CN115349515A - Cell preservation solution and cell preservation method - Google Patents
Cell preservation solution and cell preservation method Download PDFInfo
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- CN115349515A CN115349515A CN202210922177.1A CN202210922177A CN115349515A CN 115349515 A CN115349515 A CN 115349515A CN 202210922177 A CN202210922177 A CN 202210922177A CN 115349515 A CN115349515 A CN 115349515A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
An embodiment of the present invention relates to a cell preservation solution, which includes: 20mM to 50mM sucrose; 20mM to 50mM mannitol; 10 mM-50 mM lactobionic acid; 5 mM-20 mM glucose; 1 mM-3 mM adenosine; 1 mM-5 mM reduced glutathione; 1 mM-2 mM dextran-40; 10mM to 30mM potassium dihydrogen phosphate; 10 mM-30 mM potassium carbonate; 10 mM-30 mM potassium chloride; 5mM to 30mM sodium chloride; 10 mM-60 mM sodium hydroxide; 10 mM-60 mM potassium hydroxide; 0mM to 20mM vitamin E;0 mM-25mM 4-hydroxyethyl piperazine ethanesulfonic acid; and 0 to 10 percent of dimethyl sulfoxide. In another aspect, embodiments of the present invention relate to a method for preserving cells using a cell preservation solution described herein.
Description
Technical Field
The invention relates to the field of modern regenerative medicine, in particular to a cell preservation solution and a cell preservation method.
Background
Modern regenerative medicine, such as gene therapy, tumor cell therapy, novel antibody therapy, stem cell regeneration therapy and the like, is a newly developed medical field, and is characterized in that ex vivo living cells are modified to prepare therapeutic cells, and then the therapeutic cells are back-transfused/transplanted into a patient body to achieve the purpose of treatment. In general, ex vivo living cells must be cryopreserved, to ensure that viability and functionality of the cells are maintained during storage, transport. In other words, the technology of cryopreservation and cryopreservation of cells is an essential part of modern regenerative medicine.
The development of cryopreservation and cryopreservation techniques for cells is based on the intensive research and understanding of cryobiology of cells. In order to store cells efficiently by cryopreservation and freezing, the damage of cryopreservation and freezing to the cells needs to be avoided to the greatest extent according to the low-temperature biological characteristics of the cells, and the cell viability and functionality are kept. Specifically, at normal physiological temperature, intracellular fluid and extracellular fluid are in an isotonic state, and cellular metabolism is active, so that oxygen and nutrients are consumed, and a large amount of generated free radicals and peroxides are removed to maintain a normal physiological state. However, in the cryopreserved and frozen state, cells may suffer different degrees of damage, affecting normal physiological activities and even death. The damage caused to cells by cryopreservation, freezing, etc. can be manifold, such as osmotic damage, mechanical damage, chemical damage, etc. For example, during cold storage and freezing temperature reduction, since the extracellular interstitial environment may first form ice crystals, interstitial fluid is concentrated, osmotic pressure is increased, water in cells passes through cell membranes, exudation is performed, electrolyte concentration in cells is increased, pH change is caused, protein denaturation, lysosome destruction, membrane protein function loss, membrane leakage rupture and the like are caused, and finally cell death is caused. Meanwhile, further cooling, ice crystals may be generated in cells, and the ice crystals may pierce and press cell membranes, organelles and the like, so that the cell membranes are mechanically damaged and die. On the other hand, a large amount of free radicals and peroxides in cells can induce apoptosis under the low-temperature refrigeration and freezing environment.
The existing cell preservation solution and cell preservation method can prevent the damage of cryopreservation and freezing to cells to a certain extent, but have the defects. For example, when the conventional cell preservation solution and cell preservation method are clinically applied, there may be a risk of adverse reactions to the human body, a risk of distortion of intracellular deoxyribonucleic acid (DNA) and protein denaturation, and a possibility of an adverse reaction of the human body to the cells after preservation. In addition, some components of the existing cell preservation solution are limited in source and expensive, and have potential risks of contamination by other microorganisms such as viruses and the like, and side effects such as hypersensitiveness to human bodies.
Disclosure of Invention
The present invention aims to provide an improved cell preservation solution and a cell preservation method.
