CN115777693A - Cell cryopreservation liquid, preparation method, application method and application - Google Patents
Cell cryopreservation liquid, preparation method, application method and application Download PDFInfo
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Abstract
The invention discloses a cell cryopreservation solution, a preparation method, an application method and application. The cell freezing medium has simple and clear components, and consists of a freezing medium A and a freezing medium B; the frozen stock solution A consists of disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, poloxamer 188, an impermeable protective agent, ectoin, an antioxidant and DMSO; the frozen stock solution B consists of an apoptosis inhibitor. The apoptosis inhibitor in the freezing medium is added into a culture medium after cells are recovered. The invention also discloses a preparation method and application of the frozen stock solution, and the cells frozen by the frozen stock solution have high recovery survival rate and wide application range.
Description
Technical Field
The invention relates to the technical field of cell preservation, in particular to a cell cryopreservation solution, a preparation method, an application method and application.
Background
Cell cryopreservation is a technique for storing biologically living cells and tissues at temperatures below-80 ℃, particularly in low temperature environments below-140 ℃, and long term storage of cells, tissues and other biological materials has proven to be a very successful technique. However, during cryopreservation, cells are exposed to severe stresses of physical and chemical changes, among which are: metabolic decoupling/coupling, ionic imbalance, energy deficiency, protease (caspase) activation, membrane phase transitions and alterations, production and accumulation of free radicals, cellular acidosis, cytoskeletal disintegration, protein denaturation, water coagulation, cell volume changes, and high osmotic pressure. These stressors are considered to be the promoters of apoptosis and necrosis. The manifestation of cell death by cryopreservation may take several hours to days and cell death increases in the first 6 hours after thawing. Apoptosis and necrosis activity predominated 24 hours after thawing, followed by a decrease in the percentage of cell death at 48 hours, and the time of cell death after cryopreservation was termed cryopreservation-induced delayed cell death (cidcd).
Despite the significant advances in cell and tissue cryopreservation technologies currently on the market for research and therapeutic applications, late death of cells after thawing remains a significant drawback facing cryobiology. Under these circumstances, scientists are focusing on a new field of apoptosis and necrosis inhibitors that target and inhibit various proteins involved in the cell death cascade (e.g., caspases, proteases and kinases), prevent the transmission of apoptotic signals, result in reduced intensity of apoptosis and necrosis in cryopreserved cells and tissues, increase cell viability, and maintain cell function, thereby improving overall cryopreservation efficiency.
Disclosure of Invention
The traditional freezing technology mainly focuses on protecting structural components of cells by using a permeable protective agent and an impermeable protective agent and improving the cell recovery survival rate, but the frozen stock solution disclosed by the invention is optimized and improved on the traditional method, so that the frozen stock solution which is simple and clear in components, high in frozen cell survival rate and wide in application range is provided, the principle that various apoptosis channels are activated in the freezing preservation process of the cells to trigger cell death is particularly focused, strategy change is further carried out on the application method, and in the key period of 6-24h after cell recovery, cell apoptosis and necrosis inhibitors are applied to prevent the transmission of cell death related signals and further improve the cell survival rate after recovery.
The invention aims to provide a cell cryopreservation solution, a preparation method, an application method and application, and aims to overcome the technical defects of undefined components, poor cryopreservation effect and narrow application range of the cryopreservation solution in the prior art.
In order to solve the technical problems, the invention provides the following technical scheme: a cell cryopreservation solution comprises a cryopreservation solution A and a cryopreservation solution B;
the freezing solution A consists of disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, poloxamer 188, an impermeable protective agent, ectoin, an antioxidant and DMSO;
the frozen stock solution B consists of an apoptosis inhibitor;
the addition amount of the disodium hydrogen phosphate is 1g/L, the addition amount of the sodium chloride is 9g/L, the addition amount of the potassium chloride is 0.2g/L, the addition amount of the calcium chloride is 0.1g/L, the addition amount of the magnesium chloride is 0.05g/L, the addition amount of the poloxamer 188 is 0.1-10g/L, the addition amount of the impermeable protective agent is 10-100g/L, the addition amount of the ectoine is 0.005-10g/L, and the addition amount of the DMSO is 5-10%.
Preferably, the addition amount of the poloxamer 188 is 2g/L, the addition amount of the impermeable protective agent is 45g/L, the addition amount of the ectoine is 2g/L, and the addition amount of the DMSO is 5%.
