoxide were mixed evenly to prepare 10 mL of human umbilical cord Wharton's jelly tissue cryopreservation protection solution, which is the traditional protection solution in this example, and stored in a refrigerator at 4°C.

[0080] 2. Preparation of Umbilical Cord Tissue Cryopreservation Preparations

[0081] 1. Umbilical cord tissue cryopreservation preparation with high concentration β-1,3 glucan protective solution

[0082] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3 of small pieces.

[0083] Add 1 mL of umbilical cord Wharton's jelly tissue block to 2 mL of pre-prepared and refrigerated high-concentration β-1,3-glucan protective solution, mix well, and transfer 1 mL of the mixture into a 2 mL cryopreservation tube to prepare an umbilical cord tissue cryopreservation preparation, marked as A.

[0084] The above-mentioned umbilical cord tissue cryopreservation preparation was cooled using a programmed cooling apparatus and finally stored in a liquid nitrogen tank.

[0085] 2. Preparation of Umbilical Cord Tissue Cryopreservation in Medium Concentration β-1,3 Glucan Protective Solution

[0086] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors.3 of small pieces.

[0087] Add 1 mL of umbilical cord Wharton's jelly tissue block to 2 mL of pre-prepared and refrigerated medium-concentration β-1,3-glucan protective solution, mix well, and transfer 1 mL of the mixture into a 2 mL cryopreservation tube to prepare an umbilical cord tissue cryopreservation preparation, marked as B.

[0088] The above-mentioned umbilical cord tissue cryopreservation preparation was cooled using a programmed cooling apparatus and finally stored in a liquid nitrogen tank.

[0089] 3. Preparation of Umbilical Cord Tissue Cryopreservation in Low Concentration β-1,3 Glucan Protective Solution

[0090] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3 of small pieces.

[0091] Add 1 mL of umbilical cord Wharton's jelly tissue block to 2 mL of pre-prepared and refrigerated low-concentration β-1,3-glucan protective solution, mix well, and transfer 1 mL of the mixture into a 2 mL cryopreservation tube to prepare an umbilical cord tissue cryopreservation preparation, marked as C.

[0092] The above-mentioned umbilical cord tissue cryopreservation preparation was cooled using a programmed cooling apparatus and finally stored in a liquid nitrogen tank.

[0093] 4. Preparation of Umbilical Cord Tissue Cryopreservation in Protective Solution without β-1,3 Glucan

[0094] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3 of small pieces.

[0095] 1 mL of umbilical cord Wharton's jelly tissue block was added to 2 mL of pre-prepared and refrigerated protective solution without β-1,3 glucan, and mixed evenly. 1 mL of the tissue block suspension of the mixture was transferred to a 2 mL cryopreservation tube to prepare an umbilical cord tissue cryopreservation preparation, which was marked as D.

[0096] The above-mentioned umbilical cord tissue cryopreservation preparation was cooled using a programmed cooling apparatus and finally stored in a liquid nitrogen tank.

[0097] 5. Umbilical cord tissue cryopreservation preparations with traditional protective solutions

[0098] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3 of small pieces.

[0099] 1 mL of umbilical cord Wharton's jelly tissue block was added to 2 mL of pre-prepared and refrigerated traditional protective solution, mixed evenly, and 1 mL of the tissue block suspension of the mixture was transferred to a 2 mL cryopreservation tube to prepare an umbilical cord tissue cryopreservation preparation, marked as E.

[0100] The above-mentioned umbilical cord tissue cryopreservation preparation was cooled using a programmed cooling apparatus and finally stored in a liquid nitrogen tank.

[0101] 3. Test of the effect of protective liquid

[0102] 1. Resuscitation and culture of mesenchymal stem cells after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution

[0103] After 30 days of cryopreservation, the umbilical cord tissue cryopreservation preparations in high concentration β-1,3 glucan protective solution, medium concentration β-1,3 glucan protective solution, and low concentration β-1,3 glucan protective solution were taken out of the liquid nitrogen tank and quickly placed in a 37°C water bath for recovery. After the umbilical cord tissue cryopreservation preparations were thawed, they were transferred to new centrifuge tubes, and then the following operations were performed:

