Cell preservation solution and application thereof

Abstract

The invention provides a cell preservation solution and application thereof in low-temperature preservation and transportation of cells. The cell preservation solution is prepared from a preservation main body solution, an adenosine triphosphate injection, a compound amino acid injection, a compound vitamin B injection and a human serum albumin injection. Reagents adopted in the invention are injections, and animal serum is not used, so that clinical requirements are met. On the basis of ensuring the safety, the invention also provides a formula capable of supplying energy to cells and effectively prolonging the preservation time of the cells, so that long-time long-distance transportation is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cell preservation solution and application thereof. Background Art

[0002] With the continuous development of the times, countries around the world have begun to conduct large-scale cell research and have made certain progress. In recent years, with a large number of media broadcasts of some achievements of cell therapy, the cell industry has gradually entered people's field of vision. Cell therapy refers to the extraction of immune cells from human blood, or the extraction of pluripotent mesenchymal stem cells from human tissues (such as fat, dental pulp, etc.) and neonatal tissues (such as umbilical cord, placenta) for in vitro amplification or differentiation, and then making cell preparations into a certain number and returning them to the human body, so that the cells act on the body. These cells can not only be used to treat some diseases, such as using mesenchymal stem cells to treat body injuries or using autologous immune cells to treat cancer, but also can be used for medical cosmetology, for example, mesenchymal stem cells or autologous immune cells can be used to remove impurities in the body and improve physical condition to achieve the effect of "rejuvenation".

[0003] However, as clinical trials continue, some medical accidents have occurred, such as immune rejection caused by the use of allogeneic cells, or the use of animal serum during cell proliferation, which leads to the injection of non-blood-borne microorganisms into the human body, which may pose certain risks to subsequent life. Therefore, various countries have begun to introduce various bills, prohibiting the use of serum-cultured cells to be injected into the human body, so as to protect the subjects and limit the "abuse" of cells.

[0004] Since cells are living things, each individual unit can metabolize. Therefore, after in vitro expansion, they cannot be sterilized according to the injection standard and then returned to the human body. In particular, neonatal tissues cannot be fully extracted into blood bags and then separated in a biosafety cabinet like immune cells. Neonatal tissues are exposed to the air when they are taken out of the delivery room, and the placenta contains a large number of microorganisms. There is a risk of umbilical cord contamination when operating the umbilical cord connected to the placenta. If antibiotics are added, it does not meet clinical requirements. Therefore, a strict set of operating procedures is required to extract cells and then return them to the human body after expansion. Most laboratories have a fairly mature system for the extraction and expansion of stem cells, but few laboratories have explored what kind of cell preparations should be made to be transported at low temperature for a longer time and closer to the injection standard. Most clinical studies directly use sodium chloride injection to resuspend the cells after expansion, and then return them to the human body after short-distance transportation. Sodium chloride injection is only used as an injection solvent to maintain the steady state of cell osmotic pressure and cannot provide energy to the cells. In addition, low-temperature transportation will cause certain damage to the cells and affect their activity, so the cells cannot be transported at low temperature for a long time.

[0005] Therefore, there is an urgent need in the art for a cell preservation solution for preserving cells, which can not only provide energy to the cells but also ensure that the cells remain active after long-term low-temperature transportation. Summary of the invention

[0006] As described above, in view of the defects existing in the prior art, the purpose of the present invention is to provide a cell preservation solution, which uses components that provide energy to cells, is more conducive to long-term low-temperature storage and transportation of cells, and can be closer to the injection standard.

[0007] Therefore, in a first aspect, the present invention provides a cell preservation solution, the cell preservation solution comprising:

[0008] (1) Adenosine triphosphate or adenosine triphosphate disodium 8.5-30 μg / mL,

[0009] (2) Complex amino acids 5-15 mg / mL,

[0010] (3) B complex vitamins 0.185-0.555 mg / mL,

[0011] (4) human serum albumin 3%-8%, and

[0012] (5) Any of the following components:

[0013] a) Compound electrolyte injection,

[0014] b) glucose injection, or

[0015] c) Glucose and sodium chloride injection.