Preferably, the impermeable protective agent is any one of hydroxyethyl starch, dextran 40 and dextran 70.
Preferably, the antioxidant is any one of vitamin E, vitamin C and carnosine.
Preferably, the addition amount of the vitamin E in the antioxidant is 0.005g/L, the addition amount of the vitamin C is 0.1g/L, and the addition amount of the carnosine is 0.02g/L.
Preferably, the apoptosis inhibitor is any one of Rock inhibitor Y-27632 and Caspase inhibitor z-VAD-fmk.
Preferably, the final concentration of the Rock inhibitor Y-27632 is 2.5. Mu.M, and the final concentration of the Caspase inhibitor z-VAD-fmk is 10. Mu.M.
Preferably, the preparation process of the frozen stock solution A is as follows:
s1: preparation of impermeable protective agent: weighing the non-permeable protective agent, adding a certain volume of injection water, dissolving under high pressure, and cooling to room temperature;
s2: preparing a base solution: preparing water for injection, and sequentially adding disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride and magnesium chloride into the water for injection to be stirred and dissolved;
s3: sequentially adding poloxamer 188 and the cooled impermeable protective agent which is dissolved completely under high pressure in the step S1 into the base solution prepared in the step S2, then adding the ectoin and the antioxidant, finally adding DMSO, and stirring until all components are dissolved completely and mixed uniformly to obtain an MIX solution;
s4: filtering the MIX solution obtained in the step S3 by using a filter with the diameter of 0.2-0.22 mu m to obtain a frozen stock solution A;
the preparation method of the freezing solution B comprises the following steps:
diluting the apoptosis inhibitor in the frozen stock solution B with DMSO, performing ultrasonic assisted dissolution to obtain stock solution, subpackaging, and storing in a refrigerator at-80 deg.C.
Preferably, in the method for applying the cell cryopreservation solution, the cryopreservation solution B is added to a cell culture medium after the cells are recovered for use.
Preferably, the application of the cell frozen stock solution is applied to the cryopreservation of mammalian cells, such as Mesenchymal Stem Cells (MSC) from various tissues, peripheral Blood Mononuclear Cells (PBMC), T cells, NK cells, chinese hamster ovary cells (CHO cells), african green monkey kidney cells (Vero cells), human embryonic kidney cells (293 cells), human chronic myelogenous leukemia cells (K562 cells) and other types of cells.
Preferably, the application method of the cell freezing medium in cell preservation is as follows:
y1: digesting adherent cells in an exponential growth phase, (directly centrifuging and collecting the cells for suspension cells), sampling and counting;
y2: calculating the volume of the cell suspension to be taken according to the counting result and the freezing storage requirement of each tube of cells, and centrifuging;
y3: removing the centrifuged cell supernatant, adding the cryopreservation liquid A, gently mixing the cells and the cryopreservation liquid A by using a pipette gun, subpackaging the mixture into a cryopreservation tube, marking, putting the cryopreservation tube into a programmed cooling box, putting the cryopreservation tube into a refrigerator at the temperature of-80 ℃, preserving the refrigerator for more than 4 hours, and transferring the refrigerator into a liquid nitrogen tank for preservation;
y4: recovering the frozen cells in the step Y3, sampling and recording the number of the cells for 0h, inoculating the cells into a culture plate, and adding a culture medium of the corresponding cells;
y5: adding the freezing medium B according to the volume of the culture medium in the culture plate, and then putting the culture plate into an incubator of the corresponding cells for culture;
y6: sampling and counting after 24 hours, and recording the cell viability and the cell number of 24 hours;
y7: after further 24h of culture, the cell viability and cell number were recorded for 48 h.
Preferably, the viable cell density in the step Y2 cell suspension is 1X 10 6 --1×10 8 one/mL.
The cell cryopreservation solution with definite components disclosed by the invention is simple and definite in components, free of serum components, high in safety, unique in preparation method, novel in application method, high in recovery survival rate of cryopreserved cells, and capable of being widely used for preservation of various mammalian cells. The frozen stock solution is used for freezing Mesenchymal Stem Cells (MSC) from various tissues, peripheral Blood Mononuclear Cells (PBMC), T cells, NK cells, chinese hamster ovary cells (CHO cells), african green monkey kidney cells (Vero cells), human embryonic kidney cells (293 cells), human chronic myelogenous leukemia cells (K562 cells) and other types of cells, the recovery survival rate is over 90 percent, the preservation period of the frozen stock solution of the cells is long (over 30 months), the activity of the cells is stable after long-term preservation, and the recovery survival rate of the frozen cells after 24 months is still over 85 percent.