An aspect of an embodiment of the present invention relates to a cell preservation solution, which includes: 20mM to 50mM sucrose; 20mM to 50mM mannitol; 10mM to 50mM lactobionic acid; 5mM to 20mM glucose; 1mM to 3mM adenosine; 1 mM-5 mM reduced glutathione; 1 mM-2 mM dextran-40; 10mM to 30mM potassium dihydrogen phosphate; 10 mM-30 mM potassium carbonate; 10 mM-30 mM potassium chloride; 5mM to 30mM sodium chloride; 10 mM-60 mM sodium hydroxide; 10 mM-60 mM potassium hydroxide; 0mM to 20mM vitamin E;0 mM-25mM 4-hydroxyethyl piperazine ethanesulfonic acid; and 0 to 10 percent of dimethyl sulfoxide.
In some embodiments, the cell preservation solution comprises 0.5mM to 20mM vitamin E and 0% dimethyl sulfoxide.
In some embodiments, the cell preservation solution comprises 2mM to 5mM vitamin E.
In some embodiments, the cell preservation solution comprises 0mM to 20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
In some embodiments, the cell preservation solution comprises 0mM vitamin E and >0% dimethyl sulfoxide.
In some embodiments, the cell preservation solution comprises 0mM to 20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
In some embodiments, the cell preservation solution comprises 5% to 10% dimethylsulfoxide.
In some embodiments, the pH of the cell preservation solution is in the range of 7.0 to 8.0.
In some embodiments, the cell preservation fluid has an osmolality of 300 to 400 milliosmoles (mosm).
In some embodiments, the cell preservation solution comprises water.
Another aspect of embodiments of the present invention relates to a method of preserving cells using a cell preservation solution described herein.
In some embodiments, the cell preservation method preserves the cells at 2 ℃ to 8 ℃ or-80 ℃ to-196 ℃.
In some embodiments, the cells comprise one or more of stem cells, immune cells, tumor cells.
In some embodiments, the cells comprise human colon cancer cells.
The technical solutions of the embodiments in the present application may be arbitrarily combined as the technical conditions allow.
The present application will be further described with reference to the accompanying drawings.
Drawings
FIG. 1 is a graph showing experimental data on the metabolic activity of cells obtained in Experimental example 5.
FIG. 2 is a graph showing experimental data on cell proliferation activity obtained in Experimental example 5.
FIG. 3 is a graph showing experimental data on metabolic activity of cells obtained in Experimental example 10.
FIG. 4 is a graph showing experimental data on cell proliferation activity obtained in Experimental example 10.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the invention will be understood to cover all modifications and variations of this invention provided they come within the scope of the appended claims.
An embodiment of the present invention relates to a cell preservation solution, which includes: 20mM to 50mM sucrose; 20mM to 50mM mannitol; 10 mM-50 mM lactobionic acid; 5mM to 20mM glucose; 1 mM-3 mM adenosine; 1 mM-5 mM reduced glutathione; 1 mM-2 mM dextran-40; 10 mM-30 mM potassium dihydrogen phosphate; 10 mM-30 mM potassium carbonate; 10 mM-30 mM potassium chloride; 5mM to 30mM sodium chloride; 10 mM-60 mM sodium hydroxide; 10 mM-60 mM potassium hydroxide; 0mM to 20mM vitamin E;0 mM-25mM 4-hydroxyethyl piperazine ethanesulfonic acid; and 0 to 10 percent of dimethyl sulfoxide.
The cell preservation solution provided by the embodiment of the invention has the advantages of clear components, stable performance, safety and reliability, and can be directly used for human bodies. The cell preservation solution provided by the embodiment of the invention has a low content of dimethyl sulfoxide (DMSO) or 0, is not easy to cause adverse reaction to a human body when being applied to clinic, is not easy to cause distortion and protein denaturation of deoxyribonucleic acid (DNA) in cells, and has a low possibility of causing uncomfortable reaction of the human body to the preserved cells. In addition, the cell preservation solution provided by the embodiment of the invention has wide component sources and low price, does not contain exogenous animal serum, exogenous protein and the like, is not easy to have potential risks of virus and other microbial pollution, and is not easy to cause side reactions such as over-sensitivity to human bodies.
