CN113068684A - Cryopreservation and recovery method of human umbilical cord mesenchymal stem cells - Google Patents
Cryopreservation and recovery method of human umbilical cord mesenchymal stem cells Download PDFInfo
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- CN113068684A CN113068684A CN202110247535.9A CN202110247535A CN113068684A CN 113068684 A CN113068684 A CN 113068684A CN 202110247535 A CN202110247535 A CN 202110247535A CN 113068684 A CN113068684 A CN 113068684A
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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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- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
Abstract
The invention relates to the technical field of stem cells, in particular to a cryopreservation and recovery method of human umbilical cord mesenchymal stem cells. The method comprises a human umbilical cord mesenchymal stem cell cryopreservation method, a recovery method and a tracking and recording platform, wherein the umbilical cord mesenchymal stem cells are preserved through cryopreservation and recovery, each process from collection to transportation, detection to culture, and cryopreservation to recovery is strictly monitored in quality, a unique corresponding record with a buckled loop is formed, and the uniqueness and traceability of a stem cell sample are ensured. And the method also can provide an important basis of a powerful theoretical basis for researching the characteristics of morphology, growth characteristics, cell cycle, immunophenotype and the like of the umbilical cord mesenchymal stem cells, establishing a human umbilical cord mesenchymal stem cell bank and clinical application.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a cryopreservation and recovery method of human umbilical cord mesenchymal stem cells.
Background
Umbilical cord Mesenchymal Stem Cells (MSCs) refer to a multifunctional Stem cell existing in umbilical cord tissue of a newborn, and have high differentiation potential and can be differentiated in multiple directions. It has wide clinical application prospect in the aspects of tissue engineering of bones, cartilages, muscles, tendons, ligaments, nerves, livers, endothelia, cardiac muscles and the like.
The application of the inactivated umbilical cord serum culture system can successfully amplify human umbilical cord mesenchymal stem cells, and the cultured cells have the basic characteristics of the mesenchymal stem cells, thereby providing a theoretical basis for establishing a mesenchymal stem cell bank and clinical application.
The MSCs are separated from human umbilical cords, the cell content and the proliferation capacity are superior to those of bone marrow MSCs, the immunogenicity is lower than that of bone marrow MSCs, and the method has the advantages of convenient material acquisition, no ethical conflict and the like, so the method is more and more concerned by researchers.
The umbilical cord mesenchymal stem cells have stronger immunoregulation function, can be used for treating autoimmune diseases such as lupus erythematosus and scleroderma, reducing immunological rejection reaction after cell or organ transplantation and improving the success rate of cell or organ transplantation; the hematopoietic recovery function can be promoted, and compared with single hematopoietic stem cell transplantation, the mesenchymal stem cell and hematopoietic stem cell co-transplantation can remarkably improve the treatment effect of diseases such as leukemia, refractory anemia and the like; can repair damaged or diseased tissue organ, and can be used for treating degenerative diseases of bone and muscle, cardiovascular and cerebrovascular diseases, liver diseases, brain and spinal nerve injury, senile dementia, etc.
Cell cryopreservation is a technique for placing cells in a low-temperature environment, reducing cell metabolism and facilitating long-term storage. Cell cryopreservation is one of the main methods for cell preservation, and plays a role in cell seed preservation. In the process of passage and daily maintenance of cell culture, a great deal of cost is needed in culture instruments, culture solutions and various preparation works, and once the cells leave a living body and start primary culture, various biological characteristics of the cells gradually change and continuously and newly change along with the increase of the passage times and the change of in vitro environmental conditions. Therefore, it is necessary to freeze and store the cells in time. The cells can be stored for one year in a refrigerator at-70 ℃ after being frozen; the cells were stored in liquid nitrogen at a temperature of-196 ℃ for a theoretically unlimited storage time. The cells are stored in liquid nitrogen at the temperature of-196 ℃ by using a freezing technology at a low temperature, so that the cells can be temporarily separated from a growth state and the cell characteristics can be stored, and the cells are recovered for experiments when needed. Moreover, a certain amount of cells are preserved appropriately, so that the cells can be prevented from being lost due to contamination or other accidents of the cultured cells, and the function of preserving the cells is achieved. In addition, some cells may be purchased, hosted, exchanged, and shipped in their cryopreserved form. When the cells are frozen, a protective agent, namely glycerol or dimethyl sulfoxide (DMSO) with the final concentration of 5 percent or 15 percent, is added into the culture medium, so that the freezing point of the solution can be lowered, and in addition, water in the cells can permeate out under the condition of slow freezing, the formation of ice crystals is reduced, and the cells are prevented from being damaged.
