CN111676192A - Umbilical cord-derived mesenchymal stem cell culture method - Google Patents
Umbilical cord-derived mesenchymal stem cell culture method Download PDFInfo
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
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Abstract
The application provides a method for culturing mesenchymal stem cells from umbilical cord, which comprises the steps of recovering cryopreserved stem cells, and adding a culture medium to culture the stem cells; centrifuging a culture solution containing cells to be passaged, and adding the culture solution into the obtained cell suspension for passage; centrifuging a culture solution containing cells to be reinfused, adding saline water into the sediment after centrifugation, uniformly mixing, pouring the sediment into a reinfusion blood bag, sealing the opening of the reinfusion blood bag, labeling the opening of the reinfusion blood bag, and conveying the reinfusion blood bag to a reinfusion center; centrifuging a culture solution containing cells to be cryopreserved, adding the cryopreserved solution into the centrifuged sediment, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the cryopreservation tube in a program cooling box, cooling for 12h, and transferring into liquid nitrogen. The mesenchymal stem cells with high homology are efficiently separated and cultured in vitro, and flow cytometry detection results show that the stem cells provided by the application have specific immunological phenotypes of the mesenchymal stem cells, namely CD73, CD105 and CD90 are positive, CD34, CD45 and HLA-DR are not expressed, the expression rate is up to more than 95%, and the purity is excellent.
Description
Technical Field
The application relates to the technical field of biological cell culture, in particular to a method for culturing mesenchymal stem cells from an umbilical cord.
Background
Stem cells have been the focus of research by scientists since their discovery, have the ability to self-renew for life and can differentiate into specific cell types to form tissues and organs. Due to the characteristics of self-replication, high proliferation and multidirectional differentiation of stem cells, the stem cells become a hotspot of research in the research field of regenerative medicine, and have potential and application prospects. Adult stem cells are a kind of cells with multidirectional differentiation potential existing in postnatal tissues, and a large number of research results show that the corresponding stem cells can be separated from epithelia of various tissues such as heart, skin, ovary, prostate and the like. Human umbilical cord blood-derived mesenchymal stem cells are adult stem cells with a multidirectional differentiation characteristic and can be used for repairing various tissue injuries. Compared with other types of mesenchymal stem cells, the umbilical cord mesenchymal stem cells are easy to obtain in vitro and have large obtaining amount, the obtained cells are relatively original, the body damage can not be caused, the cells are easy to accept by patients, and simultaneously the immune rejection reaction and the ethical problems can not be caused, so that the human umbilical cord blood-derived mesenchymal stem cells become a potential adult stem cell bank member in tissue engineering and become a research hotspot in the field of current stem cell tissue engineering.
With the progress of research, it is found that mesenchymal stem cells have self-renewal and multi-directional differentiation capabilities, and simultaneously the generated microenvironment has important significance on both homing and cell proliferation and differentiation of hematopoietic stem cells. However, the efficiency of the existing mesenchymal stem cell culture method is not ideal, and the expression rate of the cultured mesenchymal stem cells is not high; the culture process is not standardized, and when the immunological phenotype of the cultured cells is detected after the culture process is finished, the required immunological phenotype can not be obtained, namely the cultured cells are not mesenchymal stem cells, so that the time and the energy of researchers are wasted, and experimental materials are wasted.
Disclosure of Invention
The application provides a mesenchymal stem cell culture method of an umbilical cord source, which aims to solve the problems that the efficiency of the existing mesenchymal stem cell culture method is not ideal and the expression rate of the cultured mesenchymal stem cells is not high; the culture process is not standard, and when the immunological phenotype of the cultured cells is detected after the culture process is finished, the required immunological phenotype can not be obtained, namely the cultured cells are not mesenchymal stem cells, so that the time and the energy of researchers are wasted, and experimental materials are wasted.