[0104] 5 mL of normal saline was added, and the mixture was centrifuged at 500 g for 5 minutes. The supernatant was discarded to remove the cryopreservation protection solution. 1 mL of umbilical cord Wharton's jelly tissue block was transferred to a T75 cell culture bottle, 10 mL of complete culture medium was added, and the mixture was placed in a carbon dioxide incubator at 37°C containing 5% CO2 for culture. After the cells crawled out, the complete culture medium was replaced, and after the cell confluence reached 80%, 1 mL of 0.25% trypsin was used to digest the cells. After the cells fell off, 4 mL of complete culture medium was added to terminate the digestion, and the cell suspension was transferred to a centrifuge tube, and the mixture was centrifuged at 500 g for 5 minutes. The supernatant was discarded, and the cells were resuspended with 1 mL of complete culture medium. The cells were P0 generation cells, which were counted with a cell counter, and the cell morphology was observed with an inverted microscope.

[0105] Take P0 cells and test cell activity and proliferation ability. Cell activity can be tested using a cell counter; subculture the cells to be tested to a 96-well plate for culture, and test the cell proliferation rate using a CCK-8 cell proliferation kit. For specific steps, refer to the kit instructions.

[0106] Take 4×10 5Transfer the P0 generation cells to a new T75 cell culture flask, add 10 mL of complete culture medium, and culture in a carbon dioxide incubator at 37°C containing 5% CO2; replace the complete culture medium after the cells crawl out, and after the cell confluence reaches 80%, use 1 mL of 0.25% trypsin to digest the cells; after the cells fall off, add 4 mL of complete culture medium to terminate the digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the cells with 1 mL of complete culture medium, i.e., P1 generation cells, and count them using a cell counter.

[0107] P1 cells were taken and cell surface markers were detected, including positive markers CD73, CD90, CD105, and negative markers CD34, CD45, CD11b, and CD19. Human mesenchymal stem cell detection kit (BD Human MSC Analysis Kit (562245)) was used for detection. For specific detection steps, refer to the kit instructions.

[0108] 2. Resuscitation and culture of mesenchymal stem cells after cryopreservation in protective solution without β-1,3-glucan

[0109] After 30 days of cryopreservation, the umbilical cord tissue cryopreservation preparation without β-1,3 glucan protective solution was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for recovery. After the umbilical cord tissue cryopreservation preparation was thawed, it was transferred to a centrifuge tube, 5 mL of normal saline was added, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cryopreservation protective solution was removed; 1 mL of umbilical cord Wharton's jelly tissue block was transferred to a T75 cell culture bottle, 10 mL of complete culture medium was added, and it was placed in a carbon dioxide incubator at 37°C containing 5% CO2 for culture; after the cells crawled out, the complete culture medium was replaced, and after the cell confluence reached 80%, 1 mL of 0.25% pancreatic enzyme was used to digest the cells; after the cells fell off, 4 mL of complete culture medium was added to terminate the digestion, the cell suspension was transferred to a centrifuge tube, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended with 1 mL of complete culture medium, i.e., P0 generation cells, counted with a cell counter, and the cell morphology was observed with an inverted microscope.

[0110] P0 cells were taken and the cell activity and proliferation ability were detected according to the same method as "1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution".

[0111] Take 4×10 5Transfer the P0 generation cells to a new T75 cell culture flask, add 10 mL of complete culture medium, and culture in a carbon dioxide incubator at 37°C containing 5% CO2; replace the complete culture medium after the cells crawl out, and after the cell confluence reaches 80%, use 1 mL of 0.25% trypsin to digest the cells; after the cells fall off, add 4 mL of complete culture medium to terminate the digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the cells with 1 mL of complete culture medium, i.e., P1 generation cells, and count them using a cell counter.

[0112] P1 cells were taken and cell surface markers were detected according to the same method as in “1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution”.

[0113] 3. Recovery and culture of mesenchymal stem cells after cryopreservation in traditional protective solution

[0114] After 30 days of cryopreservation, the umbilical cord tissue cryopreservation preparation in the traditional protective solution was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for recovery. After the umbilical cord tissue cryopreservation preparation was thawed, it was transferred to a centrifuge tube, 5 mL of normal saline was added, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cryopreservation protective solution was removed; 1 mL of umbilical cord Wharton's jelly tissue block was transferred to a T75 cell culture bottle, 10 mL of complete culture medium was added, and it was placed in a carbon dioxide incubator at 37°C containing 5% CO2 for culture; after the cells crawled out, the complete culture medium was replaced, and after the cell confluence reached 80%, 1 mL of 0.25% pancreatin was used to digest the cells; after the cells fell off, 4 mL of complete culture medium was added to terminate the digestion, the cell suspension was transferred to a centrifuge tube, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended with 1 mL of complete culture medium, i.e., P0 generation cells, counted with a cell counter, and the cell morphology was observed with an inverted microscope.