Cell recovery is the process opposite to cell cryopreservation, namely the process of cell recovery growth, and is to culture the cells after thawing the frozen cells in liquid nitrogen or a refrigerator at-70 ℃. When the normal temperature state is recovered, the morphological structure of the cell is kept normal, and the biochemical reaction can be recovered. Different from cell freezing, the temperature rise in the cell recovery process is fast, so that water is prevented from entering cells in the unfreezing process to form ice crystals, and the survival of the cells is prevented from being influenced.
The basic principle of cell freezing and recovery is slow freezing and fast thawing, and experiments prove that the cell activity can be preserved to the maximum extent. At present, glycerol or dimethyl sulfoxide is mostly used as a protective agent for cell freezing storage, the glycerol or dimethyl sulfoxide can improve the permeability of a cell membrane to water, and slow freezing can enable water in cells to seep out of the cells and reduce the formation of ice crystals in the cells, so that cell damage caused by the formation of ice crystals is reduced. The cell is revived by adopting a rapid thawing method, so that extracellular crystals can be thawed within a short time, and the damage to the cells caused by the water permeating into the cells to form intracellular recrystallization due to slow thawing can be avoided.
Disclosure of Invention
The invention aims to provide a cryopreservation and recovery method of umbilical cord mesenchymal stem cells, which realizes the preservation of the umbilical cord mesenchymal stem cells through cryopreservation and recovery. And also provides an important basis of a powerful theoretical basis for researching the characteristics of the umbilical cord mesenchymal stem cell morphology, the growth characteristics, the cell cycle, the immunophenotype and the like, establishing a human umbilical cord mesenchymal stem cell bank and clinical application.
In order to achieve the purpose, the invention provides the following technical scheme:
a cryopreservation and recovery method of human umbilical cord mesenchymal stem cells comprises the following steps:
d1 ultraviolet irradiation of the cell chamber and the workbench for 20-30 min; preheating culture solution, frozen stock solution, 9% sodium chloride injection, pancreatin, calf serum, DMSO, and constant temperature water bath box at 40 deg.C for use; collecting cells in logarithmic growth phase, and changing liquid one day before freezing;
preparing a freezing culture solution containing 10% of DMSO or glycerol and 10-20% of calf serum; preparing 9% sodium chloride injection, trypsin and DMSO;
d2 taking the pre-frozen cells, removing old culture solution, and washing with 9% sodium chloride injection;
d3 removing 9% sodium chloride injection, adding appropriate amount of trypsin to digest the cells grown in monolayer;
d4 adding culture medium to stop digestion, blowing to suspend cells, transferring to a centrifuge tube, sampling, counting, centrifuging at 1200rpm for 10 min;
d5 removing trypsin, adding a proper amount of prepared frozen stock solution, lightly blowing and beating the cells by using a suction pipe to make the cells uniform, counting, and adjusting the final density of the cells in the frozen stock solution to be 5 × 106/ml-1 × 107/ml;
d6 subpackaging the cells into frozen tubes, wherein each tube contains 1-2.5 ml of cells;
d7 controlling the freezing speed, and transferring the freezing tube into a liquid nitrogen container for storage when the storage standard is met;
d8 indicates the cell name, the freezing time and the operator on the freezing tube.
Further, in the step D7, placing the freezing tube filled with the cells into a 4 ℃ medicine storage box for precooling for 10-30min, transferring the freezing tube into a-80 ℃ ultra-low temperature medicine storage box in a program cooling box under the protection of a low temperature sample transfer vehicle, storing for 4-72 h, and transferring to a gas phase liquid nitrogen storage tank; or the like, or, alternatively,
d7, placing the freezing tube filled with the cells into a 4 ℃ medicine storage box for precooling for 10-30min, placing the freezing tube into a-20 ℃ medicine storage box for 0.5-1h, then placing the freezing tube into a-70 ℃ medicine storage box for 8-16h, taking out the freezing tube, and transferring the freezing tube into a liquid nitrogen container; or the like, or, alternatively,
the control mode of the freezing temperature in the step D7 is the cooling rate of-1 to-2 ℃/min; when the temperature reaches below minus 25 ℃, the temperature is increased to minus 5 ℃ to minus 10 ℃/min; when the temperature is reduced to-100 ℃, the mixture is transferred to a gas-phase liquid nitrogen storage tank.