The application provides a method for culturing umbilical cord-derived mesenchymal stem cells, which comprises the recovery of stem cells, the passage of the stem cells, the reinfusion or cryopreservation of the stem cells, wherein:
and (3) recovering the stem cells: resuscitating the frozen stem cells in a water bath, separating the stem cells from the frozen stock solution through centrifugal operation, and adding a culture medium to culture the stem cells;
passage of stem cells: pouring a culture solution containing cells to be passaged into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, adding the culture solution into cell precipitates after centrifugation for suspension, and adding the culture solution into the obtained cell suspension for passage;
back transfusion of stem cells: pouring a culture solution containing cells to be transfused back into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding saline into the precipitate, mixing the precipitate with the saline, pouring the precipitate into a blood bag for transfusing back, sealing and labeling the mixture, and conveying the mixture to a transfusing center;
freezing and storing stem cells: pouring a culture solution containing cells to be cryopreserved into a collection bottle, adding saline and pancreatin digestive juice to clean the collection bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding the cryopreserved solution to the centrifuged precipitate, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the tube in a program cooling box for cooling for 12 hours, and transferring the cell suspension into liquid nitrogen.
Optionally, the specific steps of the resuscitation of the stem cells include:
taking out the frozen stem cells from the liquid nitrogen tank, dissolving the thawed and frozen stem cells by using a water bath kettle, completing thawing, transferring the stem cells to a purification workbench of an operation room, transferring the stem cells in the frozen tube into a centrifugal tube by using a pipette, centrifuging for 5min at 1500 rpm, preparing a culture bottle, adding a serum-free culture medium into the culture bottle, completing centrifugation, discarding supernatant, leaving cell sediment, adding the serum-free culture medium into the cell sediment by using the pipette, beating to suspension, adding a cell suspension into the culture bottle, pasting a label on the culture bottle, and placing the culture bottle in an incubator for culture.
Optionally, the method further includes:
the serum-free culture medium is a prepared human mesenchymal stem cell culture medium, namely, a human mesenchymal stem cell additive is added into the basic culture medium, and the additive components comprise albumin, insulin, transferrin and other nutritional components.
Optionally, the specific steps of passaging the stem cells include:
transferring cells needing to be passaged to a purification workbench, preparing a culture bottle, a collecting bottle and a centrifugal bottle, pouring a culture solution containing the cells needing to be passaged into the collecting bottle, adding saline water into the culture bottle for cleaning, pouring a cleaning solution into the centrifugal bottle, adding pancreatin digestive juice, shaking the culture bottle, flatly placing the culture bottle until the cells are sufficiently digested, adding the culture solution to stop digestion, pouring the digestive juice and the culture solution into a centrifugal tube, pouring the saline water into the culture bottle for cleaning, pouring the cleaning solution into the centrifugal bottle, centrifuging for 5min at a speed of 500G, discarding supernatant to leave cell precipitates, adding the culture solution, using a pipette to suspend cell liquid, flatly placing the cells into an incubator after uniformly mixing, and sticking labels.
Optionally, the method further includes:
the pancreatin digestive juice is prepared by mixing pancreatin and saline water in a ratio of 1: 1.
Optionally, the specific steps of the back transfusion of the stem cells include:
preparing a centrifuge tube and a collection bottle, pouring cell sap into the collection bottle, adding saline water into the culture bottle for cleaning, pouring cleaning liquid into the centrifuge bottle, adding pancreatin digestive juice into the culture bottle for digestion for 2min till full digestion, adding culture solution to stop digestion, pouring mixed liquid of the culture solution and the digestive juice into the centrifuge bottle, centrifuging at the speed of 500G for 5min, discarding supernatant liquid to leave cell precipitate, adding saline water for mixing uniformly, injecting the mixture into a blood transfusion bag by using an injector, sealing by a heat sealing machine, returning a pasting label to a transfusion center, and reserving a cell sap sample.
Optionally, the specific steps of the cryopreservation of the stem cells include:
preparing a centrifuge tube and a collection bottle, pouring cell sap into the collection bottle, adding saline water into the culture bottle for cleaning, pouring cleaning liquid into the centrifuge bottle, adding pancreatin digestive juice into the culture bottle for digestion for 2min until the cell sap is fully digested, adding culture solution to stop the digestion, pouring mixed liquid of the culture solution and the digestive juice into the centrifuge bottle, centrifuging for 5min at the speed of 500G, removing supernate to leave cell precipitates, adding freezing solution for mixing uniformly, adding cell suspension into a freezing tube, sealing by a plastic packaging machine, placing the freezing tube in a programmed cooling box, placing the freezing tube at the temperature of minus 20 ℃ overnight in liquid nitrogen, and transferring the cell suspension into liquid nitrogen for sample retention.
