CN115678842A - Optimized culture medium, kit and tissue adherent culture method for human umbilical cord-derived mesenchymal stem cells - Google Patents

Optimized culture medium, kit and tissue adherent culture method for human umbilical cord-derived mesenchymal stem cells Download PDF

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CN115678842A
CN115678842A CN202211488828.7A CN202211488828A CN115678842A CN 115678842 A CN115678842 A CN 115678842A CN 202211488828 A CN202211488828 A CN 202211488828A CN 115678842 A CN115678842 A CN 115678842A
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金倞
雷欣华
梁磊
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Beijing Jizhongke Biotechnology Co ltd
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Abstract

The invention relates to an optimized culture medium, a kit and a tissue adherent culture method for human umbilical cord-derived mesenchymal stem cells, and belongs to the technical field of stem cell culture. The optimized culture medium comprises: basal medium, serum replacement and supplements. The optimized culture medium can ensure that the MSC can still maintain the specific cell phenotype and multidirectional differentiation potential after multiple times of passage amplification, furthest shorten the time from the separation of cells from tissues to the adaptation of the process to an in vitro culture environment, furthest improve the cell yield in unit time, shorten the time between generations of culture, further improve the cell performance and meet the supervision requirement of clinical application, can better amplify human mesenchymal stem cells in vitro, furthest improve the in vitro amplification efficiency of the MSC and reduce the difference of cells in batches.

Description

Optimized culture medium, kit and tissue adherent culture method for human umbilical cord-derived mesenchymal stem cells
The priority of the application of Chinese patent with patent office 202111467116.2 entitled "an optimized culture medium, kit and tissue adherent culture method of human umbilical cord-derived mesenchymal stem cells" filed on 03.12.2021 is claimed, and the entire content of the application is incorporated by reference.
Technical Field
The invention relates to the technical field of stem cell culture, in particular to an optimized culture medium, a kit and a tissue adherent culture method for human umbilical cord-derived mesenchymal stem cells.
Background
Stem cells are present in various organs and tissues of the body, they have regenerative potential, and throughout the human life cycle, such cells play an important role in repairing damaged tissues in the body. This has led to a great interest in Adult Stem Cells (ASCs), especially since the use of ASCs, unlike Embryonic Stem Cells (ESCs), does not pose ethical and ethical difficulties. Several studies have reported the isolation of ASCs, such as Mesenchymal Stromal Cells (MSCs), hematopoietic Stem Cells (HSCs) and other diverse progenitor cells, including various adult sources ranging from Bone Marrow (BM) to Adipose Tissue (AT) and dental pulp. However, most (1) ASCs present in adult ecosites are limited, and (2) their extraction often involves painful processes with potential donor site morbidity and tissue invasiveness. In addition, (3) donor age and environmental stress may also play an important role in determining the quality and biological activity of isolated cells. Meanwhile, (4) ASCs also show limited proliferative capacity and differentiation potential during in vitro culture.
To overcome these shortcomings of current ASCs, new sources for stem cell isolation have been sought. These attempts have evolved to isolate stem cells from perinatal sources, including cord blood, umbilical cord tissue, placenta, amnion, and amniotic fluid. These (1) sources are of widespread interest because of their readily available and abundant availability. In addition, (2) perinatal tissues can be obtained in a non-invasive manner, from which stem cells are extracted that are more primitive than ASCs isolated from adult sources. They were isolated from tissues obtained at birth and (3) were not considered to undergo minimal changes in the genome due to aging and environmental stress.
However, the reported characteristics of stem cells from perinatal sources vary widely in their potential for self-renewal and differentiation. The existing extraction process conditions of umbilical cord source mesenchymal stem cells generally adopt umbilical cord tissues as a whole, and a tissue block adherence method is used for obtaining the mesenchymal stem cells, but (1) the self-renewal and differentiation potentials of the cells obtained from different tissue positions are different, (2) the doubling time of the cells is different, although the phenotype of the cells harvested at the same time is consistent, the proliferation times and the functions of the cells crawled out from different tissue blocks during harvesting are different, and (3) the functions of the whole cell preparation can be reduced along with the increase of the passage times. At present, a high-efficiency culture method of umbilical cord-derived mesenchymal stem cells which can be used for clinical treatment is still lacking.
Disclosure of Invention
The invention aims to provide an optimized culture medium, a kit and a tissue adherence culture method for human umbilical cord derived mesenchymal stem cells. The optimized culture medium can ensure that MSC can still meet the requirements of clinical application supervision after multiple subculture amplification.
The invention provides an optimized culture medium of human umbilical cord-derived mesenchymal stem cells, which comprises the following components: basal media, serum replacement and supplements; the basic culture medium comprises DMEM-low glucose and L-glutamine; the serum replacement comprises Knockout TM A serum replacement; the supplement comprises recombinant human FGF2 protein, recombinant human FGF4 protein, recombinant human PDGF AB protein, recombinant human EGF protein, recombinant human VEGF protein, recombinant human HGF protein, recombinant human TGF-beta 1, recombinant human BMP-3 protein and nicotinamide mononucleotide.