In the embodiments of the present invention, unless otherwise specifically indicated, numerical values may include errors such as a metering error, an accuracy error, a measurement error, and the like, for example, an error within a range of plus or minus 5%. For example, 10% may comprise values in the range of 10% × (1 ± 5%), i.e. values in the interval 9.5% to 10.5%.
In embodiments of the present invention, unless specifically indicated otherwise, a numerical range may include any subrange therein, e.g., 0% to 10% may include 5% to 10%,0% to 5%,3% to 8%, etc.
The sucrose can be used as an impermeable extracellular cryoprotectant to protect cell membranes from the effects of cold shock. The sucrose may also be an energy source for cellular metabolism during cellular storage.
The mannitol can be used as an extracellular impermeable substance to improve extracellular osmotic pressure and can also be used as an effective hydroxide radical scavenger.
The lactobionic acid may act as an impermeable anion, and may be used to counteract cell swelling during cold temperatures. The lactobionic acid may be biocompatible, may be a strong chelator of calcium and iron, and may help to reduce cell damage due to calcium influx and free radical formation.
The glucose can be the main energy source for cell metabolism during cell storage, and carbon dioxide and water are generated in mitochondria through anaerobic glycolysis pathways or aerobic and tricarboxylic acid cycles, adenosine Triphosphate (ATP) is released, and energy is provided for cell life activities.
The adenosine can be a substrate for Adenosine Triphosphate (ATP) regeneration within the mitochondria of cells and can be involved in providing energy for cell life activities.
The reduced glutathione can be an important antioxidant and hydroxyl radical scavenger in cells, protects sulfydryl in molecules such as proteins and enzymes in the cells, and protects the damage of radicals to cell membranes. The reduced glutathione can also be a cofactor of glutathione peroxidase, and can promote the metabolism of lipid peroxide and hydrogen peroxide.
The dextran-40 can be an impermeable extracellular cryoprotectant, and can offset colloid osmotic pressure generated by proteins and other metabolites with negative charges in cells at low temperature, so that the cells are prevented from being cracked due to water absorption expansion.
The vitamin E can be an antioxidant, and can scavenge intracellular oxygen free radicals at low temperature, thereby preventing damage to cell membrane by free radicals. The vitamin E may also inhibit nitric oxide release. The vitamin E may also protect cells from freeze-induced apoptosis.
The 4-hydroxyethyl piperazine ethanesulfonic Acid (HEPES) can be a large Sulfonic Acid biological pH liquid, has excellent buffering capacity at low temperature, prevents acidosis, does not permeate into cells, and helps prevent osmotic swelling.
The dimethyl sulfoxide (DMSO) can be a permeable cell protective agent, can quickly penetrate a cell membrane to enter a cell, lowers the freezing point, delays the freezing storage process, improves the ion concentration in the cell, and reduces the formation of ice crystals in the cell, thereby reducing the cell damage. The content and concentration percentage of the dimethyl sulfoxide may be volume percentage, that is, 0% to 10% may mean that the number of ml of the dimethyl sulfoxide in 100ml of the cell preservation solution is 0 to 10.
The potassium dihydrogen phosphate, the potassium carbonate, the potassium chloride, the sodium hydroxide and the potassium hydroxide can be electrolytes respectively, and the pH level in cells and the sodium-potassium ion pump of the cells can be maintained to be effective at low temperature.
The cell preservation solution of the embodiment of the present application may be used for short-term cryopreservation of cells at a low temperature of, for example, 2 to 8 ℃ to maintain cell viability and functionality, or for long-term freezing of cells at a deep low temperature of, for example, -80 to-196 ℃ to maintain cell viability and functionality.
In some embodiments, the cell preservation solution comprises 0.5mM to 20mM vitamin E and 0% dimethyl sulfoxide.
Thus, it is possible to contribute to the preservation of the cell viability and functionality of the cell preservation solution during the preservation of cells at, for example, 2 ℃ to 8 ℃ under cryopreservation for up to, for example, 5 days, 7 days, and more. Moreover, the content of dimethyl sulfoxide (DMSO) in the cell preservation solution is 0, so that when the cell preservation solution is applied to clinical application, the risk of adverse reaction on a human body is not easy to exist, intracellular deoxyribonucleic acid (DNA) distortion and protein denaturation are not easy to cause, and the possibility of uncomfortable reaction of the human body on the preserved cells is low.
