CN117946968A - Isolation culture method of clinical-grade high-expression CD146+ mesenchymal stem cells - Google Patents
Isolation culture method of clinical-grade high-expression CD146+ mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly discloses a separation culture method for high-expression CD146+ mesenchymal stem cells. The separation culture method comprises the following steps: umbilical cord blood vessels separated from isolated human umbilical cord tissues and Wharton's jelly tissues around the umbilical cord blood vessels are used as culture objects to carry out tissue mass adherence culture. The isolated culture method provided by the invention has the advantages of clear process and controllable quality, can be used for preparing the human umbilical cord perivascular high-expression CD146 mesenchymal stem cells meeting clinical requirements in large scale and standardized production in vitro, and lays a foundation for clinical research and application.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a separation culture method of clinical-grade high-expression CD146+ mesenchymal stem cells.
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) are an undifferentiated pluripotent cell derived from embryonic developmental early stage germ layers, have high self-renewal capacity and multipotency and have low immunogenicity, and are important seed cells for regenerative medicine and tissue engineering. MSCs were found in bone marrow at the earliest time, and then found in adipose tissue, skeletal muscle, placenta, dental pulp, periodontal ligament, joint synovium, endometrium, umbilical cord and umbilical cord blood, and the like, and it was suggested that MSCs may exist in all tissues having blood vessels. MSCs are considered as a reservoir of repair cells in humans, capable of mobilizing, proliferating and differentiating into appropriate cell types in response to specific signals, and currently, MSCs have been widely used in clinical research and application for degenerative diseases, immune system diseases, nervous system diseases, kidney injury, liver injury, endocrine system and other multi-system diseases.
Umbilical Cord Mesenchymal Stem Cells (UCMSCs) are perinatal stem cells derived from neonatal umbilical cord tissue, which are more primitive than other adult tissue-derived MSCs, and which have a lower proliferation and differentiation capacity than embryonic stem cells but significantly higher than adult stem cells. Compared with adult stem cells, umbilical cord mesenchymal stem cells have higher cloning efficiency, stronger proliferation capacity, shorter doubling time and stronger immunosuppressive capacity. Meanwhile, the umbilical cord mesenchymal stem cells are abundant in source, the umbilical cord is obtained from medical waste after the perinatal healthy puerpera is produced, the materials are convenient, the collection process is relatively simple, no wound exists, and the umbilical cord is less polluted by viruses and bacteria by the placenta barrier, so that the medical ethical dispute is small.
The international society for cell and gene therapy (ISCT) defines a set of mesenchymal stem cell minimum standards: (1) MSCs grown under classical culture conditions have adherence; (2) High expression of CD105, CD73 and CD90, lack of CD45, CD34, CD11b, CD19 and HLA-DR expression; (3) Can induce differentiation into osteoblasts, adipocytes and chondrocytes in vitro. In addition to these minimum criteria, other phenotypes and surface markers for classification of MSC subpopulations have been proposed, including stage-specific embryonic antigen-4 (SSEA-4), platelet-derived growth factor receptor-alpha (PDGFR-alpha), stem cell antigen-1 (Sca-1), nestin, CD44, CD146, CD166, CD271, and the like. MSCs mediate immune modulatory effects, and respond to the functions of a number of effector cells of the adaptive and innate immunity, including T cells, B cells, NK cells, monocytes, macrophages, dendritic cells, neutrophils, and mast cells. In addition, paracrine production of its trophic factors and a wide range of immunomodulatory and anti-inflammatory functions are now widely accepted as the most likely mechanisms of therapeutic activity of MSCs.
Human leukocyte differentiation antigen CD146 (cluster ofdifferentiation 146,146, abbreviated as CD 146) is type I transmembrane glycoprotein, and is not Ca 2+ dependent cell adhesion molecule, belonging to immunoglobulin superfamily. CD146, also known as Melanoma Cell Adhesion Molecule (MCAM), is a member of the immunoglobulin gene superfamily found in human melanoma cells, and CD146 has been demonstrated to be highly expressed in several cell types, including vascular endothelial cells, smooth muscle cells and pericytes from a variety of tissues, playing an important role in vascular endothelial cell activity and angiogenesis. In most tissues, the perivascular distribution of mesenchymal stem cells suggests a correlation between mesenchymal stem cells and pericytes during development. Mesenchymal stem cells have an intrinsic link with pericytes and the learner speculates that all mesenchymal stem cells are pericytes. Studies have shown that MSCs and pericytes actually have similar phenotypic markers (e.g., CD146, NG2, PDGF-R beta), perivascular niches, differentiation potential, and functions related to tissue homeostasis and immunomodulation.