Preferably, in the optimized culture medium, the volume ratio of the basal medium, the serum substitute and the supplement is 90:9:1.
preferably, in the optimized medium: the mass concentration of L-glutamine is 6mM, the mass concentration of recombinant human FGF2 protein is 40ng/mL, the mass concentration of recombinant human FGF4 protein is 10ng/mL, the mass concentration of recombinant human PDGFAB protein is 4ng/mL, the mass concentration of recombinant human EGF protein is 2ng/mL, the mass concentration of recombinant human VEGF protein is 0.2ng/mL, the mass concentration of recombinant human HGF protein is 0.2ng/mL, the mass concentration of recombinant human TGF-beta 1 is 0.5ng/mL, the mass concentration of recombinant human BMP-3 protein is 0.2ng/mL, and the mass concentration of nicotinamide mononucleotide is 5mM.
The invention also provides a kit for culturing human umbilical cord-derived mesenchymal stem cells, which comprises the optimized culture medium and trypLE in the technical scheme TM Express digestive enzyme and Nunclon TM Delta culture dishes.
The invention also provides a tissue adherent culture method of the human umbilical cord-derived mesenchymal stem cells based on the kit of the technical scheme, which comprises the following steps:
placing the umbilical cord sample in a TrypLE TM Express is digested in digestive enzyme solution to obtain digested umbilical cord tissue;
placing the digested umbilical cord tissue in Nunclon containing the optimized culture medium of the technical scheme TM And performing adherent culture in a Delta culture dish to obtain the human umbilical cord-derived mesenchymal stem cells.
Preferably, the source of the umbilical cord sample comprises: umbilical cord-placental junction tissue and/or umbilical cord tissue; the umbilical cord tissue comprises an umbilical cord lining and/or umbilical cord Wharton's jelly.
Preferably, the time of digestion is 5min.
Preferably, the method further comprises the following steps after the adherent culture: when the cell fusion degree of adherent culture reaches 70-90%, trypLE is used TM Express digestive enzyme solution is used for digestive culture and centrifugation to obtain primary cells.
Preferably, the digestion culture time is 3 to 4min.
Preferably, when the obtained primary cells are subcultured, the culture is performed using the optimized medium.
The invention provides an optimized culture medium for human umbilical cord-derived mesenchymal stem cells. The culture medium comprises: basal medium, serum replacement and supplements. The optimized culture medium can ensure that the MSC can still maintain the specific cell phenotype and multidirectional differentiation potential after multiple times of passage amplification, furthest shorten the time from the separation of cells from tissues to the adaptation of the process to an in vitro culture environment, furthest improve the cell yield in unit time, shorten the time between generations of culture, further improve the cell performance and meet the supervision requirement of clinical application, can better amplify human mesenchymal stem cells in vitro, furthest improve the in vitro amplification efficiency of the MSC and reduce the difference of cells in batches.
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FIG. 1 is a schematic view of a portion of an umbilical cord collection provided by the present invention;
FIG. 2 is a schematic illustration of the anatomy of an umbilical cord provided by the present invention;
FIG. 3 is a schematic diagram of tissue adherence inoculation provided by the present invention;
FIG. 4 is a comparison of cell-climbing status of CPJ/WJ/CL tissues provided by the present invention at Day6/12/18 time points;
FIG. 5 is the preparation of UCMSC21041101 umbilical cord derived mesenchymal stem cells P1 generation cells (10 bottles);
FIG. 6 is a 20-flask preparation of umbilical cord-derived mesenchymal stem cells P1 generation cells of UCMSC21042101 provided by the present invention;
FIG. 7 is a table showing the P1 generation cell preparation (30 flasks) of UCMSC21062101 umbilical cord-derived mesenchymal stem cells provided by the present invention;
FIG. 8 shows the mesenchymal cell creep-out state of the umbilical cord WJ tissue under different conditions provided by the present invention;
FIG. 9 shows the UC-MSC optimized media combination of the Zhongke and Dake as the MSC media combination provided by the present invention;
FIG. 10 shows the state of the P2/P5/P10 generation sub-umbilical cord source MSC under the mirror of two culture systems provided by the present invention;
FIG. 11 shows the total number of cells obtained from the transfer to P10 generation after the same number of cells are inoculated into a T-25 flask under two culture conditions provided by the present invention;
FIG. 12 shows phenotypic identification of umbilical cord mesenchymal stem cells P10 under optimized culture conditions provided by the present invention;
FIG. 13 shows the identification of differentiation capacity of umbilical cord-derived mesenchymal stem cells P2/P5/P10 generation-by-generation three-line under the optimized culture conditions provided by the present invention.
Detailed Description
The invention provides an optimized culture medium of human umbilical cord-derived mesenchymal stem cells, which comprises the following components in percentage by weight: basal media, serum replacement and supplements; the basic culture medium comprises DMEM-low glucose; the serum replacement comprises Knockout TM A serum replacement; the supplement comprises L-glutamine, recombinant human FGF2 protein, recombinant human FGF4 protein, recombinant human PDGF AB protein, recombinant human EGF protein, recombinant human VEGF protein, recombinant human HGF protein, recombinant human TGF-beta 1, recombinant human BMP-3 protein and nicotinamide mononucleotide.
In the present invention, the volume ratio of the basal medium, the serum replacement and the supplement in the optimized medium is preferably 90:9:1.
in the present invention, in the optimized medium: the mass concentration of L-glutamine is 6mM, the mass concentration of recombinant human FGF2 protein is 40ng/mL, the mass concentration of recombinant human FGF4 protein is 10ng/mL, the mass concentration of recombinant human PDGFAB protein is 4ng/mL, the mass concentration of recombinant human EGF protein is 2ng/mL, the mass concentration of recombinant human VEGF protein is 0.2ng/mL, the mass concentration of recombinant human HGF protein is 0.2ng/mL, the mass concentration of recombinant human TGF-beta 1 is 0.5ng/mL, the mass concentration of recombinant human BMP-3 protein is 0.2ng/mL, and the mass concentration of nicotinamide mononucleotide is 5mM.