The cell cryopreservation solution contains various salt ions, can effectively maintain the balance of key ions inside and outside cells, and maintain the stability of a pH environment, the poloxamer 188 is a cell membrane stabilizer, the antioxidant can remove free radicals and reduce stress injury in the cell cryopreservation process, the impermeable protective agent belongs to macromolecular substances, cannot penetrate cells, is combined with permeable protective components to form a hypertonic environment outside the cells, and reduces the number of cells dead due to solute damage, the ectoine and DMSO have strong water binding capacity and reduce the damage of ice crystals during cell cryopreservation, in addition, the ectoine is extracted from halophilic bacteria, so that the cell cryopreservation solution has strong capacity of resisting the external hostile environment, and the cell cryopreservation solution also plays a role.
The activity of the frozen cells after recovery is improved to the maximum extent by combining the components, the apoptosis inhibitor is a protein, a series of stress reactions caused by low temperature in the process of low-temperature preservation of the cells can cause the activation of apoptosis signal substances or pathways, and the apoptosis inhibitor can prevent the activation and transmission of the death signals.
Compared with the prior art, the invention has the following beneficial effects:
1) The frozen stock solution has simple components, only has more than ten components, has definite components, and all the components are known and common components;
2) The preparation method is unique, and the macromolecular non-permeable protective agent is dissolved under high pressure, so that the macromolecular non-permeable protective agent is dissolved more fully, the preparation process is smoother, and the problems of turbidity rise or difficulty in filtration increase caused by incomplete dissolution are solved;
3) The application method is novel, the frozen cell survival rate of the frozen stock solution A in the invention is no worse than the frozen stock solution on the market, but the frozen stock solution B is added after recovery, so the cell recovery survival rate is further improved;
4) The frozen cells are wide in range and free from influence on the efficacy after recovery, and the frozen cells can be even promoted to adhere to the wall by adding the frozen liquid B to certain adherent cells;
5) The components of the frozen stock solution are simple and clear, so that the frozen stock solution has long shelf life and the frozen cells have long preservation time.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph of growth of hUC-MSC cells cryopreserved in a cryopreservation solution at 48h after recovery (100-fold microscopic imaging) in example 1 of the present invention;
FIG. 2 is a graph of growth of hUC-MSC cells cryopreserved in control frozen stock solution 1 (conventional cell frozen stock solution) at 48h after recovery (100-fold microscopic imaging);
FIG. 3 is a graph of growth of hUC-MSC cells cryopreserved in control frozen stock solution 2 (commercial cell frozen stock solution with clear composition) at 48h after recovery (100-fold microscopic imaging);
FIG. 4 is a comparison graph of cell viability and viable cell count of hUC-MSC cells cryopreserved by the cryopreservation solution of the invention, the control group cryopreservation solution 1 (traditional cell cryopreservation solution) and the control group cryopreservation solution 2 (commercial cell cryopreservation solution with clear components) at 0h, 24h and 48h after recovery;
FIG. 5 is a graph showing the growth of frozen T cells in the frozen stock solution at 48h after recovery (100-fold microscopic imaging) according to example 1 of the present invention;
FIG. 6 is a graph of growth of T cells cryopreserved in control frozen stock solution 1 (conventional cell frozen stock solution) at 48h after recovery (100-fold microscopic imaging);
FIG. 7 is a graph showing the growth of T cells cryopreserved in control frozen stock solution 2 (cell frozen stock solution with a clear commercial composition) at 48h after recovery (100-fold microscopic imaging);
FIG. 8 is a graph showing the comparison between the cell viability rates and viable cell counts at 0h, 24h and 48h after recovery of T cells cryopreserved in the frozen stock solution of the present invention, control frozen stock solution 1 (conventional cell frozen stock solution) and control frozen stock solution 2 (commercial cell frozen stock solution with a clear composition).
