CN112522791A - Construction method of human umbilical cord mesenchymal stem cell bank - Google Patents
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Abstract
The invention discloses a construction method of a human umbilical cord mesenchymal stem cell bank, belonging to the technical field of preparation of mesenchymal stem cells. The method comprises the steps of carrying out adherent separation on umbilical cord mesenchymal stem cells by using a tissue block method, culturing by using a serum-free culture medium, purifying the cells by using a differential adherence method, establishing a database of the umbilical cord mesenchymal stem cells, and associating the database with cryopreserved cells; the method has low production cost and high efficiency, and provides a method for quickly and efficiently obtaining the umbilical cord mesenchymal stem cells; in addition, a human umbilical cord mesenchymal stem cell bank is used for establishing a bank for a newborn fetus, and umbilical cord mesenchymal stem cells and stem cell preparations which are clinically applicable are provided for the newborn fetus, family members or authorized third parties when umbilical cord mesenchymal stem cell transplantation is needed to treat various diseases.
Description
Technical Field
The invention relates to a construction method of a human umbilical cord mesenchymal stem cell bank, belonging to the technical field of preparation of mesenchymal stem cells.
Background
The method for separating, extracting and culturing Mesenchymal Stem Cells (MSCs) is simple, the proliferation capacity is strong, and the MSCs from autologous tissue sources are selected for treatment, so that the ethical requirements are met; in addition, MSCs have low immunogenicity and low tumorigenicity, and have strong anti-inflammatory, scar-inhibiting, and immunosuppressive effects. Wherein the umbilical cord mesenchymal stem cells (hMSCs) are non-directional stem cells existing in umbilical cord Wharton's jelly and perivascular tissues. This population of cells is a pluripotent stem cell derived from the early developmental mesoderm with high self-renewal and pluripotency potential, able to maintain its original phenotype without spontaneous differentiation, with a limited lifespan and general characteristics of stem cells; the advantages of hMSCs over other types of MSCs are: the umbilical cord is sufficient in source, easy to collect, available through a non-invasive means and not limited by ethics and laws; secondly, the umbilical cord is protected by a placenta barrier, and the probability of the components being polluted by viruses and bacteria is low; the immunogenicity of the hMSCs is lower, and the human leukocyte antigens can be tolerated to a greater extent; fourthly, the hMSCs have short in-vitro doubling time and strong amplification capacity; collected hMSCs can be used as allografting donors and can be stored at low temperature for decades and used for treating related diseases by autografting.
Currently, methods for separating umbilical cord mesenchymal stem cells include collagenase-trypsin digestion and tissue adherence, and adherent cells obtained primarily by the enzyme digestion are not single cell populations, and may contain heterogeneous cell populations with various components. The primary adherent cells contain a plurality of nuclei and small round cells, and also need to be attached for a long time for subculturing and purifying. Moreover, the use of fetal bovine serum added to a common culture medium is not beneficial to transplantation due to the existence of animal-derived components, and in addition, the establishment of a database is incomplete, and the cell source and cell safety cannot be tracked, so that a simple and effective separation method and a complete information database need to be established.
Disclosure of Invention
The invention aims to provide a method for establishing an umbilical cord mesenchymal stem cell bank, which is characterized in that umbilical cord mesenchymal stem cells are subjected to wall adhesion separation by using a tissue block method, cultured by using a serum-free culture medium and purified by using a differential wall adhesion method.
The umbilical cord mesenchymal stem cell separation method specifically comprises the following steps:
(1) collecting fetal umbilical cord tissues, flushing the tissues for 2-3 times by using normal saline, removing residual blood as far as possible, and removing umbilical arteries, umbilical veins and umbilical cord adventitia to obtain the Huatong glue.
(2) And (3) randomly and linearly scratching a culture dish to fix the Huatong glue, drying in a super clean bench for 5-10 min, adding 3-5ml of serum-free culture medium, continuously adding 2-3 ml of culture medium every 1-3 days, and observing the migration and growth conditions of the cells.