[0115] P0 cells were taken and the cell activity and proliferation ability were detected according to the same method as "1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution".

[0116] Take 4×10 5 Transfer the P0 generation cells to a new T75 cell culture flask, add 10 mL of complete culture medium, and culture in a carbon dioxide incubator at 37°C containing 5% CO2; replace the complete culture medium after the cells crawl out, and after the cell confluence reaches 80%, use 1 mL of 0.25% trypsin to digest the cells; after the cells fall off, add 4 mL of complete culture medium to terminate the digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the cells with 1 mL of complete culture medium, i.e., P1 generation cells, and count them using a cell counter.

[0117] P1 cells were taken and cell surface markers were detected according to the same method as in “1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution”.

[0118] 4. Direct primary culture of fresh umbilical cord tissue without cryopreservation to obtain umbilical cord mesenchymal stem cells

[0119] Get a fresh newborn umbilical cord sample, wash it with saline, disinfect it with medical alcohol, wash it again, cut the umbilical cord into small pieces, cut the amniotic membrane, remove the blood vessels, peel off 2g of Wharton's jelly tissue, and cut it into 1-3mm pieces with sterile scissors. 3 of small pieces.

[0120] 1 mL of umbilical cord Wharton's jelly tissue block was transferred to a T75 cell culture flask, 10 mL of complete culture medium was added, and the cells were cultured in a carbon dioxide incubator at 37°C containing 5% CO2; after the cells crawled out, the complete culture medium was replaced, and after the cell confluence reached 80%, 1 mL of 0.25% trypsin was used to digest the cells; after the cells fell off, 4 mL of complete culture medium was added to terminate the digestion, the cell suspension was transferred to a centrifuge tube, centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium, i.e., P0 generation cells, counted with a cell counter, and the cell morphology was observed using an inverted microscope.

[0121] P0 cells were taken and the cell activity and proliferation ability were detected according to the same method as "1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution".

[0122] Take 4×10 5 Transfer the P0 generation cells to a new T75 cell culture flask, add 10 mL of complete culture medium, and culture in a carbon dioxide incubator at 37°C containing 5% CO2; replace the complete culture medium after the cells crawl out, and after the cell confluence reaches 80%, use 1 mL of 0.25% trypsin to digest the cells; after the cells fall off, add 4 mL of complete culture medium to terminate the digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the cells with 1 mL of complete culture medium, i.e., P1 generation cells, and count them using a cell counter.

[0123] P1 cells were taken and cell surface markers were detected according to the same method as in “1. Recovery and mesenchymal stem cell culture after cryopreservation in high, medium and low concentration β-1,3 glucan protective solution”.

[0124] 4. Test results and analysis

[0125] 1. Observation results using an inverted microscope

[0126] In this example, microscopic observation was performed on the P0 cells cultured after thawing of umbilical cord tissue cryopreservation preparations in high concentration β-1,3 glucan protective solution, medium concentration β-1,3 glucan protective solution, low concentration β-1,3 glucan protective solution, umbilical cord tissue cryopreservation preparations in protective solution without β-1,3 glucan, and umbilical cord tissue cryopreservation preparations in traditional protective solution. Microscopic observation was also performed on the P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that had not been cryopreserved. The results are as follows Figure 1 shown.

[0127] Figure 1 Figure A shows the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in high concentration β-1,3 glucan protective solution, Figure B shows the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in medium concentration β-1,3 glucan protective solution, Figure C shows the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in low concentration β-1,3 glucan protective solution, Figure D shows the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in protective solution without β-1,3 glucan, Figure E shows the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in traditional protective solution, and Figure F shows the P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that has not been cryopreserved.

