CN114041455A - Stem cell cryopreservation solution, and preparation method and application thereof - Google Patents
Stem cell cryopreservation solution, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a stem cell cryopreservation solution, a preparation method and application thereof; the freezing solution comprises the following components in percentage by volume: 20-40% of a basic culture medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine, and 6-10.5% of galactinol or glycosides. The frozen stock solution adopts the serum-free frozen stock solution to freeze stem cells, the cells are not damaged and the possibility of animal pathogen pollution can be reduced, and the galactinol or the glucoside can be used as a stabilizer, so that the cell phenotype can be effectively protected, the activity of the stem cells under the frozen stock condition can be remarkably improved, and the physiological function and the biological function after the cells are recovered are kept.
Description
Technical Field
The invention relates to the technical field of stem cell cryopreservation, in particular to a stem cell cryopreservation solution, a preparation method and application thereof.
Background
The stem cell is a multifunctional cell which can be highly proliferated and differentiated in multiple lineages, and has the capacity of self-renewal and multi-directional differentiation. Stem cells are the most primitive cells of a living body, are the origin of all cell lines, can produce daughter cells with the same phenotype and genotype as well as the cells which are specialized and form tissues and organs of a body, and can be differentiated into progenitor cells. In adult organs, stem cells can promote tissue repair by constantly dividing and releasing growth factors, and are a potential clinical material for regenerative medicine.
The stem cells with high proliferation activity can obtain a large amount of cells after being cultured, but the freezing and recovery processes are always required on the basis of the existing large-scale amplification culture process. Currently, the clinical demand of stem cells is increasing, and the research on the cryopreservation and recovery of stem cells is receiving more and more attention.
The currently common stem cell cryopreservation method is to mix cells in a cryopreservation solution containing DMSO and animal serum, perform programmed cooling, and finally store the cells in liquid nitrogen at the temperature of-196 ℃. However, DMSO is a high-permeability solvent, can rapidly permeate into cells, improves the permeability of cell membranes to water, enables water to permeate out of the cells to form ice crystals before the cells are frozen, and is an ideal cryoprotectant for cell freezing. Because low-concentration DMSO has certain toxicity to cells at normal temperature, certain requirements on environment and operation of cell cryopreservation are met, and meanwhile, animal serum is used as a foreign substance to easily increase the possibility of pathogenic contamination of animals, the research on a stem cell cryopreservation liquid without DMSO and animal serum is necessary.
Disclosure of Invention
In view of the above problems, one of the problems to be solved by the present invention is to provide a frozen stock solution of stem cells which is nontoxic and harmless to the frozen stock of stem cells and free from contamination by foreign substances.
The second problem to be solved by the present invention is to provide a method for preparing a stem cell cryopreservation solution.
The invention also provides a method for cryopreserving stem cells.
The first technical scheme of the invention is as follows:
a stem cell freezing medium comprises the following components in percentage by volume: 20-40% of a basic culture medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine, and 6-10.5% of galactinol or glycosides.
In one embodiment, the stem cell cryopreservation solution comprises, by volume percent: 30% basal medium, 15% glycerol, 50% human serum albumin, 5% trehalose, 1% glycine or taurine, 8% galactinol or glycosides.
In one embodiment, the stem cell cryopreservation solution and the basic culture medium are stem cell serum-free culture medium, RPMI-1640, MEM or DMEM.
The invention also provides a preparation method of the stem cell frozen stock solution, which comprises the following steps:
preparing according to volume percentage: 20-40% of a basal medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine, and 6-10.5% of galactinol or glycosides;
uniformly mixing the basic culture medium, glycerol, human serum albumin and trehalose according to a proportion to prepare a mixed solution 1;
and adding the taurine or the glycine and the galactinol or the glycosides into the mixed solution 1 according to a ratio, and performing vortex dissolution, uniform mixing and sterilization treatment to obtain the stem cell frozen stock solution.