Further, the pre-frozen cells in the step D2 may be cultured cells from the proliferation stage until the formation of a dense monolayer of cells, preferably cells in the logarithmic growth phase.
Further, the culture medium was replaced within 24 hours before the cells were frozen.
Further, the resuscitation method comprises the following steps:
s1, taking out the freezing tube from the liquid nitrogen container, directly immersing the tube in a constant-temperature water bath kettle at 40 ℃, and shaking the tube from time to melt the tube as soon as possible;
s2 taking out the freezing tube from the water bath kettle with constant temperature of 40 ℃, quickly wiping the outside of the freezing tube with alcohol cotton balls, sterilizing and airing;
s3, sucking the cell suspension by a suction tube, adding the sodium chloride injection precooled at 4 ℃, and re-suspending and mixing uniformly;
s4 centrifuging at 1200rpm for 8 min;
s5, discarding supernatant, transferring the cells into culture solution containing 10% calf serum to resuspend the cells, counting, and adjusting cell density;
s6 inoculating a culture bottle, then transferring the culture bottle to an incubator, and culturing at 36-38 ℃ for 72h +/-24 h;
s7, replacing a fresh culture medium, proliferating to the required quantity, and harvesting cells;
s8 records the names, the number, the recovery time and the storage positions of the recovery cells in detail.
Further, in step S1, the cover of the constant temperature water bath is covered during the process of thawing the freezing tube; melting in a constant temperature water bath at 40 deg.C for 1-3min, and removing from 40 deg.C water bath.
Further, in step S3, the cell suspension is sucked out by a suction tube, added into a centrifuge tube and more than 10 times of culture solution is added dropwise, and the mixture is uniformly mixed in a biosafety cabinet at 4 ℃ for operation.
The system further comprises a tracking and recording platform, wherein the tracking and recording platform comprises a cell acquisition unit, a transportation tracking unit, a detection tracking unit, a cryopreservation unit, a resuscitation unit and a quality control unit;
the cell acquisition unit is used for recording and acquiring the information of the human umbilical cord mesenchymal stem cells and forming a unique coding identifier for the acquired information;
the transportation tracking unit is used for tracking and recording the whole transportation process of the collected human umbilical cord mesenchymal stem cells and forming a corresponding transportation record flow;
the detection tracking unit is used for tracking and recording the whole detection process of the human umbilical cord mesenchymal stem cells and forming a detection recording stream;
the cryopreservation unit is used for tracking and recording the whole process of cryopreservation of the human umbilical cord mesenchymal stem cells and forming a cryopreservation recording stream;
the recovery unit is used for tracking and recording the whole process of recovery of the human umbilical cord mesenchymal stem cells and forming a recovery recording stream;
and the quality control unit is used for tracking and recording the state of the human umbilical cord mesenchymal stem cells during cryopreservation and preservation, the quality detection process and a quality control report, and forming a quality control record flow.
Further, during the cryopreservation period of the cryopreservation of the human umbilical cord mesenchymal stem cells, periodically extracting a part of the cryopreserved cells to recover, and performing quality detection after recovery; and culturing the cells meeting the standard and then freezing the cells again to supplement the freezing amount.
Furthermore, the quality of the human umbilical cord mesenchymal stem cells is required to be checked before cell collection, before cryopreservation, during the freezing period and quality control period, before recovery and after recovery, and the checking items comprise one or more of sterility detection, cell activity detection, cell morphology detection, cell purity detection, product stability and safety detection.
The beneficial effect of this application:
this application is from gathering to the transportation, from detecting the cultivation, from freezing each in-process of depositing to recovering from life or consciousness, all just strict quality monitoring to each process to form the record that the loop is detained, only correspond, ensure the uniqueness and the traceability of stem cell sample. Starting from the collection of human umbilical cord mesenchymal stem cells, the related information of the stem cells, the related operation environment information, the related operation personnel information and the like are recorded in the whole process, and in key operations of detection, cryopreservation, resuscitation and the like, the simple character record is kept, and the video information can be recorded by matching with a camera. The intelligent information management and the cell traceability are realized, and a reliable basis for inquiring and tracing the cell archiving information is provided for a user.
