A glassy cryopreservation solution and uses thereof

Abstract

The application provides a glass-like cryopreservation solution and application thereof, and belongs to the technical field of tissue cryopreservation. The glass-like cryopreservation solution provided by the application contains specific final concentrations of nitrogen ketone, cellobiose and alpha-lactose, and overcomes the problem of reduced activity of stem cells in the process of umbilical cord Wharton's jelly cryopreservation in the prior art. The glass-like cryopreservation solution provided by the application can make the cryopreserved tissue well preserved at low temperature, so that the tissue can produce a low degree of dehydration in a short time but does not affect physiological properties, and is more conducive to preservation. When the tissue contains stem cells, the glass-like cryopreservation solution provided by the application can effectively avoid the problem that the stem cells in the tissue are caused to die in large quantities due to ice crystal formation in the cryopreservation process, and when the tissue is resuscitated, the stem cells can be quickly extracted, without affecting the activity of the stem cells, and without significant difference compared with fresh tissue.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue cryopreservation, and particularly relates to a glassy cryopreservation solution and application thereof. BACKGROUND

[0002] Vitrification is a rapid freezing method. Vitrification uses a high concentration of cryoprotectant to replace water in cells, and through rapid cooling, the liquid inside and outside the cells is directly converted into a viscous amorphous glassy state, thereby minimizing the formation of ice crystals inside and outside the cells. Vitrification directly converts the liquid inside and outside the cells into an amorphous glassy state to reduce the formation of ice crystals inside and outside the cells. Vitrification does not require special equipment, saving time and cost.

[0003] In the prior art, when cells are cryopreserved, a protective agent, glycerol or dimethyl sulfoxide (DMSO) with a final concentration of 5% to 15%, is added to the culture medium, which can lower the freezing point of the solution. In addition, under slow freezing conditions, water in the cells is exuded, reducing the formation of ice crystals, thereby avoiding cell damage. The "slow freezing and fast thawing" method can better ensure cell survival. According to the difference in permeability, the cryopreservation solution can be divided into two categories: permeable cryopreservation solution and non-permeable cryopreservation solution. The permeable cryopreservation solution can penetrate the cell membrane and can combine with water molecules in the solution to weaken the formation of ice crystals; the non-permeable cryopreservation solution cannot penetrate the cell membrane, but can increase the extracellular osmotic pressure, so that the water molecules in the cells are exuded outside the cells to reduce the formation of intracellular ice crystals. Since the permeable cryopreservation solution is toxic to cells, and the non-permeable cryopreservation solution can cause cells to shrink due to dehydration, the concentration and amount of use of both cannot be too high, and the two types of cryopreservation solutions are often used together in vitrification. The existing cryopreservation scheme often uses a cryopreservation scheme of 10% DMSO + 90% FBS to preserve cells, but the cryopreservation scheme has a high cost, cannot avoid the introduction of viruses from animal sources, has a risk of viral contamination of the preserved cells, and has a poor cryopreservation effect on tissues, which cannot meet the requirements of tissue cryopreservation.

[0004] Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a kind of mesenchymal stem cells isolated from Wharton's jelly of umbilical cord, and the gene expression profile thereof meets the current definition of MSCs, and also expresses a certain level of pluripotency genes such as OCT-4, SOX-2 and NANOG. In addition to being able to differentiate into osteogenic, adipogenic and chondrogenic cells, WJ-MSCs can also differentiate into endoderm and ectoderm-derived cells. In addition, WJ-MSCs do not express MHC-II and have very low immunogenicity, and exhibit immunomodulatory properties in vivo, which makes them a good substitute for allogeneic and xenogeneic transplantation in cell therapy, and has a wide clinical application prospect. When umbilical cord stem cells are cultured on a large scale, a seed bank is usually established, and when the seed bank is used up, a new umbilical cord needs to be screened. In addition, the number of cells in the seed bank established by extracting umbilical cord tissue once is limited, and therefore the direct extraction directly restricts the clinical application of umbilical cord tissue block cells. Therefore, freezing the Wharton's jelly tissue of the umbilical cord can solve the application problem of the umbilical cord stem cells.