The invention also provides a method for cryopreserving stem cells by using the stem cell cryopreservation solution, which comprises the following steps:
pre-cooling the stem cell frozen stock solution for 30 minutes at the temperature of 2-8 ℃;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 5.0 x 106～3.0×107cells/mL, then subpackaging into 2mL or 5mL freezing storage tubes;
and (3) placing the freezing tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation.
In one embodiment, the stem cell cryopreservation method further comprises a stem cell recovery step after the stem cell cryopreservation:
and taking out the freezing tube from the liquid nitrogen, and putting the tube at the temperature of 37-42 ℃ for resuscitation for 2 minutes.
In one embodiment, the stem cells are cryopreserved, and the stem cells are P1 or P3 generation stem cells.
In one embodiment, the stem cell cryopreservation method is that after the cryopreservation solution re-suspends the stem cells, the density of the stem cells is 2.0 × 107cells/ml。
Compared with the prior art, the invention has the following advantages:
1) the components are clear, and the stem cells are frozen by adopting the DMSO-free and serum-free freezing medium, so that the cells are not damaged and the possibility of animal pathogenic pollution can be reduced;
2) and the galactinol or the glucoside is used as a stabilizer, so that the phenotype of the cells can be effectively protected, and the activity of the stem cells under the condition of freezing can be remarkably improved, thereby maintaining the physiological function and the biological function of the cells after recovery.
Drawings
FIG. 1 is a graph showing the culture proliferation after the freeze-storage recovery of stem cells in examples 5,6,7, 8 and comparative example 2;
FIG. 2 is a graph showing the cell doubling time after the freeze-storage recovery of stem cells of examples 5,6,7 and 8 and comparative example 2;
FIGS. 3a, 3b, 3c, 3d are osteogenic differentiation maps of stem cells; wherein, FIGS. 3a, 3b and 3c are osteogenic differentiation charts of the stem cells obtained after the frozen stem cells are recovered for 30 days by the freezing storage method of examples 5,6 and 8, respectively; FIG. 3d is a skeletal differentiation chart of stem cells after the stem cells are thawed 30 days by using the freezing method of comparative example 2; the microscope magnification was 4 x 10;
FIGS. 4a, 4b, 4c, 4d are diagrams of chondrogenic differentiation of stem cells; wherein, FIGS. 4a, 4b and 4c are diagrams of chondrogenic differentiation of stem cells after 30 days of resuscitation by the cryopreserved stem cells according to the cryopreserving methods of examples 5,6 and 8, respectively; FIG. 4d is a cartilaginous differentiation map of stem cells after 30 days of resuscitation by cryopreserving the stem cells using the cryopreservation method of comparative example 2; the microscope magnification was 4 x 10;
FIGS. 5a, 5b, 5c and 5d are diagrams of stem cell adipogenic differentiation; wherein, FIGS. 5a, 5b and 5c are graphs of stem cell adipogenic differentiation after 30 days of recovery of the frozen stem cells in the freezing storage methods of examples 5,6 and 8, respectively; FIG. 5d is a adipose differentiation map of stem cells after 30 days of resuscitation by cryopreserving the stem cells using the cryopreservation method of comparative example 2; the microscope magnification was 4 x 10.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The stem cell freezing solution provided by the invention comprises the following components in percentage by volume: 20-40% of a basal medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine and 6-10.5% of galactinol or glycosides; wherein the glycoside includes D-glucose, D-galactose, aminosugar, salidroside, adenosine, flavonoid glycoside, anthraquinone, gastrodine, stevioside, and prunasin.
Further, in one embodiment, the stem cell cryopreservation solution preferably comprises, in terms of volume percentage: 30% basal medium, 15% glycerol, 50% human serum albumin, 5% trehalose, 1% glycine or taurine and 8% galactinol or glycosides.
In the frozen stock solution of the stem cells, the percentage contents of the components such as a basic culture medium, glycerol, human serum albumin, trehalose, glycine or taurine, galactinol or glucoside and the like can also be mass percentages, and the mass percentage of each component corresponds to the volume percentage.