Previous researches show that CD146+ can be used as a surface marker for distinguishing mesenchymal stem cell subsets, and the CD146+ mesenchymal stem cell subsets have unique biological function treatment potential, have strong immunoregulation characteristics, proliferation migration potential, angiogenesis promoting capacity, secretion capacity and the like, and have excellent treatment potential in various disease researches such as cartilage regeneration, sepsis, bone joint injury, premature ovarian failure and the like. How to extract and enrich cd146+ MSCs in clinical studies is an important focus of researchers.
At present, common enrichment and extraction methods include a magnetic bead sorting method and a flow cell sorting method, for example, chinese patent CN 114366758A discloses an application of CD146+ mesenchymal stem cell subsets in preparing medicines for preventing premature ovarian failure, the CD146 sorting microspheres are used for enriching CD146+ human umbilical cord mesenchymal stem cells, so that CD146+ MSCs are improved from 27% positive cells before sorting to 98% positive cells after sorting, but the invention does not describe the change condition of the proportion of CD146+ MSCs of cells after continuous culture after sorting, and a sufficient number of clinical grade cells are difficult to obtain by the magnetic bead enrichment method. Chinese patent CN 114703131a discloses a method for sorting cd146+ cells from serum-free cultured human placental fetal side mesenchymal stem cells, which uses a flow cell sorting technique to sort cd146+ cells from a placental cathepsin digested cell suspension, and as a result, only the sorting method is described, and no further characterization of cell performance is made.
Although the method can sort out CD146+MSCs subgroup with higher purity, the process is complex and can not meet the clinical-grade large-scale cell preparation, and meanwhile, research shows that the sorted cells have the conditions of reduced proportion of CD146+MSCs and the like in the subculture process, and research also shows that the sorted cells have the phenomenon of slow proliferation.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a separation culture method for high-expression CD146+ mesenchymal stem cells, which can be used for preparing human umbilical cord perivascular high-expression CD146+ mesenchymal stem cells meeting clinical requirements in large scale and in a standardized way in vitro.
Specifically, the invention provides a separation culture method of high-expression CD146 mesenchymal stem cells, which comprises the following steps: umbilical cord blood vessels separated from isolated human umbilical cord tissues and the surrounding Wharton's jelly tissues of the umbilical cord blood vessels are used as culture objects to carry out tissue mass adherence culture.
A schematic representation of the cross-section of human umbilical tissue is shown in FIG. 1A, and includes umbilical epidermis (Umbilical epithelium), amniotic Wharton's Jelly (Subamniotic Wharton's Jelly), intravascular Wharton's Jelly (Intervascular Wharton's Jelly), vascular Zhou Huatong Jelly (Perivascular Wharton's Jelly), umbilical vein (Umbilical Vein), and vascular artery (Umbilical Arteries). Human umbilical cord Wharton's jelly tissue contains abundant mesenchymal stem cells, and human umbilical cord mesenchymal stem cells have been successfully isolated by a tissue block enzyme digestion method or a tissue implantation method, and have been widely applied to clinic in the treatment of various diseases. The research shows that compared with the Wharton's jelly tissue at the far end of an umbilical cord blood vessel (Wharton's jelly near the epidermal end of the umbilical cord), the Wharton's jelly tissue at the periphery of the blood vessel has more abundant mesenchymal stem cell content, the Wharton's jelly tissue at the far end of the umbilical cord and the blood vessel is peeled off in vitro, and only the blood vessel and the Wharton's jelly at the periphery are reserved as a culture object for tissue block adherence culture, so that a large amount of mesenchymal stem cells (CD 146 High MSC) with high expression of CD146 can be separated, thereby ensuring the in vitro large-scale and standardized preparation of clinical-grade human umbilical cord perivascular CD146 High mesenchymal stem cell products, and laying a foundation for clinical research and application.
According to the specific embodiment of the invention, umbilical vessels are umbilical arteries and umbilical veins from which the endothelium is stripped, so that the endothelial cells of the umbilical veins can be prevented from being polluted to the greatest extent; and/or, the Wharton's jelly tissue around the umbilical cord blood vessel is Wharton's jelly tissue within a range of 2mm around the umbilical artery and the umbilical vein.
