CN112753695A - Immune cell cryopreservation method - Google Patents
Immune cell cryopreservation method Download PDFInfo
- Publication number
- CN112753695A CN112753695A CN202110003175.8A CN202110003175A CN112753695A CN 112753695 A CN112753695 A CN 112753695A CN 202110003175 A CN202110003175 A CN 202110003175A CN 112753695 A CN112753695 A CN 112753695A
- Authority
- CN
- China
- Prior art keywords
- cell
- immune cells
- frozen
- cells
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an immune cell cryopreservation method, which comprises the following steps: s1, obtaining immune cells; s2, obtaining human autologous plasma; s3, preparing a cell cryopreservation solution; and S4, performing cell cryopreservation. The method for freezing the immune cells can effectively prevent the frozen damage of the immune cells and improve the recovery survival rate of the immune cells, the resveratrol added into the cell freezing solution can promote the proliferation capacity of the recovered immune cells so as to improve the activity of the frozen immune cells after recovery, the hydroxyethyl starch, the propylene glycol and the polyethylene glycol can replace the protection effect of dimethyl sulfoxide, the use amount of the dimethyl sulfoxide is reduced, the water in the cells can not be crystallized when the cells are close to the freezing point, the damage to the cells is small, the foreign protein can not be introduced into the autologous human plasma added into the cell freezing solution, the possibility of the pollution of animal sources is reduced, and the potential safety hazard of the foreign serum is avoided.
Description
Technical Field
The invention relates to an immune cell cryopreservation method, and belongs to the technical field of cell engineering.
Background
The immune cell therapy is a new type of autoimmune anticancer therapy, and it is a method of using biotechnology and biological preparation to culture and expand the immune cells collected from the body of patient in vitro and then return them to the body of patient, so as to excite and enhance the body's autoimmune function, thus achieving the goal of treating tumor. For example, immune cells are cryopreserved, immediately thawed when needed, and reinfused.
A permeable protective agent is often adopted to reduce the damage of the freezing process to cells during cell freezing, at present, the commonly used freezing protective agent is dimethyl sulfoxide (DMSO), the substance can quickly penetrate into the cells, the permeability of cell membranes to water is improved, water is enabled to penetrate out of the cells to form ice crystals before the cells are frozen, the opportunity of forming the ice crystals in the cells is reduced, and therefore the damage of the ice crystals to the cells is reduced.
The existing cell frozen stock solution is mostly mixed with dimethyl sulfoxide and animal serum, and the dimethyl sulfoxide has high content, so that the cell frozen stock solution has considerable toxicity and is not beneficial to the body health of patients, and the animal serum contains a large amount of foreign proteins and has the risks of infectious diseases, anaphylactic reactions or immunological rejection.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an immune cell cryopreservation method which can effectively prevent the immune cells from being frozen and damaged and improve the recovery survival rate of the immune cells.
In order to achieve the above object, the present invention provides a method for preparing a frozen immune cell, comprising the following steps:
s1, obtaining immune cells
S2, obtaining human autologous plasma
S3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and is prepared by uniformly mixing the components of the cell frozen stock solution and keeping the temperature at 2-5 ℃ for 2 h;
s4, performing cell cryopreservation
Slowly adding the cell cryopreservation solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare cell cryopreservation suspension, then preserving heat at 5 ℃ for 30min, preserving at-20 ℃ for 2h, preserving at-50 ℃ for 3h, cooling to-80 ℃, preserving for 5h, then preserving at-20 ℃ for 2h, preserving at-80 ℃ for 5-8h, and finally transferring to a liquid nitrogen tank for preservation.