[0128] Figure 1 The results showed that all observed umbilical cord mesenchymal stem cells were spindle-shaped, neatly arranged, able to maintain a good growth state, and showed no significant differences in appearance. Compared with umbilical cord tissue cryopreserved in traditional protective solution (Figure E) or umbilical cord mesenchymal stem cells prepared from fresh umbilical cord tissue (Figure F), there was no significant difference in the appearance of umbilical cord mesenchymal stem cells obtained using high-concentration β-1,3 glucan protective solution, medium-concentration β-1,3 glucan protective solution, and low-concentration β-1,3 glucan protective solution (i.e., Figures A, B, and C). There was no significant difference in the appearance of umbilical cord mesenchymal stem cells obtained by resuscitation and culture of umbilical cord tissue cryopreserved in human umbilical cord Wharton's jelly tissue cryopreservation protective solution with different contents of β-1,3 glucan, i.e., Figures A to D.

[0129] 2. Activity test results of umbilical cord mesenchymal stem cells

[0130] In this example, the activity of P0 cells cultured after resuscitation of umbilical cord tissue cryopreservation preparations in high-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in medium-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in low-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in protective solution without β-1,3-glucan, and umbilical cord tissue cryopreservation preparations in traditional protective solution were detected, and the same activity detection was performed on P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that had not been cryopreserved. The results are shown in Table 1.

[0131] Table 1 Activity test results of umbilical cord mesenchymal stem cells

[0132] Group A B C D E F Live rate 98.41％ 96.37％ 94.12％ 91.80％ 89.69％ 99.15％

[0133] In Table 1, A represents P0 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a high concentration β-1,3 glucan protective solution, B represents P0 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a medium concentration β-1,3 glucan protective solution, C represents P0 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a low concentration β-1,3 glucan protective solution, D represents P0 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a protective solution without β-1,3 glucan, E represents P0 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a traditional protective solution, and F represents P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that has not been cryopreserved.

[0134] The results in Table 1 show that the activity of umbilical cord mesenchymal stem cells in groups A and F in Table 1 is higher than that in groups B, C, D, and E in Table 1. It can be seen that compared with the traditional cryopreservation protection solution in group E, the activity of umbilical cord mesenchymal stem cells obtained using the protection solution containing β-1,3 glucan in this example is higher. Moreover, as the concentration of β-1,3 glucan in the cryopreservation protection solution of human umbilical cord Wharton's jelly tissue increases from low to high, the activity of umbilical cord mesenchymal stem cells obtained by resuscitation culture is also improved, that is, the activity increases from group D to group A in sequence. Compared with group F of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue, the umbilical cord mesenchymal stem cells obtained using the high-concentration β-1,3 glucan human umbilical cord Wharton's jelly tissue cryopreservation protection solution and preparation method of this example, that is, group A, have no significant difference in cell activity.

[0135] 3. Results of the proliferation ability test of umbilical cord mesenchymal stem cells

[0136] In this example, the proliferation ability of P0 cells cultured after resuscitation was tested for umbilical cord tissue cryopreservation preparations in high-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in medium-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in low-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in protective solution without β-1,3-glucan, and umbilical cord tissue cryopreservation preparations in traditional protective solution. The same proliferation ability test was performed on P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that had not been cryopreserved. The results are as follows Figure 2 shown.

[0137] Figure 2 In the figure, curve A represents the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in high concentration β-1,3 glucan protective solution, curve B represents the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in medium concentration β-1,3 glucan protective solution, curve C represents the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in low concentration β-1,3 glucan protective solution, curve D represents the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in protective solution without β-1,3 glucan, curve E represents the P0 cells cultured after thawing of the umbilical cord tissue cryopreservation preparation in traditional protective solution, and curve F represents the P0 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that has not been cryopreserved.

[0138] Figure 2 The results show that, relative to Figure 2 The four groups B, C, D, and E in Figure 2 The growth rate of umbilical cord mesenchymal stem cells in groups A and F was faster, and the number of cells obtained in the same culture time was more. Compared with group E of traditional cryopreservation protection solution, the growth rate of umbilical cord mesenchymal stem cells obtained using high-concentration β-1,3 glucan protection solution, medium-concentration β-1,3 glucan protection solution and low-concentration β-1,3 glucan protection solution was faster and the proliferation ability was stronger; and as the concentration of β-1,3 glucan in the cryopreservation protection solution of human umbilical cord Wharton's jelly tissue increased from low to high, the growth rate of umbilical cord mesenchymal stem cells obtained by resuscitation culture gradually accelerated, that is, from group D to group A, the cell growth rate gradually accelerated. Compared with group F of umbilical cord mesenchymal stem cells obtained by direct primary culture from fresh umbilical cord tissue, there was no significant difference in the proliferation ability of umbilical cord mesenchymal stem cells (i.e., group A) obtained using the high-concentration β-1,3 glucan protection solution in this example.