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Umbilical cord mesenchymal stem cells (hUC-MSC) (ATCC, cat # PCS-500-010, lot # 70005074);
mesenchymal stem cell culture medium (manufacturer excellBio, cat # ME 000-C011);
digestive enzymes (manufacturer excellBio, cat # RF 000-N031);
t cells (self-extracted PBMC cells);
t cell culture medium (manufacturer excellBio, cat # TE 000-N041);
rock inhibitor Y-27632 (manufacturer abcam, cat # ab 120129);
caspase inhibitor z-VAD-fmk (manufacturer abcam, cat # ab 120382).
Example 1
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
The preparation method of the frozen stock solution A comprises the following steps: taking 1000mL as an example, the amount of the active ingredient,
s1: preparing a dextran 40 solution: weighing 45g of dextran 40 powder, adding injection water with the volume of 500mL, uniformly stirring, placing on a balance, weighing and recording the weight, then performing high-pressure dissolution, cooling to room temperature, weighing the weight again, and supplementing lost moisture according to the weight difference before and after high pressure;
s2: preparing a base solution: preparing 500mL of water for injection, and adding 1g of disodium hydrogen phosphate, 9g of sodium chloride, 0.2g of potassium chloride, 0.1g of calcium chloride and 0.05g of magnesium chloride in sequence for stirring and dissolving;
s3: adding 2g of poloxamer 188 into the base solution prepared in the step S2, adding 500mL of dextran 40 solution which is dissolved completely under high pressure and cooled in the step S1, then adding 2g of ectoin and 0.005g of vitamin E, finally adding 50mL of DMSO, and stirring until all components are dissolved completely and mixed uniformly to obtain an MIX solution;
s4: filtering the MIX solution obtained in the step S3 by using a filter with the diameter of 0.2-0.22 mu m to obtain a frozen stock solution A;
the preparation method of the frozen stock solution B comprises the following steps:
diluting Rock inhibitor Y-27632 in frozen stock solution B to 10mM concentration with DMSO, and using ultrasonic vibration to assist dissolving to prepare stock solution, subpackaging, and storing in a refrigerator at-80 deg.C.
The application method of the umbilical cord mesenchymal stem cells (hUC-MSC) is as follows:
y1: digesting umbilical cord mesenchymal stem cells (hUC-MSC) in an exponential growth phase, sampling and counting;
y2: according to the counting result, calculating 1 × 10 frozen stock 6 Taking the cell suspension volume of the cells in the tube into a 15mL centrifuge tube, and centrifuging;
y3: removing the centrifuged cell supernatant, adding 1mL of cryopreservation liquid A of the cell cryopreservation liquid, gently mixing the cells and the cryopreservation liquid A by using a pipette gun, subpackaging the mixture in a cryopreservation tube, marking, putting the mixture into a program cooling box, putting the box into a refrigerator at the temperature of-80 ℃, preserving for more than 4 hours, and transferring the box into a liquid nitrogen tank for preservation;
y4: resuscitating umbilical cord mesenchymal stem cells (hUC-MSC) cryopreserved by the cryopreserved solution A in the step Y3, adding 1mL of cryopreserved cell suspension into 9mL of mesenchymal stem cell culture medium, sampling and recording the number of cells for 0h, discarding supernatant after centrifugation, collecting the cells, adding 12mL of mesenchymal stem cell culture medium for resuspension of the cells, and inoculating the cells into 1 culture dish of 10cm for culture;
y5: adding Rock inhibitor Y-27632 into the culture dish to a final concentration of 2.5 mu M, shaking umbilical cord mesenchymal stem cells (hUC-MSC) in the culture dish uniformly, and culturing in a 5% carbon dioxide incubator at 37 ℃;
y6: digesting umbilical cord mesenchymal stem cells (hUC-MSC) by using digestive enzyme after 24 hours, sampling and counting, recording the cell viability and cell number of 24 hours, then centrifuging the cells, removing supernatant, re-suspending by using 10mL mesenchymal stem cell culture medium, and re-inoculating and placing into an incubator;
y7: after further culturing for 24h, three groups of umbilical cord mesenchymal stem cells (hUC-MSC) were digested with digestive enzymes, and cell viability and cell number were recorded for 48 h.