By adopting the scheme, the cell culture medium can keep osmotic pressure and acid-base balance of immune cells and provide nutrition, the human serum albumin is helpful for protecting the cell structure from being damaged, the survival period of the cells is prolonged, the carbohydrate can ensure that the proliferation capability of the cells after recovery is not influenced, the frozen stock solution containing resveratrol is used for freezing the immune cells and can promote the proliferation capability of the recovered immune cells, the hydroxyethyl starch, the propylene glycol and the polyethylene glycol can replace the protection effect of dimethyl sulfoxide, the use amount of the dimethyl sulfoxide is reduced, the water in the cells can not be crystallized when the cells are close to the freezing point, the damage to the cells is small, the human autologous plasma is similar to the serum components, the cell factors and the nutrient substances required by the growth of the immune cells are contained, the animal serum is not contained, foreign protein can not be introduced, and the possibility of the pollution of animal sources is reduced, the potential safety hazard of exogenous serum is avoided, when the immune cells are frozen, the temperature is gradually reduced to-80 ℃ firstly, the damage of the cells can be reduced, then the immune cells are placed at-20 ℃ for storage, and then the immune cells are placed at-80 ℃ for storage, so that the activity of reviving the frozen immune cells can be improved.
Preferably, in step S1, the normal saline or Ficoll separation solution is mixed with peripheral blood or cord blood, centrifuged at 2000rpm for 8-10 minutes, the middle leukocyte layer is aspirated, and the mixture is washed twice with normal saline to separate immune cells.
By adopting the scheme, the immune cells are obtained from the peripheral blood or the cord blood of the human body.
Preferably, in step S1, the temperature of centrifugation is 30-37 ℃.
By adopting the scheme, the immune cells are kept to have higher activity.
Preferably, step S2 specifically includes placing human peripheral blood or umbilical cord blood into a sterile centrifuge tube, centrifuging at 15-20 ℃ and 2000rpm for 4-6 minutes, taking the upper plasma, inactivating, transferring to a new centrifuge tube, centrifuging at 3000-5000rpm for 15-20 minutes, and collecting the supernatant to obtain human autologous plasma.
By adopting the scheme, the human autologous plasma is obtained from human peripheral blood or umbilical blood.
Preferably, in step S2, the inactivation temperature is 62-65 ℃.
By adopting the scheme, the bacteria in human peripheral blood or umbilical blood are reduced.
Preferably, in step S3, the cell frozen stock solution contains 0.1-0.4mg/mL of dimethyl sulfoxide, 0.5-0.7mL/mL of cell culture medium, 1-5mg/mL of human serum albumin, 0.3-4mg/mL of sugar, 10-50 μ g/mL of resveratrol, 6-8mg/mL of hydroxyethyl starch, 5-10mg/mL of propylene glycol, 5-10mg/mL of polyethylene glycol, and 60-70% of human autologous plasma based on the total volume of the cell frozen stock solution.
Preferably, in step S3, the cell culture medium is any one of RPMI-1640 medium, GT-T561 medium, AIM-V medium, X-VIVO15 medium, and GT-T551 medium.
Preferably, in step S3, the cell freezing medium further comprises non-essential amino acids, and the addition amount of the non-essential amino acids is 10-20 mg/mL.
By adopting the scheme, the added non-essential amino acid can ensure that the cell proliferation activity after recovery is not influenced.
Preferably, in step S3, the cell freezing medium further comprises dextran, and the dextran is added in an amount of 2-6 mg/mL.
By adopting the scheme, the dextran can maintain osmotic pressure and pH value and provide a medium environment suitable for cell survival.
Preferably, in step S4, when the cell cryopreservation suspension is prepared, the density of the cells is 5X 105Per mL-5X 106one/mL.
Compared with the prior art, the invention has the following beneficial effects:
(1) the resveratrol is added into the cell frozen stock solution, so that the proliferation capability of recovered immune cells can be promoted, and the activity of the recovered frozen immune cells can be effectively improved.
(2) The hydroxyethyl starch, the propylene glycol and the polyethylene glycol can replace the protection effect of dimethyl sulfoxide, reduce the using amount of the dimethyl sulfoxide, ensure that water in cells cannot be crystallized when the cells are close to a freezing point, and have small damage to the cells.
