CN107711823B - Cell cryopreservation liquid stored at normal temperature and application thereof - Google Patents
Cell cryopreservation liquid stored at normal temperature and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
Abstract
The invention provides a serum-free cell cryopreservation liquid suitable for mononuclear cells and capable of being preserved at normal temperature and application thereof, wherein the serum-free cell cryopreservation liquid comprises the following components: dimethyl sulfoxide at about 10% by volume; glucose in a mass to volume ratio of about 2.5 g/L; dextran with the mass-to-volume ratio of about 30-40 g/L; hydroxyethyl starch with the mass volume ratio of about 6-12 g/L; the volume percentage of the compound electrolyte solution is about 25-45%. The invention also provides a preparation method of the serum-free cell frozen stock solution and a freezing method adopting the serum-free cell frozen stock solution. The serum-free cell freezing medium can be preserved or transported at normal temperature, has high survival rate of resuscitation cells after freezing, and can be directly used for clinical or subsequent culture.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a serum-free cell cryopreservation solution which can be preserved at normal temperature and is suitable for mononuclear cells, and a preparation method and application thereof.
Background
Mononuclear cells, generally, refer to cells having only one nucleus in umbilical cord blood and peripheral blood, and include lymphocytes, dendritic cells, monocytes, and hematopoietic stem cells, and the like. Hematopoietic stem cells among mononuclear cells are often used for hematopoietic stem cell transplantation; meanwhile, the mononuclear cell can be stimulated by cytokines to induce different immune cells in vitro, and killer cells (Cytokine-induced killers, CIK) and natural killer cells (NK) induced by various cytokines have great application in the aspect of clinical treatment and tumor resistance.
On the day of the widespread use of cell culture technology in scientific research and clinical treatment, cell cryopreservation and thawing and resuscitation techniques are becoming increasingly important.
Cell cryopreservation refers to the storage of cells at ultra low temperatures. The biochemical reaction of the cell is extremely slow and even stops, but the normal structure and function can be still preserved after the recovery through long-term low-temperature preservation. The continuous research proves that the cell is preserved under the condition of liquid nitrogen ultralow temperature of-196 ℃, and is the best cryopreservation temperature at present. Because at-196 ℃ the vital activities of the cells are almost completely stopped, but the structure and function of the cells are intact after recovery.
The cells are directly frozen without a freezing protective agent, the water inside and outside the cells is frozen along with the reduction of the temperature, and the formed ice crystals can cause the damage of cell membranes and organelles to die, thereby causing the failure of cell freezing. The freezing protective agent is added in the process of freezing the cells, so that the cells can be protected from being damaged by ice crystals. The cell cryopreservation is successful because the cryoprotectant is easy to combine with water molecules in the solution, thereby lowering the freezing point and reducing the formation of ice crystals.
Cryoprotectants can be classified into two categories, permeable and impermeable, depending on whether they penetrate the cell membrane. The osmotic cryoprotectant can permeate into cells and is generally a small molecular substance, mainly comprising glycerol, DMSO, ethylene glycol, propylene glycol, acetamide, methanol and the like. The non-permeable cryoprotectant cannot permeate into cells, and is generally a macromolecular substance, mainly comprising polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, hydroxyethyl starch (HES) and the like. The most commonly used cryoprotectant at present is DMSO.
Although cells are extremely slow to metabolize when frozen, they still require some nutrients or energy to consume to maintain essential activity. Therefore, Fetal Bovine Serum (FBS), culture medium, human serum, albumin and the like are added to most of the cell freezing medium at present. Because Fetal Bovine Serum (FBS) may carry the risk of animal-derived viruses, human serum may also contain human-derived viruses, and the culture medium contains various ions, the clinical use of the prior frozen stock solution has various limitations and inconveniences.
At present, some frozen stock solutions need to be prepared to have a final concentration of 2 times before use, placed in a refrigerator at 2-8 ℃ for precooling for a period of time or for long-term storage, taken out when used, and added with an equal amount of 2 times of frozen stock solutions after the cells are resuspended by the solution, so that the final concentration of the frozen stock solutions is 1 time. The operation can not realize the on-site preparation of the frozen stock solution, and the long-term low-temperature storage leads to complex operation, is not beneficial to the quality control of products, is easy to pollute, and has the problems of easy error and the like.
Therefore, there is still a need in the art for a cell cryopreservation solution that is more convenient to store and use and that improves the cryopreservation and recovery effects of cells using the same, particularly the recovery rate and survival rate of mononuclear cells.