In some embodiments, the cell preservation solution comprises 2mM to 5mM vitamin E.
Thus, the cell preservation solution can be beneficial to scavenging oxygen free radicals in cells when the cells are preserved at low temperature of 2-8 ℃, so as to prevent the damage of the free radicals to cell membranes, inhibit the release of nitric oxide and protect the cells from cell apoptosis induced by freezing.
In some embodiments, the cell preservation solution comprises 0mM to 20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
Thus, the cell preservation solution can contribute to the preservation of cells at a low temperature of, for example, 2 ℃ to 8 ℃ and can exhibit an excellent buffering capacity to prevent acidosis, and can contribute to the prevention of osmotic swelling without permeating into the cells.
In some embodiments, the cell preservation solution comprises 0mM vitamin E and >0% dimethyl sulfoxide.
Thus, the cell preservation solution can rapidly penetrate cell membranes into cells to reduce the freezing point, delay the cryopreservation process, simultaneously improve the ion concentration in the cells and reduce the formation of ice crystals in the cells, thereby reducing cell damage and maintaining the cell survival rate and functionality in the process of cryopreservation of the cells at the deep low temperature of-80 ℃ to-196 ℃ for months, years, decades and more.
In some embodiments, the cell preservation solution comprises 0mM to 20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
Thus, the cell-preserving fluid can contribute to the preservation of cells at cryogenic temperatures, for example, from-80 ℃ to-196 ℃, and can have an excellent buffering capacity to prevent acidosis, and can contribute to the prevention of osmotic swelling without permeating into the cells.
In some embodiments, the cell preservation solution comprises 5% to 10% dimethylsulfoxide.
Therefore, the cell preservation solution can rapidly penetrate cell membranes to enter cells when the cells are preserved by deep low temperature freezing at-80 ℃ to-196 ℃, so that the freezing point is lowered, the cryopreservation process is delayed, the ion concentration in the cells is increased, the formation of ice crystals in the cells is reduced, and the cell damage is reduced.
In some embodiments, the pH of the cell preservation solution is in the range of 7.0 to 8.0.
Thus, the cell preservation solution can provide a physiological environment suitable for cells.
In some embodiments, the cell preservation fluid has an osmolality of 300 to 400 milliosmoles (mosm).
Thus, the cell preservation solution can provide a physiological environment suitable for cells. The osmotic pressure may be the sum of the number of moles of each component in the cell preservation solution.
In some embodiments, the cell preservation solution comprises water.
Thus, the cell preservation solution can provide a physiological environment suitable for cells. The cell-preserving fluid may be a mixture of components other than water in water.
Another aspect of embodiments of the present invention relates to a method of preserving cells using a cell preservation solution described herein.
The cell preservation solution used by the cell preservation method provided by the embodiment of the invention has the advantages of definite components, stable performance, safety and reliability, and can be directly used for human bodies. The cell preservation solution used by the cell preservation method provided by the embodiment of the invention has a low content of dimethyl sulfoxide (DMSO) or 0, so that when the cell preservation solution is clinically applied, the risk of adverse reaction on a human body is not easily caused, the distortion and protein denaturation of deoxyribonucleic acid (DNA) in cells are not easily caused, and the possibility of uncomfortable reaction of the human body on the preserved cells is low. In addition, the cell preservation solution used in the cell preservation method according to the embodiment of the invention has wide component sources and low price, does not contain exogenous animal serum, is not easy to have potential risks of being polluted by other microorganisms such as viruses and the like, and is not easy to cause side reactions such as over-sensitivity to human bodies.
In some embodiments, the cell preservation method preserves the cells at 2 ℃ to 8 ℃ or-80 ℃ to-196 ℃.
Thus, it is advantageous to ensure that the viability and functionality of the cells are maintained during storage and transportation for a relatively short period of time, or for a relatively long period of time. For example, the cells may be stored using the cell storage method at 2 ℃ to 8 ℃ for 5 days, 7 days, or longer. Alternatively, the cells may be cryopreserved at-80 ℃ to-196 ℃ for months, years, decades, or longer using the cell preservation method.
In some embodiments, the cell preservation method may include, after centrifugation and collection of the cells to be preserved, resuspending the cells in the cell preservation solution to achieve a cell concentration of, for example, 2X 10 6 /ml～1×10 7 And/ml, storing in a refrigerator at 2-8 deg.c for 5 days, 7 days or longer.