[0016] In a second aspect, the present invention provides a method for preserving cells, the method comprising suspending the cells in the cell preservation solution of the first aspect of the present invention and preserving the cells at 0-15°C.

[0017] In a third aspect, provided is the use of the cell preservation solution described in the first aspect of the present invention in the low-temperature preservation and transportation of cells.

[0018] The beneficial effect of the present invention is that the cell preservation solution of the present invention is an injection-grade cell preservation solution, and no animal serum is used, which meets clinical requirements. On the basis of ensuring safety, a formula is provided that can provide energy for cells and effectively prolong the storage time of cells, thereby realizing long-term long-distance transportation. DETAILED DESCRIPTION

[0019] The following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. In addition, those skilled in the art will appreciate that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0021] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value to one-tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are included in the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0022] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of..."

[0023] In order to better understand the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers and other numerical values ​​used in the specification and claims indicating quantities, percentages or ratios should be understood as being modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which may vary depending on the desired properties sought to be obtained. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0024] In a first aspect, a cell preservation solution is provided, comprising:

[0025] (1) Adenosine triphosphate or adenosine triphosphate disodium 8.5-30 μg / mL,

[0026] (2) Complex amino acids 5-15 mg / mL,

[0027] (3) B complex vitamins 0.1875-0.5625 mg / mL,

[0028] (4) human serum albumin 3%-8%, and

[0029] (5) Any of the following components:

[0030] a) Compound electrolyte injection,

[0031] b) glucose injection, or

[0032] c) Glucose and sodium chloride injection.

[0033] In one embodiment, the cell preservation solution contains 10-30 μg / mL of adenosine disodium triphosphate, preferably 20 μg / mL. The adenosine triphosphate is used to provide energy to cells, maintain cell viability, and prolong the preservation time.

[0034] In one embodiment, the working concentration of each amino acid in the composite amino acid is: L-proline 0.5-1.5 mg / mL, L-serine 0.3125-0.9375 mg / mL, L-alanine 0.4813-1.4438 mg / mL, L-arginine 0.375-1.125 mg / mL, L-histidine 0.15-0.45 mg / mL, L-tryptophan 0.0413-0.1238 mg / mL, L-valine 0.525-1.575 mg / mL, L-threonine In an optional embodiment, the composite amino acid may further comprise sodium bisulfite 0.0313-0.0938 mg / mL.

[0035] In a preferred embodiment, the working concentration of the composite amino acid is 10 mg / mL. As an example, the working concentrations of the amino acids in the cell preservation solution are: L-proline 1.0 mg / mL, L-serine 0.625 mg / mL, L-alanine 0.9625 mg / mL, L-arginine 0.75 mg / mL, L-histidine 0.3 mg / mL, L-tryptophan 0.0825 mg / mL, L-valine 1.05 mg / mL, L-threonine 0.5625 mg / mL, L-leucine 1.375 mg / mL, L-methionine 0.125 mg / mL, L-isoleucine 1.125 mg / mL, L-phenylalanine 0.125 mg / mL, L-lysine acetate 1.075 mg / mL, L-cysteine ​​hydrochloride <0.025 mg / mL, glycine 1.125 mg / mL. In an optional embodiment, the complex amino acid may further contain 0.0625 mg / mL of sodium bisulfite.

[0036] It should be understood that, in this article, when referring to the "working concentration" of a component, it refers to the final concentration of the component in the cell preservation solution, at which the component provides the best technical effect for cell preservation.

[0037] In one embodiment, the complex amino acids in the cell preservation solution are obtained by adding the complex amino acid injection solution to a) a complex electrolyte injection solution, b) a glucose injection solution or c) a glucose and sodium chloride injection solution as a preservation main solution. As an example, the complex amino acid injection solution may contain L-proline 8.0 mg / mL, L-serine 5.0 mg / mL, L-alanine 7.7 mg / mL, L-arginine 6.0 mg / mL, L-histidine 2.4 mg / mL, L-tryptophan 0.66 mg / mL, L-valine 8.4 mg / mL, L-threonine 4.5 mg / mL, L-leucine 11.0 mg / mL, L-methionine 1.0 mg / mL, L-isoleucine 9.0 mg / mL, L-phenylalanine 1.0 mg / mL, L-lysine acetate 8.6 mg / mL, L-cysteine ​​hydrochloride <0.2 mg / mL, glycine 9.0 mg / mL and sodium bisulfite 0.5 mg / mL.