According to the technical scheme, the method for culturing the umbilical cord-derived mesenchymal stem cells comprises the steps of recovering the frozen stem cells in a water bath, separating the stem cells from a frozen stock solution through centrifugal operation, and adding a culture medium to culture the stem cells; pouring a culture solution containing cells to be passaged into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, adding the culture solution into cell precipitates after centrifugation for suspension, and adding the culture solution into the obtained cell suspension for passage; pouring a culture solution containing cells to be transfused back into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding saline into the precipitate, mixing the precipitate with the saline, pouring the precipitate into a blood bag for transfusing back, sealing and labeling the mixture, and conveying the mixture to a transfusing center; pouring a culture solution containing cells to be cryopreserved into a collection bottle, adding saline and pancreatin digestive juice to clean the collection bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding the cryopreserved solution to the centrifuged precipitate, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the tube in a program cooling box for cooling for 12 hours, and transferring the cell suspension into liquid nitrogen. The mesenchymal stem cells with high homology are efficiently separated and cultured in vitro, and flow cytometry detection results show that the stem cells provided by the application have specific immunological phenotypes of the mesenchymal stem cells, namely CD73, CD105 and CD90 are positive, CD34, CD45 and HLA-DR are not expressed, the expression rate is up to more than 95%, and the purity is excellent.
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In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow phenotyping test of a suspension of umbilical cord mesenchymal stem cells provided in an embodiment of the present application;
fig. 2 is a schematic flow chart of a method for culturing umbilical cord-derived mesenchymal stem cells according to an embodiment of the present disclosure.
Detailed Description
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.
The mesenchymal stem cell is a pluripotent stem cell, has all commonalities of stem cells, namely self-renewal and multidirectional differentiation capacity, is most clinically applied, and can be combined with hematopoietic stem cells to improve the success rate of transplantation and accelerate hematopoietic reconstruction. After a patient receives a large dose of chemotherapy, the mesenchymal stem cells and the hematopoietic stem cells are input together, so that the recovery time of the blood cells of the patient can be obviously accelerated, and the chemotherapy is safe and has no adverse reaction. Mesenchymal stem cells are present not only in the bone marrow but also in skeletal muscle, periosteum and trabecula. It has wide tissue types, so it is of great clinical application value. As a potential adult stem cell bank member in tissue engineering, human umbilical cord blood-derived mesenchymal stem cells become another research hotspot in the current stem cell tissue engineering field.
Referring to fig. 1, a flow phenotyping test of umbilical cord mesenchymal stem cell suspension is provided in the examples of the present application.
As can be seen from fig. 1, in the flow type phenotype detection of the umbilical cord mesenchymal stem cells cultured by the umbilical cord-derived mesenchymal stem cell culture method provided by the present application, the expression rate of CD105 is 97.76%, the expression rate of CD90 is 98.90%, the expression rate of CD73 is 99.96%, the expression rate of CD34 is 0.30%, the expression rate of CD45 is 0.16%, and the expression rate of HLA-DR is 0.53%.
Flow cytometry detection results show that the stem cells provided by the application have specific immunological phenotypes of mesenchymal stem cells, namely positive CD73, CD105 and CD90, do not express CD34, CD45 and HLA-DR, and have the advantages of high expression rate of more than 95% and excellent purity.