Umbilical cord tissue contains many epithelial cells and other cell types in addition to mesenchymal stem cells. The isolation and expansion of the desired mesenchymal stem cells therefrom requires powerful in vitro cell culture expansion methods to obtain sufficient numbers of cells in high purity to meet basic research and clinical applications. The optimized culture medium used in the extraction and amplification method of the source umbilical cord source mesenchymal stem cells is a complete culture medium which is serum-free and does not contain animal-derived components, is suitable for the growth and amplification of the human mesenchymal stem cells, provides complete nutrition and amplification support of cell specificity for the mesenchymal stem cells, and can ensure that the MSC can still maintain the specific cell phenotype and multidirectional differentiation potential after multiple times of subculture amplification. The optimized culture medium can shorten the time from the separation of cells from tissues to the adaptation of the in vitro culture environment to the maximum extent, improve the cell yield in unit time to the maximum extent, shorten the time between subcultures, and further improve the cell performance and meet the clinical application supervision requirements. The optimized culture medium is added with a mesenchymal stem cell stimulating supplement besides a basic culture medium and a serum substitute, and can better amplify the human mesenchymal stem cells in vitro by combining the use. The specific setting of the proportion of the content cytokines in the supplement can improve the in vitro expansion efficiency of the MSC to the maximum extent and reduce the difference of cells among batches.
The invention also provides a kit for culturing human umbilical cord-derived mesenchymal stem cells, which comprises the optimized culture medium and trypLE in the technical scheme TM Express digestive enzyme and Nunclon TM Delta culture dishes.
TrypLE in the kit is used TM Express digestive enzyme digests umbilical cord-derived tissue in Nunclon TM The method has the advantages that adherent culture is carried out on a Delta culture dish, the optimized culture medium is used as the culture medium for culture, the umbilical cord mesenchymal stem cells can be stably cultured, the tissue cell climbing efficiency is improved, the mutation probability of the cells is reduced, the stable phenotype and function of the cell maintenance system are ensured, the cells with enough quantity and high purity can be finally obtained, the clinical application standard is met, the long-term passage of MSC is realized, and the three-line differentiation potential of the mesenchymal stem cells of the maintenance system is kept while the cell consistency is kept.
The invention also provides a tissue adherent culture method of the human umbilical cord derived mesenchymal stem cells based on the kit of the technical scheme, which comprises the following steps:
placing the umbilical cord sample in a TrypLE TM Express digestive enzyme solution is subjected to short-time mild digestion to obtain digested umbilical cord tissue;
placing the digested umbilical cord tissue in Nunclon containing the optimized culture medium of the technical scheme TM And performing adherent culture in a Delta culture dish to obtain the human umbilical cord-derived mesenchymal stem cells.
The invention puts the umbilical cord sample in TrypLE TM Express digestive enzyme solution is subjected to short-time mild digestion to obtain digested umbilical cord tissue. In the present invention, the source of the umbilical cord sample preferably comprises: umbilical Cord-placental Junction (CPJ) tissue and/or Umbilical Cord (UC) tissue; the umbilical cord tissue includes an umbilical Cord Lining (CL) and/or umbilical cord Wharton's Jelly (WJ). The CPJ is used as a sample for culturing, so that the produced cells are more and faster, the cell preparation time can be shortened, and the cell mutation probability can be reduced; hua-quan glue is slow, but because of the large amount of tissue, the total amount of cells is large. The embodiment of the invention proves that in unit time, compared with CL and WJ parts, the time required for separating MSC cells from CPJ tissue parts is short, the yield is higher, obvious MSC cell climbing can be observed in 3-4 days of an experiment, and more than 70% of cell confluence can be achieved in 7-10 days. Prior to said digestion, the present invention preferably shears the cord sample into pieces. The normal tissue digestion time is usually 120-180 min to release cells from the tissue for cell inoculation, in the present invention, the short digestion time is preferably 5min, and the tissue is not digested into cells but is inoculated into a tissue block, the short digestion promotes the adhesion efficiency of the tissue block on the surface of a culture dish, promotes the cell climbing efficiency from the tissue, and the operation time required for tissue treatment is shorter. In the present invention, the short digestion is carried out in an incubator at 37 ℃. In the invention, the TrypLE TM Express digestive enzymes are preferably available from Thermo Fisher, cat #: A1217702. the TrypLE of the invention TM Express digestive enzyme can be inactivated by dilution without adding protease inhibitor, and contains no animal-derived components. The invention preferably uses TrypLE preheated at 37 DEG C TM Express (10 ×) undergoes tissue shearing followed by TrypLE preheated at 37 ℃ TM Express (1 ×) underwent a mild digestion. The present invention is not particularly limited in its operation for terminating digestion, and digestion can be terminated by diluting with a culture medium.