[0038] When preparing the cell preservation solution of the present invention, an appropriate amount of the composite amino acid injection solution can be added to the preservation main solution according to the working concentration of the composite amino acid in the cell preservation solution. Amino acids, as one of the raw materials for cell metabolism, can provide energy for cells to maintain cell vitality.

[0039] In one embodiment, the working concentration of each B vitamin in the complex B vitamins is: vitamin B1 0.05-0.15 mg / mL, vitamin B2 0.005-0.015 mg / mL, vitamin B6 0.005-0.015 mg / mL, niacinamide 0.125-0.375 mg / mL, and sodium pantothenate 0.0025-0.0075 mg / mL.

[0040] In a preferred embodiment, the cell preservation solution contains complex B vitamins at a working concentration of 0.375 mg / mL. As an example, the working concentrations of each B vitamin in the cell preservation solution are: vitamin B1 0.1 mg / mL, vitamin B2 0.01 mg / mL, vitamin B6 0.01 mg / mL, nicotinamide 0.25 mg / mL, and sodium dextropantothenate 0.005 mg / mL.

[0041] In one embodiment, the complex B vitamins in the cell preservation solution are obtained by adding a complex amino acid injection to a) a complex electrolyte injection, b) a glucose injection, or c) a glucose and sodium chloride injection as a preservation main solution. As an example, the complex B vitamin injection contains vitamin B1 10 mg / mL, vitamin B2 1 mg / mL, vitamin B6 1 mg / mL, nicotinamide 25 mg / mL, and sodium pantothenate 0.5 mg / mL.

[0042] In a preferred embodiment, the working concentration of human serum albumin is 5%.

[0043] In one embodiment, the human albumin is obtained by adding human albumin injection to a) compound electrolyte injection, b) glucose injection or c) glucose sodium chloride injection as a storage main solution. As an example, the concentration of human albumin in the human albumin injection can be, for example, 20%. Human albumin is mainly used to prevent cells from adhering to the bottom of the container during transportation.

[0044] In one embodiment, the compound electrolyte injection comprises: 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride, and 0.3 g / L magnesium chloride.

[0045] In one embodiment, the glucose injection contains 50 g / L of glucose.

[0046] In one embodiment, the glucose and sodium chloride injection comprises 50 g / L glucose and 9 g / L sodium chloride.

[0047] When the composite electrolyte component, the glucose component, and the glucose and sodium chloride component are provided in the form of injection, they can not only be used to determine the concentrations of the components, but also can maintain the steady state of cell osmotic pressure.

[0048] In one embodiment, the cell preservation solution further comprises other pharmaceutical excipients. In a preferred embodiment, the pharmaceutical excipients comprise propylene glycol and / or trehalose. Propylene glycol and trehalose, as cryoprotectants replacing dimethyl sulfoxide, have the effect of reducing cell cryogenic damage.

[0049] As can be seen from the above description, the cell preservation solution of the present invention can be prepared by mixing the injection solutions of the components together, thereby providing an injection-grade cell preservation solution. Since the components used are all injection solutions, they can be used directly without re-preparation, which greatly improves safety.

[0050] However, it is understood that the preparation method of the cell preservation solution of the present invention is not limited thereto, and any other preparation method is also possible, as long as it can ensure that the obtained cell preservation solution contains the required content of each component and is injection-grade. For example, appropriate amounts of each component can be added to a suitable solvent such as sterile water or deionized water, mixed, and then filtered and sterilized to obtain an injection-grade cell preservation solution.