(3) Subculturing: and when the fusion degree of the passage cells reaches 85-95%, continuing passage culture, putting the cell suspension into a culture dish in the passage process, putting the culture dish into an incubator, standing for 5-10 min, putting the cells in the suspension into a new culture dish again, and discarding the cells which are attached to the wall.
(4) Freezing and storing: part of the cells were centrifuged at 3-4X 105Putting the solution/mL into 1mL of frozen stock solution, putting the frozen stock solution into a programmed freezing storage box, and putting the frozen stock solution into a refrigerator at the temperature of minus 80 ℃.
(5) And establishing a database of umbilical cord mesenchymal stem cells, and associating the database with the cryopreserved cells.
Further, the conditions for culturing in step (3) of the present invention are: standing at 37 deg.C for 5% CO2An incubator.
Further, the frozen stock solution in the step (4) of the invention comprises the following components: 70% serum-free medium, 20% fetal bovine serum (Thermo Fisher), 10% dimethyl sulfoxide (Sigma).
The database of the umbilical cord mesenchymal stem cells comprises the following contents:
(1) and (3) detecting the activity of the cells: counting after trypan blue staining, recording the cell number of the cryopreserved cells before and after cryopreservation, and drawing a growth curve by using an MTT method.
(2) Detection of cell infection: detecting whether the cells are polluted by bacteria, fungi and mycoplasma by using a small amount of cell culture, and detecting whether animal-derived substances exist; and detecting the presence of parvovirus.
(3) Detection of genetic diseases: and detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
(4) And (3) detecting the carcinogenicity of the cells: the carcinogenicity of the cells was detected by the formation of soft agar clones and the activity of experimental telomerase and recorded.
(5) HLA-ABC/DR match: HLA-ABC/DR phenotype was tested and documented.
(6) Recording of cell origin: record the details of the supplier and record in case.
The invention has the beneficial effects that:
(1) according to the invention, the Huatong glue is fixed by scratches, and the culture medium in primary culture is reduced, so that the tissue is better attached to a culture dish, cell migration is facilitated, and the culture medium is added subsequently instead of changing liquid, so that the growth factor in the Huatong glue is retained, and the cell growth is promoted; in the passage process, a differential wall sticking method is used to accelerate the purification rate.
(2) The method adopts a complete serum-free culture medium for culture, avoids the interference of animal-derived serum components, has high safety, achieves the clinical level library building process, and is favorable for later clinical application.
(3) The method of the invention builds a bank for the new born fetus, and provides the umbilical cord mesenchymal stem cells and stem cell preparations which are clinically applicable for the members of the person and family or the allowed third party when the umbilical cord mesenchymal stem cells are transplanted to treat various diseases.
Drawings
FIG. 1 shows that umbilical cord mesenchymal stem cells grow by climbing out of the Huatong gel block;
FIG. 2 umbilical cord mesenchymal stem cells are in a long fusiform shape and grow in a vortex-like adherent manner;
FIG. 3 adipogenic differentiation potential of umbilical cord mesenchymal stem cells;
FIG. 4 osteogenic differentiation potential of umbilical cord mesenchymal stem cells;
FIG. 5 chondrogenic differentiation potential of umbilical cord mesenchymal stem cells;
FIG. 6 is a flow cytometry detection result of the surface marker of umbilical cord mesenchymal stem cells.
Detailed Description
The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited to the above description.
The invention signs an informed consent with a donor, collects the 5-10cm umbilical cord of a healthy normal full-term delivery fetus, simultaneously collects 10mL blood sample of the donor, and detects the blood type, HA-ABC/DR match type, infectious diseases (hepatitis B, hepatitis C, cytomegalovirus, EB virus, syphilis and AIDS virus) of the donor, thereby ensuring that all indexes are normal.
The reagents such as the serum-free human mesenchymal stem cell culture medium, the cell digestive juice and the like used in the invention are all commercial products unless specially indicated.
