CN117757741A - Preparation method of umbilical cord mesenchymal stem cells - Google Patents
Preparation method of umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a preparation method of umbilical mesenchymal stem cells, which has extremely strong hormone regulation effect, can be used for treating autoimmune system diseases such as lupus erythematosus, scleroderma and the like, reducing immune rejection reflection after somatic cell or liver transplantation, and improving the passing rate of somatic cell or liver transplantation; can promote the hematopoietic function to repair, and is superior to single hematopoietic stem cell transplantation, and the mesenchymal stem cell beauty and hematopoietic stem cell co-transplantation can obviously improve the treatment actual effects of sepsis, difficult anemia treatment and other diseases.
Description
Technical Field
The invention relates to the technical field of stem cell preparation methods, in particular to a preparation method of umbilical cord mesenchymal stem cells.
Background
Some stem cells not only have multipotency, but also are easy to transfect and express exogenous genes. The stem cells subjected to in vitro genetic modification are used for treatment, so that adverse effects caused by entering a transfection vector into a receptor can be avoided. Genetically modified stem cells can function at multiple targets; alterations in stem cell potency, the transfection of stem cell target genes may alter certain characteristics of stem cells. Stem cells isolated from adults or adult animals sometimes exhibit age-related, hereditary or acquired disease-related impairment of regenerative capacity, and gene transcription or cleavage modifications may be effective to maintain, enhance or inhibit the differentiation and proliferation capacity of stem cells; the performance of the organ system is improved, the genes transferred into stem cells by stem cell progeny targets can be inherited to the progeny cells along with differentiation and passage of the stem cells, and the genes are continuously expressed in the organ system reconstructed by the stem cells transduced by the genes, so that the function of the reconstructed organ system is improved; the regeneration of damaged tissues is accelerated, and the target damaged tissues are repaired in the regeneration process through the amplification and differentiation of cells. For example, endothelial progenitor cells complete vascular remodeling, neural stem cells complete neural regeneration, hematopoietic stem cells and mesenchymal stem cells complete bone marrow remodeling, etc. Natural repair processes tend to be slow due to severe injury, disease and age factors. The gene modification can increase the characteristics of a mitosis source of cells or generate factors for inhibiting negative regulation, so that the tissue regeneration process can be accelerated; generating exogenous gene expressed tissue, implanting stem cells modified by the systemic target gene into the body to generate a substitute tissue or organ with continuous expression of the modified gene, and further providing a certain amount of target molecules for treatment. It has been found that umbilical cord mesenchymal stem cells can treat more than 80 diseases including human nervous system, immune system, digestive system, motor system and other systems, and research on anti-aging of umbilical cord mesenchymal stem cells has also made major breakthrough and application attempts, and has been predicted by expert, and the wide application of stem cell anti-aging technology will lead the life of human being to be generally over 120 years in 2050. The umbilical cord mesenchymal stem cells have very important use value as important renewable medical resources in the application fields of disease treatment and human body anti-aging, but because the umbilical cord mesenchymal stem cells only exist in the umbilical cord, if the umbilical cord is discarded as medical waste, the abnormally precious renewable medical resources of the umbilical cord mesenchymal stem cells are wasted and cannot be remedied. Therefore, it is highly necessary to save this valuable renewable medical resource in time for future use.
Disclosure of Invention
The invention solves the technical problem of overcoming the prior art and providing a preparation method of umbilical cord mesenchymal stem cells.