The cryopreservation and recovery method for the umbilical cord mesenchymal stem cells realizes the preservation of the umbilical cord mesenchymal stem cells through cryopreservation and recovery, establishes an enrichment method for the umbilical cord mesenchymal stem cells for researching the characteristics of the umbilical cord mesenchymal stem cells such as morphology, growth characteristics, cell cycle, immunophenotype and the like, and provides an important basis of powerful theoretical basis for establishing an umbilical cord mesenchymal stem cell bank and clinical application.
The cells were put into a resting state by deep cryogenic liquid nitrogen storage at-196 ℃ storage environment. The cell death rate can be improved by adopting 40 ℃ water bath for prolonging, and the cell death rate is generally between 20 and 25 percent in the recovery process. The stem cells are transported by monitoring the temperature in the whole process, so that the temperature stability in the transportation process is guaranteed.
Detailed Description
The technical solutions in the embodiments of the present invention will be described below clearly and completely in connection with the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The application discloses a cryopreservation and recovery method of human umbilical cord mesenchymal stem cells, which comprises the following steps:
d1 ultraviolet irradiation of the cell chamber and the workbench for 20-30 min; preheating culture solution, frozen stock solution, 9% sodium chloride injection, pancreatin, calf serum, DMSO, and constant temperature water bath box at 40 deg.C for use; collecting cells in logarithmic growth phase, and changing liquid one day before freezing;
preparing a freezing culture solution containing 10% of DMSO or glycerol and 10-20% of calf serum; preparing 9% sodium chloride injection, trypsin and DMSO;
d2 taking the pre-frozen cells, removing old culture solution, and washing with 9% sodium chloride injection;
d3 removing 9% sodium chloride injection, adding appropriate amount of trypsin to digest the cells grown in monolayer;
d4 adding culture medium to stop digestion, blowing to suspend cells, transferring to a centrifuge tube, sampling, counting, centrifuging at 1200rpm for 10 min;
d5 removing trypsin, adding a proper amount of prepared frozen stock solution, lightly blowing and beating the cells by using a suction pipe to make the cells uniform, counting, and adjusting the final density of the cells in the frozen stock solution to be 5 × 106/ml-1 × 107/ml;
d6 subpackaging the cells into frozen tubes, wherein each tube contains 1-2.5 ml of cells;
d7 controlling the freezing speed, and transferring the freezing tube into a liquid nitrogen container for storage when the storage standard is met;
d8 indicates the cell name, the freezing time and the operator on the freezing tube.
And D7, placing the freezing tube filled with the cells into a 4 ℃ medicine storage box for precooling for 10-30min, transferring the freezing tube into a-80 ℃ ultra-low temperature medicine storage box in a program cooling box under the protection of a low temperature sample transfer vehicle, storing for 4-72 h, and transferring to a gas phase liquid nitrogen storage tank. Or, in the step D7, placing the freezing tube filled with the cells into a 4 ℃ medicine storage box for precooling for 10-30min, placing the freezing tube into a-20 ℃ medicine storage box for 0.5-1h, then placing the freezing tube into a-70 ℃ medicine storage box for 8-16h, taking out the freezing tube, and transferring the freezing tube into a liquid nitrogen container. Optionally, the freezing temperature control mode in the step D7 is a cooling rate of-1 to-2 ℃/min; when the temperature reaches below minus 25 ℃, the temperature is increased to minus 5 ℃ to minus 10 ℃/min; when the temperature is reduced to-100 ℃, the mixture is transferred to a gas-phase liquid nitrogen storage tank. The first mode is preferably employed. Preferably, the culture medium is replaced within 24 hours before freezing the cells.
The deep low temperature liquid nitrogen is stored in a storage environment of-196 ℃, the cells enter a dormant state, and the liquid nitrogen storage is provided with a continuous power supply system and a centralized nitrogen supply system, so that the continuous and stable power and liquid nitrogen supply are realized, and the long-term stability of the deep low temperature storage condition is ensured. An alarm system and an oxygen concentration automatic monitoring and alarm system are arranged on the preservation liquid level so as to comprehensively ensure the cell safety and the personnel safety.