According to a specific embodiment of the present invention, tissue mass adherence culture comprises: culturing the tissue block in a serum-free complete medium, harvesting P 0 generation cells, inoculating the P 0 generation cells to a new serum-free complete medium, performing gradual expansion culture, and harvesting P 1 -Pn generation cells. The traditional MSC culture system has various problems, such as adding fetal bovine serum, trypsin, collagenase and the like, introducing various heterologous substances such as bovine serum, porcine pancreatin and the like and potential pathogenic factors, and has potential risks in clinical research and application, so that the serum-free culture system is particularly important in cell patent medicine and clinical research.
According to a specific embodiment of the present invention, the serum-free complete medium comprises a basal medium and additional components, the additional components comprising serum-free human platelet lysate extract, basic fibroblast growth factor and epidermal growth factor; the serum-free human platelet lysis extract is GMP grade with concentration of 2% -10% v/v, preferably 5% v/v; basic fibroblast growth factor (bFGF) can promote cell proliferation and growth and maintain the cell in an undifferentiated state, and the final concentration is 1-20 ng/mL, preferably 5ng/mL; epidermal Growth Factor (EGF) is an effective mitogenic factor that promotes proliferation of cultured cells in vitro at a final concentration of 1-20 ng/mL, preferably 4ng/mL.
According to a specific embodiment of the invention, the basal medium is αMEM or DMEM/F12, preferably αMEM, comprising amino acids, vitamins, glucose, nucleosides, inorganic salts, and the like.
According to the specific embodiment of the invention, the step-by-step expansion culture is carried out until P 5 -generation cells are harvested, so that a main cell bank (P 2 generation) and a working cell bank (P 5 generation) which meet the requirement of clinical-grade large-scale preparation can be established, the mesenchymal stem cells highly express CD146, specifically, the positive rate of the cell CD146 in the main cell bank is 40-70%, and the positive rate of the cell CD146 in the working cell bank is 30-60%.
According to a specific embodiment of the present invention, the conditions of the culture include: culturing in an incubator at 37 ℃ with 5% CO 2; ii) culturing until the cell fusion degree is 80-90%.
According to an embodiment of the invention, the density of the inoculation is 5000-15000 cells/cm 2, preferably 8000-10000 cells/cm 2.
According to a specific embodiment of the invention, the method of harvesting comprises: firstly adding a cell digestive juice, when a cell rounding part is separated from a culture vessel, adding a serum-free complete culture medium and a stop solution, centrifuging the obtained cell suspension, and re-suspending and filtering a cell precipitate obtained by centrifugation; the cell digest is a recombinant protein digest, preferably a TrypLE TM Express cell digest, other than conventional trypsin.
According to the specific embodiment of the invention, the method further comprises the steps of regulating the cell freezing solution for the harvested cells to a certain cell density and then freezing; the cell cryopreservation liquid is serum-free cell cryopreservation liquid, preferably any one of CTS TM Synth-a-FreezeTM cell cryopreservation liquid and CryoStor CS10 cell cryopreservation liquid.
The beneficial effects of the invention are as follows:
1. The method can separate high-expression CD146 mesenchymal stem cells (CD 146 High MSC), realizes large-scale and standardized production and preparation of human umbilical cord perivascular CD146 High MSC meeting clinical requirements, and has higher feasibility, durability and stability of cell therapy medicine production process compared with the current commonly used magnetic bead sorting method and flow cell sorting method.
2. The method realizes the construction of two cell libraries of the CD146 High MSC around the blood vessel of the human umbilical cord, and the positive rate of the cell CD146 in the main cell library is 40-70%. The positive rate of the cell CD146 in the working cell bank is 30-60%.
3. The human umbilical cord perivascular CD146 High MSC population obtained by the method has short doubling time, effectively overcomes spontaneous unoriented differentiation in the stem cell culture process, maintains the characteristics of stem cells, has full cell bodies, smaller cell morphology and average size of about 16 mu m, has uniform spindle shape, is spiral or fingerprint-like adherent growth, simultaneously highly expresses CD73, CD90, CD105, CD29, CD44, CD146, CD166 and HLA-ABC, does not express CD34, CD45, CD31, CD14, CD19 and HLA-DR, meets the minimum standard of mesenchymal stem cells defined by the International society of cell and gene therapy (ISCT), has the differentiation potential of adipogenic cells, osteoblasts and chondrogenic cells, can inhibit lymphocyte proliferation, regulates lymphocyte subpopulations (Th 1, th17 and Treg), and has no in-vivo and in-vitro tumorigenicity.