In order to achieve the above purpose, the present invention provides the following technical solutions: a preparation method of umbilical cord mesenchymal stem cells comprises the following steps of:
s1, preparation: fully washing the umbilical cord with physiological saline and cutting into small sections; removing arteries and veins, and tearing the Huatong glue by at least 8ml; after being fully sheared, the mixture is equally divided into 4 culture flasks of 175cm2 with 25ml of complete culture medium; standing and culturing for 7 days; liquid exchange and passage are carried out according to the growth condition on the 8 th day;
s2, liquid replacement: determining full-quantity liquid exchange or half-quantity liquid exchange according to the growth condition of cells and the color of a culture medium; removing half or whole amount of old culture medium by using a pipette, replacing the pipette, and slowly adding an equivalent amount of new culture medium on the non-cell culture surface of the culture flask;
s3, passage: adding 3ml of 0.25% pancreatin into each bottle, tapping the bottle wall after the cells are rounded, adding 10ml of stop solution (2% FBS+a-MEM) into each bottle, sucking out the cell suspension into 2 50ml centrifuge tubes, adding 10ml of physiological saline into each culture bottle, and flushing and collecting into 50ml centrifuge tubes; centrifuging at 1200rpm for 6min, and discarding the supernatant; combining the precipitate to a 1-tube, adding 40ml of physiological saline, centrifuging again, washing the precipitate, re-suspending the precipitate with 10ml of complete culture medium, filtering the precipitate by a cell sieve, flushing the screen with 5ml of complete culture medium, and counting; spreading bottles according to the cell number, enabling the cell concentration to be 1-2 multiplied by 104/ml, and culturing in a 5% CO2 incubator at 37 ℃;
s4, harvesting: adding 3ml of 0.25% pancreatin into each bottle, digesting for 1min at 37 ℃, adding 10ml of stop solution into each bottle, collecting all the liquid into a 50ml centrifuge tube, adding 10ml of physiological saline into each bottle, gently blowing, and then collecting into the 50ml centrifuge tube; centrifuging at 1200rpm for 6min, discarding supernatant, suspending cell precipitate with 16ml physiological saline, mixing, taking 1ml for counting and flow detection; adding normal saline to 40ml, taking 500 μl of supernatant as endotoxin detection, centrifuging at 1200rpm for 6min; discarding the supernatant, suspending the centrifugal precipitate with 2.5ml FBS, slowly adding 2.5ml cryopreservation mother liquor, mixing, and packaging into cryopreservation tubes with 1ml each tube; the number of the frozen cells is controlled within the range of 2-5 multiplied by 106/ml; and (5) placing the medical refrigerator at the temperature of minus 80 ℃ by using a program cooling box overnight and transferring the medical refrigerator to a liquid nitrogen tank.
Preferably, the umbilical cord mesenchymal stem cells are isolated as follows:
s1, separation of umbilical cord mesenchymal stem cells: immersing umbilical cord in physiological saline containing antibiotics after being taken down from an operating table, preserving at 4 ℃, taking out umbilical cord in the operating table, flushing residual blood in umbilical arteries and umbilical veins with D-PBS, removing the blood vessels with hemostats and scissors, shearing the umbilical cord into tissue blocks with the size of 1mm & lt 3 & gt, putting the tissue blocks into a 200mL blue cap reagent bottle, adding 30mL of type II collagenase with the mass/volume ratio of 0.1%, putting the tissue blocks into a constant temperature oscillator, continuously digesting for 6h, and filtering and collecting cells with a 100-mesh screen; adding D-Hank's solution to wash the cells for 3 times, re-suspending the cells with DMEM/F12 containing 10% fetal calf serum by volume fraction, adjusting the cell density to be (4.8X10-1X 10-4)/cm 2, inoculating into a 6-well plate, culturing in a CO2 incubator with the volume fraction of 5% at 37 ℃ for 24 hours, and changing the solution;
s2, in-vitro expansion of umbilical cord mesenchymal stem cells in different culture systems: after the umbilical cord mesenchymal stem cells of primary culture are attached, performing growth comparison experiments of 3 culture systems in two 6-hole plates; the cell density in the 1 st 6 th pore plate is 1X 10-7L-1, and the continuous adaptation method is adopted to gradually transition the cells from DMEM/F12 used in primary culture to low sugar DMEM, mesenPRORS TM Medium and method for producing sameMSCSFM3 culture system, namely, mixing culture cells with DMEM/F12 culture medium containing 10% fetal bovine serum and corresponding 3 culture mediums according to the volume ratio of 1:1, continuously reducing the quantity of the current culture medium for several generations by adopting the following mixed culture medium mode, namely, 1:2, 1:4, 1:16 and 100% replacement culture medium, wherein when the culture system is changed adaptively, the cells are passaged twice, wherein the hole 1 is DMEM/F12 culture solution in primary culture, the hole 2 is low-sugar DMEM, and the hole 3 is MesenPRORS TM Medium, hole 4 +.>MSCSFM; the cell density in the 2 nd 6 th pore plate is 2 x 10-7L-1, and each pore culture solution is set as above, so as to avoid experimental operation errors when the mesenchymal stem cells are digested and passaged at different times;
isolated culture of umbilical cord mesenchymal stem cells in vitro:
(1) After the umbilical cord white operating table is taken down, immersing the umbilical cord white operating table in 0.9% physiological saline containing antibiotics, and storing 4"C;