The application method on T cells is as follows:
z1: collecting T cells in logarithmic growth phase, sampling and counting;
z2: according to the counting result, 5X 10 frozen stocks are calculated 6 Taking the cell suspension volume of the cells in the tube into a 15mL centrifuge tube, and centrifuging;
z3: removing the centrifuged cell supernatant, adding the cryopreservation liquid A, gently mixing the T cells and the cryopreservation liquid A uniformly by using a pipette, subpackaging the mixture into a cryopreservation tube, marking, putting the cryopreservation tube into a programmed cooling box, putting the cryopreservation tube into a refrigerator at the temperature of-80 ℃, preserving for more than 4 hours, and transferring the cryopreservation tube into a liquid nitrogen tank for preservation;
z4: recovering the T cells frozen in the frozen stock solution A in the step Z3, sampling and recording the number of the cells for 0h, inoculating the cells into 1T 25 culture bottle, and adding 6mL of T cell culture medium for culture;
z5: adding Rock inhibitor Y-27632 into a T25 culture bottle to a final concentration of 2.5 μ M, and culturing T cells in a 5% carbon dioxide incubator at 37 deg.C;
z6: after 24h, gently blowing the T cells in the culture bottle by using a pipette, sampling and counting, and recording the cell viability and cell number for 24 h;
z7: after further incubation for 24h, the T cells in the flask were gently pipetted off, samples were counted, and cell viability and cell number were recorded for 48 h.
The methods of preparation and use of examples 2-16 below refer to example 1.
Example 2
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 0.1g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 3
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 10g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 4
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 10g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 5
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 100g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 6
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 0.005g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 7
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 10g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 8
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 10%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 9
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of hydroxyethyl starch is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 10
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 70 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 11
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin C is 0.1g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 12
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of carnosine is 0.02g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 13
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 40 is 45g/L, the addition amount of vitamin E is 0.005g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Caspase inhibitor z-VAD-fmk is 10 mu M.
Example 14
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of hydroxyethyl starch is 45g/L, the addition amount of vitamin C is 0.1g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Example 15
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of hydroxyethyl starch is 45g/L, the addition amount of vitamin C is 0.1g/L, the addition amount of ectoine is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Caspase inhibitor z-VAD-fmk is 10 mu M.
Example 16
The addition amount of disodium hydrogen phosphate is 1g/L, the addition amount of sodium chloride is 9g/L, the addition amount of potassium chloride is 0.2g/L, the addition amount of calcium chloride is 0.1g/L, the addition amount of magnesium chloride is 0.05g/L, the addition amount of poloxamer 188 is 2g/L, the addition amount of dextran 70 is 45g/L, the addition amount of carnosine is 0.02g/L, the addition amount of ectoin is 2g/L, the addition amount of DMSO is 5%, and the final concentration of Rock inhibitor Y-27632 is 2.5 mu M.
Control group
Preparation of control frozen stock solution 1: the frozen stock solution comprises the following components in percentage by volume: 10% of dimethyl sulfoxide and 90% of fetal calf serum (namely a traditional cell freezing medium);
control frozen stock solution 2: the cryopreservation solution is a commercially available STEMCELL brand cell cryopreservation solution (CryoStor CS 10).
Comparative example 1
Compared with the example 1, after the cells are frozen by the frozen stock solution A, the Rock inhibitor Y-27632 is not added into the culture bottle during recovery, and the distribution ratio, the preparation steps and the application method of the other components are the same as those in the example 1.
Comparative example 2
Compared with the embodiment 1, the preparation method is that the Rock inhibitor Y-27632 is directly added into the frozen stock solution A to the final concentration of 2.5 mu M after the preparation of the frozen stock solution A is finished, the Rock inhibitor Y-27632 is not added after the cells are recovered, and the distribution ratio, the preparation steps and the application method of the other components are the same as those in the embodiment 1.
Comparative example 3
Compared with the example 1, the dextran 40 is not added in the fine freezing solution A during preparation, namely the addition amount of the dextran 40 is 0g/L, and the distribution ratio, the preparation steps and the application method of the rest components are the same as those in the example 1.
Comparative example 4
Compared with the example 1, the dextran 40 is not dissolved in advance under high pressure when the frozen stock solution A is prepared, but 45g of powder is directly weighed and added into injection water for dissolution, and the proportion, the preparation steps and the application method of the other components are the same as those in the example 1.
Comparative example 5
Compared with the example 1, the frozen stock solution A is prepared by adding 11g/L of poloxamer 188, and the proportion of the other components, the preparation steps and the application method are the same as those in the example 1.
Comparative example 6
Compared with the example 1, the addition amount of the vitamin E in the frozen stock solution A during preparation is 0.06g/L, and the distribution ratio, the preparation steps and the application method of the other components are the same as those in the example 1.