After obtaining the digested umbilical cord tissue, the invention puts the digested umbilical cord sample in Nunclon containing the optimized culture medium of the technical scheme TM And performing adherent culture in a Delta culture dish to obtain the human umbilical cord-derived mesenchymal stem cells. In the present invention, the temperature of the adherent culture is preferably 37 ℃; the adherent culture is preferably performed in the presence of 5% CO by volume 2 In an incubator. The method comprises the steps of preferably paving the digested umbilical cord tissue on a culture dish, adding an optimized culture medium, preferably incubating for 3d, preferably changing the culture medium for half amount after incubation, and preferably changing a new optimized culture medium every 3d after the cells climb out. In the invention, the CPJ tissue is incubated for 3-4 days to allow the cells to obviously climb out, and the CL and WJ tissues are incubated for 7-10 days to allow the cells to obviously climb out. The first three days of incubation, if there are floating debris, the present invention preferably transfers the debris to a new Nunclon TM Attachment was performed in Delta dishes.
The invention uses TrypLE preheated at 37 ℃ before tissue adherence TM Express (1 ×) performs a mild digestion that promotes cell climbing out of the tissue; simultaneously inoculating the tissue in Nunclon TM Attachment was performed in Delta dishes, with Nunclon Delta coating, which promoted tissue adhesion to its surface. Under the combined action of the two technical steps, the tissue adhesion time is shortened, the tissue adhesion area is increased, and the UC-MSC cell yield in unit time is further improved. Test results show that the tissue digestion enzymolysis treatment and the use of the specific culture dish shorten the preparation flow time of primary cells, increase the cell yield in unit time, improve the adhesion efficiency of tissue slices in unit time, shorten the wall-adhering time of tissues on the surface of the culture dish, create a good external environment for cell climbing, further accelerate the climbing speed of cells from the tissues and finally realize the output of high-quantity cells in unit time.
In the present invention, it is preferable that the adherent culture further comprises: when the cell fusion degree of adherent culture reaches 70-90%, trypLE is used TM Express digestive enzyme solution is used for digestive culture and centrifugation to obtain primary cells. In the present invention, the digestion culture is preferably carried out for a period of time3-4 min, the digestion is preferably carried out using TrypLE Express (1X) preheated at 37 ℃. In the present invention, the centrifugation is preferably performed at 1200rpm for 5min. In the present invention, when the obtained primary cells are subcultured, the culture is performed using an optimized medium. In the subculture according to the present invention, the ratio is preferably 1X 10 4 /cm 2 Is carried out for subculture. The excess cells are preferably at 4X 10 6 The cryo-preservation was carried out at a concentration of/mL in Cryostor CS10 cell stocks.
The optimized culture medium, the kit and the tissue adherence culture method of the human umbilical cord-derived mesenchymal stem cells are further described in detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
(1) Intact tissue with Umbilical Cord specimens was collected aseptically, with UC (Umbilical Cord, UC) approximately 15cm in length, including 10-11 cm to the Umbilical Cord-placental Junction site (CPJ) and 1-3 cm of CPJ to Placenta (Fetal Placenta, FP) (differences between individual tissues). Immediately, the entire tissue sample was placed in a tissue preservation bag containing tissue preservation solution (DMEM containing 4500mg/mL glucose and antibiotic solutions (0.1% gentamicin, 0.2% streptomycin, and 0.12% penicillin)) and the tissue preservation bag was stored in a 4 ℃ tissue transport cassette and transported to the laboratory. The samples were processed 2-4 h after tissue collection, all in a sterile laboratory environment.
(2) The samples were placed in medical stainless steel trays inside a biosafety cabinet. Using a needle and syringe, the tissue surface was rinsed multiple times with 4 ℃ pre-cooled D-PBS to remove blood clots. Ensure that the samples remain wet when immersed in D-PBS during processing and do not allow the tissue to dry out. To maintain sterility, the samples were aseptically processed throughout the sample processing using sterile D-PBS and surgical tools.
(3) The samples were carefully examined to identify different anatomical regions: UC, CPJ and FP. UC is parsed first. The fetal end of the UC is held by forceps and a first cut is carefully made at the tip of the CPJ with a pair of scissors. A second cut was made below the CPJ, separating the CPJ tissue (1.5-2.5 cm) from the FP. The isolated UC, CPJ and FP were placed in separate petri dishes for further processing. (the umbilical cord collection section is schematically shown in FIG. 1, and includes the tissue at the junction of placenta and umbilical cord tissue (top right) and umbilical cord tissue (bottom right)).
(4) The UC is cut longitudinally with the aid of scissors and forceps, and is further dissected into two regions: cord Lining (CL) and Wharf's Jelly (WJ). The vessels and surrounding WJ were fully exposed without disturbing the epithelium. The umbilical cord anatomy is schematically illustrated in fig. 2, and includes the removed arterial vessels and the retained Wharton's jelly and umbilical cord lining.
(5) The WJ was scraped from the blood vessels and endothelium under the amniotic membrane with a scalpel, and then the blood vessels were removed. Ensure that any remaining perivascular WJ below and around the blood vessel is collected and place the collected WJ in a separate culture dish.
(6) The remaining tissue, the Cord Lining (CL), was collected in a separate petri dish.
(7) After isolation of the tissues representing CL, WJ, CPJ, D-PBS was replaced with 5mL of cryptLE Express (10X) and each tissue was cut into 2mm pieces using scissors.
(8) The tissue pieces were incubated in 37 ℃ incubator for 5min in 37 ℃ pre-warmed TrypLE Express (1 ×) solution to perform short-term marginal digestion of the samples. The partial digestion process was observed by visualizing the release of cells using a phase contrast microscope.