In the above-mentioned frozen stock solution for stem cells, the basic medium may be a serum-free medium for stem cells, RPMI-1640, MEM, DMEM, or another medium for stem cells.
The invention also provides a preparation method of the stem cell frozen stock solution, which comprises the following steps:
s1, preparing according to volume percentage: 20-40% of a basal medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine and 6-10.5% of galactinol or glycosides;
s2, uniformly mixing the basic culture medium, glycerol, human serum albumin and trehalose according to a proportion to prepare a mixed solution 1;
and S3, adding taurine or glycine and galactoside inositol or glycosides into the mixed solution 1 according to the proportion, and performing vortex dissolution, uniform mixing and sterilization treatment to obtain the stem cell frozen stock solution.
The invention also provides a method for cryopreserving stem cells by using the stem cell cryopreservation solution, which comprises the following steps:
s11, pre-cooling the stem cell frozen stock solution for 30 minutes at the temperature of 2-8 ℃;
s12, resuspending the stem cells by using the frozen and precooled stem cell frozen stock solution, and adjusting the density of the P1 or P3 stem cells to 5.0 x 106～3.0×107cells/mL, then subpackaging into 2mL or 5mL freezing storage tubes;
s13, placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation.
In one embodiment, after the stem cells are cryopreserved, the method further comprises a stem cell recovery step:
s14, taking out the freezing tube from the liquid nitrogen, and resuscitating the tube at the temperature of 37-42 ℃ for 2 minutes.
In one embodiment, after the cryopreservation solution resuspends the stem cells, the density of the stem cells is preferably 2.0 × 107cells/ml。
First, preparation of stem cell freezing medium
Example 1
Adding 26ml of a stem cell serum-free culture medium, 13ml of glycerol, 48ml of human serum albumin and 5ml of trehalose into a measuring cup, and stirring and uniformly mixing to obtain a mixed solution 1;
adding 1ml of taurine and 7ml of galactinol into the mixed solution 1, and obtaining a stem cell frozen stock solution for later use after vortex dissolution, uniform mixing and sterilization treatment at 56 ℃.
Example 2
Adding 20ml of PRMI-1640 culture medium, 10ml of glycerol, 60ml of human serum albumin and 2ml of trehalose into a measuring cup, and stirring and uniformly mixing to obtain a mixed solution 1;
adding 2ml of glycine and 6ml of galactinol into the mixed solution 1, and performing vortex dissolution, uniform mixing and 56 ℃ sterilization treatment to obtain a stem cell frozen stock solution for later use.
Example 3
Adding 400ml of DMEN culture medium, 150ml of glycerol, 300ml of human serum albumin and 40ml of trehalose into a measuring cup, stirring and uniformly mixing to obtain a mixed solution 1;
adding 5ml of glycine and 105ml of glycosides into the mixed solution 1, dissolving by vortex, mixing uniformly, and sterilizing at 56 ℃ to obtain a stem cell frozen stock solution for later use.
Example 4
In this embodiment, the components of the frozen stock solution do not contain galactinol or glycosides.
Adding 33ml of stem cell serum-free medium, 13ml of glycerol, 48ml of human serum albumin and 5ml of trehalose into a measuring cup, and stirring and uniformly mixing to obtain a mixed solution 1;
and adding 1ml of taurine into the mixed solution 1, and performing vortex dissolution, uniform mixing and 56 ℃ sterilization treatment to obtain a stem cell frozen stock solution for later use.
Comparative example 1
A stem cell cryopreservation liquid comprises the following components in percentage by volume: 80% DMEN medium, 10% FBS and 10% DMSO; namely, 800ml of DMEN culture medium, 100ml of FBS and 100ml of DMSO are uniformly mixed and sterilized at 56 ℃ to obtain the stem cell frozen stock solution.
Second, freezing and recovering stem cells
Example 5
Precooling the stem cell frozen stock solution prepared in the example 1 at 5 ℃ for 30 minutes;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 1.0 x 107cells/mL, then subpackaging into 2mL freezing tubes;
placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation;
the cryopreserved tubes were removed from the liquid nitrogen and placed at 40 ℃ for 2 minutes to resuscitate the stem cells.