Disclosure of Invention
The present invention has been made to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a cell serum-free frozen stock solution which can be used as it is or stored at room temperature, particularly a cell serum-free frozen stock solution suitable for use in mononuclear cells. The cell freezing solution and the cell freezing method have high cell recovery rate and survival rate after cell recovery by freezing and recovering, and keep good cell activity.
Specifically, the invention provides a serum-free cell cryopreservation liquid which is characterized by comprising the following components: dextran with the mass-volume ratio of about 30-40 g/L.
In one aspect of the invention, the invention provides a serum-free cell cryopreservation solution, which is characterized by comprising the following components:
dimethyl sulfoxide at about 10% by volume;
glucose in a mass to volume ratio of about 2.5 g/L;
dextran with the mass-to-volume ratio of about 30-40 g/L;
hydroxyethyl starch with the mass volume ratio of about 6-12 g/L;
the volume percentage of the compound electrolyte solution is about 25-45%.
In yet another aspect of the present invention, the mass to volume ratio of dextran in the serum-free cell culture solution of the present invention is about 30-35 g/L.
In one aspect of the invention, the serum-free cell freezing medium comprises the following components in volume fraction:
about 10% dimethylsulfoxide;
about 30-40% dextran solution with mass volume ratio of 100 g/L;
about 10-20% of hydroxyethyl starch solution with the mass volume ratio of 60 g/L;
about 5% by mass/volume of an aqueous glucose solution at a mass/volume ratio of 50 g/L;
the balance is about 25-45% of compound electrolyte solution.
In yet another aspect of the invention, the dextran solution in the serum-free cell freezing medium can be 0.9% sodium chloride solution.
In yet another aspect of the invention, the dextran in the serum-free cell cryopreservation solution is a low molecular dextran, such as dextran 40.
In yet another aspect of the invention, the hydroxyethyl starch solution in the serum-free cell freezing medium can be 0.9% sodium chloride solution in solute.
The compound electrolyte solution (or called compound electrolyte injection) used in the invention is a common clinical reagent, usually used as a supplement source of water and electrolyte, and contains electrolyte components compatible with blood and blood components, and can be infused before or after transfusion, or used as a diluent of blood cells. In yet another aspect of the present invention, the complex electrolyte solution used in the present invention may have the following composition: each 1000ml of the beverage contains 5.26g of sodium chloride, 5.02g of sodium gluconate, 3.68g of sodium acetate, 0.37g of potassium chloride and 0.30g of magnesium chloride; or each 1000ml of the solution contains 2.34g of sodium chloride, 0.75g of potassium chloride, 2.24g of sodium lactate and 27.00g of glucose.
In yet another aspect of the invention, the serum-free cell cryopreservation solution is suitable for cryopreservation of mononuclear cells, such as umbilical cord blood mononuclear cells or peripheral blood mononuclear cells. In yet another aspect of the present invention, the serum-free cell cryopreservation solution is suitable for cryopreserving cells induced to form mononuclear cells, such as killer cells or natural killer cells.
In one aspect of the invention, the serum-free cell culture medium is suitable for ready use. In yet another aspect of the invention, the serum-free cell cryopreservation solution is suitable for storage at room temperature. The inventor of the application unexpectedly finds that the cell freezing solution can obtain good effect no matter the cell freezing solution is used as it is or is taken out after being stored at normal temperature and used immediately, thereby avoiding the step of precooling before freezing and storing the cells in the operation of using the cell freezing solution, improving the working efficiency, proving that the recovery rate and survival rate of the frozen and recovered cells are high, and preserving good cell activity. Therefore, the serum-free cell freezing medium is not limited by low-temperature preservation, can be preserved or transported at normal temperature, and has important practical significance to the field. Without being limited by a particular theory, the inventor believes that the serum-free cell cryopreservation solution contains dextran with high concentration in a particular range, so that the cell osmotic pressure is effectively improved, the water in cytoplasm is sucked out, the ice crystal formed by the cells in the cryopreservation process is reduced, and meanwhile, the aggregation and the damage of the cells caused by the dextran with excessively high concentration can be avoided, so that the recovery survival rate of the cells is improved and the activity is maintained as a whole.