(9) To the partially digested sample, 5mL of MSC complete medium was added to dilute the TrypLE Express solution. The contents were transferred to a 50mL centrifuge tube and the partially digested tissue mass was allowed to stand for 3min.
(10) Carefully aspirate the supernatant, including single cells (which do not amplify efficiently), and spread 15-20 partially digested tissue pieces on 100mm Nunclon TM Delta petri dish and 5mL of MSC optimization medium was added. At 37 ℃,5% CO 2 Incubate under conditions for 3 days without moving the dish during which time to facilitate adhesion of the tissue pieces. Tissue anchorage inoculation scheme is shown in fig. 3, including CPJ tissue (left), WJ tissue (middle) and CL tissue (bottom).
(11) After 3 days of culture, 3m of the culture medium was aspiratedL, culturing the supernatant, adding 3mL of new MSC optimized culture medium for half liquid exchange, and observing whether the explant has cell growth; under normal conditions, cell growth is respectively started after 3-4 days of CPJ cell incubation and 7-10 days of CL or WJ cell incubation, and cell climbing-out can be obviously observed from adherent explants. From this time on, the culture medium was changed every 3 days with new MSC optimization. The explant reaches 70% -90% confluence 7-10 days after the initial cell growth. The non-adherent tissue mass did not produce any cell growth until it was adhered to the plastic. Care must be taken not to touch the adherent tissue to avoid separation of adherent tissue fragments. If there were any floating debris after the first 3 days of culture, they could be transferred to a new 100mm Nunclon TM Attachment was performed in Delta dishes. FIG. 4 is a comparison of cell crawl-out status of CPJ/WJ/CL tissues at Day6/12/18 time points. It can be seen that the cells in the CPJ tissue part have climbed out of the tissue at the earliest, the D12 has reached the cell density for enrichment, and then the WJ tissue part, and the mesenchymal stem cells in the two groups have a growth state with uniform growth at the D18 time. However, the CL tissue site has a slower relative cell climbing speed and a smaller number of cells than the CPJ tissue and the WJ tissue.
(12) For cell harvest and cell passage, 4 mlptype Express (1 ×)/petri dish was incubated at 37 ℃ for 3-4 min to digest the isolated cells. After digestion, trypLE Express was diluted with 4mL of MSC-optimized medium and centrifuged at 1200rpm for 5min to harvest cells. Ensure that the petri dish is shaken slightly while adding TrypLE Express to distribute it evenly.
(13) The cells obtained, considered as passage 0 (P0), were resuspended in MSC-optimized medium and counted using a cell counter and counted at 1 × 10 4 /cm 2 For scale-up, characterization and cryopreservation. The excess cells may be present at 4X 10 6 The concentration of the cryo-preservation solution per mL was frozen in Cryostor CS10 cell frozen stock solution.
Example 2
Verification that the umbilical cord mesenchymal stem cell culture method has stability capable of being repeatedly applied
Based on the method conditions of example 1, stable isolation and expansion of mesenchymal stem cells at the respective sites of CPJ/WJ/CJ was achieved on umbilical cord tissue from 3 different tissue donor sources.
(1) UCMSC21041101 preparation of umbilical cord-derived mesenchymal stem cell P1 generation cell
Extraction and amplification of human umbilical cord-derived mesenchymal stem cells (P0 generation) by tissue adherence method of example 1, in which UC length is 19cm and CPJ length is 2.6cm, 4T-175 culture flasks (2X 10) of P1 generation CPJ-derived MSC were inoculated on day 18 of experiment 6 Flask), inoculated with 4T-175 culture flasks (2X 10) of MSC derived from WJ of P1 generation 6 Flask), inoculated with 2T-175 culture flasks (2X 10) of CJ-derived MSC generation P1 6 Bottle). Figure 5 shows UCMSC21041101 umbilical cord-derived mesenchymal stem cell P1 generation cell preparation (10 flasks).
(2) Preparation of UCMSC21042101 umbilical cord-derived mesenchymal stem cell P1 generation cell
Extraction and expansion of human umbilical cord-derived mesenchymal stem cells (P0 generation) by tissue adherence method of example 1, in which UC length is 27cm, CPJ length is 3.1cm, experiment day 19, 5T-175 culture flasks (2X 10) of P1 generation CPJ-derived MSC were inoculated 6 Flask), inoculated with 10T-175 flasks (2X 10) of MSC derived from WJ of the P1 generation 6 Flask), inoculated with P1-generation CJ-derived MSC 5T-175 flasks (2X 10) 6 Bottle). Figure 6 shows UCMSC21042101 umbilical cord-derived mesenchymal stem cell generation P1 cell preparation (20 flasks).
(3) Preparing UCMSC21062101 umbilical cord-derived mesenchymal stem cells P1 generation cells;
extraction and amplification of human umbilical cord-derived mesenchymal stem cells (P0 generation) by tissue adherence method of example 1, in which UC length was 17.4cm and CPJ length was 3.2cm, 5T-175 flasks (2X 10) of P1 generation CPJ-derived MSC were inoculated on day 22 of the experiment 6 Flask), inoculated with 17T-175 culture flasks (2X 10) of MSC from WJ of P1 generation 6 Flask), inoculated with 8T-175 culture flasks (2X 10) of CJ-derived MSC generation P1 6 Bottle). Figure 7 shows UCMSC21062101 umbilical cord-derived mesenchymal stem cell P1 generation cell preparation (30 flasks).