Example 6
Precooling the stem cell frozen stock solution prepared in the embodiment 2 at 2 ℃ for 30 minutes;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 5.0 x 106cells/mL, then subpackaging into 2mL freezing tubes;
placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation;
the cryopreserved tubes were removed from the liquid nitrogen and placed at 37 ℃ for 2 minutes for stem cell recovery.
Example 7
Precooling the stem cell frozen stock solution prepared in the embodiment 3 for 30 minutes at 8 ℃;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 3.0 x 107cells/mL, then subpackaging into 5mL freezing tubes;
placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation;
the cryopreserved tubes were removed from the liquid nitrogen and placed at 42 ℃ for 2 minutes to resuscitate stem cells.
Example 8
Precooling the stem cell frozen stock solution prepared in the embodiment 4 at 5 ℃ for 30 minutes;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 1.0 x 107cells/mL, then subpackaging into 2mL freezing tubes;
placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation;
the cryopreserved tubes were removed from the liquid nitrogen and placed at 40 ℃ for 2 minutes to resuscitate the stem cells.
Comparative example 2
Precooling the stem cell frozen stock solution prepared in the comparative example 1 at 5 ℃ for 30 minutes;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 1.0 x 107cells/mL, then subpackaging into 2mL freezing tubes;
placing the cryopreservation tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation;
the cryovial was removed from the liquid nitrogen and placed at 40 ℃ for 2 min to resuscitate the stem cells.
Third, analysis of test results
(I) detection of Stem cell Activity
Examples 4, 5, and 6 and comparative example 2 after the stem cells were cryopreserved for 30 days and recovered, the recovered stem cells were cultured for 7 days, and the number of cells was counted every day, and the statistical results are shown in table 1 and fig. 1.
TABLE 1 growth proliferation after Stem cells are resuscitated Table of numbers, × 104cells
As shown in fig. 1 and table 1, after the stem cells were thawed for 30 days by freezing, the stem cells were cultured for 7 days, and the test results were:
1) the frozen stock solutions of examples 5,6 and 7 contained galactinol or glycosides, while the frozen stock solution used in example 8 did not contain a cardiac polymer compound; from the test results, the galactosyl inositol or the glucoside inositol can effectively protect the cell phenotype and improve the activity of the stem cells under the condition of freezing storage, thereby maintaining the physiological function and the biological function of the recovered cells and being beneficial to the rapid growth of the recovered cells; therefore, after the frozen stock solution stem cells of the freezing and storing methods of examples 5,6 and 7 are recovered, the cells grow relatively fast;
2) comparative example 2 is a conventional cell cryopreservation solution and does not contain galactinol or glycosides; therefore, in view of the influence of the freezing medium on cell growth and expansion corresponding to each of examples 5,6,7 and comparative example 2, after the frozen stem cells of the freezing medium provided by the invention are recovered, the cells grow and expand relatively quickly; even if the embodiment 8 is compared with the comparative example 2, the cell amplification growth of the frozen cells of the freezing medium of the invention is better than that of the comparative example 2 after the cells are recovered; the invention adopts the frozen stem cells of the frozen stock solution without DMSO and serum, has no damage to the cells and can well maintain the activity of the cells.
In general, after the frozen stem cells of the frozen stock solution are recovered, the cells are expanded and grown faster, and the cell expansion amount is obviously faster particularly after 3 days, as can be seen from the cell count of the frozen stem cells in the table 1 every day.
In addition, taking the example of counting the number of cells at intervals of 2 days in table 1, the counted number of cells was substituted into the formula DT ═ t × [ lg2 ]/(lgNt-lgN0)](t is time/hour; NtCell number indicating the culture time t hours; n is a radical of0Representing the number of cells at hour 0), the doubling time required for cell proliferation within one week (within 7 days) was calculated, and the results are shown in table 2 and fig. 2.