In yet another aspect of the present invention, there is provided the use of the serum-free cell cryopreservation solution of the present invention described above in cryopreservation and/or recovery after cryopreservation of cells. In yet another aspect of the invention, the cell is a mononuclear cell, for example, a cord blood mononuclear cell or a peripheral blood mononuclear cell. In yet another aspect of the invention, the cell is a cell in which formation of a monocyte is induced, such as a killer cell or a natural killer cell.
In one aspect of the present invention, there is provided a method for cryopreserving mononuclear cells (e.g., umbilical cord blood and peripheral blood mononuclear cells), comprising the steps of:
(1) separating to obtain mononuclear cells;
(2) preparing the serum-free cell cryopreservation solution of the invention as described above;
(3) resuspending the mononuclear cells by using the serum-free cell cryopreservation solution, then subpackaging the mononuclear cells in a cryopreservation tube, and carrying out programmed temperature reduction and then carrying out cryopreservation at a low temperature.
In still another aspect of the present invention, in the method for cryopreserving mononuclear cells, the mononuclear cell cryopreserving solution prepared in step 2 is stored at room temperature before step 3.
In yet another aspect of the present invention, the method for cryopreserving the mononuclear cells further comprises a step of resuscitating the cryopreserved mononuclear cells.
In still another aspect of the present invention, in the cell cryopreservation process of step (3) of the above-described method for cryopreserving mononuclear cells, the density of the mononuclear cells is 1X 106/～5×107/ml。
In one aspect of the present invention, there is provided a method for preparing the serum-free cell cryopreservation solution of the present invention, which comprises the steps of mixing the following components: in terms of volume fraction, the amount of the active ingredient,
about 10% dimethylsulfoxide;
about 30-40% dextran solution with mass volume ratio of 100 g/L;
about 10-20% of hydroxyethyl starch solution with the mass volume ratio of 60 g/L;
about 5% by mass/volume of an aqueous glucose solution at a mass/volume ratio of 50 g/L;
the balance is about 25-45% of compound electrolyte solution.
In still another aspect of the present invention, the above preparation method is performed at normal temperature. Preferably, the prepared serum-free cell cryopreservation solution can be directly used for cryopreservation and/or recovery after cryopreservation of cells.
In one aspect of the present invention, there is also provided a use of the mononuclear cell obtained after cryopreservation and recovery of the above-mentioned cryopreservation method for mononuclear cells (e.g., umbilical cord blood and peripheral blood mononuclear cells) according to the present invention, or a killer cell or natural killer cell obtained by induced differentiation of the mononuclear cell, or a killer cell or natural killer cell obtained by cryopreservation and recovery of a killer cell or natural killer cell obtained by induced differentiation of the mononuclear cell using the serum-free cell cryopreservation solution of the present invention in the preparation of a medicament for treating tumors. The cell freezing medium is ready for use or taken out for immediate use after being stored at normal temperature, and the cells frozen and recovered by the cell freezing medium have high recovery rate and survival rate and good cell activity. For example, umbilical cord blood and peripheral blood mononuclear cells maintain good activity after freezing and reviving with the cell cryopreservation solution of the present invention, can be efficiently differentiated into killer cells or natural killer cells after induction, and the killer cells or natural killer cells can effectively kill tumor cells.
The "about" as used herein represents an acceptable difference between a particular operation and a theoretically calculated value directly during production or deployment. In general, those skilled in the art will appreciate that "about" is intended to mean, for example, ± 0.01, ± 0.05, ± 0.1, ± 0.5, ± 1, ± 2, ± 5, or ± 10.
Drawings
FIG. 1 is a graph of the viability of cells cryopreserved by mononuclear cells using exemplary different compositions of the cryopreservation solution and method of the invention;
FIG. 2 viability and CD34+ detection of cord blood mononuclear cells before and after cryopreservation using exemplary cryopreservation solutions and methods of the invention;
FIG. 3 is a 40X picture of CIK cells expanded by resuscitation of peripheral blood mononuclear cells using an exemplary cryopreservation solution and cryopreservation method of the present invention;
FIG. 4 is a 40X photograph of CIK cells obtained by resuscitating expanded mononuclear cells from umbilical cord blood cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the present invention;
FIG. 5 is a photograph 40X of expanded NK cells recovered from peripheral blood mononuclear cells cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the present invention;
FIG. 6 is a 40X picture of cord blood mononuclear cell resuscitation expanded NK cells cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the present invention;
FIG. 7 is a graph of a CIK surface marker detection map of peripheral blood mononuclear cell resuscitation expansion cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the invention;
FIG. 8 is a graph of CIK surface marker detection by resuscitating expanded mononuclear cells from cord blood cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the invention;
FIG. 9 is a detection map of NK surface markers of peripheral blood mononuclear cell resuscitation expansion cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the invention;
FIG. 10 is a graph of the recovery expanded NK surface marker detection of cord blood mononuclear cells cryopreserved using an exemplary cryopreservation solution and cryopreservation method of the invention.