4. The human umbilical cord perivascular CD146 High MSC obtained by the method disclosed by the invention has no progressive nodule formation in an in-vivo nude mouse subcutaneous inoculation tumorigenicity test, no clone formation is found in an in-vitro soft agar clone formation test, and good biological safety is reflected.
5. The number of the primary cells isolated and cultured by the method is obviously higher than that of the primary cells isolated and cultured from the Wharton's jelly at the far end of the umbilical cord blood vessel, and the cells of the main cell bank and the working cell bank have shorter Population Doubling Time (PDT) and stronger proliferation migration capacity than that of mesenchymal stem cells derived from the Wharton's jelly at the far end of the umbilical cord blood vessel.
6. The human umbilical cord perivascular CD146 High MSC obtained by the method of the invention shows good osteoblast, adipogenic cell and chondroblast three-line differentiation capability in the aspect of biological effectiveness, has lymphocyte proliferation inhibition capability in the aspect of immunological reaction, can inhibit lymphocyte subpopulation Th1 and Th17 proliferation, and lymphocyte subpopulation Treg proliferation promotion capability.
7. The serum-free culture system adopted by the method comprises a basic culture medium, a GMP-grade serum-free human platelet lysis extract, recombinant cytokines, recombinant cell digestive juice and clinical serum-free cell frozen stock solution, the defect of adding animal-derived serum is avoided, raw materials and reagents used in the culture process meet the GMP grade, the potential risks of pollution and zoonotic infection of heterologous substances such as bovine serum, porcine-derived trypsin and the like are overcome, and the cultured cells are ensured to meet the requirements of clinical research and application.
Drawings
FIG. 1A is a schematic representation of a cross-section of an umbilical cord;
FIG. 1B is a cellular STR identification map;
FIG. 1C is a chromosome karyotype analysis;
FIG. 1D is a Master Cell Bank (MCB) and Working Cell Bank (WCB) morphology (100X);
FIG. 1E is a cell diameter distribution;
FIG. 2A is an osteoblast, adipogenic, chondrogenic differentiation assay;
FIG. 2B shows the detection of high expression positive markers of human umbilical cord mesenchymal stem cells by flow cytometry;
FIG. 3A is a CFSE staining assay for inhibition of lymphocyte proliferation by CD146 High MSC;
FIG. 3B is a Th1 modulation assay of lymphocyte subpopulations by CD146 High MSC;
FIG. 3C is a Th17 modulation assay of lymphocyte subpopulations by CD146 High MSC;
FIG. 3D shows the results of inhibition of lymphocyte proliferation by CD146 High MSC;
FIG. 3E shows the Th1 proliferation inhibition results of CD146 High MSC on lymphocyte subpopulations;
FIG. 3F shows the inhibition of Th17 proliferation of lymphocyte subpopulations by CD146 High MSC;
FIG. 3G is a CD146 High MSC on lymphocyte subpopulation Treg modulation assay;
fig. 3H is the results of enhancement of Treg proliferation of lymphocyte subpopulations by CD146 High MSCs;
FIGS. 4A and 4B are CD146 positive rate flow detection results of human umbilical cord perivascular CD146 High MSC and CD146 Low MSC in vascular distal Wharton's jelly tissue;
FIG. 4C is a graph showing the cell growth curves of CD146 High MSC and CD146 Low MSC in the working cell pool;
FIG. 4D is a chart (40X) of the morphology of primary cells (P0) of human umbilical cord perivascular CD146 High MSC and CD146 Low MSC in the vascular distal Wharton's jelly tissue;
FIG. 4E is cell Population Doubling Time (PDT) for CD146 High MSC and CD146 Low MSC in the working cell pool;
FIG. 4F shows the average number of primary cells per dish that can be harvested for human umbilical cord perivascular CD146 High MSC and for CD146 Low MSC in the vascular distal Wharton's jelly tissue (P0);
FIGS. 4G and 4H are graphs showing CD146 positive rate comparisons of human umbilical cord vascular Zhou Huatong's gum to all Wharton's jelly mesenchymal stem cells of the umbilical cord;
FIG. 5A is a graph of scratch test 0h, 6h, 12h, 24h cell healing for working cell banks CD146 High MSC and CD146 Low MSC (100X);
FIG. 5B shows the results of scratch tests 0h, 6h, 12h, 24h cell healing rates for CD146 High MSC and CD146 Low MSC;
FIG. 5C is a soft agar clone format test (100X);
fig. 5D is a subcutaneous inoculation of nude mice for neoplasia detection.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. 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.