[0005] The existing cryoprotectant cannot effectively avoid the large number of cell deaths or significant reduction in activity of stem cells in the tissue caused by ice crystal formation during the freezing process of the Wharton's jelly of the umbilical cord. SUMMARY

[0006] The purpose of the present application is to provide a glass-like cryoprotectant which can solve the problem of reduced activity of stem cells during the freezing process of the Wharton's jelly of the umbilical cord.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a glass-like cryoprotectant, which contains nitrogen ketone, cellobiose and alpha-lactose in the glass-like cryoprotectant;

[0009] The final volume concentration of the nitrogen ketone in the glass-like cryoprotectant is 3-5%;

[0010] The final concentration of the cellobiose in the glass-like cryoprotectant is 0.1-0.5 mol / L;

[0011] The final concentration of the alpha-lactose in the glass-like cryoprotectant is 0.1-0.5 mol / L.

[0012] Preferably, the glass-like cryoprotectant contains a serum substitute;

[0013] The volume of the serum substitute accounts for 10-30% of the total volume of the glass-like cryoprotectant.

[0014] Preferably, the glass-like cryoprotectant uses a balanced salt solution as a base solvent.

[0015] Preferably, the balanced salt solution is one or more of Eargle's buffer, Hank's buffer and phosphate buffer.

[0016] Preferably, the glassy cryopreservation solution contains dimethyl sulfoxide.

[0017] Preferably, the final volume concentration of dimethyl sulfoxide in the glassy cryopreservation solution is 3-5%.

[0018] The application also provides the use of the above glassy cryopreservation solution in the glassy cryopreservation of cells or tissues.

[0019] Preferably, the tissue is umbilical cord Wharton's jelly tissue.

[0020] Preferably, the volume ratio of the umbilical cord Wharton's jelly tissue to the glassy cryopreservation solution is 1:3-8.

[0021] Technical effects and advantages of the application:

[0022] The glassy cryopreservation solution provided by the application contains nitrogen ketone, cellobiose and alpha-lactose, and through the complexing of components, the cryopreserved tissue can be well preserved at low temperature, so that the tissue can produce a low degree of dehydration in a short time but does not affect the physiological properties, and is more conducive to preservation. When the tissue contains stem cells, the glassy cryopreservation solution provided by the application can effectively avoid the problem that the stem cells in the tissue are caused to die in large quantities due to the formation of ice crystals during the cryopreservation process, and when the tissue is recovered, the stem cells therein can be quickly extracted, without affecting the activity of the stem cells, and without significant difference compared with fresh tissue. The glassy cryopreservation solution of the application can greatly improve the permeability of the tissue, avoid the formation of ice crystals, provide a microenvironment suitable for the preservation of cells in the tissue, reduce the mechanical damage to the cells, and avoid the damage and death of the cells caused by the decrease or increase of temperature after preservation. DETAILED DESCRIPTION

[0023] The application provides a glassy cryopreservation solution, which contains nitrogen ketone, cellobiose and alpha-lactose, the final volume concentration of the nitrogen ketone in the glassy cryopreservation solution is 3-5%, preferably 3.5-4.5%, the final concentration of the cellobiose in the glassy cryopreservation solution is 0.1-0.5 mol / L, preferably 0.2-0.4 mol / L, and the final concentration of the alpha-lactose in the glassy cryopreservation solution is 0.1-0.5 mol / L, preferably 0.2-0.4 mol / L, in the application, the glassy cryopreservation solution preferably takes balanced salt solution as a base solvent, the balanced salt solution is preferably one or more of Eargle's buffer, Hank's buffer and phosphate buffer, and the balanced salt solution is further preferably Hank's buffer, and the nitrogen ketone, cellobiose and alpha-lactose are diluted in the Hank's buffer.

[0024] In the application, the glassy cryopreservation solution preferably contains serum substitutes, the volume of the serum substitutes preferably accounts for 10-30% of the total volume of the glassy cryopreservation solution, and the volume of the serum substitutes is further preferably 15-25%; the glassy cryopreservation solution preferably contains dimethyl sulfoxide, and the final volume concentration of the dimethyl sulfoxide in the glassy cryopreservation solution is preferably 3-5%, and the final volume concentration of the dimethyl sulfoxide in the glassy cryopreservation solution is further preferably 3.5-4.5%.

[0025] The application also provides application of the above glassy cryopreservation solution in cell or tissue glassy cryopreservation, and the tissue is preferably umbilical cord Wharton's jelly tissue; in the glassy cryopreservation process, the volume ratio of the umbilical cord Wharton's jelly tissue to the glassy cryopreservation solution is preferably 1:3-8, and the volume ratio of the umbilical cord Wharton's jelly tissue to the glassy cryopreservation solution is further preferably 1:4-6; the glassy cryopreservation solution is preferably stored in a 4 DEG C environment for standby, and preferably used within one week after being prepared.