Detailed Description
The invention will be further described below by means of specific embodiments
Example 1 method for isolating human peripheral blood mononuclear cells
Human anticoagulated peripheral blood was collected from volunteers (all signed informed consent). Separating mononuclear cells from human peripheral blood using a lymphocyte separation medium, comprising the steps of:
1.1 sample Whole blood count, transfer the anticoagulated peripheral blood with pipette into 50ml centrifuge tube, centrifuge for 15 minutes at 900 g.
1.2 after centrifugation, taking the supernatant blood plasma for heat inactivation at 56 ℃, and taking the supernatant for later use after centrifugation. Precipitating blood cells, adding 0.9% sodium chloride for injection and dilution (blood volume: diluent: lymphocyte separation solution: 1:1), mixing well, slowly adding into 50ml centrifuge tube filled with 15ml lymphocyte separation solution, and making interface clear.
1.3700 g were centrifuged for 20 minutes.
1.4 centrifugation, carefully handling the tube until a buffy coat is visible in the middle of the tube, carefully aspirating the buffy coat with a pipette and transferring the buffy coat to another tube.
1.5 the aspirated buffy coat is washed by adding 0.9% sodium chloride injection and centrifuged at 600g for 10 minutes.
1.6 centrifugation, and supernatant removal, peripheral blood mononuclear cells were obtained.
EXAMPLE 2 preparation of serum-free cell cryopreservation solution that can be stored at Normal temperature
Invention frozen stock solution 1: according to the volume percentage, 10% of dimethyl sulfoxide, 20% of dextran 40 injection, 10% of hydroxyethyl starch injection, 5% of glucose injection and the balance of compound electrolyte injection are prepared to obtain the serum-free cell freezing solution.
Invention frozen stock solution 2: according to the volume percentage, 10% of dimethyl sulfoxide, 25% of dextran 40 injection, 10% of hydroxyethyl starch injection, 5% of glucose injection and the balance of compound electrolyte injection are prepared to obtain the serum-free cell freezing solution.
Invention frozen stock solution 3: according to the volume percentage, 10% of dimethyl sulfoxide, 30% of dextran 40 injection, 10% of hydroxyethyl starch injection, 5% of glucose injection and the balance of compound electrolyte injection are prepared to obtain the serum-free cell freezing solution.
Invention frozen stock solution 4: according to the volume percentage, 10% of dimethyl sulfoxide, 35% of dextran 40 injection, 10% of hydroxyethyl starch injection, 5% of glucose injection and the balance of compound electrolyte injection are prepared to obtain the serum-free cell freezing solution.
Invention frozen stock solution 5: according to the volume percentage, 10% of dimethyl sulfoxide, 40% of dextran 40 injection, 10% of hydroxyethyl starch injection, 5% of glucose injection and the balance of compound electrolyte injection are prepared to obtain the serum-free cell freezing solution.
Wherein the dextran 40 injection (Hospira Inc, cat No. 0409-: dextran-40 mass volume ratio is 100g/L, 0.9% sodium chloride solution.
Wherein the hydroxyethyl starch injection (Hospira Inc, with the product number of 0409-7248-143): the mass volume ratio of the hydroxyethyl starch is 60g/L, and the mass volume ratio of the hydroxyethyl starch is 0.9 percent of sodium chloride solution.
Wherein the glucose injection (Zhejiang Dubang, Chinese medicine standard character: H33021289): an aqueous glucose solution containing 50g/L of glucose.
The compound electrolyte injection (Sichuan Kelun, Chinese medicine standard character H20113476) comprises the following components: each 1000ml of the beverage contains 5.26g of sodium chloride, 5.02g of sodium gluconate, 3.68g of sodium acetate, 0.37g of potassium chloride and 0.30g of magnesium chloride.
According to the volume percentage of the comparison frozen stock solution 1, 10 percent of dimethyl sulfoxide, 10 percent of dextran 40 injection, 5 percent of glucose injection and 75 percent of FBS (Gibco, product number: 10100-147-FBS) are uniformly mixed to prepare the comparison cell frozen stock solution 1.