TABLE 2 cell culture doubling time Table
The data in table 1 show that statistical analysis shows that there is significant difference, p is less than 0.05; among them, the cell doubling time of example 6 was the shortest, superior to that of the other groups, with p < 0.05.
The above results show that:
1) the cell doubling time significance of the groups of examples 5,6,7 and 8 is better than that of the group of comparative example 2;
2) the cell doubling times of the groups of examples 5,6 and 7 were significantly better than those of the group of example 8.
The above test results mainly occur because the frozen stock solutions used in examples 5,6 and 7 contain galactinol or glycosides, and the frozen stock solution used in example 8 does not contain galactinol or glycosides, which can effectively protect cell phenotype and improve the activity of stem cells under the condition of frozen stock, thereby maintaining the physiological function and biological function of the recovered cells and being beneficial to the rapid growth of the recovered cells.
The frozen stem cells in the comparative example 2 are recovered and cultured to the 2 nd day, the cell proliferation doubling time is obviously longer than that of the three examples (5,6 and 7), and the doubling time of the stem cell culture in the comparative example 2 in the first 5 days is longer than that of the examples (5,6 and 7), so that the adaptation period required after the frozen stem cells in the comparative example 2 are recovered is longer, and the early growth condition after the recovery culture is slightly poor, because the frozen stem cells in the frozen stock solution without DMSO and serum are not damaged, the cell activity can be well maintained; meanwhile, the galactinol or the glucoside can effectively protect the cell phenotype and improve the activity of the stem cells under the condition of cryopreservation, thereby maintaining the physiological function and the biological function of the recovered cells, being beneficial to the rapid growth of the recovered cells and further shortening the multiplication time of cell proliferation.
After the 6 th day of the stem cell recovery culture, the doubling time of the three examples (5,6,7) begins to increase because the cell culture reaches a certain cell density, and the cells are slowly grown due to mutual contact and mutual inhibition, which further illustrates that the cell recovery growth of the three examples (5,6,7) is faster, and at the moment, a cell culture bottle or culture bag with a larger space needs to be replaced.
(II) cell survival rate test
The experimental method comprises the following steps: the frozen stem cell suspensions of examples 5,6,7, 8 and comparative example 2 were centrifuged to remove the supernatant, and then the frozen stem cell suspension was added to mix, dispensed, and frozen. The freezing time and the survival rate of the cells are shown in Table 3.
TABLE 3 Stem cell cryopreservation time and survival Table
The data in table 3 are statistically different, p is <0.05, and among them, the cell viability rate of example 5 is the highest, better than that of other groups, p is < 0.05.
Table 3 the results show that:
1) examples 5,6,7 had higher cell viability rates after cryopreservation relative to comparative example 2;
2) examples 5,6 and 7 showed higher cell viability after cryopreservation than example 8.
The above test results are mainly due to the fact that the cryopreservation solution used in examples 5,6 and 7 contains galactinol or glycosides, which can effectively protect cell phenotype and improve the activity of stem cells under cryopreservation conditions, so that physiological functions and biological functions of the recovered cells are maintained, and rapid growth of the recovered cells is facilitated.
(III) cell flow assay
The frozen stem cells were cultured for 3 days after being thawed 30 days, and after the frozen stem cells were examined by a flow cytometer, the surface markers CD90, CD73, CD105, CD44, CD34, CD14, CD19, CD45, etc. were examined by the cells, and the examination results are shown in table 4.
TABLE 4 flow results after 3 days of resuscitative culture
Table 4 shows that the positive rate of CD90, CD73, CD105 and CD44 is more than or equal to 95 percent; the positive rates of CD34, CD14, CD19 and CD45 are less than or equal to 5%, and HLA-DR (CD34, CD14, CD19 and CD45) is negative mixture. Meanwhile, the difference of the change of the cells on the surface markers is small before and after the cells are frozen, and the phenotype is relatively stable. It is further described that the galactinol or glycosides of the frozen stock solution are effective in protecting the cell phenotype and increasing the activity of the stem cells under the frozen stock conditions.