[0026] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0027] The reagents in the examples are from the following sources:

[0028] HANK's solution, brand: Merck, article number: H6648;

[0029] DMSO, brand: Solarbio, article number: D8371;

[0030] Nitrogen ketone, brand: Aladdin, article number: A104375;

[0031] PALL Ultroser™ G serum substitute, brand: PALL, article number: 15950-017;

[0032] Cellobiose, brand: Merck, article number: C7252;

[0033] Alpha-lactose, purchased from Shanghai Yuye Biotechnology Co., Ltd., item number: S11047.

[0034] Example 1

[0035] Solution 1 preparation: cellobiose and alpha-lactose were added to HANK'S buffer so that the final concentration of cellobiose and alpha-lactose was 0.5 mol / L; DMSO and azacycloalkane were added to the system so that the final volume concentration of DMSO and azacycloalkane was 7.5%.

[0036] Solution 2 preparation: PALL Ultroser™ G serum substitute was dissolved in HANK'S buffer so that the final volume concentration of PALL Ultroser™ G serum substitute was 50%.

[0037] Solution 1 and solution 2 were mixed thoroughly at a volume ratio of 1:1 to obtain a glassy cryopreservation solution, which was placed at 4°C for standby.

[0038] Example 2

[0039] Solution 1 preparation: cellobiose and alpha-lactose were added to HANK'S buffer so that the final concentration of cellobiose and alpha-lactose was 1 mol / L; DMSO and azacycloalkane were added to the system so that the final volume concentration of DMSO and azacycloalkane was 10%.

[0040] Solution 2 preparation: PALL Ultroser™ G serum substitute was dissolved in HANK'S buffer so that the final volume concentration of PALL Ultroser™ G serum substitute was 20%.

[0041] Solution 1 and solution 2 were mixed thoroughly at a volume ratio of 1:1 to obtain a glassy cryopreservation solution, which was placed at 4°C for standby.

[0042] Example 3

[0043] Solution 1 preparation: cellobiose and alpha-lactose were added to HANK'S buffer so that the final concentration of cellobiose and alpha-lactose was 0.2 mol / L; DMSO and azacycloalkane were added to the system so that the final volume concentration of DMSO and azacycloalkane was 6%.

[0044] Solution 2 preparation: PALL Ultroser™ G serum substitute was dissolved in HANK'S buffer so that the final volume concentration of PALL Ultroser™ G serum substitute was 60%.

[0045] Solution 1 and solution 2 were mixed thoroughly at a volume ratio of 1:1 to obtain a glassy cryopreservation solution, which was placed at 4°C for standby.

[0046] Comparative Example 1

[0047] Formulation of cryopreservation solution: 10% DMSO + 40% FBS + 50% DMEM / F12.

[0048] Comparative Example 2

[0049] Formulation of cryopreservation solution: 10% DMSO + 90% FBS.

[0050] Comparative Example 3

[0051] The difference compared with Example 1 is only that cellobiose is replaced by trehalose at the same final concentration.

[0052] Comparative Example 4

[0053] The difference compared with Example 1 is only that azone is replaced by mannitol (a pyrrolidone osmotic promoter) at the same final concentration.

[0054] Comparative Example 5

[0055] The difference compared with Example 1 is only that cellobiose is replaced by maltose (both are disaccharides) at the same final concentration.

[0056] Comparative Example 6

[0057] Mix 10% dimethyl sulfoxide, 5% human serum albumin, 10% FetalClone III, and 75% DMEM / F12 as a control cryopreservation protective agent.

[0058] Comparative Example 7

[0059] Mix 10 parts dimethyl sulfoxide, 5 parts glycerol, 10 parts polyethylene glycol, 5 parts bovine serum albumin, 3 parts casein, and 1 part gelatin as a control cryopreservation protective agent.

[0060] Experimental Example 1

[0061] Obtain umbilical cord Wharton's jelly:

[0062] ① After sterilizing the storage bottle containing the umbilical cord, place it on the clean bench, and use a Pasteur pipette to take about 3 mL of the solution soaked in the umbilical cord, and divide it into two EP tubes, 1.5 mL per tube, one for sample retention, and one for bacterial and fungal testing in the quality control department.