Comparing the frozen stock solution 2: according to the volume percentage, 10% of dimethyl sulfoxide, 2% of dextran 40 injection, 5% of glucose injection and 83% of compound electrolyte injection are prepared to obtain the contrast cell frozen stock solution 2.
Comparing the frozen stock solution 3: according to volume percentage, 10% of dimethyl sulfoxide, 5% of glucose injection, 30% of hydroxyethyl starch injection and 55% of compound electrolyte injection are prepared into the contrast cell frozen stock solution 3.
EXAMPLE 3 Effect of dextran 40 injection concentration ratio in serum-free cell freezing Medium
1. Frozen stocks 1-5 of the invention were prepared as in example 2, i.e., the dextran 40 injection solutions contained therein were 20%, 25%, 30%, 35%, 40% v/v, respectively.
2. Adding the freezing medium into the peripheral blood mononuclear cell sediment to make the cell concentration 1 × 106And/ml, sucking and beating by a pipette at a slow speed and mixing uniformly.
3. The cell suspension was dispensed into 1ml vials.
4. And (4) sticking labels on the cryopreservation tubes, wherein the labels comprise related information such as sample numbers, cell numbers, cryopreservation liquid batch numbers, cryopreservation dates, operators and the like.
5. Placing the frozen tube in a programmed cooling box filled with isopropanol, storing for 4-12 h at-80 deg.C in a refrigerator, and transferring into a liquid nitrogen tank the next day.
6. After the cells are frozen for one month, the cells are recovered to detect the survival rate.
The results of the detection are shown in FIG. 1. FIG. 1 is the ordinate of the survival rate of the resuscitated cells. The results show that the cell viability rate of the frozen and recovered cells is the highest for the dextran in the frozen stock solution of the mononuclear cells within the concentration range of 30-35%.
EXAMPLE 4 Single Nuclear cell cryopreservation test (in a ready-to-use cryopreservation solution)
4.1 cryopreservation of human peripheral blood mononuclear cells
The cryopreservation method of human umbilical cord blood or peripheral blood mononuclear cells comprises the following specific steps:
1. the invention frozen stock solution 3, the invention frozen stock solution 4 and the invention frozen stock solution 5 were prepared according to example 2 before starting the experiment, and the next step was immediately performed after the preparation.
2. The peripheral blood mononuclear cell pellet isolated in example 1 was taken.
3. Adding freezing medium into the mononuclear cell sediment to make the cell concentration be 1X 107～5×107And/ml, sucking and beating by a pipette at a slow speed and mixing uniformly.
4. The cell suspension was dispensed into 1ml vials.
5. And (4) sticking labels on the cryopreservation tubes, wherein the labels comprise related information such as sample numbers, cell numbers, cryopreservation liquid batch numbers, cryopreservation dates, operators and the like.
6. Placing the frozen tube in a programmed cooling box filled with isopropanol, storing for 4-12 h at-80 deg.C in a refrigerator, and transferring into a liquid nitrogen tank the next day.
4.2 Resuscitation of human peripheral blood mononuclear cells
The recovery method of the mononuclear cells comprises the following specific steps:
1. waiting for the temperature of the constant-temperature water bath box to rise to 37 ℃; the cell cryopreservation tube is taken out of the liquid nitrogen, immediately put into warm water at 37 ℃ and gently shaken, and the cell cryopreservation tube is flicked by fingertips while shaking until ice blocks are dissolved.
2. Dipping 75% ethanol with a dust-free cloth, wiping the freezing tube and disinfecting; and opening the cover of the freezing tube, slowly dripping 1ml of pre-cooling culture medium with the temperature of 4 ℃ into the freezing tube, slightly blowing, uniformly mixing, and standing for about 5 min.
3. Transferring into a centrifuge tube containing 10ml of precooled culture medium, centrifuging for 10min at 300g, and removing supernatant;
4. 5ml of the precooled culture medium is added by a 5ml pipette, the suspension is gently blown and uniformly mixed, and 0.3ml of the suspension is sampled and counted.
4.3 calculating the total number and the survival rate of the cells, and counting the loss rate of the cryopreserving resuscitation on the cells.
The formulations of inventive and comparative cryopreservation solutions 3-5 and 1-3 of example 2 were followed to prepare 10 frozen and resuscitated batches of mononuclear cells (10 peripheral blood batches) using the methods and procedures of 4.1 and 4.2, respectively. And (4) calculating the total number and the survival rate of the cells subjected to freezing and thawing, and counting the loss rate of the cells subjected to freezing and thawing.