(2) Taking out the umbilical cord in the super clean bench, flushing and cleaning residual blood in umbilical arteries and umbilical veins by using normal saline, removing the blood vessels by using hemostats and scissors, and cutting the umbilical cord into tissue blocks with the size of 1mm & lt 3 >
(3) Adding type II collagenase with the mass/volume ratio of 0.1% until the collagenase completely covers the tissue blocks, placing the tissue blocks in an incubator for continuous digestion for 6 hours, and filtering and collecting cells by a 100-mesh screen;
(4) Washing the cells 3 times with physiological saline at 2000rpm for 8min each;
(5) Resuspension cells with DMEM/F12 medium containing 10% fbs, adjusting cell density to 1x106cells/ml, inoculating into t.25 flask, and placing in 37; C. culturing in a saturated humidity and 5% C02 incubator;
(6) After 3 days, the total amount is changed into MesenPRORSrMMedium culture solution, cells and impurities which are not adhered are discarded, and then the solution is changed once every 3 days according to the growth condition of the cells;
preferably, in vitro expansion of umbilical cord mesenchymal stem cells
(1) When the cells obtained by the culture method grow to 80% and fuse, digesting the cells by using a mixed solution of 0.25% trypsin and 0.02% EDTA, observing the cells under a microscope, controlling the digestion time, and stopping digestion when cytoplasm is retracted, and adding a DMEM/F12 culture solution containing 10% FBS;
(2) The cell suspension was centrifuged at 1000rpm for 3min and the primary cells were spun at 1:1, carrying out passage according to the proportion, and marking as generation P1;
(3) The culture solution after the next passage is replaced by MesenPRORSa-MMedium culture solution; the generation P2 is followed by the following steps of 1:3 or l:4, proportion passage; the above procedure was repeated until the cells fused with each other, filling the bottom of the flask.
Compared with the prior art, the invention has the beneficial effects that: the invention has extremely strong hormone regulation effect, can be used for treating the diseases of the immune system such as lupus erythematosus, scleroderma and the like, reduces the immune rejection reaction after somatic cell or liver transplantation, and improves the passing rate of somatic cell or liver transplantation; can promote the hematopoietic function to repair, and is superior to single hematopoietic stem cell transplantation, and the mesenchymal stem cell beauty and hematopoietic stem cell co-transplantation can obviously improve the treatment actual effects of sepsis, difficult anemia treatment and other diseases.
Drawings
FIG. 1 is a comparative diagram of stem cell preparation according to the present invention;
fig. 2 is a flow chart of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-2, the present invention provides a technical solution: a preparation method of umbilical cord mesenchymal stem cells comprises the following steps of:
s1, preparation: fully washing the umbilical cord with physiological saline and cutting into small sections; removing arteries and veins, and tearing the Huatong glue by at least 8ml; after being fully sheared, the mixture is equally divided into 4 culture flasks of 175cm2 with 25ml of complete culture medium; standing and culturing for 7 days; liquid exchange and passage are carried out according to the growth condition on the 8 th day;
s2, liquid replacement: determining full-quantity liquid exchange or half-quantity liquid exchange according to the growth condition of cells and the color of a culture medium; removing half or whole amount of old culture medium by using a pipette, replacing the pipette, and slowly adding an equivalent amount of new culture medium on the non-cell culture surface of the culture flask;
s3, passage: adding 3ml of 0.25% pancreatin into each bottle, tapping the bottle wall after the cells are rounded, adding 10ml of stop solution (2% FBS+a-MEM) into each bottle, sucking out the cell suspension into 2 50ml centrifuge tubes, adding 10ml of physiological saline into each culture bottle, and flushing and collecting into 50ml centrifuge tubes; centrifuging at 1200rpm for 6min, and discarding the supernatant; combining the precipitate to a 1-tube, adding 40ml of physiological saline, centrifuging again, washing the precipitate, re-suspending the precipitate with 10ml of complete culture medium, filtering the precipitate by a cell sieve, flushing the screen with 5ml of complete culture medium, and counting; spreading bottles according to the cell number, enabling the cell concentration to be 1-2 multiplied by 104/ml, and culturing in a 5% CO2 incubator at 37 ℃;
s4, harvesting: adding 3ml of 0.25% pancreatin into each bottle, digesting for 1min at 37 ℃, adding 10ml of stop solution into each bottle, collecting all the liquid into a 50ml centrifuge tube, adding 10ml of physiological saline into each bottle, gently blowing, and then collecting into the 50ml centrifuge tube; centrifuging at 1200rpm for 6min, discarding supernatant, suspending cell precipitate with 16ml physiological saline, mixing, taking 1ml for counting and flow detection; adding normal saline to 40ml, taking 500 μl of supernatant as endotoxin detection, centrifuging at 1200rpm for 6min; discarding the supernatant, suspending the centrifugal precipitate with 2.5ml FBS, slowly adding 2.5ml cryopreservation mother liquor, mixing, and packaging into cryopreservation tubes with 1ml each tube; the number of the frozen cells is controlled within the range of 2-5 multiplied by 106/ml; and (5) placing the medical refrigerator at the temperature of minus 80 ℃ by using a program cooling box overnight and transferring the medical refrigerator to a liquid nitrogen tank.