The application discloses a cryopreservation and recovery method of human umbilical cord mesenchymal stem cells, which comprises the following steps:
s1, taking out the freezing tube from the liquid nitrogen container, directly immersing the tube in a constant-temperature water bath kettle at 40 ℃, and shaking the tube from time to melt the tube as soon as possible;
s2 taking out the freezing tube from the water bath kettle with constant temperature of 40 ℃, quickly wiping the outside of the freezing tube with alcohol cotton balls, sterilizing and airing;
s3, sucking the cell suspension by a suction tube, adding the sodium chloride injection precooled at 4 ℃, and re-suspending and mixing uniformly;
s4 centrifuging at 1200rpm for 8 min;
s5, discarding supernatant, transferring the cells into culture solution containing 10% calf serum to resuspend the cells, counting, and adjusting cell density;
s6 inoculating a culture bottle, then transferring the culture bottle to an incubator, and culturing at 36-38 ℃ for 72h +/-24 h;
s7, replacing a fresh culture medium, proliferating to the required quantity, and harvesting cells;
s8 records the names, the number, the recovery time and the storage positions of the recovery cells in detail.
Wherein, in step S1, the cover of the constant temperature water bath kettle is covered in the process of unfreezing the freezing tube; melting in a constant temperature water bath at 40 deg.C for 1-3min, and removing from 40 deg.C water bath. The cell death rate can be improved by prolonging the water bath time at 40 ℃, and the cell death rate is generally between 20 and 25 percent in the recovery process. In step S3, the cell suspension is sucked out by a suction tube, added into a centrifuge tube and more than 10 times of culture solution is added dropwise, and the mixture is uniformly mixed in a biological safety cabinet at 4 ℃ for operation.
And judging whether the cell resuscitation is successful or not, wherein the cell adherence rate and the cell survival rate after resuscitation need to be considered. Cell viability remaining cells in the tube were cryopreserved and viability of the resuscitated cells was detected by trypan blue staining. Cells that appear blue are dead cells, and living cells are not stained. And counting the cells by using a cell counting plate and a counter to obtain the cell survival rate. If more than 95% of the cells are attached to the wall after recovery and the activity of the cells is good, the recovery process of the cells has no problem. The revived cells are restored to normal growth, achieve normal growth characteristics, such as cell population doubling time, and the like, and are then frozen to supplement the cell stock quantity.
The application discloses a cryopreservation and resuscitation method of human umbilical cord mesenchymal stem cells, which further comprises a tracking and recording platform, wherein the tracking and recording platform comprises a cell acquisition unit, a transportation tracking unit, a detection tracking unit, a cryopreservation unit, a resuscitation unit and a quality control unit;
the cell acquisition unit is used for recording and acquiring the information of the human umbilical cord mesenchymal stem cells and forming a unique coding identifier for the acquired information;
the transportation tracking unit is used for tracking and recording the whole transportation process of the collected human umbilical cord mesenchymal stem cells and forming a corresponding transportation record flow; the stem cells are transported by adopting whole-process temperature monitoring, and when the samples are collected at the moment, the system is started immediately, so that the temperature change can be accurately sensed, and the temperature stability in the transportation process is guaranteed; ensuring that the sample must be transported to a laboratory within 36 hours;
the detection tracking unit is used for tracking and recording the whole detection process of the human umbilical cord mesenchymal stem cells and forming a detection recording stream;
the cryopreservation unit is used for tracking and recording the whole process of cryopreservation of the human umbilical cord mesenchymal stem cells and forming a cryopreservation recording stream;
the recovery unit is used for tracking and recording the whole process of recovery of the human umbilical cord mesenchymal stem cells and forming a recovery recording stream;
the quality control unit is used for tracking and recording the state of the human umbilical cord mesenchymal stem cells during cryopreservation and storage, the quality detection process and a quality control report, and forming a quality control record flow; in each process from collection to transportation, from detection to culture, from cryopreservation to resuscitation, each process is strictly quality monitored, and a unique corresponding record with a buckled loop is formed, so that the uniqueness and traceability of the stem cell sample are ensured. Starting from the collection of human umbilical cord mesenchymal stem cells, the related information of the stem cells, the related operation environment information, the related operation personnel information and the like are recorded in the whole process, and in key operations of detection, cryopreservation, resuscitation and the like, the simple character record is kept, and the video information can be recorded by matching with a camera. The intelligent information management and the cell traceability are realized, and a reliable basis for inquiring and tracing the cell archiving information is provided for a user.