[0051] The cell preservation solution of the present invention can effectively maintain the activity of cells, preserve them for a longer period of time, and realize long-distance transportation to more distant areas.

[0052] In a second aspect, the present invention provides a method for preserving cells, the method comprising suspending the cells in the cell preservation solution of the first aspect of the present invention and preserving the cells at 0-15°C.

[0053] In a preferred embodiment, the cells are stored at 2-8°C.

[0054] In one embodiment, the cell is a stem cell. In a preferred embodiment, the cell is a mesenchymal stem cell. In a more preferred embodiment, the cell is a placental mesenchymal stem cell, an adipose mesenchymal stem cell, or a spinal cord mesenchymal stem cell.

[0055] In addition, if the cell density is too low, the amount of cells will be insufficient and the therapeutic effect will not be achieved. If the cell density is too high, the cells will aggregate and the cell preservation solution will not be able to protect more cells. In addition, there will be a risk of blockage when the cells are reinfused. Therefore, in one embodiment, the density of the cells in the cell preservation solution is in the range of 1×10 6 -5×10 6 cells / mL, preferably 2×10 6 cells / mL.

[0056] In a third aspect, provided is the use of the cell preservation solution of the first aspect of the present invention in the low-temperature preservation and transportation of cells.

[0057] In one embodiment, the low temperature storage is storage at 0-15°C, preferably storage at 2-8°C.

[0058] Example

[0059] The embodiments of the present invention will be described in detail below in conjunction with examples. It will be appreciated by those skilled in the art that the following examples are illustrative only and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not indicate manufacturers are conventional products that can be obtained commercially.

[0060] (1) Names and manufacturers of injection solutions used in the examples

[0061] Table 1. Names and manufacturers of injection solutions used

[0062]

[0063]

[0064] The injection additives used in the examples are all finished products from manufacturers and can be used directly after opening without the need for re-formulation or secondary processing.

[0065] (2) Preparation of cell preparations in stage 1

[0066] Phase 1 was divided into 4 control groups. The details of the control group groups are shown in Table 2.

[0067] Table 2. Grouping of the control group

[0068] Group formula Normal saline group Sodium chloride injection + 5% human albumin Compound electrolyte injection group Compound electrolyte injection + 5% human albumin Glucose injection group Glucose injection + 5% human albumin Glucose and sodium chloride injection group Glucose and Sodium Chloride Injection + 5% Human Albumin

[0069] Preparation method: Pipette 2 mL of 20% human albumin into a 15 mL centrifuge tube for each group, then add 6 mL of the corresponding injection solution and mix well.

[0070] The mesenchymal stem cells tested were cultured to P5 using serum-free stem cell complete medium. After digestion and counting, the cells were plated at 2×10 6 The cells were divided into 16 15mL centrifuge tubes, 4 tubes for each group, 16 tubes for 4 groups in total. After centrifugation, the supernatant was removed, and 2mL / tube of the above injection solution was added to the group for resuspending, and then the cells were stored in a 2℃-8℃ refrigerator, and 1 tube was taken out from each group at 8h, 24h, 48h and 72h to test the cell viability, sterility test, surface markers and adhesion of each group of cell preparations after 24h of culture.

[0071] The detailed information of each tube of cells is shown in Table 3.

[0072] Table 3. Detailed information of cells

[0073]

[0074]

[0075] Cell viability determination: Trypan blue counting method was used to test the cell viability of each group of cell preparations.

[0076] Sterility test: The culture method was used to test the sterility of each group of cell preparations.

[0077] Surface marker assay: Flow cytometry was used to detect the surface markers of each group of cell preparations.

[0078] Adhesion after 24 hours of culture: Each group of cell preparations was stained using Cell Counting Kit-8, and then the absorbance was measured using an ELISA reader to determine the adhesion of each group of cell preparations after 24 hours of culture.