In some embodiments, the cell preservation method may include culturing the cells to be preserved in a culture dish to a desired number, sucking out the cell culture fluid, adding the cell preservation fluid, and storing in a refrigerator at 2-8 ℃ for 5 days, 7 days, or more.
In some embodiments, the cell preservation method may comprise, after collecting the cells to be preserved by centrifugation, resuspending the cells in the cell preservation solution to a cell concentration of 2X 10 6 /ml～1×10 7 Transferring into freezing tube, and placingCooling at-80 deg.C at a cooling rate of about 1 deg.C/min, and transferring into liquid nitrogen (-196 deg.C) for long-term storage overnight. The storage time can reach decades.
In some embodiments, the cells comprise one or more of stem cells, immune cells, tumor cells.
In this way, it may help to ensure that one or more of the stem cells, the immune cells, the tumor cells remain active and functional during storage, transport.
In some embodiments, the cells comprise human colon cancer cells.
In this way, it can help to ensure that the human colon cancer cells remain viable and functional during storage, transport.
The cell preservation method and the method for rewarming the cryopreserved cell and the cryopreserved cell preservation solution according to the embodiment of the present application may include: after the cells which are refrigerated and frozen are collected by centrifugation, the cell preservation solution is sucked, the cells are resuspended by using the cell culture solution, and the cells are continuously cultured.
The method for detecting a function of a cell stored by the method for storing a cell and the method for detecting a function of a cell stored by cold storage or freeze storage of a cell storage solution according to an embodiment of the present application may include: the preserved cells are cultured for 1 day at a temperature below that of the original cells, and then incubated with, for example, ALAMAR BLUE TM The cell activity detection reagent detects the cell metabolic activity. In 1 day after cell rewarming, hysteresis apoptosis phenomenon may occur, and cells tend to be stable after 1 day, so that the cell activity can be more truly reflected by detection after 1 day.
When the cells preserved by the cell preservation method and the cell preservation solution in cold storage and freeze preservation need to be clinically used, the cells can be directly returned to the body after being resuspended.
The experimental examples in this application are mainly used to help understanding the embodiments of the present invention, and are not intended to limit the scope of the claims. As can be seen by referring to the experimental examples described later, the cell preservation solution of the embodiment of the application has the advantages of simple formula, low cost and performance superior to that of the commercially available products.
Experimental examples 1 to 4
Sucrose, mannitol, lactobionic acid, glucose, adenosine, reduced glutathione, dextran-40, potassium dihydrogen phosphate, potassium carbonate, potassium chloride, sodium chloride, potassium hydroxide, vitamin E, 4-hydroxyethylpiperazine ethanesulfonic acid (4- (2-hydroxyethaneyl) piperazine-1-ethanesulfonic acid, HEPES) were weighed according to the components and concentrations of the cell preservation solutions corresponding to experimental examples 1-4 shown in table 1 below, and prepared into 4 mixed solutions with purified water, each solution was subjected to pH titration with sodium hydroxide until 7.6,0.2um sterile filtration with sterile filtration membranes, and the filtrate obtained by filtration was the 4 cell preservation solutions of experimental examples 1-4.
TABLE 1
Experimental example 5
To be compared with those commercially available
Preservation solution quality control of the cell preservation solutions of experimental examples 1-4 was performed by a comparative method with respect to the standard.
Specifically, a test cell line of human colon cancer cells (HCT 116 WT) was cultured in a petri dish, digested with trypsin solution, the cell suspension was collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cell preservation solution samples of test examples 1 to 4 and the supernatant were used, respectively
Resuspend cells in stock solution samples (control) at 2X 10 6 The cell concentration of the tube is divided into sterile freezing tubes, and the tubes are placed in a refrigerator at 4 ℃ for cold storage for 5 days.
Then, a portion of the frozen tube was removed, the cells were collected by centrifugation, the preservation solution was aspirated, the cells were resuspended in cell culture medium, and the percentage of viable cells was morphologically measured using a Countess 3 automatic cytometer, and the results are shown in Table 2 below. It can be seen that the survival rate of the cells preserved using the cell preservation solution samples of experimental examples 1 to 4 was as high as 94% or more, and was higher than that of the cells preserved using the control group. However, the cell preservation solution of the embodiment of the invention is simpler and lower in cost than the formula of the comparison group.