[0139] 4. Surface marker detection results of umbilical cord mesenchymal stem cells

[0140] In this example, surface markers were detected on P1 cells cultured after resuscitation of umbilical cord tissue cryopreservation preparations in high-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in medium-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in low-concentration β-1,3-glucan protective solution, umbilical cord tissue cryopreservation preparations in protective solution without β-1,3-glucan, and umbilical cord tissue cryopreservation preparations in traditional protective solution. The same surface markers were detected on P1 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that had not been cryopreserved. The results are shown in Table 2.

[0141] Table 2 Surface marker detection results of umbilical cord mesenchymal stem cells

[0142]

[0143] In Table 2, A represents P1 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a high concentration β-1,3 glucan protective solution, B represents P1 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a medium concentration β-1,3 glucan protective solution, C represents P1 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a low concentration β-1,3 glucan protective solution, D represents P1 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a protective solution without β-1,3 glucan, E represents P1 cells cultured after thawing of an umbilical cord tissue cryopreservation preparation in a traditional protective solution, and F represents P1 cells of umbilical cord mesenchymal stem cells obtained by direct primary culture of fresh umbilical cord tissue that has not been cryopreserved.

[0144] The results in Table 2 showed that the umbilical cord mesenchymal stem cells in the six groups A, B, C, D, E, and F were able to highly express the positive markers of mesenchymal stem cells CD73, CD90, and CD105, and did not express the negative markers of mesenchymal stem cells CD11b, CD19, CD34, CD45, and HLA-DR.

[0145] The umbilical cord mesenchymal stem cells obtained by resuscitation culture using the high-concentration β-1,3-glucan human umbilical cord Wharton's jelly tissue cryopreservation protection solution and preparation method (i.e., Group A) of this example, the human umbilical cord Wharton's jelly tissue cryopreservation protection solution using different concentrations of β-1,3-glucan (i.e., Groups B, C, and D) or the traditional cryopreservation protection solution (i.e., Group E), and the primary culture of fresh umbilical cord tissue (i.e., Group F) can all meet the standards for mesenchymal stem cell surface markers.

[0146] The above results show that the protective solution containing β-1,3 glucan in this case can improve the protective effect of cryopreserved tissues and the activity and proliferation ability of cells after resuscitation; especially the protective solution with high concentration of β-1,3 glucan, its activity and proliferation ability are equivalent to the umbilical cord mesenchymal stem cells directly cultured from fresh umbilical cord tissue that has not been cryopreserved, and there is no significant difference between the two. Moreover, with the increase of β-1,3 glucan concentration, the protective effect of the protective solution on the cryopreserved tissue is enhanced, and the activity and proliferation ability of the cells after resuscitation are also enhanced accordingly. It shows that the use of β-1,3 glucan can increase the stability of the cryopreservation protective solution, improve the protective effect of the cryopreserved tissue and the viability of the cells after resuscitation.

[0147] In addition, the protective solution of complete culture medium was used in this example. Compared with the traditional protective solution without complete culture medium, the activity and proliferation ability of the revived cells after cryopreservation using the protective solution of this example were also improved, probably because the complete culture medium can provide nutrition for umbilical cord tissue and cells. Moreover, the complete culture medium used in the protective solution of this example is consistent with the culture medium used for subsequent resuscitation and culture, which increases the convenience of preparation and application of cryopreservation protective solution.

[0148] The protective solution in this example does not contain fetal bovine serum, autologous umbilical cord blood serum and human albumin, which improves the safety and reliability of clinical application and avoids the quality uniformity problems caused by these components; in addition, there is no need to use expensive human albumin, which reduces the production cost of the protective solution.

[0149] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A protective liquid, characterized in that: The protective solution includes a cryopreservation agent and a complete culture medium; The cryopreservation agent is composed of an osmotic protective agent and an impermeable protective agent, wherein the osmotic protective agent includes at least one of dimethyl sulfoxide, glycerol, ethylene glycol and propylene glycol; the impermeable protective agent is composed of at least one of hydroxyethyl starch, sucrose, polyethylene glycol and trehalose, and β-1,3 glucan; the concentration of the β-1,3 glucan in the protective solution is 0.1 mg / mL-9 mg / mL; The complete medium consists of serum-free basal medium, serum replacement and L-glutamine; Furthermore, the protective solution does not contain fetal bovine serum, autologous umbilical cord blood serum and human serum albumin.