[0079] Table 4. Test reagent names and manufacturers

[0080] name Manufacturer 0.4% Trypan blue Gibco Aerobic culture bottle Biomerieux Anaerobic culture bottle Biomerieux Mesenchymal stem cell flow cytometry kit BD CellCountingKit-8 Blue Sky

[0081] Table 5. Equipment name, model and manufacturer

[0082]

[0083]

[0084] Table 6. Test methods and indicators

[0085]

[0086] The test results of Examples 1-6 are shown in Tables 7-12, respectively, wherein the umbilical cord mesenchymal stem cells used in each example are derived from different newborns, so there are certain differences between samples. Note: The reagents and consumables used in each example for testing are all of the same batch number, and the instruments used are all of the same manufacturer and factory number.

[0087] Table 7. Test results of Example 1

[0088]

[0089]

[0090] Table 8. Test results of Example 2

[0091]

[0092] Table 9. Test results of Example 3

[0093]

[0094]

[0095] Table 10. Test results of Example 4

[0096]

[0097] Table 11. Test results of Example 5

[0098]

[0099] Table 12. Test results of Example 6

[0100]

[0101]

[0102] From the above results, it can be seen that the stem cells used in Examples 1, 3 and 6 grew relatively slowly during the cell culture process, and the fusion degree after 72 hours of culture did not reach more than 90%, while the stem cells used in Examples 2, 4 and 5 were in good condition during the culture process, and the fusion degree after 72 hours of culture reached more than 95%; the cell viability between the control groups within 24 hours of storage time did not change much, and the absorbance indicating the adhesion did not decrease significantly; after 48 hours, the cell viability and the absorbance indicating the adhesion decreased significantly, among which the decrease in the physiological saline control group with the lowest nutrient was particularly Obviously, in this group, the cell viability of Example 1, Example 3 and Example 6 with relatively poor cell quality after 72 hours of storage was less than 80%; the cell viability of the compound electrolyte injection group in the 6 examples was significantly higher than that of the control group of the same period. Even if the cell quality was poor, the cell viability after 72 hours of storage was still close to 90%, and the absorbance indicating the adhesion was also significantly higher than that of the control group of the same period, proving that the compound electrolyte injection group was relatively more suitable as the main storage solution for stem cell preparations among the 4 control groups. Therefore, the compound electrolyte injection group was selected for the 2-stage experiment when both the sterility test and the surface markers were qualified.

[0103] (3) Preparation of 2-stage cell preparations

[0104] The 2nd stage was divided into 2 control groups, and the specific grouping details are shown in Table 13.

[0105] Table 13. Grouping of control group

[0106]

[0107] Preparation method:

[0108] The compound electrolyte injection group was prepared as follows: 2 mL of 20% human albumin was drawn into a 15 mL centrifuge tube, and then 6 mL of the compound electrolyte injection was added and mixed.

[0109] The preferred group was prepared as follows: 2 mL of 20% human albumin was drawn into a 15 mL centrifuge tube, and then adenosine triphosphate disodium injection was added to make the final concentration of adenosine triphosphate disodium 20 μg / mL, compound amino acid injection was added to make the final concentration of total amino acids 10 mg / mL, and vitamin B complex injection was added to make the total concentration of vitamin B 0.375 mg / mL, and finally a compound electrolyte solution was added to make the volume to 8 mL and mixed.

[0110] The cell preparation and testing methods in Phase 2 are the same as those in Phase 1.

[0111] The test results of Examples 7-12 are shown in Tables 14-19, respectively. The umbilical cord mesenchymal stem cells used in each example are derived from different newborns, so there are certain differences between samples.

[0112] Table 14. Test results of Example 7

[0113]

[0114] Table 15. Test results of Example 8

[0115]

[0116] Table 16. Test results of Example 9

[0117]

[0118] Table 17. Test results of Example 10

[0119]

[0120] Table 18. Test results of Example 11

[0121]

[0122] Table 19. Test results of Example 12

[0123]

[0124] As shown in the test results of the above embodiments, Example 8 grew relatively slowly during the recovery culture process, and the counted cell amount and viability were less than those of other embodiments. The proliferation conditions of other embodiments during the culture process were good and there was no significant difference. In the preferred group, which added energy-providing adenosine triphosphate disodium injection, compound amino acid injection and complex vitamin B injection on the original basis, the cell viability was significantly higher than that of the compound electrolyte injection group in the same period, and the viability did not decrease significantly after storage for 48 hours. Even in Example 8 with poor cell quality, the cell viability after storage for 72 hours was still able to maintain above 90%. In addition, in terms of absorbance indicating cell adhesion, the preferred group was significantly higher than the compound electrolyte injection group, and the value was significantly higher than any control group in the same period of stage 1; and the sterility test and surface markers were both qualified.