Comparative example 7
Compared with the example 1, the addition amount of ectoin in the frozen stock solution A during preparation is 11g/L, and the distribution ratio, preparation steps and application method of the other components are the same as those in the example 1.
Comparative example 8
Compared with example 1, the frozen stock solution A is not added with ectoin during preparation, namely the addition amount of ectoin is 0g/L, and the distribution ratio, preparation steps and application method of the rest components are the same as those of example 1.
Experiment 1
According to the preparation and application methods of the frozen stock solutions in examples 1-16, control frozen stock solution 1 and control frozen stock solution 2 and comparative examples 1-8, the freezing and thawing work of the frozen stock solutions in all groups on umbilical mesenchymal stem cells (hUC-MSC) is carried out simultaneously, the freezing and thawing work of the frozen stock solutions in liquid nitrogen tanks is carried out simultaneously for 1 month, 6 months, 12 months and 24 months, the cell viability rates are recorded, and the results are shown in the following table:
| group of | 1 month | 6 months old | 12 months old | 24 months |
| Example 1 | 99.6% | 98.9% | 96.4% | 94.2% |
| Example 2 | 95.3% | 94.5% | 90.1% | 86.6% |
| Example 3 | 92.6% | 89.7% | 85.3% | 81.6% |
| Example 4 | 92.1% | 90.3% | 87.3% | 83.3% |
| Example 5 | 93.7% | 92.5% | 88.8% | 84.9% |
| Example 6 | 96.4% | 94.2% | 92.2% | 90.7% |
| Example 7 | 93.8% | 90.4% | 87.0% | 80.5% |
| Example 8 | 97.3% | 95.8% | 93.4% | 90.0% |
| Example 9 | 96.9% | 95.2% | 91.7% | 88.6% |
| Example 10 | 97.8% | 96.6% | 93.1% | 91.3% |
| Example 11 | 95.7% | 92.2% | 90.4% | 89.8% |
| Example 12 | 96.2% | 93.4% | 92.1% | 90.2% |
| Example 13 | 97.3% | 96.9% | 94.0% | 92.9% |
| Example 14 | 96.5% | 92.3% | 90.1% | 87.3% |
| Example 15 | 94.2% | 91.3% | 88.5% | 84.8% |
| Example 16 | 94.0% | 90.7% | 86.8% | 83.2% |
| Control frozen stock solution 1 | 97.1% | 95.4% | 92.4% | 90.3% |
| Control frozen stock solution 2 | 94.3% | 92.9% | 87.8% | 82.4% |
| Comparative example 1 | 96.7% | 94.9% | 91.8% | 89.6% |
| Comparative example 2 | 96.4% | 95.2% | 91.3% | 90.1% |
| Comparative example 3 | 83.3% | 76.0% | 68.4% | 50.2% |
| Comparative example 4 | 91.0% | 86.5% | 80.1% | 74.5% |
| Comparative example 5 | 91.2% | 88.3% | 84.1% | 78.5% |
| Comparative example 6 | 92.8% | 90.7% | 85.2% | 80.5% |
| Comparative example 7 | 91.1% | 87.5% | 84.6% | 80.1% |
| Comparative example 8 | 85.1% | 79.0% | 70.5% | 62.3% |
Experiment 2
According to the preparation and application methods of the frozen stock solutions in the examples 1-16, the control frozen stock solution 1 and the control frozen stock solution 2 and the frozen stock solutions in the comparative examples 1-8, the freezing and recovery work of the frozen stock solutions of all groups on T cells is simultaneously carried out, the freezing and recovery work of the frozen stock solutions of all groups in a liquid nitrogen tank is simultaneously carried out for 1 month, 6 months, 12 months and 24 months, the cell viability rate is recorded, and the results are shown in the following table:
| group of | 1 month | 6 months old | 12 months old | 24 months |
| Example 1 | 93.7% | 92.1% | 90.9% | 87.8% |
| Example 2 | 87.6% | 85.9% | 82.1% | 78.6% |
| Example 3 | 83.4% | 81.5% | 77.3% | 70.3% |
| Example 4 | 78.9% | 76.4% | 68.7% | 60.6% |
| Example 5 | 84.8% | 83.3% | 80.5% | 75.2% |
| Example 6 | 89.9% | 88.8% | 87.5% | 83.6% |
| Example 7 | 80.8% | 79.4% | 75.3% | 68.9% |
| Example 8 | 90.3% | 89.8% | 86.4% | 82.0% |
| Example 9 | 90.6% | 88.9% | 86.8% | 81.4% |
| Example 10 | 91.7% | 90.7% | 87.8% | 83.2% |