Referring to fig. 2, a schematic flow chart of a method for culturing umbilical cord-derived mesenchymal stem cells according to an embodiment of the present disclosure is shown. As can be seen from fig. 2, the present application provides a method for culturing umbilical cord-derived mesenchymal stem cells, comprising the steps of recovering stem cells, passaging stem cells, returning stem cells or freezing stem cells, wherein:
and (3) recovering the stem cells: resuscitating the frozen stem cells in a water bath, separating the stem cells from the frozen stock solution through centrifugal operation, and adding a culture medium to culture the stem cells;
passage of stem cells: pouring a culture solution containing cells to be passaged into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, adding the culture solution into cell precipitates after centrifugation for suspension, and adding the culture solution into the obtained cell suspension for passage;
back transfusion of stem cells: pouring a culture solution containing cells to be transfused back into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding saline into the precipitate, mixing the precipitate with the saline, pouring the precipitate into a blood bag for transfusing back, sealing and labeling the mixture, and conveying the mixture to a transfusing center;
freezing and storing stem cells: pouring a culture solution containing cells to be cryopreserved into a collection bottle, adding saline and pancreatin digestive juice to clean the collection bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding the cryopreserved solution to the centrifuged precipitate, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the tube in a program cooling box for cooling for 12 hours, and transferring the cell suspension into liquid nitrogen.
Optionally, the specific steps of the resuscitation of the stem cells include:
taking out the frozen stem cells from the liquid nitrogen tank, dissolving the thawed and frozen stem cells by using a water bath kettle, completing thawing, transferring the stem cells to a purification workbench of an operation room, transferring the stem cells in the frozen tube into a centrifugal tube by using a pipette, centrifuging for 5min at 1500 rpm, preparing a culture bottle, adding a serum-free culture medium into the culture bottle, completing centrifugation, discarding supernatant, leaving cell sediment, adding the serum-free culture medium into the cell sediment by using the pipette, beating to suspension, adding a cell suspension into the culture bottle, pasting a label on the culture bottle, and placing the culture bottle in an incubator for culture.
The purification workbench creates a clean environment by setting the flow direction of clean air, and is widely applicable to scientific research and production departments which need local clean and sterile working environments, such as medicine and health, biological pharmacy, food, medical science experiments, aseptic microorganism inspection, plant tissue culture inoculation and the like. The purification workbench used in the application can provide a local high-cleanliness working environment, and has good effects on improving the stem cell culture condition, improving the quality of cultured stem cells and increasing the yield of the stem cells.
The water bath pot is mainly used for distilling, drying, concentrating and warm-soaking chemical drugs or biological products in a laboratory, can also be used for constant-temperature heating and other temperature tests, and is a necessary tool for biology, heredity, viruses, aquatic products, environmental protection, medicine, sanitation, laboratories, analysis rooms and educational and scientific research. The water bath that uses in this application can provide homothermal recovery environment for the stem cell of cryopreserving, and the better faster recovery of stem cell of help cryopreserving.
In practical application, the time for dissolving and recovering the frozen stem cells by using a water bath can be 90 seconds, the temperature of the water bath can be 37 ℃, the model of the culture bottle can be T175, the amount of serum-free culture medium added into the culture bottle can be 22ml, and the amount of cell suspension added into each culture bottle can be 10 ml.
Optionally, the method further includes:
the serum-free culture medium is a prepared human mesenchymal stem cell culture medium, namely, a human mesenchymal stem cell additive is added into the basic culture medium, and the additive components comprise albumin, insulin, transferrin and other nutritional components.
Albumin, also known as albumin, is the most important protein in human plasma and maintains the nutrition and osmotic pressure of the body. The concentration reaches 38-48 g/L and accounts for about 50% of the total protein of plasma. The liver synthesizes about 12 g-20 g of albumin each day, and the mature albumin is a single polypeptide chain containing 585 amino acid residues and has an oval molecular shape.
Insulin is a protein hormone secreted by islet beta cells in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc., and is the only hormone in the body that reduces blood sugar and promotes glycogen, fat and protein synthesis.
Transferrin, also known as transferrin, is the major iron-containing protein in plasma, responsible for carrying iron absorbed by the digestive tract and released by the degradation of erythrocytes, in the form of a TRF-Fe3+ complex into the bone marrow for the production of mature erythrocytes.
The serum-free culture medium contains the abundant nutrient substances, so that the recovered stem cells after centrifugation can successfully survive and grow in the serum-free culture medium.