[0125] (4) Adenosine triphosphate disodium injection and compound amino acid injection have a synergistic effect

[0126] The inventors found in the test that adenosine triphosphate disodium injection and compound amino acid injection may have a synergistic effect, so they verified it. The experimental grouping is shown in Table 20.

[0127] Table 20. Grouping of control group

[0128]

[0129]

[0130] Preparation method:

[0131] The blank control group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection was added to make the total concentration of vitamin B 0.375 mg / ml, and finally compound electrolyte injection was added to make up to 8 mL and mixed.

[0132] The adenosine triphosphate disodium injection group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then 80 μL of complex vitamin B injection (to make the total vitamin B concentration 0.375 mg / ml) and adenosine triphosphate disodium injection (to make the final concentration of adenosine triphosphate disodium 20 μg / mL) were added, and finally, compound electrolyte injection was added to make up to 8 mL and mixed.

[0133] The compound amino acid injection group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally compound electrolyte injection was added to make up to 8 mL and mixed.

[0134] The preferred group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), adenosine triphosphate disodium injection (total adenosine triphosphate disodium final concentration was 20 μg / mL) and compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally compound electrolyte injection was added to make up to 8 mL and mixed.

[0135] The preparation and testing methods of the cell preparations were the same as those in Phase 1.

[0136] The test results of Examples 13-18 are shown in Tables 21-26, respectively. The umbilical cord mesenchymal stem cells used in each example are derived from different newborns, so there are certain differences between samples.

[0137] Table 21. Test results of Example 13

[0138]

[0139]

[0140] Table 22. Test results of Example 14

[0141]

[0142]

[0143] Table 23. Test results of Example 15

[0144]

[0145] Table 24. Test results of Example 16

[0146]

[0147]

[0148] Table 25. Test results of Example 17

[0149]

[0150] Table 26. Test results of Example 18

[0151]

[0152]

[0153] As shown in the test results of the above examples, Example 13 and Example 18 grew relatively slowly during the recovery culture process, and the counted cell amount and viability were less than those of other examples, while the proliferation conditions of other examples were good during the culture process, with no significant difference; in terms of cell viability, there was not much difference in the cell viability of all examples within 24 hours, and the cell viability decreased significantly after 48 hours of storage, especially the blank control group without the addition of adenosine triphosphate disodium injection and compound amino acid injection had a lower cell viability after 72 hours of storage than the control group of the same period, while the cell viability of the preferred group after 48 hours of storage was significantly higher than that of the control group of the same period, even the cell state of Example 13 and Example 18 was poor, and the cell viability could still reach about 90% after 72 hours of storage; in terms of absorbance indicating the adhesion condition, the absorbance of all examples within 24 hours was not much different, and began to decline significantly after 48 hours, the adenosine triphosphate disodium injection group and the compound amino acid injection group were similar in the same period, and the preferred group was significantly better than the control group of the same period; in addition, the sterility test of all groups was negative, and the surface markers were qualified.

[0154] In summary, the test results of the four control groups were: preferred group > adenosine triphosphate disodium injection group = compound amino acid injection group > blank control group, proving that adenosine triphosphate disodium injection and compound amino acid injection have a synergistic effect.

[0155] (5) Effects of glucose injection and glucose sodium chloride injection as the main storage solution

[0156] The composite electrolyte injection of the above preferred group (4) was replaced with glucose injection and glucose sodium chloride injection respectively, and the tests were carried out at the same time. The experimental grouping is shown in Table 27.