[0063] ② Pour 100 mL of pre-cooled cell washing solution into a 15 cm glass culture dish, and place the umbilical cord in it with flat forceps, wash the blood stains on the surface of the umbilical cord, and cut off the ropes wrapped around the ends of the umbilical cord together with 1-2 cm of the umbilical cord with surgical scissors, clamp the end of the umbilical cord with hemostats, and squeeze out the blood in the blood vessels with flat forceps.

[0064] ③ Use flat forceps to transfer the umbilical cord to a new 15 cm glass culture dish, pour in 100 mL of pre-cooled 75% alcohol, soak for 1-2 min, and keep stirring the umbilical cord. Use flat forceps to transfer the umbilical cord to a new 15 cm glass culture dish, pour in 100 mL of PBS, and wash off the alcohol.

[0065] ④ Use straight head surgical scissors to cut the umbilical cord into small pieces about 1-2 cm long.

[0066] ⑤ Take two pairs of tissue forceps, clamp a small piece of umbilical cord, and peel off the vein. Then peel off the two arteries, discard the outer membrane of the umbilical cord and blood vessels, and pay attention to reserve the tissue around the blood vessels. Tear the obtained tissue into small pieces.

[0067] ⑥ Take out the Wharton's jelly tissue, cut it into small pieces with a volume of 1-3 mm 3 , and wash it again with sterile dissection solution. Centrifuge at 1500 rpm for 5 min; discard the supernatant;

[0068] Mix the umbilical cord Wharton's jelly with the cell freezing solution of Example 1, Comparative Examples 1-7, and commercial freezing solution (GMP grade vitrification cell freezing solution; brand: Youkang, product number: NC1011) at a volume ratio of 5:1; each tube contains 1.8 mL. Freeze 12 tubes of each type of cell.

[0069] Place the freezing tubes containing the umbilical cord Wharton's jelly into the freezing box and put them into the -80°C refrigerator. After 48 hours, transfer them into liquid nitrogen.

[0070] Resuscitate the tissues of each group at 1 week, 1 month, 2 months, and 3 months, respectively. Resuscitate 3 tubes of each type of freezing system. Take the cells out of the liquid nitrogen and quickly dissolve them in water at 37°C. Transfer the cell suspension to a T75 culture bottle containing Youkang umbilical cord complete culture medium. The volume ratio of tissue pieces to complete culture medium is 1:10. Transfer the tissue pieces to the cell culture bottle, flatten them, and place them in a CO2 volume fraction of 5% at 37°C. After the Wharton's jelly tissue pieces adhere, change the culture medium every 2 days. Observe the cell growth under an inverted microscope (new cells have the characteristic of adherent growth). When typical fibroblast-like cells, which are spindle-shaped or star-shaped, are observed under a microscope and arranged in parallel or spiral, they can be separated.

[0071] According to the above method, separate the mesenchymal stem cells in the tissue, and count the time, number of clones, cell morphology, and cell purity of the mesenchymal stem cells obtained in each type of freezing scheme.

[0072] Table 1 Time (days) for extracting umbilical cord stem cells in each type of freezing scheme

[0073]

[0074] * indicates that compared with fresh tissue, P < 0.05, there is a significant difference.

[0075] From the above table, the time for extracting umbilical cord mesenchymal stem cells from fresh tissue is 5-7 days; compared with fresh tissue, the time for extracting umbilical cord mesenchymal stem cells from the umbilical cord tissue of the present application examples 1-3 frozen for 1 week, 1 month, 2 months and 3 months has no significant difference; while the extraction time of cells from the comparative examples 1-7 and the finished product cryopreservation solution is more than 8 days after being frozen for 1 month, and the longer the freezing time, the longer the extraction time; and compared with fresh tissue, there is a significant difference, P < 0.05.

[0076] Experimental Example 2

[0077] The umbilical cord mesenchymal stem cells cultured for 10 days after the tissues in each group were frozen for 1 month were recovered and cultured in T75 bottles, each 3 bottles, and the cell tissue blocks were discarded. The number of colonies was counted under an inverted microscope and marked to prevent repeated counting.

[0078] The umbilical cord mesenchymal stem cells cultured for 10 days were taken, 0.25% trypsin was used for digestion, and 5mL of cell suspension was resuspended. 20μL of cell suspension was taken, 20μL of 0.2% trypan blue dye was added, and after mixing evenly, 20μL of the suspension was taken and added to a cell counting plate. The countstar was used to detect the cell viability (repeated 3 times), and the results are shown in Table 2.

[0079] Table 2: Number of colonies, total number and viability of umbilical cord stem cells extracted within 10 days under different types of cryopreservation schemes

[0080]

[0081] * indicates that compared with fresh tissue, P < 0.05, there is a significant difference.