| Example 11 | 89.3% | 87.5% | 84.1% | 79.0% |
| Example 12 | 89.7% | 88.7% | 85.9% | 81.3% |
| Example 13 | 92.5% | 91.2% | 88.0% | 85.8% |
| Example 14 | 88.6% | 86.3% | 81.7% | 76.5% |
| Example 15 | 89.1% | 87.8% | 82.4% | 80.6% |
| Example 16 | 90.3% | 89.4% | 88.6% | 83.7% |
| Control frozen stock solution 1 | 88.2% | 85.9% | 80.4% | 75.9% |
| Control frozen stock solution 2 | 85.1% | 83.6% | 75.7% | 68.3% |
| Comparative example 1 | 89.4% | 86.2% | 82.3% | 78.5% |
| Comparative example 2 | 89.9% | 85.2% | 83.8% | 79.4% |
| Comparative example 3 | 73.3% | 70.1% | 65.6% | 58.7% |
| Comparative example 4 | 89.0% | 88.7% | 85.2% | 80.3% |
| Comparative example 5 | 83.3% | 80.0% | 72.6% | 64.8% |
| Comparative example 6 | 84.5% | 82.2% | 79.4% | 70.8% |
| Comparative example 7 | 80.6% | 78.9% | 70.5% | 62.6% |
| Comparative example 8 | 74.5% | 71.2% | 66.8% | 59.4% |
Results and analysis
From the data in the table above, it is clear that the following conclusions can be drawn:
compared with a comparative example, in the experiment 1 and the experiment 2, by utilizing the cryopreservation solution provided by the invention, a better preservation effect is obtained through a unique formula combination design, the survival rate of the preserved cells is obviously improved, the survival rate of the cells after being preserved for 24 months is still more than 90% on umbilical cord mesenchymal stem cells (hUC-MSC), and the survival rate on T cells is still more than 85%, and is obviously superior to that of the cryopreservation solution of a control group.
The cell cryopreservation effect is closely related to the component design, the dosage concentration and the component interaction of the cryopreservation solution, and for example, under the condition of the component combination and the dosage concentration of the embodiment 1, the cell preservation with higher efficiency is realized; examples 2 to 8 respectively carry out dosage adjustment tests within application ranges on various concentrations of a single material, examples 9 to 16 respectively carry out adjustment within application ranges on single material or combination and collocation of a plurality of materials, comparative examples 3 to 8 respectively carry out dosage tests beyond ranges on various materials, compared with example 1, under the conditions of different dosage concentrations and interaction of different components, the fluctuation of the generated cell preservation effect is larger, the large dosage or overdose use of individual materials greatly reduces the cell cryopreservation effect, and the importance of the composition combination design and dosage concentration of the cryopreservation liquid is further verified.
In addition, comparative examples 1-2, in which the order of adding the frozen stock solution A and the frozen stock solution B of the present invention was adjusted, will be described in more detail, in which the cell viability rate of the frozen stock solution A of the present invention was no less than the freezing effect of the frozen stock solution on the market, but the cell recovery viability rate was further improved by adding the frozen stock solution B after recovery.
The results shown in the attached figures 1-3 show that the hUC-MSC frozen by the cell freezing solution has normal adherence capability and can well maintain the original form.
The results in FIG. 4 show that hUC-MSC cells cryopreserved by using the cell cryopreserving liquid of the present invention show higher survival rate and faster proliferation rate.
The results of FIGS. 5-7 show that T cells cryopreserved using the cell cryopreserving solution of the present invention maintain their original growth pattern (clump growth) after recovery.