[0157] Table 27. Grouping of control group

[0158]

[0159] Preparation method:

[0160] The blank control group was prepared as follows: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), adenosine triphosphate disodium injection (total adenosine triphosphate disodium final concentration was 20 μg / mL) and compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally physiological saline was added to make up to 8 mL and mixed.

[0161] The glucose injection group was prepared as follows: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), adenosine triphosphate disodium injection (total adenosine triphosphate disodium final concentration was 20 μg / mL) and compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally glucose injection was added to make up to 8 mL and mixed.

[0162] The glucose and sodium chloride injection group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), adenosine triphosphate disodium injection (total adenosine triphosphate disodium concentration was 20 μg / mL) and compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally glucose and sodium chloride injection were added to make up to 8 mL and mixed.

[0163] The preferred group was prepared by the following method: 2 mL of 20% human albumin was aspirated into a 15 mL centrifuge tube, and then vitamin B complex injection (total vitamin B concentration was 0.375 mg / ml), adenosine triphosphate disodium injection (total adenosine triphosphate disodium final concentration was 20 μg / mL) and compound amino acid injection (total amino acid final concentration was 10 mg / mL) were added, and finally compound electrolyte injection was added to make up to 8 mL and mixed.

[0164] The preparation and testing methods of the cell preparations were the same as those in Phase 1.

[0165] The test results of Examples 19-24 are shown in Tables 27-32, respectively. The umbilical cord mesenchymal stem cells used in each example are from the same source as the umbilical cord mesenchymal stem cells used in (4) above. Based on the same reasons mentioned above, there are certain differences between the samples.

[0166] Table 28. Test results of Example 19

[0167]

[0168] Table 29. Test results of Example 20

[0169]

[0170]

[0171] Table 30. Test results of Example 21

[0172]

[0173]

[0174] Table 31. Test results of Example 22

[0175]

[0176] Table 32. Test results of Example 23

[0177]

[0178]

[0179] Table 33. Test results of Example 24

[0180]

[0181] As shown in the test results of the above embodiments, Example 19 and Example 24 grew relatively slowly during the recovery culture process, and the counted cell amount and viability were less than those of other embodiments, while the proliferation conditions of other embodiments were good during the culture process, with no significant difference; in terms of cell viability, there was not much difference in the cell viability of all embodiments within 24 hours, and the cell viability decreased significantly after 48 hours of storage, especially the blank control group using physiological saline as the main storage solution after 72 hours of storage. The cell viability of the preferred group was significantly higher than that of the control group in the same period, and even the cell state of Example 19 and Example 24 was poor. The cell viability can still reach about 90% after 72 hours of storage; in terms of absorbance indicating the adhesion condition, the absorbance of all embodiments within 24 hours was not much different, and began to decline significantly after 48 hours. The glucose and sodium chloride injection group and the glucose injection group were similar in the same period, and the preferred group was significantly better than the control group in the same period; in addition, the sterility tests of all groups were negative, and the surface markers were qualified.

[0182] In summary, the test results of the four control groups are: preferred group > glucose injection group = glucose sodium chloride injection group > blank control group, among which the cell viability of the glucose injection group and the glucose sodium chloride injection group within 48 hours is still greater than 90%, indicating that in addition to the compound electrolyte injection as the main preservation solution of the cell preservation solution for short-distance transportation, glucose injection or glucose sodium chloride injection can also be selected as the main preservation solution of the cell preservation solution.

Claims

1. A cell preservation solution, comprising: (1) Adenosine triphosphate or adenosine triphosphate disodium 8.5-30 μg / mL, (2) complex amino acids 5-15 mg / mL, preferably 10 mg / mL, (3) B complex vitamins 0.1875-0.5625 mg / mL, preferably 0.375 mg / mL, (4) human serum albumin 3%-8%, and (5) Any of the following components: a) Compound electrolyte injection, b) glucose injection, or c) Glucose and sodium chloride injection. 2 . The cell preservation solution according to claim 1 , wherein the cell preservation solution comprises 10-30 μg / mL of adenosine disodium triphosphate, preferably 20 μg / mL.