Optionally, the specific steps of passaging the stem cells include:
transferring cells needing to be passaged to a purification workbench, preparing a culture bottle, a collecting bottle and a centrifugal bottle, pouring a culture solution containing the cells needing to be passaged into the collecting bottle, adding saline water into the culture bottle for cleaning, pouring a cleaning solution into the centrifugal bottle, adding pancreatin digestive juice, shaking the culture bottle, flatly placing the culture bottle until the cells are sufficiently digested, adding the culture solution to stop digestion, pouring the digestive juice and the culture solution into a centrifugal tube, pouring the saline water into the culture bottle for cleaning, pouring the cleaning solution into the centrifugal bottle, centrifuging for 5min at a speed of 500G, discarding supernatant to leave cell precipitates, adding the culture solution, using a pipette to suspend cell liquid, flatly placing the cells into an incubator after uniformly mixing, and sticking labels.
The above 500G refers to the separation factor of the centrifuge, which is related to the rotational speed and diameter of the centrifuge, and the rotational speed of the centrifuge can be calculated if the diameter of the centrifuge is known. The diameter is generally measured on the model of the centrifuge, for example, LW450 is 450 for model LW, and GK800 is 800 for model GK.
The separation factor of the centrifuge is calculated by the formula that the separation factor Fr is r × (2 pi n/60)2/g；
In the above formula: fr is 500, radius r is m, G is gravity acceleration 9.8, pi is 3.14, n is rotation speed in rpm, and the rotation speed equivalent to 500G as mentioned in the present application can be calculated by the formula.
In practical application, the source of the stem cells is umbilical cord, and the obtained primary generation cells can be marked as P0Passage to P4The infusion can be done, for example, transferring P3 from P2, and transferring 10 bottles P3 from each bottle P2. The naming rule of the tag may be: 008[ umbilical cord number ]]P2, ①, time.
Optionally, the method further includes:
the pancreatin digestive juice is prepared by mixing pancreatin and saline water in a ratio of 1: 1.
Trypsin in pancreatin digestive juices is a serine protease from the PA family, present in the digestive system of many vertebrates, where proteins are hydrolyzed. Trypsin is formed in the small intestine and is activated when its zymogen forms a pancreas-derived trypsinogen, which cleaves the peptide chain predominantly on the carboxy side of the amino acids lysine or arginine.
In the stem cell in vitro culture step of the present application, some stem cells adhere to the side and bottom of the culture flask, so that the protein adhering the cultured stem cells to the culture flask is cut using trypsinized fluid, thereby collecting all the cultured stem cells.
Optionally, the specific steps of the back transfusion of the stem cells include:
preparing a centrifuge tube and a collection bottle, pouring cell sap into the collection bottle, adding saline water into the culture bottle for cleaning, pouring cleaning liquid into the centrifuge bottle, adding pancreatin digestive juice into the culture bottle for digestion for 2min till full digestion, adding culture solution to stop digestion, pouring mixed liquid of the culture solution and the digestive juice into the centrifuge bottle, centrifuging at the speed of 500G for 5min, discarding supernatant liquid to leave cell precipitate, adding saline water for mixing uniformly, injecting the mixture into a blood transfusion bag by using an injector, sealing by a heat sealing machine, returning a pasting label to a transfusion center, and reserving a cell sap sample.
The heat sealing machine is equipment applying high-frequency medium heating technology to plastic packaging, and has application range of disposable blood transfusion bags, extracorporeal drainage bags, emergency stretcher, PVC oxygen bags, medical clinical identification belts and the like. Use the heat-sealing machine to seal for the blood bag of reinfusion in this application, prevent that the cell sap of cultivateing from revealing, and safe nontoxic can not pollute the cell sap.
In practical applications, when the cells can be passaged or returned, but the cell culture is sufficient or the object is not returned, the stem cells can be frozen and restored for further passaging or returning. Before the operation of the reinfusion or the cryopreservation, the condition of cell growth is observed under an inverted microscope, and whether the condition of reinfusion and cryopreservation is met or not is judged. The syringe used may be 50ml and the reinfusion bag used may be 200 ml.
In practical application, the culture solution needs to be tested for fungi, bacteria, mycoplasma and endotoxin in advance, and the culture solution can be sent to a feedback center after being tested to be qualified.