(3) The human autologous plasma added into the cell frozen stock solution is similar to serum components, contains cell factors and nutrient substances required by immune cell growth, does not contain animal serum, does not introduce exogenous protein, reduces the possibility of animal pathogen pollution, and avoids potential safety hazards of exogenous serum.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
An immune cell cryopreservation method comprises the following steps:
s1, obtaining immune cells
Mixing normal saline, Ficoll separating medium and peripheral blood or cord blood, centrifuging at 2000rpm for 8 min at 37 deg.C, sucking the middle leucocyte layer, washing with normal saline twice, and separating to obtain immunocyte;
s2, obtaining human autologous plasma
Placing human peripheral blood or cord blood in an aseptic centrifuge tube, centrifuging for 4 minutes at the rotation speed of 2000rpm at 15 ℃, inactivating upper layer plasma at 62 ℃, transferring to a new centrifuge tube, centrifuging for 15 minutes at the rotation speed of 3000rpm, and sucking supernatant to obtain human autologous plasma;
s3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and the cell frozen stock solution is obtained by uniformly mixing the components of the cell frozen stock solution and then preserving the heat for 2 hours at the temperature of 2 ℃;
the content of dimethyl sulfoxide in the cell frozen stock solution is 0.1mg/mL, the content of a cell culture medium is 0.5mL/mL, the content of human serum albumin is 5mg/mL, the content of saccharides is 0.3mg/mL, the content of resveratrol is 50 mug/mL, the content of hydroxyethyl starch is 6mg/mL, the content of propylene glycol is 10mg/mL, the content of polyethylene glycol is 10mg/mL, and the content of human autologous plasma is 60% of the total volume of the cell frozen stock solution;
the cell culture medium is RPMI-1640 culture medium;
the cell frozen stock solution also comprises non-essential amino acid, and the addition amount of the non-essential amino acid is 10 mg/mL;
the cell freezing solution also comprises dextran, and the addition amount of the dextran is 6 mg/mL;
s4, performing cell cryopreservation
Slowly adding the cell frozen stock solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare a cell frozen stock suspension, wherein the density of the cells in the cell frozen stock suspension is 5 multiplied by 105Keeping the temperature at 5 ℃ for 30min, storing at-20 ℃ for 2h, storing at-50 ℃ for 3h, cooling to-80 ℃, storing for 5h, storing at-20 ℃ for 2h, storing at-80 ℃ for 8h, and transferring to a liquid nitrogen tank for storing.
Example 2
An immune cell cryopreservation method comprises the following steps:
s1, obtaining immune cells
Mixing normal saline, Ficoll separating medium and peripheral blood or cord blood, centrifuging at 2000rpm for 10 min at 30 deg.C, sucking the middle leucocyte layer, washing with normal saline twice, and separating to obtain immunocyte;
s2, obtaining human autologous plasma
Placing human peripheral blood or cord blood in a sterile centrifuge tube, centrifuging for 6 minutes at the rotating speed of 2000rpm at 20 ℃, inactivating upper layer plasma at 65 ℃, transferring to a new centrifuge tube, centrifuging for 20 minutes at the rotating speed of 5000rpm, and sucking supernatant to obtain human autologous plasma;
s3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and is prepared by uniformly mixing the components of the cell frozen stock solution and keeping the temperature at 5 ℃ for 2 hours;
the content of dimethyl sulfoxide in the cell frozen stock solution is 0.4mg/mL, the content of a cell culture medium is 0.7mL/mL, the content of human serum albumin is 1mg/mL, the content of saccharides is 4mg/mL, the addition amount of resveratrol is 50 mug/mL, the content of hydroxyethyl starch is 8mg/mL, the content of propylene glycol is 5mg/mL, the content of polyethylene glycol is 5mg/mL, and the content of human autologous plasma is 70% of the total volume of the cell frozen stock solution;
the cell culture medium is GT-T561 culture medium;
the cell frozen stock solution also comprises non-essential amino acid, and the addition amount of the non-essential amino acid is 17 mg/mL;
the cell freezing solution also comprises dextran, and the addition amount of the dextran is 2 mg/mL;
s4, performing cell cryopreservation
Slowly adding the cell frozen stock solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare a cell frozen stock suspension, wherein the density of the cells in the cell frozen stock suspension is 5 multiplied by 106Keeping the temperature at 5 ℃ for 30min, storing at-20 ℃ for 2h, storing at-50 ℃ for 3h, cooling to-80 ℃, storing for 5h, storing at-20 ℃ for 2h, storing at-80 ℃ for 5h, and transferring to a liquid nitrogen tank for storing.