In the following examples, fresh human umbilical cord was derived from a full term caesarean section fetal umbilical cord and informed consent was obtained from both parents, and human-derived specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and treponema pallidum infection were excluded by maternal peripheral blood and cord blood tests through health screening (including general examination, current medical history, past medical history, family medical history of both couples).
Example 1 preparation of clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cells (CD 146 High MSC) and construction of Main cell Bank
1. Primary cell isolation culture:
(1) The products required by the experiment are prepared, and the experimental apparatus such as ophthalmic scissors, forceps, hemostatic forceps, surgical knife and the like are cleaned, sterilized and dried and heated in advance to remove the heat source.
(2) Collecting fresh umbilical cord tissue of a healthy donor, ligating a section of the umbilical cord by using a surgical thread, extruding the umbilical cord along the section of the umbilical cord, removing residual umbilical cord blood, ligating the other end of the umbilical cord, flushing the residual blood stain and dirt on the surface of the umbilical cord with 75% medical alcohol and 0.9% sodium chloride injection, placing the umbilical cord into an umbilical cord protection solution, and transporting the umbilical cord to a cell preparation center at the temperature of 2-8 ℃ for no more than 24 hours.
(3) Taking out umbilical cord tissue in a biosafety cabinet, soaking and cleaning the umbilical cord tissue in DPBS containing gentamicin for 3-5 times in a 150mm culture dish, cleaning and soaking for 3-5 min each time, and replacing a new culture dish.
(4) Placing the cleaned umbilical cord tissue into a new culture dish, cutting off the ligature parts at the two ends of the umbilical cord tissue by a scalpel, removing hemolysis or edema yellowing parts, cutting the rest parts into small sections of 1-2 cm, transferring the small sections into a 50mL centrifuge tube, vibrating and cleaning for 3-5 times for 3-5 min each time, and replacing the new centrifuge tube.
(5) Placing the cleaned umbilical cord tissue into a culture dish, cutting longitudinally along the umbilical cord epidermis with an ophthalmic scissors, removing the umbilical cord epidermis and the Wharton's jelly at the far end of the umbilical cord blood vessel with a surgical knife, and only retaining the umbilical cord blood vessel and surrounding Wharton's jelly tissue.
(6) The umbilical vein was cut longitudinally along the umbilical vein with an ophthalmic scissors, and the umbilical vein intima was peeled off with forceps while preserving the umbilical vein and umbilical artery and the Wharton's jelly tissue within a range of 2mm around the umbilical vein and umbilical artery.
(7) The stripped umbilical cord tissue is cut into tissue blocks with uniform size of 1-3 mm 3 by a surgical knife, and uniformly spread in culture dishes with 100mm or 150mm at intervals of 0.5 cm.
(8) The petri dish is placed upside down into a 5% CO 2, and placed in a 37 ℃ incubator for 30-60 min, then 15-30 mL of complete medium (basic culture solution alpha MEM, cat# 12571063, gibco, 5% Elite Gro TM -Advanced-GMP Grade human platelet lysis extract, cat# EPAGMP-500, elite cell,5ng/mLbFGF cat# AFL3718-02,R&D systems,4ng/mL EGF, cat# 236-GMP-200, R & D systems) is slowly added.
(9) Cells were cultured by resting in a 5% CO 2, 37℃incubator, and the day was noted as d 0 days. Half the volume was changed once on day d 4, after which the solution was changed every 3 days and the cell growth was observed.
2. Primary cells were harvested and subcultured:
(1) And observing the growth condition of cells around the tissue block by an inverted microscope, wherein the cell is climbed out in a large amount around the cells about 9-15 days, and the primary cells can be harvested.