The results in FIG. 8 show that T cells cryopreserved using the cell cryopreserving solution of the present invention exhibit higher survival rates and faster proliferation rates.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Furthermore, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process method article or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process method article or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A cell freezing solution is characterized by consisting of a freezing solution A and a freezing solution B;
the freezing solution A consists of disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, poloxamer 188, an impermeable protective agent, ectoin, an antioxidant and DMSO;
the frozen stock solution B consists of an apoptosis inhibitor;
the addition amount of the disodium hydrogen phosphate is 1g/L, the addition amount of the sodium chloride is 9g/L, the addition amount of the potassium chloride is 0.2g/L, the addition amount of the calcium chloride is 0.1g/L, the addition amount of the magnesium chloride is 0.05g/L, the addition amount of the poloxamer 188 is 0.1-10g/L, the addition amount of the impermeable protective agent is 10-100g/L, the addition amount of the ectoine is 0.005-10g/L, and the addition amount of the DMSO is 5-10%.
2. The cell cryopreservation solution of claim 1, wherein the poloxamer 188 is added in an amount of 2g/L, the impermeable protectant is added in an amount of 45g/L, the ectoine is added in an amount of 2g/L, and the DMSO is added in an amount of 5%.
3. The cell cryopreservation solution of claim 1, wherein the impermeable protectant is any one of hydroxyethyl starch, dextran 40 and dextran 70.
4. The cell cryopreservation solution of claim 1 wherein the antioxidant is any one of vitamin E, vitamin C and carnosine.
5. The cell cryopreservation solution of claim 4, wherein the antioxidant comprises 0.005g/L vitamin E, 0.1g/L vitamin C, and 0.02g/L carnosine.
6. The cell cryopreservation solution of claim 1 wherein the apoptosis inhibitor is any one of Rock inhibitor Y-27632 and Caspase inhibitor z-VAD-fmk.
7. The cell culture medium of claim 6, wherein the final concentration of Rock inhibitor Y-27632 is 2.5 μ M and the final concentration of Caspase inhibitor z-VAD-fmk is 10 μ M.
8. The method of preparing a cell cryopreservation solution according to claim 1,
the preparation process of the freezing solution A is as follows:
s1: preparation of the impermeable protective agent: weighing the non-permeable protective agent, adding a certain volume of injection water, dissolving under high pressure, and cooling to room temperature;
s2: preparing a base solution: preparing water for injection, and sequentially adding disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride and magnesium chloride into the water for injection to be stirred and dissolved;
s3: sequentially adding poloxamer 188 and the cooled impermeable protective agent which is dissolved completely under high pressure in the step S1 into the base solution prepared in the step S2, then adding the ectoin and the antioxidant, finally adding DMSO, and stirring until all components are dissolved completely and mixed uniformly to obtain an MIX solution;
s4: filtering the MIX solution obtained in the step S3 by using a filter with the diameter of 0.2-0.22 mu m to obtain a frozen stock solution A;
the preparation method of the frozen stock solution B comprises the following steps:
diluting the apoptosis inhibitor in the frozen stock solution B with DMSO, performing ultrasonic assisted dissolution to obtain stock solution, packaging, and storing in a refrigerator at-80 deg.C.
9. The method of claim 1, wherein the frozen stock solution B is added to a cell culture medium after the cells are thawed.
10. The use of the cell cryopreservation solution of claim 1, wherein the cell cryopreservation solution is used for cryopreservation of mammalian cells.
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| CN118028238A (en) * | 2024-04-09 | 2024-05-14 | 四川省肿瘤医院 | Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method |
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| US20160135446A1 (en) * | 2013-06-13 | 2016-05-19 | Biomatrica, Inc. | Cell stabilization |
| CN114007416A (en) * | 2019-04-30 | 2022-02-01 | 明尼苏达大学董事会 | Compositions and methods for cryopreservation of cells |
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| US20160135446A1 (en) * | 2013-06-13 | 2016-05-19 | Biomatrica, Inc. | Cell stabilization |
| CN114007416A (en) * | 2019-04-30 | 2022-02-01 | 明尼苏达大学董事会 | Compositions and methods for cryopreservation of cells |
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| CN117178980A (en) * | 2023-09-08 | 2023-12-08 | 苏州依科赛生物科技股份有限公司 | Immune cell low-temperature frozen stock solution and application thereof |
| CN117178980B (en) * | 2023-09-08 | 2024-04-16 | 苏州依科赛生物科技股份有限公司 | Immune cell low-temperature frozen stock solution and application thereof |
| CN118028238A (en) * | 2024-04-09 | 2024-05-14 | 四川省肿瘤医院 | Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method |
| CN118028238B (en) * | 2024-04-09 | 2024-06-11 | 四川省肿瘤医院 | Human colorectal cancer organoid resuscitation liquid, culture liquid and resuscitation method |
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