Preferably, the umbilical cord mesenchymal stem cells are isolated as follows:
s1, separation of umbilical cord mesenchymal stem cells: immersing umbilical cord in physiological saline containing antibiotics after being taken down from an operating table, preserving at 4 ℃, taking out umbilical cord in the operating table, flushing residual blood in umbilical arteries and umbilical veins with D-PBS, removing the blood vessels with hemostats and scissors, shearing the umbilical cord into tissue blocks with the size of 1mm & lt 3 & gt, putting the tissue blocks into a 200mL blue cap reagent bottle, adding 30mL of type II collagenase with the mass/volume ratio of 0.1%, putting the tissue blocks into a constant temperature oscillator, continuously digesting for 6h, and filtering and collecting cells with a 100-mesh screen; adding D-Hank's solution to wash the cells for 3 times, re-suspending the cells with DMEM/F12 containing 10% fetal calf serum by volume fraction, adjusting the cell density to be (4.8X10-1X 10-4)/cm 2, inoculating into a 6-well plate, culturing in a CO2 incubator with the volume fraction of 5% at 37 ℃ for 24 hours, and changing the solution;
s2, in-vitro expansion of umbilical cord mesenchymal stem cells in different culture systems: after the umbilical cord mesenchymal stem cells of primary culture are attached, performing growth comparison experiments of 3 culture systems in two 6-hole plates; the cell density in the 1 st 6 th pore plate is 1X 10-7L-1, and the continuous adaptation method is adopted to gradually transition the cells from DMEM/F12 used in primary culture to low sugar DMEM, mesenPRORS TM Medium and method for producing sameMSCSFM3 culture system, namely, mixing culture cells with DMEM/F12 culture medium containing 10% fetal bovine serum and corresponding 3 culture mediums according to the volume ratio of 1:1, continuously reducing the quantity of the current culture medium for several generations by adopting the following mixed culture medium mode, namely, 1:2, 1:4, 1:16 and 100% replacement culture medium, wherein when the culture system is changed adaptively, the cells are passaged twice, wherein the hole 1 is DMEM/F12 culture solution in primary culture, the hole 2 is low-sugar DMEM, and the hole 3 is MesenPRORS TM Medium, hole 4 +.>MSCSFM; the cell density in the 2 nd 6 th pore plate is 2 x 10-7L-1, and each pore culture solution is set as above, so as to avoid experimental operation errors when the mesenchymal stem cells are digested and passaged at different times;
isolated culture of umbilical cord mesenchymal stem cells in vitro:
(1) After the umbilical cord white operating table is taken down, immersing the umbilical cord white operating table in 0.9% physiological saline containing antibiotics, and storing 4"C;
(2) Taking out the umbilical cord in the super clean bench, flushing and cleaning residual blood in umbilical arteries and umbilical veins by using normal saline, removing the blood vessels by using hemostats and scissors, and cutting the umbilical cord into tissue blocks with the size of 1mm & lt 3 >
(3) Adding type II collagenase with the mass/volume ratio of 0.1% until the collagenase completely covers the tissue blocks, placing the tissue blocks in an incubator for continuous digestion for 6 hours, and filtering and collecting cells by a 100-mesh screen;
(4) Washing the cells 3 times with physiological saline at 2000rpm for 8min each;
(5) Resuspension cells with DMEM/F12 medium containing 10% fbs, adjusting cell density to 1x106cells/ml, inoculating into t.25 flask, and placing in 37; C. culturing in a saturated humidity and 5% C02 incubator;
(6) After 3 days, the total amount is changed into MesenPRORSrMMedium culture solution, cells and impurities which are not adhered are discarded, and then the solution is changed once every 3 days according to the growth condition of the cells;