Example 1
A construction method of a human umbilical cord mesenchymal stem cell bank specifically comprises the following steps:
(1) flushing the collected umbilical cord with normal saline for 3 times in a super clean bench, and carrying out blunt separation on umbilical artery, umbilical vein and umbilical cord adventitia to obtain huatong glue; random straight scratches were made on 10cm petri dishes (Corning) using a disposable scalpel to immobilize the huatong glue.
(2) Cutting the Huatong glue into tissue blocks with the size of 0.5cm, and fixing the tissue blocks in the scratches by using a scalpel; and drying the culture dish with the fixed tissue in a super clean bench for 5min to ensure that the Huatong glue is more tightly adhered to the culture dish.
(3) Adding 4ml of mesenchymal stem cell serum-free medium (Jingmeng) into a culture dish, and placing at 37 ℃ and 5% CO2In the incubator, 2ml of the culture medium is added every 3 days, the cell climbing growth condition is observed (see figure 1), and the long spindle cell migration growth is seen after 7-10 days of tissue inoculation and surrounds the tissue block from figure 1.
(4) When the fusion degree of cells around the tissue block is about 90%, digesting for 3-5min at 37 ℃ by using 2ml of cell digestive juice (Jingmeng), observing the cells to be round under a microscope, and shaking the culture dish to show that the cells fall off; adding 2ml of serum-free culture medium to terminate the reaction, collecting the solution in a 15ml centrifuge tube, centrifuging the solution at 1000rpm for 3min, and collecting cells; the liquid is changed every other day when the liquid is passed according to the ratio of 1:3 and is marked as P1.
(5) And when the fusion degree of the passage cells reaches 95%, continuing passage culture, putting the cell suspension into a culture dish in the passage process, putting the culture dish into an incubator, standing for 5min, putting the cells in the suspension into a new culture dish again, abandoning the cells which are attached to the wall, and after passage, obtaining the umbilical cord mesenchymal stem cells which grow in a vortex shape and have the shape rule shown in figure 2.
(6) And centrifuging a part of the cells to 3-4 × 105Putting the solution into 1mL of a frozen stock solution, wherein the frozen stock solution comprises the following components: 70% serum-free medium, 20% fetal bovine serum (Thermo Fisher), 10% dimethyl sulfoxide (Sigma); placing into a programmed freezing box (Corning), and placing in a refrigerator at-80 deg.C.
(7) And (3) rapidly melting the frozen cells in a 37 ℃ water bath, adding a culture medium preheated to 37 ℃ in the same volume, centrifuging for 3min at 1000rpm, discarding the supernatant, adding 8ml of a serum-free culture medium, transferring to a 10cm culture dish, and observing the adherent growth condition of the cells the next day.
Identification of multidirectional induced differentiation potential of umbilical cord mesenchymal stem cells prepared by the embodiment
Inoculating the P3 generation umbilical cord mesenchymal stem cells into a six-well plate, wherein each well is inoculated with 1 × 105Cells, 2mL complete medium per well, 3 replicates per sample set, every two daysReplacing the culture medium once, respectively adding 2mL osteogenic and adipogenic induction culture mediums (Cyagen) when the cell fusion degree is 70%, replacing the induction culture medium once every 2-3 days by taking a completely-added culture medium as a blank control, continuously culturing for 2-3 weeks, removing the induction culture medium and washing the induction culture medium once by PBS, adding 2mL paraformaldehyde solution (4%, v/v) for fixing for 30min, removing the paraformaldehyde solution and washing the induction culture medium twice by PBS, and dyeing by using a dyeing solution, wherein the osteogenic induction dyeing time is 2-3 min; the adipogenic induction dyeing time is 30min, then the dyeing solution is removed, 1ml of PBS solution is added for washing once, and finally the mixture is placed under an inverted microscope for observation and photo taking, wherein the formation of circular lipid drops can be seen as shown in figure 3, the visible result shown in figure 4 shows that mineralized nodule deposition can be seen, and alizarin red dyeing is red; after 2-3 weeks of induction culture, the umbilical cord mesenchymal stem cells can be subjected to adipogenic and osteogenic induced differentiation.