Optionally, the specific steps of the cryopreservation of the stem cells include:
preparing a centrifuge tube and a collection bottle, pouring cell sap into the collection bottle, adding saline water into the culture bottle for cleaning, pouring cleaning liquid into the centrifuge bottle, adding pancreatin digestive juice into the culture bottle for digestion for 2min until the cell sap is fully digested, adding culture solution to stop the digestion, pouring mixed liquid of the culture solution and the digestive juice into the centrifuge bottle, centrifuging for 5min at the speed of 500G, removing supernate to leave cell precipitates, adding freezing solution for mixing uniformly, adding cell suspension into a freezing tube, sealing by a plastic packaging machine, placing the freezing tube in a programmed cooling box, placing the freezing tube at the temperature of minus 20 ℃ overnight in liquid nitrogen, and transferring the cell suspension into liquid nitrogen for sample retention.
The liquid nitrogen is inert, colorless, odorless, non-corrosive, nonflammable, extremely low in temperature, has a liquid nitrogen temperature of-196 ℃ at normal pressure, and can be used as a deep refrigerant. The very low temperature of liquid nitrogen is used in this application to allow immediate freezing of the resulting cell suspension without destroying cell viability.
Two types of cryopreservation tubes can be used for cryopreservation of stem cells: the surface area of the thin straw is larger, the recovery time is shorter, the activity of stem cells can be ensured, and the thin straw is better than the rough straw.
In practical applications, the cell suspension added to each vial may be 1 ml.
According to the technical scheme, the method for culturing the umbilical cord-derived mesenchymal stem cells comprises the steps of recovering the frozen stem cells in a water bath, separating the stem cells from a frozen stock solution through centrifugal operation, and adding a culture medium to culture the stem cells; pouring a culture solution containing cells to be passaged into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, adding the culture solution into cell precipitates after centrifugation for suspension, and adding the culture solution into the obtained cell suspension for passage; pouring a culture solution containing cells to be transfused back into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding saline into the precipitate, mixing the precipitate with the saline, pouring the precipitate into a blood bag for transfusing back, sealing and labeling the mixture, and conveying the mixture to a transfusing center; pouring a culture solution containing cells to be cryopreserved into a collection bottle, adding saline and pancreatin digestive juice to clean the collection bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding the cryopreserved solution to the centrifuged precipitate, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the tube in a program cooling box for cooling for 12 hours, and transferring the cell suspension into liquid nitrogen. The mesenchymal stem cells with high homology are efficiently separated and cultured in vitro, and flow cytometry detection results show that the stem cells provided by the application have specific immunological phenotypes of the mesenchymal stem cells, namely CD73, CD105 and CD90 are positive, CD34, CD45 and HLA-DR are not expressed, the expression rate is up to more than 95%, and the purity is excellent.
The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.
Claims (7)
1. A method for culturing umbilical cord-derived mesenchymal stem cells is characterized by comprising the steps of recovering stem cells, passaging the stem cells, and returning or freezing the stem cells, wherein:
and (3) recovering the stem cells: resuscitating the frozen stem cells in a water bath, separating the stem cells from the frozen stock solution through centrifugal operation, and adding a culture medium to culture the stem cells;
passage of stem cells: pouring a culture solution containing cells to be passaged into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, adding the culture solution into cell precipitates after centrifugation for suspension, and adding the culture solution into the obtained cell suspension for passage;
back transfusion of stem cells: pouring a culture solution containing cells to be transfused back into a collection bottle, adding saline and pancreatin digestive juice to clean the culture bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding saline into the precipitate, mixing the precipitate with the saline, pouring the precipitate into a blood bag for transfusing back, sealing and labeling the mixture, and conveying the mixture to a transfusing center;
freezing and storing stem cells: pouring a culture solution containing cells to be cryopreserved into a collection bottle, adding saline and pancreatin digestive juice to clean the collection bottle, adding the culture solution to stop digestion, pouring the culture solution and the digestive juice into a centrifuge bottle for centrifugation, taking the centrifuged precipitate, adding the cryopreserved solution to the centrifuged precipitate, uniformly mixing, adding the obtained cell suspension into a cryopreservation tube, placing the tube in a program cooling box for cooling for 12 hours, and transferring the cell suspension into liquid nitrogen.