TABLE 2
| Sample(s) | Cell viability% (morphology) |
| Experimental example 1 | 94.7 |
| Experimental example 2 | 95.3 |
| Experimental example 3 | 96.2 |
| Experimental example 4 | 96.6 |
| Comparison group | 94.4 |
On the other hand, the cell preservation solution samples of Experimental examples 1-4 were prepared at 4000 cells/well and
cells of the storage solution samples (comparative group) stored at 4 ℃ for 5 days were transferred to 96-well culture dishes for recovery and culture, and after 1 day, ALAMAR BLUE was used TM The cell activity detection reagent detects the cell metabolic activity, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording the cell proliferation activity. Subsequently, ALAMAR BLUE is removed TM Cell viabilityAdding fresh culture medium, recovering culture, and repeating ALAMAR BLUE 3 days later TM The cell activity detection reagent is used for detecting cell metabolic activity experiments, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording cell proliferation activity. Then, the ALAMAR BLUE is removed TM Adding fresh culture solution into cell viability detection reagent solution, recovering culture, and repeating ALAMAR BLUE 5 days later TM The cell activity detection reagent is used for detecting cell metabolic activity experiments, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording cell proliferation activity.
The above ALAMAR BLUE TM Cell metabolic activity experimental data obtained by cell activity detection reagent detection experiments after 1 day, 3 days and 5 days of resuscitation are shown in figure 1, and cell proliferation activity experimental data recorded by a Saudies IncuCyte real-time living cell imaging analyzer is shown in figure 2. Referring to FIG. 1, the cell metabolic functions preserved with the cell preservation fluid samples of experimental examples 1 to 4 were effectively restored, and the number of absorption units was more as the number of days of resuscitation increased. As shown in FIG. 2, the cells preserved in the samples of the cell stocks of Experimental examples 1-4 were normal in growth, differentiation and proliferation, indicating that the cryopreserved cells were efficiently recovered and that the degree of cell fusion was higher with the number of days of recovery. And it can be seen that the performance of the cell preservation solution of the embodiment of the invention can be better than that of the comparative group.
Experimental examples 6 to 9
Sucrose, mannitol, lactobionic acid, glucose, adenosine, reduced glutathione, dextran-40, potassium dihydrogen phosphate, potassium carbonate, potassium chloride, sodium chloride, potassium hydroxide, 4-hydroxyethylpiperazine ethanesulfonic acid (4- (2-hydroxyethaneyl) piperazine-1-ethanesulfonic acid, HEPES), and dimethyl sulfoxide were weighed according to the components and concentrations of the cell preservation solutions corresponding to experimental examples 6 to 9 shown in table 3 below, and prepared into 4 kinds of mixed solutions with purified water, each solution was subjected to pH titration with sodium hydroxide until 7.6,0.2um sterile filtration with sterile filtration membranes, and the filtrate obtained by filtration was the 4 kinds of cell preservation solutions of experimental examples 6 to 9.
TABLE 3
Experimental example 10
By and on the market
CS5 cell cryopreservation solution and
the quality of the cell preservation solutions of experimental examples 6 to 9 was examined by comparing the CS10 cell frozen stock solutions with standards.
Specifically, the test cell line HCT116WT was cultured in a petri dish, digested with trypsin solution, the cell suspension was collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded, and the cell preservation solution samples of examples 6 to 9 and the supernatant were used, respectively
CS5 cell cryopreservation liquid sample (comparative group 1) and
CS10 cell lysate samples (control 2) resuspended cells at 2X 10 6 The cell concentration of the tube/tube is divided into sterile freezing tubes, the tubes are placed at the temperature of minus 80 ℃ and cooled at the cooling rate of about 1 ℃/min, and the tubes are moved into liquid nitrogen (-196 ℃) for freezing and preservation after the night.