Example 3
An immune cell cryopreservation method comprises the following steps:
s1, obtaining immune cells
Mixing normal saline, Ficoll separating medium and peripheral blood or cord blood, centrifuging at 2000rpm for 10 min at 35 deg.C, sucking the middle leucocyte layer, washing with normal saline twice, and separating to obtain immunocyte;
s2, obtaining human autologous plasma
Placing human peripheral blood or cord blood in an aseptic centrifuge tube, centrifuging for 4 minutes at the rotating speed of 2000rpm at 20 ℃, inactivating upper plasma at 64 ℃, transferring to a new centrifuge tube, centrifuging for 17 minutes at the rotating speed of 4000rpm, and sucking supernatant to obtain human autologous plasma;
s3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and the cell frozen stock solution is obtained by uniformly mixing the components of the cell frozen stock solution and then preserving the heat for 2 hours at the temperature of 4 ℃;
the content of dimethyl sulfoxide in the cell frozen stock solution is 0.2mg/mL, the content of a cell culture medium is 0.6mL/mL, the content of human serum albumin is 3mg/mL, the content of saccharides is 2mg/mL, the content of resveratrol is 30 mu g/mL, the content of hydroxyethyl starch is 7mg/mL, the content of propylene glycol is 8mg/mL, the content of polyethylene glycol is 8mg/mL, and the content of human autologous plasma is 65% of the total volume of the cell frozen stock solution;
the cell culture medium is an AIM-V culture medium;
the cell freezing medium also comprises non-essential amino acid, and the addition amount of the non-essential amino acid is 14 mg/mL;
the cell freezing solution also comprises dextran, and the addition amount of the dextran is 4 mg/mL;
s4, performing cell cryopreservation
Slowly adding the cell frozen stock solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare a cell frozen stock suspension, wherein the density of the cells in the cell frozen stock suspension is 2 multiplied by 106Keeping the temperature at 5 ℃ for 30min, storing at-20 ℃ for 2h, storing at-50 ℃ for 3h, cooling to-80 ℃, storing for 5h, storing at-20 ℃ for 2h, storing at-80 ℃ for 6h, and transferring to a liquid nitrogen tank for storing.
Example 4
An immune cell cryopreservation method comprises the following steps:
s1, obtaining immune cells
Mixing normal saline, Ficoll separating medium and peripheral blood or cord blood, centrifuging at 2000rpm for 8 min at 30 deg.C, sucking the middle leucocyte layer, washing with normal saline twice, and separating to obtain immunocyte;
s2, obtaining human autologous plasma
Placing human peripheral blood or cord blood in an aseptic centrifuge tube, centrifuging for 6 minutes at the rotating speed of 2000rpm at 20 ℃, inactivating upper layer plasma at 65 ℃, transferring to a new centrifuge tube, centrifuging for 20 minutes at the rotating speed of 4000rpm, and sucking supernatant to obtain human autologous plasma;
s3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and is prepared by uniformly mixing the components of the cell frozen stock solution and keeping the temperature at 3 ℃ for 2 hours to obtain the cell frozen stock solution;
the content of dimethyl sulfoxide in the cell frozen stock solution is 0.3mg/mL, the content of a cell culture medium is 0.7mL/mL, the content of human serum albumin is 3mg/mL, the content of saccharides is 3mg/mL, the content of resveratrol is 10 mu g/mL, the content of hydroxyethyl starch is 8mg/mL, the content of propylene glycol is 10mg/mL, the content of polyethylene glycol is 5mg/mL, and the content of human autologous plasma is 60% of the total volume of the cell frozen stock solution;
the cell culture medium is GT-T551 culture medium;
the cell frozen stock solution also comprises non-essential amino acid, and the addition amount of the non-essential amino acid is 20 mg/mL;
the cell freezing solution also comprises dextran, and the addition amount of the dextran is 6 mg/mL;
s4, performing cell cryopreservation
Slowly adding the cell frozen stock solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare a cell frozen stock suspension, wherein the density of the cells in the cell frozen stock suspension is 5 multiplied by 105Keeping the temperature at 5 ℃ for 30min, storing at-20 ℃ for 2h, storing at-50 ℃ for 3h, cooling to-80 ℃, storing for 5h, storing at-20 ℃ for 2h, storing at-80 ℃ for 7h, and transferring to a liquid nitrogen tank for storing.