Example 3
The invention compares short-term TrypLE before tissue inoculation by the following experiment TM Express enzymatic treatment and application Nunclon TM Tissue cell climbing efficiency of a Delta culture dish instead of an uncoated cell culture dish.
The experiment is totally provided with 4 experimental groups, namely an enzymolysis coating culture group, an enzymolysis non-coating culture group, a non-enzymolysis coating culture group and a non-enzymolysis non-coating culture group, wherein culture tissues come from the same donor WJ part, and are cultured for 6 days under the condition of 10mLUC-MSC optimized culture medium, liquid changing operation is not carried out in the period, the cell climbing state of each culture dish tissue part is checked under a microscope on the 6 th day, and the result is shown in figure 8. FIG. 8 shows the mesenchymal cell climbing-out state of umbilical cord WJ tissue under different conditions.
According to the results of the cell state under the Day6 mirror, after the tissue is treated by enzymolysis for a short time, the tissue is treated by Nunclon TM Delta culture dish will present uniform diffusion state (figure 8 (1)), and non-coating culture dish can see the existence of cell blank area (figure 8 (2)), which shows Nunclon TM The Delta culture dish can obviously increase the adhesion efficiency of cells and increase the cell amplification area; in addition, the same is true for Nunclon TM Delta culture is carried out on a culture dish, cells after enzymolysis treatment of the tissues show a uniform growth and proliferation state (figure 8 (1)) climbing out from the tissues to the periphery, cells without enzymolysis treatment of the tissues show a growth state (figure 8 (3)) with relatively large individual morphology difference, and the amplification range of the sub-generation cells is limited due to the large individual cells, so that the number of the cells is relatively less (figure 8 (1)), and the climbing-out number of the mesenchymal stem cells can be increased due to the short-time enzymolysis treatment. The results ((2) in fig. 8) and ((3) in fig. 8) can be compared with ((4) in fig. 8) to reach the same conclusion.
The comparison of (1 in FIG. 8) with (4 in FIG. 8) shows that the TrypLE is used to determine whether the TrypLE is a good candidate for the comparison TM Express tissue enzymolysis treatment and cell inoculation in 100mmNunclon TM The step of Delta culture dish can shorten the time of the climbing out of the mesenchymal stem cells, and further increase the climbing out number of the mesenchymal stem cells in unit time. The cells in the same part have consistent doubling time, and the short-term number of amplification is more, which means that the number of amplification times of the cells is less for obtaining the same number of cells in unit time, so that the mutation probability of the cells is reduced, and the stable phenotype and function of the cells can be ensured.
Example 4
The UC-MSC optimized culture medium used by the invention can realize long-term passage of MSC, maintain the cell consistency and maintain the trilineage differentiation potential of mesenchymal stem cells.
The optimized medium used in the present invention is a serum-free medium (SFM) specifically configured for the growth and expansion of human Mesenchymal Stem Cells (MSCs). Compared with a combined culture system of a commercial conventional basal culture medium supplemented with a serum substitute, the optimized culture medium is added with a mesenchymal stem cell stimulating supplement, so that the growth of the MSC can be better promoted, and the consistency is improved. Using the optimized media of the present invention, human-derived MSCs can be expanded for over 10 generations while still maintaining a very strong trilineage mesodermal differentiation potential (i.e., the ability to differentiate into osteoblast, chondroblast, and adipoblastic lineages).
The optimized culture medium components of the invention are shown in the table 1.
TABLE 1 optimized UC-MSC Medium (500 mL) composition
Figure BDA0003963983670000121
Note: the total volume of a basal medium consisting of DMEM-lowglucose and L-glutamine is 450mL; when the subsequent supplement is prepared, the solvent used in the preparation is DMEM-lowglucose, namely the total volume of the supplement after various supplementary factors (recombinant human FGF2 protein, recombinant human FGF4 protein, recombinant human PDGFAB protein, recombinant human EGF protein, recombinant human VEGF protein, recombinant human HGF protein, recombinant human TGF-beta 1, recombinant human BMP-3 protein and nicotinamide mononucleotide) are dissolved in DMEM-lowglucose is 5mL.
DMEM-low glucose (Thermo Fisher # 11885) was used as the basal medium for UC-MSC culture. The source information of each component in the optimized culture medium is as follows:
l-glutamine (Thermo Fisher # A2916801);
Knockout TM serum replacement (Knockout) TM SR)(
Figure BDA0003963983670000122
Human Platelet Lysate(Clinical Grade)，SARTORIUS，PLTGOLD100GMP)；
Recombinant human FGF2 protein (Animal Free) (ab 179489);
recombinant human FGF4 protein (Animal Free) (ab 222368);
recombinant human PDGFAB protein (Animal Free) (ab 179496);
recombinant human EGF protein (Animal Free) (ab 9697);
recombinant human VEGF 165A protein (Animal Free) (ab 179624);
recombinant human HGF protein (ab 105061);
recombinant human TGF-beta 1 protein (Animal Free) (ab 217396);
recombinant human BMP3 protein (ab 97412);
Nicotinamide mononucleotide，KeyNAD+intermediate(ab223879)；
the invention takes the CPJ tissue position with the most abundant MSC cells as a cell source, continuously expands the tissue P0 generation mesenchymal stem cells on respective culture systems to P10 generation cells, obtains the total number of the cells by microscopic examination of the cell state of each generation, and compares the respective culture efficiency and cell function evaluation. Meanwhile, the three-line differentiation capacity of the P2/P5/P10 generation mesenchymal stem cells and the cell phenotype of the P10 generation mesenchymal stem cells are detected to verify whether the mesenchymal stem cells on the optimized culture medium system still maintain the clinically required cell phenotype and differentiation capacity after multiple passages.