After 2 months of preservation, the freezing tube is taken out, the temperature is rapidly rewarming is carried out under the environment of 37 ℃, the rewarming is stopped when the ice crystals are about to be completely melted, after centrifugal collection, the cell preservation solution and the freezing solution are sucked off, the cells are re-suspended by using the cell culture solution, and the percentage of the living cells is morphologically detected by using a Countess 3 automatic cell counter. The results are shown in table 4 below, and it can be seen that the survival rate of the cells preserved using the cell preservation solution samples of experimental examples 6 to 9 was as high as 95% or more, and the specific shoulder was even higher than that of the cells preserved using the comparative groups 1, 2. However, the cell preservation solution of the examples of the present invention is simpler and less costly than the formulations of comparative groups 1 and 2.
TABLE 4
| Sample(s) | Cell viability% (morphology) |
| Experimental example 6 | 95.7 |
| Experimental example 7 | 97.2 |
| Experimental example 8 | 96.9 |
| Experimental example 9 | 95.7 |
| |
94.9 |
| Comparative group 2 | 95.6 |
On the other hand, the cells cryopreserved for 2 months in the cell stock samples of Experimental examples 6-9 and comparative groups 1 and 2 were transferred to 96-well culture dishes at 4000 cell/well for recovery culture, and after 1 day, ALAMAR BLUE was used TM The cell activity detection reagent detects the cell metabolic activity, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording the cell proliferation activity. Subsequently, ALAMAR BLUE is removed TM Adding fresh culture solution into cell viability detection reagent solution, recovering culture, and repeating ALAMAR BLUE 3 days later TM The cell activity detection reagent is used for detecting cell metabolic activity experiments, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording cell proliferation activity. Then, the ALAMAR BLUE is removed TM Adding fresh culture solution into cell viability detection reagent solution, recovering culture, and repeating ALAMAR BLUE 5 days later TM The cell activity detection reagent is used for detecting cell metabolic activity experiments, and a Sadolis IncuCyte real-time living cell imaging analyzer is synchronously used for recording cell proliferation activity.
The above ALAMAR BLUE TM Cell metabolic activity experimental data obtained by detecting the cell activity detection reagent for 1 day, 3 days and 5 days of resuscitation are shown in figure 3, and cell proliferation activity experimental data recorded by a Saudies IncuCyte real-time living cell imaging analyzer for 1 day, 3 days and 5 days of resuscitation are shown in figure 4. Referring to FIG. 3, the cell metabolic functions preserved with the cell preservation solution samples of experimental examples 6 to 9 were effectively restored, and the number of absorption units was more as the number of days of resuscitation was increased. As shown in FIG. 4, the cells preserved in the samples of the cell stocks of examples 6 to 9 were normal in growth, differentiation and proliferation, indicating that the frozen cells were efficiently revived and that the degree of cell fusion was higher as the number of days of revival increased. And it can be seen that the performance of the cell preservation solution of the embodiment of the present invention can be better than that of the comparative groups 1 and 2.
The various embodiments described above and shown in the drawings are illustrative of the invention and are not exhaustive of the invention. Any modifications made to the present invention by a person of ordinary skill in the relevant art within the scope of the basic technical idea of the present invention are within the scope of the present invention.
Claims (14)
1. A cell preservation solution, comprising:
20mM to 50mM of sucrose;
20mM to 50mM mannitol;
10 mM-50 mM lactobionic acid;
5 mM-20 mM glucose;
1 mM-3 mM adenosine;
1 mM-5 mM reduced glutathione;
1 mM-2 mM dextran-40;
10 mM-30 mM potassium dihydrogen phosphate;
10 mM-30 mM potassium carbonate;
10 mM-30 mM potassium chloride;
5mM to 30mM sodium chloride;
10 mM-60 mM sodium hydroxide;
10 mM-60 mM potassium hydroxide;
0mM to 20mM vitamin E;
0 mM-25mM 4-hydroxyethyl piperazine ethanesulfonic acid; and
0 to 10 percent of dimethyl sulfoxide.
2. The cell preservation solution according to claim 1, comprising 0.5mM to 20mM vitamin E and 0% dimethyl sulfoxide.
3. The cell preservation solution according to claim 2, comprising 2mM to 5mM vitamin E.
4. The cell preservation solution according to any one of claims 1 to 3, comprising 0mM to 20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
5. The cell preservation solution according to claim 1, comprising 0mM vitamin E and >0% dimethyl sulfoxide.
6. The cell preservation solution according to claim 5, comprising 0 mM-20mM 4-hydroxyethylpiperazine ethanesulfonic acid.