Recovering immune cells: taking out immune cells preserved in liquid nitrogen for 10 days, 30 days and 60 days, quickly placing the immune cells in water bath at 40 ℃ for 3 minutes, oscillating until cell suspension is completely melted, carrying out trypan blue staining on the thawed immune cells, and then calculating the activity.
Test example 1 Effect of resveratrol, hydroxyethyl starch, propylene glycol and polyethylene glycol on test results
Comparative examples 1 to 4: in the comparative experiments corresponding to examples 1-4, except that resveratrol was not added in step S3, the other experimental steps were identical to the respective steps of the corresponding examples, and then immune cell resuscitation was performed, and the specific experimental results are detailed in table 1.
Comparative examples 5 to 8: in comparative experiments corresponding to comparative examples 1 to 4, except that hydroxyethyl starch, propylene glycol and polyethylene glycol added in step S3 were replaced with dimethyl sulfoxide of equal mass, the other experimental steps were identical to the respective steps of the corresponding comparative examples, and then immune cell resuscitation was performed, and the specific experimental results are detailed in table 1.
Comparative examples 9 to 12: the experimental procedures were exactly the same as those of the corresponding examples except that cryo-stock CS10 series of cell frozen stocks obtained from Stemcell, canada were used, and then immune cells were recovered, and the detailed experimental results are shown in table 1.
TABLE 1 results of calculation of the immunocyte Activity of examples 1 to 4 and comparative examples 1 to 12
As can be seen from Table 1, compared with examples 1-4, the activity of the immune cells of comparative examples 1-4 after recovery after cryopreservation is obviously lower than that of corresponding examples 1-4, which indicates that the resveratrol added in examples 1-4 can effectively improve the activity of the frozen immune cells after recovery;
compared with comparative examples 1-4, the activity of the immune cells of comparative examples 5-8 after cell recovery after cryopreservation is basically the same as that of the corresponding comparative examples 1-4, which shows that the dimethyl sulfoxide in the step S3 is replaced by hydroxyethyl starch, propylene glycol and polyethylene glycol with equal mass, so that the freezing damage of the immune cells can be effectively prevented, and the activity of the immune cells after recovery is basically unchanged;
compared with the examples 1-4, the activity of the immune cells in the comparative examples 9-12 after being frozen is obviously lower than that of the immune cells in the examples 1-4, the activity of the immune cells in the example 3 after being frozen for 10 days is 99.0%, the activity of the immune cells after being frozen for 30 days is 98.9%, and the activity of the immune cells after being frozen for 60 days is 98.5%, which shows that the activity of the immune cells frozen by the freezing method is higher after being frozen.
Test example 2 Effect of freezing temperature program on test results
Comparative examples 13 to 16: in the comparative experiments corresponding to examples 1-4, the freezing temperature procedure in step S4 is to put the prepared cell suspension directly into-80 ℃ for preservation, and finally transfer the cell suspension into a liquid nitrogen tank for preservation, wherein the preservation time is the same as that in the corresponding examples, the other experimental steps are completely the same as those in the corresponding examples, and then immune cells are recovered, and the specific experimental results are detailed in table 2.
TABLE 2 calculation results of the immunocyte activities of examples 1 to 4 and comparative examples 13 to 16
As can be seen from Table 2, compared with comparative examples 13-16, the activity of the immune cells of examples 1-4 after recovery after freezing is significantly higher than that of the corresponding comparative examples 13-16, which illustrates that the freezing temperature program of step S4 in examples 1-4 can effectively improve the activity of the frozen immune cells after recovery.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. The immune cell cryopreservation method is characterized by comprising the following steps of:
s1, obtaining immune cells
S2, obtaining human autologous plasma
S3, preparing cell cryopreservation liquid
The cell frozen stock solution comprises dimethyl sulfoxide, a cell culture medium, human serum albumin, saccharides, resveratrol, hydroxyethyl starch, propylene glycol, polyethylene glycol and human autologous plasma, and is prepared by uniformly mixing the components of the cell frozen stock solution and keeping the temperature at 2-5 ℃ for 2 h;
s4, performing cell cryopreservation
Slowly adding the cell cryopreservation solution prepared in the step S3 into the immune cells obtained in the step S1 to prepare cell cryopreservation suspension, then preserving heat at 5 ℃ for 30min, preserving at-20 ℃ for 2h, preserving at-50 ℃ for 3h, cooling to-80 ℃, preserving for 5h, then preserving at-20 ℃ for 2h, preserving at-80 ℃ for 5-8h, and finally transferring to a liquid nitrogen tank for preservation.