Counting cells after conventional digestion before chondrogenic induction differentiation experiment; mixing 3-4X 105Transferring the cells into a 15mL centrifuge tube, centrifuging for 4min at 250g, sucking the supernatant, adding 0.5mL premix, resuspending the precipitate obtained by the previous centrifugation step to clean the human umbilical cord mesenchymal stem cells, and centrifuging for 5min at 150g at room temperature. The resulting pellet was resuspended in 0.5mL of complete medium for chondrogenic differentiation of human umbilical cord mesenchymal stem cells (Cyagen). Centrifuge at 150g for 5min at room temperature. The centrifuge tube cap was unscrewed to facilitate gas exchange and placed at 37 ℃ with 5% CO2Cultured in an incubator. When the cell mass is gathered (generally 24h or 48h later, actually according to the cell growth condition), the bottom of the centrifugal tube is flicked to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid. Cells were replaced with fresh chondrogenic differentiation complete medium every 2-3d, approximately 0.5mL chondrogenic differentiation complete medium per tube, calculated from the start of inoculation. After the liquid is changed, the bottom of the centrifugal tube is flicked to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid; slightly unscrewing the centrifugal tube cover, putting at 37 deg.C and 5% CO2The incubator continues to induce culture; after a typical 21-28 day incubation period, the chondrocytes can be formalin-fixed and paraffin-embedded, and finally stained with safranin O, and the staining observed microscopically, as shown in FIG. 5, with visible red chondroitin sulfate。
According to the analysis, the human umbilical cord mesenchymal stem cells prepared by the method can be induced to differentiate into adipogenesis, osteogenesis and chondrogenesis.
The surface marker of umbilical cord mesenchymal stem cells detected by the flow cytometer prepared in the embodiment
Selecting third generation umbilical cord mesenchymal stem cells with good growth condition and 70-80% fusion degree, digesting with digestive enzyme (Jingmeng), collecting into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, washing with PBS for 3 times, counting cells, and adjusting concentration to 1 × 106Per mL, 12 EP tubes were added at 1X 106After centrifugation, 100. mu.l of PBS is added for resuspension, 10. mu.l of each of CD29-APC, CD44-PE, CD73-90, CD90-FITC, CD105-PerCP, CD45-FITC and HLA-DR-APC antibody and respective isotype control are added, mixed evenly and incubated for 30min at 4 ℃ in the dark. After centrifugation to remove supernatant and 3 washes with PBS, the resuspended cells were examined using a flow cytometer.
The results are shown in FIG. 6, and the expression rates of the markers are: CD29, CD44, CD73, CD90 and CD105 are strongly positively expressed, and the expression rates are 99.84%, 95.68%, 96.93%, 99.79% and 97.76% in sequence; HLA-DR and CD45 are expressed negatively, the expression rate is 0.72 percent and 0.59 percent in sequence, and the standard of the mesenchymal stem cells is met.
Example 2
Establishment of umbilical cord mesenchymal stem cell database
(1) And (3) detecting the activity of the cells: counting after trypan blue staining, recording the cell number of the cryopreserved cells before and after cryopreservation, and drawing a growth curve by using an MTT method.
(2) Detection of cell infection by a small amount of cell culture, detecting whether or not it is contaminated with bacteria, fungi and mycoplasma, and detecting whether or not there is a substance of animal origin; and detecting the presence of parvovirus.
(3) Detection of genetic diseases: and detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
(4) And (3) detecting the carcinogenicity of the cells: the carcinogenicity of the cells was detected by the formation of soft agar clones and the activity of experimental telomerase and recorded.
(5) HLA-ABC/DR match: HLA-ABC/DR phenotype was tested and documented.
(6) Recording of cell origin: record the details of the supplier and record in case.