Counting results show that the recovery survival rate of the cells recovered by the invention frozen stock solutions 3, 4 and 5 is above 94% on average, the cell loss rate is within 20%, the freezing effect is excellent, and details are shown in table 1. The cell survival rate of the cryopreserved mononuclear cells before and after the cryopreservation and the detection of CD34+ are compared, and as shown in figure 1 and figure 2, the cell survival rate after the cryopreservation is 95.27%, and the CD34+ is 0.44, which is not obviously different from that before the cryopreservation.
The recovery survival rate and the cell loss rate of the cells prepared by the existing preparation method compared with those of the frozen stock solution 1-3 are obviously inferior to those of the frozen stock solution prepared by the invention.
TABLE 1
| Detecting items | Total number before frozen | Total number after resuscitation | Rate of loss | Rate of resuscitation |
| Invention frozen stock solution 3 | 3x107 | 2.71x107 | 9.6％ | 96.7％ |
| Invention frozen stock solution 4 | 3x107 | 2.55x107 | 15％ | 94.9％ |
| Invention frozen stock solution 5 | 3x107 | 2.64x107 | 12％ | 95.8％ |
| Comparative frozen stock solution 1 | 3x107 | 1.53x107 | 49％ | 78.8％ |
| Contrast frozen stock solution 2 | 3x107 | 1.35x107 | 55％ | 83.4％ |
| Contrast frozen stock solution 3 | 3x107 | 1.48x107 | 50.3％ | 86.7％ |
EXAMPLE 5 Single Nuclear cell cryopreservation test (after three months of cryopreservation at ambient temperature with freezing medium)
5.1 cryopreservation of human peripheral blood mononuclear cells
The human peripheral blood mononuclear cell cryopreservation method comprises the following specific steps:
1. invention frozen stock solution 3, invention frozen stock solution 4 and invention frozen stock solution 5 were prepared in advance as in example 2 two months before the start of the experiment.
2. The peripheral blood mononuclear cell pellet isolated in example 1 was taken.
3. Adding freezing medium into the mononuclear cell sediment to make the cell concentration be 1X 107～5×107And/ml, sucking and beating by a pipette at a slow speed and mixing uniformly.
4. The cell suspension was dispensed into 1ml vials.
5. And (4) sticking labels on the cryopreservation tubes, wherein the labels comprise related information such as sample numbers, cell numbers, cryopreservation liquid batch numbers, cryopreservation dates, operators and the like.
6. Placing the frozen tube in a programmed cooling box filled with isopropanol, storing for 4-12 h at-80 deg.C in a refrigerator, and transferring into a liquid nitrogen tank the next day.
5.2 Resuscitation of human peripheral blood mononuclear cells
The recovery method of the mononuclear cells comprises the following specific steps:
1. waiting for the temperature of the constant-temperature water bath box to rise to 37 ℃; the cell cryopreservation tube is taken out of the liquid nitrogen, immediately put into warm water at 37 ℃ and gently shaken, and the cell cryopreservation tube is flicked by fingertips while shaking until ice blocks are dissolved.
2. Dipping 75% ethanol with a dust-free cloth, wiping the freezing tube and disinfecting; and opening the cover of the freezing tube, slowly dripping 1ml of pre-cooling culture medium with the temperature of 4 ℃ into the freezing tube, slightly blowing, uniformly mixing, and standing for about 5 min.
3. Transferring into a centrifuge tube containing 10ml of precooled culture medium, centrifuging for 10min at 300g, and removing supernatant;
4. 5ml of the precooled culture medium is added by a 5ml pipette, the suspension is gently blown and uniformly mixed, and 0.3ml of the suspension is sampled and counted.
5.3 calculating the total number and the survival rate of the cells, and counting the loss rate of the cryopreserving resuscitation on the cells.
The formulations of inventive and comparative cryopreservation solutions 3-5 and 1-3 of example 2 were followed to prepare 10 frozen and resuscitated batches of mononuclear cells (10 peripheral blood batches) using the methods and procedures of 5.1 and 5.2, respectively. And (4) calculating the total number and the survival rate of the cells subjected to freezing and thawing, and counting the loss rate of the cells subjected to freezing and thawing.