[0082] From Table 2, in terms of the number of colonies, the number of colonies of umbilical cord mesenchymal stem cells extracted within 10 days using the cryopreservation solution of the present application examples 1-2 was 30, which had no significant difference compared with the number of colonies of cells extracted from fresh tissue, which was 33; while the number of colonies of cells extracted within 10 days using the finished product cryopreservation solution of the comparative examples 1-7 was 20, 18 and 19 respectively, which had a significant difference compared with the number of colonies of cells extracted from fresh tissue.

[0083] In terms of cell viability, the viability of cells extracted by each type of cryopreservation scheme was higher than 95%; and there was no significant difference in the viability of cells extracted from fresh tissue.

[0084] In terms of the number of cells extracted, the number of umbilical cord mesenchymal stem cells extracted within 10 days by the present application was 1.53×10 7cells, which was significantly different from the number of cells extracted from fresh tissue (1.75 x 10 7 cells, which was significantly different from the number of cells extracted from fresh tissue (1.75 x 10 7 cells, which was significantly different from the number of cells extracted from fresh tissue (1.75 x 10 7 cells, which was significantly different from the number of cells extracted from fresh tissue (1.75 x 10 7 cells, which was significantly different from the number of cells extracted from fresh tissue (1.75 x 10

[0085] Detection of cell surface antigens: the stem cells extracted from each freezing formula in each group were resuspended with 10% FBS + PBS and adjusted to a cell suspension of 1 x 10 6 cells / mL, 5 μL of monoclonal antibodies against human CD73, CD105, CD90, CD45, CD19, CD11b, CD34 and HLA-DR were added to 200 μL of the cell suspension, and incubated at room temperature for 20 min in the dark, while a same type control group was set up, centrifuged at 1500 r / min for 5 min, the supernatant was discarded, washed twice with PBS containing 10% FBS, resuspended with 500 μL of 1640 basic medium and then detected on a machine, and the results are shown in Table 3.

[0086] Table 3 Detection of surface antigens of umbilical cord stem cells extracted by various freezing schemes

[0087]

[0088] According to the Minimum Criteria for the Identification of Mesenchymal Stem Cells issued by ISCT in 2006, CD73, CD90 and CD105 are positively expressed, and the expression should be ≥ 95%; CD11b, CD19, CD34, CD45 and HLA-DR are negatively expressed, and the expression should be ≤ 2.0%. As shown in Table 3, the positive expression of stem cells from various sources is higher than 95%; the negative expression is lower than 2.0%; which indicates that the purity of the umbilical cord mesenchymal stem cells meets the standard.

[0089] As can be seen from the above examples, the glass-like freezing solution provided by the present application can effectively avoid the problem of death or reduced activity of umbilical cord mesenchymal stem cells caused by ice crystal formation during the freezing process of the umbilical cord Wharton's jelly tissue, and the umbilical cord mesenchymal stem cells can be quickly extracted after the umbilical cord Wharton's jelly tissue is recovered, without affecting the activity of the umbilical cord mesenchymal stem cells, and without significant difference compared with fresh tissue.

[0090] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A vitreous cryopreservation solution, characterized in that, The vitreous cryopreservation solution consists of azone, cellobiose, α-lactose, serum substitute, balanced salt solution, and dimethyl sulfoxide; The cryopreservation solution for the glass samples uses a balanced salt solution as the base solvent. The final volume concentration of azone in the glass sample cryopreservation solution is 3-5%; The final concentration of cellobiose in the glass sample cryopreservation solution is 0.1~0.5 mol / L; The final concentration of α-lactose in the glass cryopreservation solution is 0.1~0.5 mol / L; The volume of the serum substitute accounts for 10-30% of the total volume of the vitreous cryopreservation solution; The final volume concentration of the dimethyl sulfoxide in the glass sample cryopreservation solution is 3-5%; The balanced salt solution is one or more of Eargle's buffer, Hank's buffer, and phosphate buffer; The serum substitute is PALL Ultroser™ G serum substitute.

2. The application of the vitreous cryopreservation solution according to claim 1 in the cryopreservation of tissue vitreous samples, characterized in that, The tissue in question is Wharton's jelly tissue from the umbilical cord.

3. The application according to claim 2, characterized in that, The volume ratio of the umbilical cord Wharton's jelly tissue to the vitreous cryopreservation solution was 1:3~8.

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