(IV) cell recovery osteogenesis, chondrogenesis and adipogenesis differentiation function detection
(1) Cell recovery osteogenic differentiation assay
FIGS. 3a, 3b, 3c, 3d are osteogenic differentiation maps of stem cells; wherein, FIGS. 3a, 3b and 3c are osteogenic differentiation charts of the stem cells obtained after the frozen stem cells are recovered for 30 days by the freezing storage method of examples 5,6 and 8, respectively; FIG. 3d is a skeletal differentiation chart of stem cells after the stem cells are thawed 30 days by using the freezing method of comparative example 2; the microscope magnification was 4 x 10.
As can be seen from fig. 3a, 3b, 3c and 3d, osteogenic differentiation of the stem cells is more obvious after the stem cells are cryopreserved by the cryopreservation method of examples 5,6 and 8 for 30 days, as indicated by an arrow "→" in fig. 3a, 3b and 3c, the stem cells are differentiated into bone tissue blocks, while the osteogenic differentiation of the stem cells is not obvious after the stem cells are cryopreserved by the cryopreservation method of comparative example 2 for 30 days, and the differentiation of the stem cells into bone tissue is not observed at all, which is because the invention adopts the frozen stem cells of the DMSO-free and serum-free cryopreservation liquid, which has no damage to the cells and can well preserve the activity of the cells. Meanwhile, since the frozen stock solution in example 8 does not contain galactinol or glycosides, the cells transformed from stem cells into bone tissue grow relatively slowly, and the cell expansion time is longer and the expressed bone tissue is smaller than those in examples 5 and 6.
(2) Cell recovery cartilage differentiation detection
FIGS. 4a, 4b, 4c, 4d are diagrams of chondrogenic differentiation of stem cells; wherein, FIGS. 4a, 4b and 4c are diagrams of chondrogenic differentiation of stem cells after 30 days of resuscitation by the cryopreserved stem cells according to the cryopreserving methods of examples 5,6 and 8, respectively; the microscope magnification was 4 x 10; FIG. 4d is a cartilaginous differentiation map of stem cells after 30 days of resuscitation by cryopreserving the stem cells using the cryopreservation method of comparative example 2.
As shown in FIGS. 4a, 4b, 4c and 4d, the stem cells were differentiated into cartilage after cryopreservation and recovery, but the stem cells were significantly chondrogenic after 30 days of recovery by the cryopreservation methods in examples 5 and 6; for example, the density of the formed cartilage is denser; in the comparative example 2, the density of the stem cells divided into cartilage is loosened after the stem cells are recovered by freezing for 30 days; this shows that the growth rate of stem cells into cartilage after the freeze-stored stem cells are recovered for 30 days in the freeze-storage methods of examples 5 and 6 is higher than the rate of stem cells dividing into cartilage after the freeze-stored stem cells are recovered for 30 days in the freeze-stored method of comparative example 2, because the freeze-stored stem cells in the freeze-stored method of the invention without DMSO and serum are not damaged, and the activity of the cells can be well preserved. Meanwhile, since the frozen stock solution of example 8 does not contain galactinol or glycosides, the cells transformed from stem cells into cartilage tissue grow relatively slowly, and the cell expansion time is longer than that of examples 5 and 6, and the result is that the cartilage tissue is loose.
(3) And detecting the differentiation of the cells into fat
FIGS. 5a, 5b, 5c and 5d are diagrams of stem cell adipogenic differentiation; wherein, FIGS. 5a, 5b and 5c are graphs of stem cell adipogenic differentiation after 30 days of recovery of the frozen stem cells in the freezing storage methods of examples 5,6 and 8, respectively; FIG. 5d is a adipose differentiation map of stem cells after 30 days of resuscitation by cryopreserving the stem cells using the cryopreservation method of comparative example 2; the microscope magnification was 4 x 10.