2. The umbilical cord-derived mesenchymal stem cell culture method according to claim 1, wherein the specific steps of recovering the stem cells comprise:
taking out the frozen stem cells from the liquid nitrogen tank,
using a water bath kettle to dissolve and resuscitate the frozen stem cells,
after the recovery is finished, transferring the stem cells to a purification workbench of an operation room,
transferring the stem cells in the cryopreserved tube into a centrifugal tube by using a pipette, centrifuging for 5min at 1500 rotation speed,
preparing a culture bottle, adding a serum-free culture medium into the culture bottle,
centrifuging, removing supernatant, collecting cell precipitate,
adding serum-free culture medium into the cell sediment by using a pipette, blowing the mixture to be suspended,
adding the cell suspension into the culture flask,
and sticking a label on the culture bottle, and placing the culture bottle in an incubator for culture.
3. The method of claim 2, further comprising:
the serum-free culture medium is a prepared human mesenchymal stem cell culture medium, namely, a human mesenchymal stem cell additive is added into the basic culture medium, and the additive components comprise albumin, insulin, transferrin and other nutritional components.
4. The umbilical cord-derived mesenchymal stem cell culture method of claim 1, wherein the specific steps of passaging the stem cells comprise:
transferring the cells to be passaged to a clean bench,
preparing a culture bottle, a collecting bottle and a centrifugal bottle,
the culture fluid containing the cells to be passaged is poured into a collection bottle,
adding saline water into the culture bottle for cleaning, pouring the cleaning solution into a centrifugal bottle,
adding pancreatin digestive juice, shaking the culture flask,
the culture bottle is flatly placed until the digestion is fully carried out, the culture solution is added to stop the digestion,
pouring the digestive juice and the culture solution into a centrifuge tube,
pouring saline water to clean the culture bottle, pouring cleaning solution into a centrifugal bottle, centrifuging at the speed of 500G for 5min,
discarding supernatant, leaving cell precipitate, adding culture solution,
suspending the cell suspension by using a pipette, uniformly mixing, horizontally placing into an incubator, and sticking a label.
5. The method of claim 4, further comprising:
the pancreatin digestive juice is prepared by mixing pancreatin and saline water in a ratio of 1: 1.
6. The umbilical cord-derived mesenchymal stem cell culture method of claim 1, wherein the specific steps of the back transfusion of the stem cells comprise:
preparing a centrifuge tube and a collecting bottle, pouring the cell sap into the collecting bottle,
adding saline water into the culture bottle for cleaning, pouring the cleaning solution into a centrifugal bottle,
adding pancreatin digestive juice into the culture bottle, digesting for 2min, adding culture solution to stop digestion,
pouring the mixed solution of the culture solution and the digestive juice into a centrifuge bottle, centrifuging for 5min at the speed of 500G,
discarding supernatant, retaining cell precipitate, adding saline water, mixing,
injecting the blood transfusion bag by using a syringe, sealing by using a heat sealing machine, returning the adhesive label to the transfusion center, and reserving the cell sap.
7. The umbilical cord-derived mesenchymal stem cell culture method of claim 1, wherein the specific steps of the cryopreservation of the stem cells comprise:
preparing a centrifuge tube and a collecting bottle, pouring the cell sap into the collecting bottle,
adding saline water into the culture bottle for cleaning, pouring the cleaning solution into a centrifugal bottle,
adding pancreatin digestive juice into the culture bottle, digesting for 2min, adding culture solution to stop digestion,
pouring the mixed solution of the culture solution and the digestive juice into a centrifuge bottle, centrifuging for 5min at the speed of 500G,
discarding supernatant, retaining cell precipitate, adding frozen solution, mixing,
adding the cell suspension into the cryopreservation tube, sealing by a plastic packaging machine,
the cryopreservation tubes are placed in a programmed cooling box, placed at-20 ℃ overnight and transferred into liquid nitrogen, and the cell suspension is reserved.
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