Further, during the cryopreservation period of the cryopreservation of the human umbilical cord mesenchymal stem cells, periodically extracting a part of the cryopreserved cells to recover, and performing quality detection after recovery; and culturing the cells meeting the standard and then freezing the cells again to supplement the freezing amount. The quality of the human umbilical cord mesenchymal stem cells is tested before cell collection, before cryopreservation, during the quality control period of the cryopreservation period, before recovery and after recovery, and the test items comprise one or more of sterility test, cell activity test, cell morphology test, cell purity test, product stability and safety test.
The preservation of umbilical cord mesenchymal stem cells is realized through cryopreserving and resuscitating to this application, from gathering to the transportation, from detecting the cultivation, from cryopreserving to each in-process of resuscitating, all carry out strict quality monitoring to each process to form the record that the ring detained, only correspond, ensure the uniqueness and the traceability of stem cell sample. And also provides an important basis of a powerful theoretical basis for researching the characteristics of the umbilical cord mesenchymal stem cell morphology, the growth characteristics, the cell cycle, the immunophenotype and the like, establishing a human umbilical cord mesenchymal stem cell bank and clinical application.
While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A cryopreservation and recovery method of human umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
d1 ultraviolet irradiation of the cell chamber and the workbench for 20-30 min; preheating culture solution, frozen stock solution, 9% sodium chloride injection, pancreatin, calf serum, DMSO, and constant temperature water bath box at 40 deg.C for use; collecting cells in logarithmic growth phase, and changing liquid one day before freezing;
preparing a freezing culture solution containing 10% of DMSO or glycerol and 10-20% of calf serum; preparing 9% sodium chloride injection, trypsin and DMSO;
d2 taking the pre-frozen cells, removing old culture solution, and washing with 9% sodium chloride injection;
d3 removing 9% sodium chloride injection, adding appropriate amount of trypsin to digest the cells grown in monolayer;
d4 adding culture medium to stop digestion, blowing to suspend cells, transferring to a centrifuge tube, sampling, counting, centrifuging at 1200rpm for 10 min;
d5 removing trypsin, adding a proper amount of prepared frozen stock solution, lightly blowing and beating the cells by using a suction pipe to make the cells uniform, counting, and adjusting the final density of the cells in the frozen stock solution to be 5 × 106/ml-1 × 107/ml;
d6 subpackaging the cells into frozen tubes, wherein each tube contains 1-2.5 ml of cells;
d7 controlling the freezing speed, and transferring the freezing tube into a liquid nitrogen container for storage when the storage standard is met;
d8 indicates the cell name, the freezing time and the operator on the freezing tube.
2. The cryopreservation and resuscitation method for human umbilical cord mesenchymal stem cells according to claim 1, wherein in the step D7, the cryopreservation tube filled with the cells is placed in a 4 ℃ medicine storage box for precooling for 10-30min, is placed in a programmed cooling box, is transferred to a-80 ℃ ultra-low temperature medicine storage box under the protection of a low temperature sample transfer cart, is stored for 4-72 hours, and is transferred to a gas phase liquid nitrogen storage tank; or the like, or, alternatively,
d7, placing the freezing tube filled with the cells into a 4 ℃ medicine storage box for precooling for 10-30min, placing the freezing tube into a-20 ℃ medicine storage box for 0.5-1h, then placing the freezing tube into a-70 ℃ medicine storage box for 8-16h, taking out the freezing tube, and transferring the freezing tube into a liquid nitrogen container; or the like, or, alternatively,
the control mode of the freezing temperature in the step D7 is the cooling rate of-1 to-2 ℃/min; when the temperature reaches below minus 25 ℃, the temperature is increased to minus 5 ℃ to minus 10 ℃/min; when the temperature is reduced to-100 ℃, the mixture is transferred to a gas-phase liquid nitrogen storage tank.