The experiment was performed with two culture system test groups in total, as shown in fig. 9 (fig. 9 is the UC-MSC optimized medium combination of beijing giraldisco (midge family) and daceae is the MSC medium combination):
culture system 1 (optimized medium of the invention): basic medium for MSC of Zhongke (450 mL) + serum replacement for MSC of Zhongke (45 mL) + medium supplement for UC-MSC of Zhongke (5 mL).
Culture system 2 (existing commercial medium): daceae is MSC basal medium (480 mL) + daceae is serum replacement (20 mL).
Based on respective culture systems, the same tissue donor source CPJ position is taken, the treatment is carried out according to the process flow, 25 small tissue blocks with uniform size are inoculated in each culture dish, the cell digestion, the passage and the liquid change operation are carried out at the same time point in the period so as to reduce the interference of other factors on the experiment, and the cells are cultured to P10 generations.
Meanwhile, the cell status of the two experimental groups at P0/P5/P10 passage is detected under a microscope, as shown in FIG. 10 (FIG. 10: the status of the MSC from the source of the P2/P5/P10 passage under the two culture systems):
according to the comparison of cell morphology under the P2 generation mirror, it can be found that hMSC cultured in the optimized MSC culture medium shows more primitive morphological characteristics compared with the contrast culture system. Dake is the flat shape of hMSC cultured in the culture medium, which can reach 1.0-3.0 × 10 4 Individual cell/cm 2 The hMSC cultured in the optimized culture medium has much smaller cell volume, is in a spindle shape, and has a shear density reaching>5.0×10 4 Individual cell/cm 2 。
By comparing the state of P2/P5/P10 generation cells in each group, the morphological characteristics of the uniform mesenchymal stem cells can be maintained until the P10 generation cells under the condition of the medium optimized by the Chinese family, and the size difference between the P10 generation cells, partial cellular vacuolation and poor morphological consistency are found under the condition of the medium optimized by the Dakoku family.
The above results demonstrate that the optimized culture system can maintain the normal growth state of the cells in long-term cell culture and passaging.
Meanwhile, in order to test the cell expansion efficiency of different culture systems, 2.5X 10 4 Flask inoculum cells in T-25 flasks containing different media, 3 parallel samples per group were counted at passage P1/P3/P5/P7/P10 to calculate the total amount of cells obtained from the harvested cell volume. The results are shown in FIG. 11 (FIG. 11: total number of cells obtained by transferring to P10 generation after the same number of cells were inoculated in T-25 flask under both culture conditions).
As shown by the results, the same number of cells (0.25X 10) were inoculated in each of the two culture systems 6 T25), the total cell mass in the culture system of the middle family reaches 3.403 +/-0.165 x 10 when the cell count is detected in the P1 generation 6 And Dake is the total under the culture systemThe cell amount is 0.383. + -. 0.025X 10 6 The results show that the cell proliferation of the family system is faster in the initial stage of cell culture, and the higher cell number is obtained in P1, and then the passage is carried out by the same cell inoculation number until the P10 generation, and the total 7.993 +/-0.276 x 10 is obtained under the middle culture system 6 The cells are cultured in culture system to obtain 4.300 +/-0.096X 10 6 The total number of the cells obtained under the culture system of the midkine is about 2 times that of the cells obtained under the culture system of the midkine, which shows that the total number of the cells obtained under the culture medium condition of the midkine is extremely advantageous relative to the culture system of the midkine along with the increase of the culture time.
Based on the above experimental results, scale-up experiments were performed on T-175 plates at 2X 10 6 The cells/T175 were inoculated with the same tissue-derived P4-generation umbilical cord-derived mesenchymal stem cells, 10 flasks were inoculated per culture system, and with cell confluence reaching 90% as the cell collection end point, the calculation time, culture medium consumption and labor cost for obtaining 2 billion cells in the midkine culture system and daceae as culture systems were calculated, the results are shown in table 2:
TABLE 2 comparison of time and cost to expand to 2 hundred million cells
Expansion to 2 hundred million cell conditions Medium science optimized UC-MSC culture medium Dake is a basic culture medium plus Xuezhi
Number of cells inoculated in a single flask 2×10 6 2×10 6
Time to 90% of cell confluence 4 days 5 days
T-175 cells are required for the number of target cells 20 bottle 40 bottles
Volume of culture medium required for culture 25mL×20＝500mL 30mL×40＝1200mL
Total length of operation 4～5h 7～8h
Expanding the production of hmscs is crucial for any living cell therapy. According to the experimental results, the total amount of the culture medium required to produce 2 hundred million cells was reduced by 58%, the surface area was reduced by 50%, the time was shortened by 18%, and the time energy was reduced by 40% using the optimized MSC stem cell culture medium compared to the culture system in which daceae added hemotizin as the basal medium.
The results show that under the optimized condition of adding the supplement to the basic culture medium of the MSC of the traditional Chinese medicine under high cell density, the high-efficiency hMSC amplification can be realized, the using amount of the culture medium is reduced, the culture interface surface area required by cell culture is reduced, and the preparation time of the cell preparation is saved.