(7) Establishing an umbilical cord mesenchymal stem cell database: after normal umbilical cord mesenchymal stem cells are preserved, a database of the umbilical cord mesenchymal stem cells is established, wherein the database comprises the first six items of information, and the association with the cryopreserved cells is established.
According to the method, umbilical cord mesenchymal stem cells are subjected to adherent separation by using a tissue block method, cultured by using a serum-free culture medium, purified by using a differential adherence method, and associated with cryopreserved cells; the method has low production cost and high efficiency, and provides a method for quickly and efficiently obtaining the umbilical cord mesenchymal stem cells; in addition, the human umbilical cord mesenchymal stem cell bank is built for a newborn fetus, and provides clinically applicable umbilical cord mesenchymal stem cells and stem cell preparations for the fetus, family members or permitted third parties when umbilical cord mesenchymal stem cell transplantation is needed to treat various diseases.
Claims (4)
1. A method for constructing a human umbilical cord mesenchymal stem cell bank is characterized by comprising the following steps:
(1) collecting fetal umbilical cord tissues, flushing the tissues for 2-3 times by using normal saline, removing residual blood, and removing umbilical arteries, umbilical veins and umbilical cord adventitia to obtain huatong glue;
(2) randomly and linearly scratching a culture dish to fix the Huatong glue, drying the Huatong glue in a super clean bench for 5-10 min, adding 3-5ml of serum-free culture medium, continuously adding 2-3 ml of culture medium every 2-3 days, and observing the migration and growth conditions of cells;
(3) subculturing: when the fusion degree of the passage cells is 85-95%, continuing passage culture, putting the cell suspension into a culture dish in the passage process, putting the culture dish into an incubator, standing for 5-10 min, putting the cells in the suspension into a new culture dish again, and discarding the cells adhered to the wall;
(4) freezing and storing: part of the cells were centrifuged at 3-4X 105Putting the solution/mL into 1mL of frozen stock solution, putting the frozen stock solution into a programmed freezing storage box, and putting the frozen stock solution into a refrigerator at the temperature of minus 80 ℃;
(5) and establishing a database of umbilical cord mesenchymal stem cells, and associating the database with the cryopreserved cells.
2. The method for constructing a human umbilical cord mesenchymal stem cell bank according to claim 1, wherein the method comprises the following steps: the culture conditions in the step (3) are as follows: standing at 37 deg.C for 5% CO2An incubator.
3. The method for constructing a human umbilical cord mesenchymal stem cell bank according to claim 1, wherein the method comprises the following steps: the frozen stock solution in the step (4) comprises the following components: 70% of mesenchymal stem cell serum-free culture medium, 20% of fetal bovine serum and 10% of dimethyl sulfoxide.
4. The method for constructing a human umbilical cord mesenchymal stem cell bank according to claim 1, wherein the method comprises the following steps: the database of umbilical cord mesenchymal stem cells comprises the following:
(1) and (3) detecting the activity of the cells: counting after trypan blue staining, recording the cell number of the cryopreserved cells before and after cryopreservation, drawing a growth curve by using an MTT method, and recording;
(2) detection of cell infection: detecting whether the cells are polluted by bacteria, fungi and mycoplasma by using a small amount of cell culture, and detecting whether animal-derived substances exist; detecting whether parvovirus exists or not, and recording;
(3) detection of genetic diseases: detecting whether the frozen cells have genetic diseases by using a molecular genetics method, and recording;
(4) and (3) detecting the carcinogenicity of the cells: detecting the carcinogenicity of the cells by utilizing the cloning formation of soft agar and the activity of experimental telomerase, and recording;
(5) HLA-ABC/DR match: detecting HLA-ABC/DR phenotype and recording the phenotype;
(6) recording of cell origin: record the details of the supplier and record in case.
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| CN113712995A (en) * | 2021-07-20 | 2021-11-30 | 河北医科大学 | Application of neural stem cell and umbilical cord mesenchymal stem cell in spinal cord injury |
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