As can be seen from fig. 5a, 5b, 5c and 5d, the stem cells can be differentiated into fat after frozen and restored, but the fat forming effect of the stem cells after frozen and restored for 30 days by the frozen and restored method in examples 5,6 and 8 is obvious, as shown in fig. 5a, 5b and 5c, the arrow "→" in the figure indicates fat tissue mass, which is large and easy to identify; in contrast, in comparative example 2, the arrow "→" in the figure indicates a mass of adipose tissue, which is small and not easily recognizable. The relative size of stem cell adipogenic block, the growth speed of stem cell adipogenic block on the surface, large adipogenic block, fast stem cell differentiation, small adipogenic block, slow stem cell differentiation, because the invention adopts the freezing storage of stem cells without DMSO and serum, no damage to cells, and good cell activity preservation. Meanwhile, since the frozen stock solution in example 8 does not contain galactinol or glycosides, the cells transformed from stem cells into adipose tissue grow relatively slowly, the cell expansion time is longer and the adipose tissue expressed is smaller compared to examples 5 and 6.
Therefore, as can be seen from the function tests of cell recovery osteogenesis, chondrogenesis and adipogenesis differentiation, the frozen stock solution and the frozen stock method provided by the invention have the advantage that the growth speed of the stem cells is better than the recovery effect of the stem cells in comparative example 2 after the frozen stock of the stem cells is recovered.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (8)
1. A stem cell freezing medium is characterized by comprising the following components in percentage by volume: 20-40% of a basal medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine and 6-10.5% of galactinol or glycosides.
2. The stem cell cryopreservation solution of claim 1, comprising, in volume percent: 30% basal medium, 15% glycerol, 50% human serum albumin, 5% trehalose, 1% glycine or taurine, 8% galactinol or glycosides.
3. The stem cell cryopreservation liquid of claim 1 or 2, wherein the basal medium is a stem cell serum-free medium, RPMI-1640, MEM or DMEM.
4. Stem cell cryopreservation solution A method for preparing a stem cell cryopreservation solution according to claim 1, comprising the steps of:
preparing according to volume percentage: 20-40% of a basal medium, 10-20% of glycerol, 30-60% of human serum albumin, 2-6% of trehalose, 0.5-2% of glycine or taurine, and 6-10.5% of galactinol or glycosides;
uniformly mixing the basic culture medium, glycerol, human serum albumin and trehalose according to a proportion to prepare a mixed solution 1;
and adding the taurine or the glycine and the galactinol or the glycosides into the mixed solution 1 according to a ratio, and performing vortex dissolution, uniform mixing and sterilization treatment to obtain the stem cell frozen stock solution.
5. A stem cell cryopreservation method is characterized by comprising the following steps:
pre-cooling the stem cell freezing medium of claim 1 for 30 minutes at 2-8 ℃;
resuspending the stem cells with the cryopreserved precooled stem cell cryopreserving liquid, and adjusting the density of the stem cells to 5.0 x 106～3.0×107cells/mL, then subpackaging into 2mL or 5mL freezing storage tubes;
and (3) placing the freezing tube filled with the stem cells at-80 ℃ for 12 hours, and then placing the tube in liquid nitrogen for preservation.
6. The stem cell cryopreservation method of claim 5, further comprising a stem cell recovery step after the stem cell cryopreservation:
and taking out the freezing tube from the liquid nitrogen, and putting the tube at the temperature of 37-42 ℃ for resuscitation for 2 minutes.
7. The method for cryopreserving stem cells according to claim 5, wherein the stem cells are P1 or P3 generation stem cells.
8. The method for cryopreserving stem cells according to claim 5, wherein the density of the stem cells after the cryopreservation solution resuspends the stem cells is 2.0 x 107cells/ml。
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| CN114958724A (en) * | 2022-06-26 | 2022-08-30 | 依雨大健康科技(广州)有限公司 | Production method of stable primary hepatocyte kit |
| CN115281180A (en) * | 2022-07-04 | 2022-11-04 | 四川大学华西医院 | mRNA-platelet compound frozen stock solution and preparation method thereof |
| CN115281184A (en) * | 2022-10-08 | 2022-11-04 | 四川大学华西医院 | Mesenchymal stem cell composite cryopreservation solution as well as preparation method and application thereof |
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