2. The method according to claim 1, wherein step S1 comprises mixing normal saline and Ficoll separating medium with peripheral blood or cord blood, centrifuging at 2000rpm for 8-10 min, sucking the middle leukocyte layer, washing with normal saline twice, and separating to obtain immune cells.
3. The method for cryopreserving an immune cell according to claim 2, wherein the temperature of centrifugation in step S1 is 30-37 ℃.
4. The method of claim 1, wherein the step S2 comprises placing human peripheral blood or umbilical cord blood into a sterile centrifuge tube, centrifuging at 2000rpm at 15-20 ℃ for 4-6 minutes, inactivating the upper plasma layer, transferring to a new centrifuge tube, centrifuging at 5000rpm for 15-20 minutes, and collecting the supernatant to obtain autologous plasma.
5. The method for cryopreserving the immune cells, according to claim 4, wherein the inactivation temperature is 62-65 ℃ in step S2.
6. The method of claim 1, wherein in step S3, the cell frozen solution contains 0.1-0.4mg/mL of dimethyl sulfoxide, 0.5-0.7mL/mL of cell culture medium, 1-5mg/mL of human serum albumin, 0.3-4mg/mL of sugar, 10-50 μ g/mL of resveratrol, 6-8mg/mL of hydroxyethyl starch, 5-10mg/mL of propylene glycol, 5-10mg/mL of polyethylene glycol, and the total volume of human autologous plasma is 60-70% of the total volume of the cell frozen solution.
7. The method of claim 1, wherein in step S3, the cell culture medium is any one of RPMI-1640 medium, GT-T561 medium, AIM-V medium, X-VIVO15 medium, GT-T551 medium.
8. The method according to claim 1, wherein in step S3, the frozen cell stock solution further comprises non-essential amino acids, and the amount of the non-essential amino acids added is 10-20 mg/mL.
9. The method of claim 1, wherein in step S3, the frozen cell stock solution further comprises dextran, and the dextran is added in an amount of 2-6 mg/mL.
10. The method of claim 1, wherein the cell density of the frozen cell suspension prepared in step S4 is 5X 105Per mL-5X 106one/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110003175.8A CN112753695A (en) | 2021-01-04 | 2021-01-04 | Immune cell cryopreservation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110003175.8A CN112753695A (en) | 2021-01-04 | 2021-01-04 | Immune cell cryopreservation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112753695A true CN112753695A (en) | 2021-05-07 |
Family
ID=75698954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110003175.8A Pending CN112753695A (en) | 2021-01-04 | 2021-01-04 | Immune cell cryopreservation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112753695A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181901A (en) * | 2021-12-08 | 2022-03-15 | 杭州中赢生物医疗科技有限公司 | Method for in vitro induced amplification and cryopreservation of immune cells |
| CN114521549A (en) * | 2022-02-28 | 2022-05-24 | 四川中科博瑞生物科技有限公司 | Immune cell cryopreservation liquid and application thereof, cell cryopreservation method and cell recovery method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013138239A1 (en) * | 2012-03-14 | 2013-09-19 | Membrane Protective Technologies, Inc. | System and substances for cryopreservation of viable cells |
| CN107047541A (en) * | 2017-05-31 | 2017-08-18 | 东莞市保莱生物科技有限公司 | A kind of immunocyte frozen stock solution and immunocyte cryopreservation methods |
| CN107094753A (en) * | 2017-05-31 | 2017-08-29 | 东莞市保莱生物科技有限公司 | A kind of candidate stem cell frozen stock solution and candidate stem cell cryopreservation methods |
| CN110447636A (en) * | 2019-08-07 | 2019-11-15 | 广东万海细胞生物科技有限公司 | A kind of frozen stock solution of peripheral blood mononuclear cells, cryopreservation methods and method for resuscitation |
-
2021
- 2021-01-04 CN CN202110003175.8A patent/CN112753695A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013138239A1 (en) * | 2012-03-14 | 2013-09-19 | Membrane Protective Technologies, Inc. | System and substances for cryopreservation of viable cells |