preferably, in vitro expansion of umbilical cord mesenchymal stem cells
(1) When the cells obtained by the culture method grow to 80% and fuse, digesting the cells by using a mixed solution of 0.25% trypsin and 0.02% EDTA, observing the cells under a microscope, controlling the digestion time, and stopping digestion when cytoplasm is retracted, and adding a DMEM/F12 culture solution containing 10% FBS;
(2) The cell suspension was centrifuged at 1000rpm for 3min and the primary cells were spun at 1:1, carrying out passage according to the proportion, and marking as generation P1;
(3) The culture solution after the next passage is replaced by MesenPRORSa-MMedium culture solution; the generation P2 is followed by the following steps of 1:3 or l:4, proportion passage; the above procedure was repeated until the cells fused with each other, filling the bottom of the flask.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A preparation method of umbilical cord mesenchymal stem cells comprises the preparation method of the mesenchymal stem cells and is characterized by comprising the following steps:
s1, preparation: fully washing the umbilical cord with physiological saline and cutting into small sections; removing arteries and veins, and tearing the Huatong glue by at least 8ml; after being fully sheared, the mixture is equally divided into 4 culture flasks of 175cm2 with 25ml of complete culture medium; standing and culturing for 7 days; liquid exchange and passage are carried out according to the growth condition on the 8 th day;
s2, liquid replacement: determining full-quantity liquid exchange or half-quantity liquid exchange according to the growth condition of cells and the color of a culture medium; removing half or whole amount of old culture medium by using a pipette, replacing the pipette, and slowly adding an equivalent amount of new culture medium on the non-cell culture surface of the culture flask;
s3, passage: adding 3ml of 0.25% pancreatin into each bottle, tapping the bottle wall after the cells are rounded, adding 10ml of stop solution (2% FBS+a-MEM) into each bottle, sucking out the cell suspension into 2 50ml centrifuge tubes, adding 10ml of physiological saline into each culture bottle, and flushing and collecting into 50ml centrifuge tubes; centrifuging at 1200rpm for 6min, and discarding the supernatant; combining the precipitate to a 1-tube, adding 40ml of physiological saline, centrifuging again, washing the precipitate, re-suspending the precipitate with 10ml of complete culture medium, filtering the precipitate by a cell sieve, flushing the screen with 5ml of complete culture medium, and counting; spreading bottles according to the cell number, enabling the cell concentration to be 1-2 multiplied by 104/ml, and culturing in a 5% CO2 incubator at 37 ℃;
s4, harvesting: adding 3ml of 0.25% pancreatin into each bottle, digesting for 1min at 37 ℃, adding 10ml of stop solution into each bottle, collecting all the liquid into a 50ml centrifuge tube, adding 10ml of physiological saline into each bottle, gently blowing, and then collecting into the 50ml centrifuge tube; centrifuging at 1200rpm for 6min, discarding supernatant, suspending cell precipitate with 16ml physiological saline, mixing, taking 1ml for counting and flow detection; adding normal saline to 40ml, taking 500 μl of supernatant as endotoxin detection, centrifuging at 1200rpm for 6min; discarding the supernatant, suspending the centrifugal precipitate with 2.5ml FBS, slowly adding 2.5ml cryopreservation mother liquor, mixing, and packaging into cryopreservation tubes with 1ml each tube; the number of the frozen cells is controlled within the range of 2-5 multiplied by 106/ml; and (5) placing the medical refrigerator at the temperature of minus 80 ℃ by using a program cooling box overnight and transferring the medical refrigerator to a liquid nitrogen tank.