The counting results show that the cell recovery survival rate of the cell recovered from the mononuclear cells of 10 batches of peripheral blood frozen for 2 months by using the frozen stock solutions 3, 4 and 5 of the invention is above 90% on average, the cell loss rate is within 20% and the freezing effect is excellent, and the details are shown in table 2. And obviously, the frozen stock solution is not suitable for being stored at normal temperature.
TABLE 2
| Detecting items | Total number before frozen | Total number after resuscitation | Rate of loss | Rate of resuscitation |
| Invention frozen stock solution 3 | 3x107 | 2.51x107 | 16.33％ | 92.45％ |
| Invention frozen stock solution 4 | 3x107 | 2.61x107 | 13％ | 91.33％ |
| Invention frozen stock solution 5 | 3x107 | 2.49x107 | 17％ | 95.8％ |
| Comparative frozen stock solution 1 | 3x107 | 1.13x107 | 62.33％ | 64.8％ |
| Contrast frozen stock solution 2 | 3x107 | 1.05x107 | 65％ | 75.4％ |
| Contrast frozen stock solution 3 | 3x107 | 1.09x107 | 63.67％ | 78.7％ |
Example 6 Induction of mononuclear cells into CIK cells after recovery
The induction experiment of the mononuclear cell to the CIK cell comprises the following specific steps:
1. after freezing the recovered mononuclear cells obtained in example 5, 1640 medium was added to the cells at a density of 2X 106Resuspend the cell pellet, inoculate in T75 cell culture flask, add rhIFN-r 1000U/ml and 10% autologous plasma on day 1, then place in 37 ℃, 5% CO2 incubator to culture.
2. After culturing for 24 hours, rhIL-2500U/ml and CD3McAb 50ng/ml were added to the culture medium, and the culture was continued.
3. Fresh culture medium was supplemented every three days later and rhIL-2 was supplemented.
4. On day 14, the cell status was observed, and CIK cells were collected, as shown in fig. 3 and 4 (fig. 3 and 4 are 40-fold micrographs of CIK expanded after cryopreservation of mononuclear cells derived from peripheral blood and umbilical cord blood, respectively), the cell status was very good, and there were many cell masses that proliferated. And the amplified CIK is subjected to cell surface antigen CD3+ CD8+, CD3+ CD56+ detection and killing activity experiment aiming at tumor cells.
5. The flow detection result shows that after the CIK cells are cultured for 14 days, the expanded CD3+ CD8+ of the peripheral blood mononuclear cells is 62.23%, and the expanded CD3+ CD56+ cells are 26.05%; cord blood mononuclear cell expansion was 85.02% for CD3+ CD8+ and 28.05% for CD3+ CD56+ cells (fig. 7 and 8); the induction effect was good.
6. The killing activity of the CIK on tumor cell melanoma, breast cancer and bladder cancer cells is 70-90% when the killing activity of the CIK on the tumor cell melanoma, breast cancer and bladder cancer cells is 20:1, the effect is good, and the result is shown in table 3.
TABLE 3
| CIK/tumor cells | Melanoma cells | Breast cancer cell | Bladder cancer cell |
| Effective target ratio of 1:20 | 72.53％ | 86.74％％ | 81.89％ |
Example 7 Induction of mononuclear cells into NK cells after Resuscitation
The specific method of inducing the mononuclear cells to the NK cells is as follows:
1. the mononuclear cells recovered after the cryopreservation in example 4 were added to 1640 medium to make the cell density 2X 106Resuspending the cell pellet, inoculating it into a T75 cell culture flask, adding 500-2000U/mL IL-2, 5-50ng/mL IL-21, 5-50ng/mL IL-15 and 10% autologous plasma on day 1, and then placing it at 37 ℃ and 5% CO2Culturing in an incubator.
2. After culturing for 24 hours, rhIL-2500U/ml and CD3McAb 50ng/ml were added to the culture medium, and the culture was continued.
3. Fresh medium was replenished every three days later and the corresponding factors and plasma were replenished.
4. On day 14, NK cells were collected and observed for cell status (fig. 5 and 6 are photomicrographs of NK cells expanded after cryopreservation and recovery of peripheral blood and cord blood-derived mononuclear cells, 40-fold respectively), which was very good in cell status and numerous in cell mass for cell proliferation. And performing cell surface antigen CD3-CD16+ CD56+ detection on the amplified NK and killing activity experiments aiming at tumor cells.
4. The flow detection result shows that after 14 days of NK cell culture, the expansion of the peripheral blood mononuclear cells is 30.49 percent of CD3-CD16+ CD56 +; the expansion of cord blood mononuclear cells was 35.88% of CD3-CD16+ CD56+ (FIG. 9 and FIG. 10), showing good induction.