| CN107047541A (en) * | 2017-05-31 | 2017-08-18 | 东莞市保莱生物科技有限公司 | A kind of immunocyte frozen stock solution and immunocyte cryopreservation methods |
| CN107094753A (en) * | 2017-05-31 | 2017-08-29 | 东莞市保莱生物科技有限公司 | A kind of candidate stem cell frozen stock solution and candidate stem cell cryopreservation methods |
| CN110447636A (en) * | 2019-08-07 | 2019-11-15 | 广东万海细胞生物科技有限公司 | A kind of frozen stock solution of peripheral blood mononuclear cells, cryopreservation methods and method for resuscitation |
Non-Patent Citations (1)
| Title |
|---|
| 邱佳裔 等: ""细胞、组织冻存方法及应用的研究进展"", 《中国生物制品学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181901A (en) * | 2021-12-08 | 2022-03-15 | 杭州中赢生物医疗科技有限公司 | Method for in vitro induced amplification and cryopreservation of immune cells |
| CN114181901B (en) * | 2021-12-08 | 2024-02-02 | 杭州中赢生物医疗科技有限公司 | Method for in vitro induction expansion and cryopreservation of immune cells |
| CN114521549A (en) * | 2022-02-28 | 2022-05-24 | 四川中科博瑞生物科技有限公司 | Immune cell cryopreservation liquid and application thereof, cell cryopreservation method and cell recovery method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110050782B (en) | Stem cell cryopreservation solution and preparation method and cryopreservation method thereof | |
| KR920005684B1 (en) | Synthetic plasma-free transfusible platelet storage medium | |
| AU693339B2 (en) | Cryopreservation solution | |
| JP2000516963A (en) | Erythrocyte long-term preservation method using oxygen removal and additives | |
| US5309723A (en) | Method of freezing red blood cells | |
| CN112753695A (en) | Immune cell cryopreservation method | |
| WO2001045503A2 (en) | Cryopreservation compositions and methods for peripheral blood lymphocytes | |
| CN102669087A (en) | Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method | |
| CN112400863A (en) | Clinical NK cell cryopreservation liquid and cryopreservation method | |
| CN112544611B (en) | Cell cryopreservation agent and cell cryopreservation method | |
| CN104739865A (en) | Method for preparing placenta hematopoietic stem cell preparation | |
| CN112806354A (en) | Immune cell cryopreservation liquid as well as preparation method and application thereof | |
| CN107711823B (en) | Cell cryopreservation liquid stored at normal temperature and application thereof | |
| CN108812643B (en) | Preparation and cryopreservation method and application of human placental chorionic villus tissue | |
| CN113854280B (en) | Low-temperature preservation solution and preparation method and application thereof | |
| CN113925049B (en) | Cell preservation solution for maintaining cell activity and preparation method and application thereof | |
| CN111602648A (en) | Immune cell serum-free cryopreservation liquid and cryopreservation method | |
| CN115802889A (en) | Low temperature storage of biological samples | |
| EP3863649A1 (en) | Oxygenation media for ex-vivo preservation of organs and tissues | |
| CN108812642B (en) | Systematic method for preparing placenta tissue according to structural hierarchy and cryopreserving and application | |
| CN114009425B (en) | Immune cell vitrification cryopreservation protective solution and cryopreservation method thereof | |
| CN111449053A (en) | Immune cell storage liquid and preparation and application methods thereof | |
| CN115005199A (en) | Cryopreservation solution and cryopreservation method for natural killer cells and application of cryopreservation solution and cryopreservation method | |
| CN108812641B (en) | Preparation and cryopreservation method and application of human placenta villus tissue | |
| EP0061277A2 (en) | Anaerobic method for preserving whole blood, tissue and components containing living mammalian cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210507 |
|
| RJ01 | Rejection of invention patent application after publication |