7. The cell preservation solution according to claim 5 or 6, comprising 5% to 10% dimethylsulfoxide.
8. The cell preservation solution according to any one of claims 1 to 7, wherein the pH is in the range of 7.0 to 8.0.
9. The cell preservation solution according to any one of claims 1 to 8, wherein the osmotic pressure is 300 to 400 mOsmol.
10. The cell preservation solution according to any one of claims 1 to 9, comprising water.
11. A method for cell preservation comprising preserving cells with the cell preservation solution according to any one of claims 1 to 10.
12. The method for preserving cells according to claim 11, wherein the cells are preserved at 2 ℃ to 8 ℃ or-80 ℃ to-196 ℃.
13. A method for cell preservation according to claim 11 or 12, wherein the cells comprise one or more of stem cells, immune cells, tumour cells.
14. A method of cell preservation according to any one of claims 11 to 13, wherein the cells comprise human colon cancer cells.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN202210922177.1A CN115349515A (en) | 2022-08-02 | 2022-08-02 | Cell preservation solution and cell preservation method |
| PCT/CN2022/120197 WO2024026996A1 (en) | 2022-08-02 | 2022-09-21 | Cell preservation solution and cell preservation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210922177.1A CN115349515A (en) | 2022-08-02 | 2022-08-02 | Cell preservation solution and cell preservation method |
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| CN202210922177.1A Pending CN115349515A (en) | 2022-08-02 | 2022-08-02 | Cell preservation solution and cell preservation method |
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| CN (1) | CN115349515A (en) |
| WO (1) | WO2024026996A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115843782A (en) * | 2022-12-27 | 2023-03-28 | 南京三生生物技术股份有限公司 | Cell preservation solution, application of cell preservation solution in cell storage and method for transferring refrigerated cells into cryopreservation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060078872A1 (en) * | 2004-10-12 | 2006-04-13 | Atsushi Taguchi | Cell-preservation liquid |
| CN104542578A (en) * | 2015-02-05 | 2015-04-29 | 广州赛莱拉干细胞科技股份有限公司 | Cell preservation solution and preparation method and applications thereof |
| CN109845728A (en) * | 2019-03-25 | 2019-06-07 | 西安医斯美生物科技有限公司 | The adipose tissue frozen stock solution and cryopreservation methods of clinical application rank |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| GB0822246D0 (en) * | 2008-12-05 | 2009-01-14 | Reneuron Ltd | Composition |
| JP2022501053A (en) * | 2018-09-25 | 2022-01-06 | ボーン、セラピューティクス、エスアーBone Thrapeutics Sa | Methods for Differentiating Mesenchymal Stem Cells |
| US20210371828A1 (en) * | 2018-10-12 | 2021-12-02 | Reneuron Limited | Induced pluripotent cell comprising a controllable transgene for conditional immortalization |
| PL3685665T3 (en) * | 2019-01-23 | 2023-10-16 | Carnamedica Sp. Z O.O. | Use of a solution comprising peg for the preservation of stem cells |
| CN114457003A (en) * | 2020-11-10 | 2022-05-10 | 维思尔治疗有限公司 | Methods of preconditioning vascular cells for transduction, methods of transduction, and methods of preserving transduced cells |
-
2022
- 2022-08-02 CN CN202210922177.1A patent/CN115349515A/en active Pending
- 2022-09-21 WO PCT/CN2022/120197 patent/WO2024026996A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060078872A1 (en) * | 2004-10-12 | 2006-04-13 | Atsushi Taguchi | Cell-preservation liquid |
| CN104542578A (en) * | 2015-02-05 | 2015-04-29 | 广州赛莱拉干细胞科技股份有限公司 | Cell preservation solution and preparation method and applications thereof |
| CN109845728A (en) * | 2019-03-25 | 2019-06-07 | 西安医斯美生物科技有限公司 | The adipose tissue frozen stock solution and cryopreservation methods of clinical application rank |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115843782A (en) * | 2022-12-27 | 2023-03-28 | 南京三生生物技术股份有限公司 | Cell preservation solution, application of cell preservation solution in cell storage and method for transferring refrigerated cells into cryopreservation |
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| WO2024026996A1 (en) | 2024-02-08 |
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