5. The killing activity of NK to the tumor cell K562, the ratio of NK to K562 cell is 20: the killing rate can reach 68.96% when 1 is used, and the detection results showing good killing activity are shown in Table 4.
TABLE 4
| NK/K562 | Repetition of 1 | Repetition 2 | Repetition of 3 | Mean value |
| Killing rate (effective target ratio 1:20) | 71.35％ | 66.21％ | 69.33％ | 68.96％ |
Example 8
CIK or NK cells amplified in example 6 and example 7 are added into the cryopreservation solution 4 of the invention, and are recovered after one month of cryopreservation to detect the survival rate. The cryopreservation and recovery procedures were as described in example 4, methods and procedures 4.1 and 4.2.
As shown in Table 5, the mean survival rate of high-density CIK immune cells after cryopreservation is 89%, and the mean survival rate of NK immune cells is 91.5%, so that the cryopreservation effect is good.
TABLE 5
| Cryopreserving cells | Repetition of 1 | Repetition 2 | Repetition of 3 | Mean value |
| CIK | 91.61％ | 87.28％ | 89.58％ | 89.49％ |
| NK | 86.61％ | 92.11％ | 87.89％ | 88.87％ |
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, polymer chemistry, biotechnology and the like, and it will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Many modifications and variations are possible in light of the above teaching and are therefore within the scope of the invention.
The unit "degree" of temperature as used herein refers to degrees celsius, i.e., degrees celsius, unless otherwise indicated.
Claims (13)
1. A serum-free cell cryopreservation liquid is characterized by comprising the following components:
dimethyl sulfoxide at about 10% by volume;
glucose in a mass to volume ratio of about 2.5 g/L;
dextran with the mass-to-volume ratio of 30-40 g/L;
hydroxyethyl starch with the mass volume ratio of 6-12 g/L;
the volume percentage of the compound electrolyte solution is 25-45%.
2. The serum-free cell cryopreservation solution of claim 1, characterized by comprising the following components in volume fraction:
about 10% dimethylsulfoxide;
30-40% of dextran solution with the mass volume ratio of 100g/L, wherein the solvent of the solution is 0.9% of sodium chloride solution;
10-20% of hydroxyethyl starch solution with the mass volume ratio of 60g/L, wherein the solvent of the solution is 0.9% of sodium chloride solution;
about 5% by mass/volume of an aqueous glucose solution at a mass/volume ratio of 50 g/L;
the balance is 25-45% of compound electrolyte solution.
3. The serum-free cell cryopreservation solution of claim 1, wherein the mass to volume ratio of the dextran is 30-35 g/L.
4. The serum-free cell cryopreservation solution of claim 1, wherein the cells are mononuclear cells or cells induced to form mononuclear cells.
5. The serum-free cell cryopreservation solution of claim 4, wherein the cells are umbilical cord blood mononuclear cells or peripheral blood mononuclear cells.
6. The serum-free cell cryopreservation solution of claim 4, wherein the cells are killer cells or natural killer cells.
7. The serum-free cell culture medium according to claim 1, which is a serum-free cell culture medium stored at ordinary temperature.
8. Use of the serum-free cell cryopreservation solution according to any one of claims 1 to 6 in the cryopreservation of cells.
9. A cryopreservation method of mononuclear cells is characterized by comprising the following steps:
(1) separating to obtain mononuclear cells;
(2) preparing the serum-free cell cryopreservation solution of any one of claims 1 to 7;
(3) resuspending the mononuclear cells by using the serum-free cell cryopreservation solution, then subpackaging the mononuclear cells in a cryopreservation tube, and carrying out programmed temperature reduction and then carrying out cryopreservation at a low temperature.
10. The cryopreservation method of claim 9, wherein: wherein, before the step (3), the mononuclear cell frozen stock solution prepared in the step (2) is preserved at normal temperature.
11. The cryopreservation method of claim 9, wherein: the method also comprises the step of recovering the frozen mononuclear cells.
12. The cryopreservation method of claim 9, wherein: in the process of freezing and storing the cells in the step (3), the density of the mononuclear cells is 1 multiplied by 106Per/ml-5 × 107One per ml.
13. The serum-free cell cryopreservation solution of claim 9, wherein the cells are umbilical cord blood mononuclear cells or peripheral blood mononuclear cells.
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