2. The protective liquid according to claim 1, characterized in that: The concentration of the β-1,3 glucan in the protective solution is 0.45 mg / mL, 2.25 mg / mL or 9 mg / mL.

3. The protective liquid according to claim 1, characterized in that: The volume ratio of the serum-free basal medium, serum replacement and L-glutamine is 97:2:1; Alternatively, the volume ratio of the serum-free basal medium, serum replacement and L-glutamine is 95:4:

1.

4. The protective liquid according to claim 1, characterized in that: The volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservation agent is 1:1, and the volume ratio of the complete culture medium to the cryopreservation agent is 9:1; Alternatively, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservative is 3:1, and the volume ratio of the complete culture medium to the cryopreservative is 4:1; Alternatively, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservation agent is 4:1, and the volume ratio of the complete culture medium to the cryopreservation agent is 3:1; Alternatively, the volume ratio of the non-permeable protective agent to the permeable protective agent in the cryopreservation agent is 9:1, and the volume ratio of the complete culture medium to the cryopreservation agent is 1:

1.

5. The protective liquid according to claim 1, characterized in that: The protective solution consists of 5-20 volumes of an osmotic protective agent, 2-50 volumes of a non-osmotic protective agent, 0.2-1 volumes of L-glutamine, 1-10 volumes of a serum replacement, and 45-90 volumes of a serum-free basal medium.

6. The protective liquid according to claim 5, characterized in that: The protective solution consists of 5 volumes of an osmotic protective agent, 45 volumes of a non-osmotic protective agent, 0.5 volumes of L-glutamine, 1 volume of a serum replacement, and 48.5 volumes of a serum-free basal medium.

7. The protective liquid according to any one of claims 1 to 6, characterized in that: The osmotic protective agent is dimethyl sulfoxide.

8. The protective liquid according to claim 7, characterized in that: The non-permeable protective agent consists of hydroxyethyl starch and beta-1,3 glucan.

9. The protective liquid according to claim 7, characterized in that: The L-glutamine is clinical-grade L-glutamine, the serum substitute is clinical-grade serum substitute, and the serum-free basal medium is clinical-grade basal medium.

10. The method for preparing the protective solution according to any one of claims 1 to 9, characterized in that: The following steps are included: A. Mix serum-free basal medium, serum replacement and L-glutamine evenly to prepare complete medium and store at 2-8℃; B. Add the permeable protective agent to the non-permeable protective agent containing β-1,3 glucan, mix well, prepare a cryopreservative, and store at 2-8°C; C. Evenly mix the complete culture medium prepared in step A and step B with the cryopreservation agent to obtain the protective solution, and store it at 2-8°C.

11. The preparation method according to claim 10, characterized in that: The non-permeable protective agent containing β-1,3 glucan is prepared with physiological saline.

12. Use of the protective solution according to any one of claims 1 to 9 in cryopreservation of tissues or cells.

13. Use of the protective solution according to claim 12 in cryopreservation of tissues or cells, characterized in that: The tissue is human umbilical cord Wharton's jelly tissue.

14. A method for cryopreservation of human umbilical cord Wharton's jelly tissue, characterized in that: The following steps are included: A. Clean and disinfect the fresh umbilical cord sample, peel off the Wharton's jelly tissue, and cut the Wharton's jelly tissue into small pieces; B. adding the Wharton's jelly tissue block obtained in step A to the protective solution according to any one of claims 1 to 9, mixing and dispensing into cryopreservation tubes to prepare an umbilical cord tissue cryopreservation preparation; C. The umbilical cord tissue cryopreservation preparation obtained in step B is subjected to programmed cooling and stored in a liquid nitrogen tank.

15. The cryopreservation method according to claim 14, characterized in that: In the umbilical cord tissue cryopreservation preparation, the volume ratio of Wharton's jelly tissue to the protective solution is 1:

2.

16. The cryopreservation method according to claim 15, characterized in that: Cut the Wharton's jelly tissue into small pieces of 1-3 mm in size. 3 The specification of the freezing tube is 2 mL.

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