3. The cell preservation solution according to claim 1 or 2, wherein: The working concentration of each amino acid in the composite amino acid is: L-proline 0.5-1.5mg / mL, L-serine 0.3125-0.9375mg / mL, L-alanine 0.4813-1.4438mg / mL, L-arginine 0.375-1.125mg / mL, L-histidine 0.15-0.45mg / mL, L-tryptophan 0.0413-0.1238mg / mL, L-valine 0.525-1.575mg / mL, L-threonine 0.2813-0.8438mg / mL, L-leucine 0.6875-2.0625mg / mL, L-methionine 0.0625-0.1875mg / mL, L-isoleucine 0.5625-1.6875mg / mL, L-phenylalanine 0.0625-0.1875mg / mL, L- Lysine acetate 0.5375-1.6125 mg / mL, L-cysteine ​​hydrochloride <0.0375 mg / mL, glycine 0.5625-1.6875 mg / mL; Preferably, the working concentration of each amino acid in the composite amino acid is: L-proline 1.0 mg / mL, L-serine 0.625 mg / mL, L-alanine 0.9625 mg / mL, L-arginine 0.75 mg / mL, L-histidine 0.3 mg / mL, L-tryptophan 0.0825 mg / mL, L-valine 1.05 mg / mL, L-threonine 0.5625 mg / mL, L-leucine 1.375 mg / mL, L-methionine 0.125 mg / mL, L-isoleucine 1.125 mg / mL, L-phenylalanine 0.125 mg / mL, L-lysine acetate 1.075 mg / mL, L-cysteine ​​hydrochloride <0.025 mg / mL, and glycine 1.125 mg / mL.

4. The cell preservation solution according to any one of claims 1 to 3, wherein: The working concentrations of the B vitamins in the B vitamin complex are: vitamin B1 0.05-0.15 mg / mL, vitamin B2 0.005-0.015 mg / mL, vitamin B6 0.005-0.015 mg / mL, niacinamide 0.125-0.375 mg / mL, sodium dextropantothenate 0.0025-0.0075 mg / mL; Preferably, the working concentrations of the B vitamins in the cell preservation solution are: vitamin B1 0.1 mg / mL, vitamin B2 0.01 mg / mL, vitamin B6 0.01 mg / mL, niacinamide 0.25 mg / mL, and sodium dextropantothenate 0.005 mg / mL.

5. The cell preservation solution according to any one of claims 1 to 4, wherein the working concentration of human albumin is 5%.

6. The cell preservative solution according to any one of claims 1 to 5, wherein the cell preservative solution is prepared by using components (1) to (4) in the form of an injection solution.

7. The cell preservation solution according to any one of claims 1 to 6, wherein: The composite electrolyte injection contains: 5.26 mg / mL sodium chloride, 5.02 mg / mL sodium gluconate, 3.68 mg / mL sodium acetate, 0.37 mg / mL potassium chloride, and 0.3 mg / mL magnesium chloride; The glucose injection contains 50 mg / mL of glucose; The glucose and sodium chloride injection contains 50 mg / mL of glucose and 9 mg / mL of sodium chloride.

8. The cell preservation solution according to any one of claims 1 to 7, wherein the cell preservation solution further comprises other pharmaceutical excipients; preferably, the pharmaceutical excipients comprise propylene glycol and / or trehalose.

9. A method for preserving cells, comprising suspending the cells in a cell preserving solution according to any one of claims 1 to 8, and preserving the cells at 0-15°C, preferably 2-8°C.

10. The method according to claim 9, wherein the cells are stem cells; preferably, the cells are mesenchymal stem cells; more preferably, the cells are placental mesenchymal stem cells, adipose mesenchymal stem cells, spinal cord mesenchymal stem cells.

11. The method according to claim 9 or 10, wherein the density of the cells in the cell preservation solution is in the range of 1×10 6 -5×10 6 cells / mL, preferably 2×10 6 cells / mL.

12. Use of the cell preservation solution according to any one of claims 1 to 8 in cell cryopreservation and transportation.

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