MSCs for clinical use need to be kept in a single cell composition, i.e., extremely high cell purity, to ensure safety. MSCs have a unique morphology and express a specific set of CD (cluster of differentiation) molecules. The phenotypic patterns clinically used to identify MSCs cells require expression of CD44, CD73, CD90 and CD105 and lack of CD34, CD45 and HLA-DR antigens. The identification result of the P10 generation UC-MSC flow phenotype detected by the flow cytometry is shown in fig. 12 (fig. 12, identification of the umbilical cord mesenchymal stem cell P10 cell phenotype under the optimized culture condition), the expression ratios of the mesenchymal cell surface markers CD44, CD73, CD90 and CD105 are 99.7%, 99.9%, 99.7% and 97.9%, respectively; while negative markers were expressed at 0.95%.
The results show that the purity of the UC-MSC obtained under the optimized process condition until P10 generation is extremely high, and the UC-MSC meets the clinical application standard.
UC-MSC cultured under optimized culture process condition retains its trilineage differentiation potential
The invention verifies whether the MSCs of high generation obtained after multiple passages still have the same three-line differentiation capacity as the cells of low generation under the condition of an optimized culture system.
Prior to performing differentiation experiments, human umbilical cord-derived MSCs were expanded to P10 passages in optimized medium. Subsequent use of mesenchymal stem cells corresponding adipogenesis (StemPro) TM Adipogenic differentiation kit, thermo FisherA 1007001), osteogenesis (StemPro) TM Osteogenic differentiation kit, thermo Fisher A1007201, chondrogenic (StemPro) TM Chondrogenesis differentiation kit, thermo Fisher a 1007101), differentiation induction kit, culturing according to the operating manual, staining adipocytes, osteocytes and chondrocytes with oil red O, alizarin red and toluidine blue, respectively, and evaluating differentiation ability of UC-MSC. The results are shown in figure 13 (figure 13, identification of differentiation capacity of umbilical cord-derived mesenchymal stem cells P2/P5/P10 generation-by-generation three-line under optimized culture conditions).
As can be seen from fig. 13, the P2/P5/P10 generation secondary umbilical cord mesenchymal stem cells obtained under the condition of the optimized culture system all have the ability to differentiate into adipocytes, osteocytes, and chondrocytes, which indicates that the stability of MSC cell function can be still ensured after multiple passages by amplifying the MSCs under the condition of the optimized culture system.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (10)

1. Human umbilical cord source intermittence chargerAn optimized culture medium for plastid stem cells, said optimized culture medium comprising: basal medium, serum replacement and supplements; the basic culture medium comprises DMEM-low glucose and L-glutamine; the serum replacement comprises Knockout TM A serum replacement; the supplement comprises recombinant human FGF2 protein, recombinant human FGF4 protein, recombinant human PDGF AB protein, recombinant human EGF protein, recombinant human VEGF protein, recombinant human HGF protein, recombinant human TGF-beta 1, recombinant human BMP-3 protein and nicotinamide mononucleotide.
2. The optimized culture medium of claim 1, wherein the optimized culture medium comprises a basal medium, a serum replacement and a supplement in a volume ratio of 90:9:1.
3. the optimized culture medium of claim 1, wherein in the optimized culture medium: the mass concentration of the L-glutamine is 6mM, the mass concentration of the recombinant human FGF2 protein is 40ng/mL, the mass concentration of the recombinant human FGF4 protein is 10ng/mL, the mass concentration of the recombinant human PDGF AB protein is 4ng/mL, the mass concentration of the recombinant human EGF protein is 2ng/mL, the mass concentration of the recombinant human VEGF protein is 0.2ng/mL, the mass concentration of the recombinant human HGF protein is 0.2ng/mL, the mass concentration of the recombinant human TGF-beta 1 is 0.5ng/mL, the mass concentration of the recombinant human BMP-3 protein is 0.2ng/mL, and the mass concentration of the nicotinamide mononucleotide is 5mM.
4. A kit for culturing human umbilical cord-derived mesenchymal stem cells, wherein the kit comprises the optimized culture medium of any one of claims 1 to 3, trypLE TM Express digestive enzyme and Nunclon TM Delta culture dish.
5. A tissue adherence culture method of human umbilical cord-derived mesenchymal stem cells based on the kit of claim 4, comprising the following steps:
the umbilical cord sample is placed in a TrypLE TM Express digestive enzyme solution for digestionThe umbilical cord tissue of (a);
placing the digested umbilical cord tissue in Nunclon containing the optimized culture medium according to any one of claims 1 to 3 TM And performing adherent culture in a Delta culture dish to obtain the human umbilical cord-derived mesenchymal stem cells.
6. The tissue adherent culture method of claim 5, wherein the source of the umbilical cord sample comprises: umbilical cord-placental junction tissue and/or umbilical cord tissue; the umbilical cord tissue includes an umbilical cord lining and/or umbilical cord Wharton's jelly.
7. The tissue adherent culture method of claim 5, wherein the time for digestion is 5min.
8. The method of adherent tissue culture of claim 6, further comprising, after the adherent culture: when the cell fusion degree of adherent culture reaches 70-90%, trypLE is used TM Express digestive enzyme solution is used for digestive culture and centrifugation to obtain primary cells.
9. The tissue adherent culture method of claim 8, wherein the digestion culture is performed for 3 to 4min.
10. The culture method according to claim 8, wherein the culture is performed using the optimized medium when the obtained primary cells are subcultured.
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