2. The method for preparing umbilical cord mesenchymal stem cells according to claim 1, wherein: the umbilical cord mesenchymal stem cells were isolated as follows:
s1, separation of umbilical cord mesenchymal stem cells: immersing umbilical cord in physiological saline containing antibiotics after being taken down from an operating table, preserving at 4 ℃, taking out umbilical cord in the operating table, flushing residual blood in umbilical arteries and umbilical veins with D-PBS, removing the blood vessels with hemostats and scissors, shearing the umbilical cord into tissue blocks with the size of 1mm & lt 3 & gt, putting the tissue blocks into a 200mL blue cap reagent bottle, adding 30mL of type II collagenase with the mass/volume ratio of 0.1%, putting the tissue blocks into a constant temperature oscillator, continuously digesting for 6h, and filtering and collecting cells with a 100-mesh screen; adding D-Hank's solution to wash the cells for 3 times, re-suspending the cells with DMEM/F12 containing 10% fetal calf serum by volume fraction, adjusting the cell density to be (4.8X10-1X 10-4)/cm 2, inoculating into a 6-well plate, culturing in a CO2 incubator with the volume fraction of 5% at 37 ℃ for 24 hours, and changing the solution;
s2, in-vitro expansion of umbilical cord mesenchymal stem cells in different culture systems: after the umbilical cord mesenchymal stem cells of the primary culture are attached, 3 culture systems are carried out in two 6-hole platesIs a growth comparison experiment of (2); the cell density in the 1 st 6 th pore plate is 1X 10-7L-1, and the continuous adaptation method is adopted to gradually transition the cells from DMEM/F12 used in primary culture to low sugar DMEM, mesenPRORS TM Medium and method for producing sameMSCSFM3 culture system, namely, mixing culture cells with DMEM/F12 culture medium containing 10% fetal bovine serum and corresponding 3 culture mediums according to the volume ratio of 1:1, continuously reducing the quantity of the current culture medium for several generations by adopting the following mixed culture medium mode, namely, 1:2, 1:4, 1:16 and 100% replacement culture medium, wherein when the culture system is changed adaptively, the cells are passaged twice, wherein the hole 1 is DMEM/F12 culture solution in primary culture, the hole 2 is low-sugar DMEM, and the hole 3 is MesenPRORS TM Medium, hole 4 +.>MSCSFM; the cell density in the 2 nd 6 th pore plate is 2 x 10-7L-1, and each pore culture solution is set as above, so as to avoid experimental operation errors when the mesenchymal stem cells are digested and passaged at different times;
isolated culture of umbilical cord mesenchymal stem cells in vitro:
(1) After the umbilical cord white operating table is taken down, immersing the umbilical cord white operating table in 0.9% physiological saline containing antibiotics, and storing 4"C;
(2) Taking out the umbilical cord in the super clean bench, flushing and cleaning residual blood in umbilical arteries and umbilical veins by using normal saline, removing the blood vessels by using hemostats and scissors, and cutting the umbilical cord into tissue blocks with the size of 1mm & lt 3 >
(3) Adding type II collagenase with the mass/volume ratio of 0.1% until the collagenase completely covers the tissue blocks, placing the tissue blocks in an incubator for continuous digestion for 6 hours, and filtering and collecting cells by a 100-mesh screen;
(4) Washing the cells 3 times with physiological saline at 2000rpm for 8min each;
(5) Resuspension cells with DMEM/F12 medium containing 10% fbs, adjusting cell density to 1x106cells/ml, inoculating into t.25 flask, and placing in 37; C. culturing in a saturated humidity and 5% C02 incubator;
(6) After 3 days, the whole culture solution is replaced by MesenPRORSrMMedium culture solution, cells and impurities which are not adhered are discarded, and then the solution is replaced every 3 days according to the growth condition of the cells.
3. The method for preparing umbilical cord mesenchymal stem cells according to claim 1, wherein: in vitro expansion of umbilical cord mesenchymal stem cells
(1) When the cells obtained by the culture method grow to 80% and fuse, digesting the cells by using a mixed solution of 0.25% trypsin and 0.02% EDTA, observing the cells under a microscope, controlling the digestion time, and stopping digestion when cytoplasm is retracted, and adding a DMEM/F12 culture solution containing 10% FBS;
(2) The cell suspension was centrifuged at 1000rpm for 3min and the primary cells were spun at 1:1, carrying out passage according to the proportion, and marking as generation P1;
(3) The culture solution after the next passage is replaced by MesenPRORSa-MMedium culture solution; the generation P2 is followed by the following steps of 1:3 or l:4, proportion passage; the above procedure was repeated until the cells fused with each other, filling the bottom of the flask.
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