3. The cryopreservation and recovery method of human umbilical cord mesenchymal stem cells according to claim 2, wherein the pre-cryopreserved cells in the step D2 can be cultured cells from a proliferation stage to before a compact monolayer of cells is formed, preferably cells in a logarithmic growth phase.
4. The cryopreservation and recovery method of human umbilical cord mesenchymal stem cells according to claim 3, wherein the culture solution is replaced within 24 hours before the cells are cryopreserved.
5. The cryopreservation and recovery method of human umbilical cord mesenchymal stem cells according to any one of claims 2, wherein the recovery method comprises the following steps:
s1, taking out the freezing tube from the liquid nitrogen container, directly immersing the tube in a constant-temperature water bath kettle at 40 ℃, and shaking the tube to melt the tube as soon as possible without any time;
s2, taking out the freezing tube from the constant temperature water bath kettle at 40 ℃, and quickly wiping the outside of the freezing tube with an alcohol cotton ball to sterilize and air the freezing tube;
s3, sucking the cell suspension by a suction tube, adding sodium chloride injection precooled at 4 ℃, and re-suspending and uniformly mixing;
s4 centrifuging at 1200rpm for 8 min;
s5, discarding supernatant, transferring the cells into culture solution containing 10% calf serum to resuspend the cells, counting, and adjusting cell density;
s6 inoculating a culture bottle, then transferring the culture bottle to an incubator, and culturing at 36-38 ℃ for 72h +/-24 h;
s7, replacing a fresh culture medium, proliferating to the required quantity, and harvesting cells;
s8 records the names, the number, the recovery time and the storage positions of the recovery cells in detail.
6. The cryopreservation and recovery method for human umbilical cord mesenchymal stem cells according to any one of claims 5, wherein in step S1, the cover of the constant temperature water bath kettle is covered during the process of thawing the cryopreservation tube; melting in a constant temperature water bath at 40 deg.C for 1-3min, and removing from 40 deg.C water bath.
7. The cryopreservation and recovery method of human umbilical cord mesenchymal stem cells according to any one of claims 6, wherein in step S3, the cell suspension is sucked out by a suction tube, added into a centrifuge tube and added with more than 10 times of culture solution dropwise, and the mixture is mixed uniformly and operated in a biosafety cabinet at 4 ℃.
8. The cryopreservation and resuscitation method for human umbilical cord mesenchymal stem cells according to any one of claims 5-7, further comprising a tracking and recording platform, wherein the tracking and recording platform comprises a cell collection unit, a transportation tracking unit, a detection tracking unit, a cryopreservation unit, a resuscitation unit and a quality control unit;
the cell acquisition unit is used for recording and acquiring the information of the human umbilical cord mesenchymal stem cells and forming a unique coding identifier for the acquired information;
the transportation tracking unit is used for tracking and recording the whole transportation process of the collected human umbilical cord mesenchymal stem cells and forming a corresponding transportation record flow;
the detection tracking unit is used for tracking and recording the whole detection process of the human umbilical cord mesenchymal stem cells and forming a detection recording stream;
the cryopreservation unit is used for tracking and recording the whole process of cryopreservation of the human umbilical cord mesenchymal stem cells and forming a cryopreservation recording stream;
the recovery unit is used for tracking and recording the whole process of recovery of the human umbilical cord mesenchymal stem cells and forming a recovery recording flow;
and the quality control unit is used for tracking and recording the state of the human umbilical cord mesenchymal stem cells during cryopreservation and the quality detection process and quality control reports and forming a quality control record flow.
9. The cryopreservation and recovery method for human umbilical cord mesenchymal stem cells according to any one of claims 8, wherein during the cryopreservation period of the cryopreservation of the human umbilical cord mesenchymal stem cells, a part of the cryopreserved cells is periodically extracted for recovery, and quality detection is performed after recovery; and culturing the cells meeting the standard and then freezing the cells again to supplement the freezing amount.
10. The cryopreservation and recovery method of human umbilical cord mesenchymal stem cells according to claim 9, wherein the human umbilical cord mesenchymal stem cells are subjected to quality inspection before cell collection, before cryopreservation, during the quality control period of the cryopreservation period, before recovery and after recovery, and the inspection items comprise one or more of sterility inspection, cell activity inspection, cell morphology inspection, cell purity inspection, product stability and safety inspection.
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