(2) The tissue mass was lifted off the dish with a pipette, and the tissue mass was pipetted off, washed 2 times with 15 to 20mL of DPBS, and then digested 3 to 5min at room temperature with 6mL of recombinant cell digest TrypLE TM Express (cat# 12604039, gibco) preheated at 37℃added, and when the rounded cell portion was off the dish, 10mL of complete medium was added and digestion was stopped with 30mL of DPBS.
(3) The cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 1000rpm for 5min.
(4) The supernatant was discarded, the cells were resuspended in DPBS, filtered through a 40 μm cell screen, and a small count was taken and the remaining cells were centrifuged at 1000rpm for 5min.
(5) The supernatant was discarded, the cells were resuspended in complete medium, and the cells were inoculated into 175cm 2 flasks at 10000cells/cm 2, fed to 40mL, and placed in a 5% CO 2, 37℃incubator for P1 generation.
(6) And (5) sequentially subculturing the P2-generation cells.
3. Master cell bank cell cryopreservation:
(1) Observing the growth condition of cells by an inverted microscope, and harvesting and freezing and storing the P2 generation when the cell fusion degree reaches 80-90%.
(2) In a biosafety cabinet, the original culture solution is sucked and removed, after the original culture solution is washed for 2 times by 15-20 mL of DPBS, 6mL of recombinant cell digestive solution TrypLE TM Express preheated at 37 ℃ is added for digestion for 3-5 min at room temperature, when the rounded cell part is separated from a culture dish, 10mL of complete culture medium is added, and 30mL of DPBS is added for stopping digestion.
(3) The cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 1000rpm for 5min.
(4) The supernatant was discarded, and the cells were resuspended with DPBS and centrifuged at 1000rpm for 5min.
(5) The supernatant was discarded, the cells were resuspended with DPBS, a small count was taken, and the remaining cells were centrifuged at 1000rpm for 5min.
(6) The supernatant was discarded and the cells were resuspended in cell cryopreservation solution CryoStor CS10 (cat. No. 100-1061, STEMCELL).
(7) The cell density is adjusted to 3X 10 6 cells/mL, the cell is transferred into a 2mL cell freezing tube, and the 3X 10 6 cells/tube is placed into a program cooling instrument and then transferred into a liquid nitrogen tank for long-term storage.
Example 2 clinical grade human umbilical cord perivascular CD146 High mesenchymal Stem cell (CD 146 High MSC) working library construction
1. Cell resuscitating and culturing the main cell bank:
(1) The cells were removed from the main cell bank liquid nitrogen tank and thawed by slow shaking in a 37 ℃ water bath.
(2) The cells were transferred to a 15mL centrifuge tube, pre-added with 10mL of complete medium, and centrifuged at 1000rpm for 5min.
(3) The supernatant was discarded, the cells were resuspended in complete medium, and the cells were inoculated into 175cm 2 flasks at a density of 8000-10000 cells/cm 2, fed to 40mL, and placed in a 5% CO 2, 37℃incubator.
2. And (3) cell subculture:
(1) Observing the growth condition of the cells by an inverted microscope, and harvesting the cells and passaging when the cell fusion degree reaches 80-90%.
(2) In a biosafety cabinet, the original culture solution is sucked and removed, after the original culture solution is washed for 2 times by 15-20 mL of DPBS, 6mL of recombinant cell digestive solution TrypLE TM Express preheated at 37 ℃ is added for digestion for 3-5 min at room temperature, when the rounded cell part is separated from a culture dish, 10mL of complete culture medium is added, and 30mL of DPBS is added for stopping digestion.
(3) The cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 1000rpm for 5min.
(4) The supernatant was discarded, the cells were resuspended in 0.9% sodium chloride injection, a small count was taken, and the remaining cells were centrifuged at 1000rpm for 5min.
(5) The supernatant is discarded, cells are inoculated into 175cm 2 culture flasks according to 8000-10000 cells/cm 2 density, the culture is supplemented to 40mL, and the culture is placed into a culture box with 5% CO 2 and 37 ℃.
(6) The cells were passaged sequentially to P5-generation cells.
3. Cell cryopreservation in working library
(1) Observing the growth condition of cells by an inverted microscope, and harvesting and freezing and storing the P5 generation when the cell fusion degree reaches 80-90%.
(2) In a biosafety cabinet, the original culture solution is sucked and removed, after the original culture solution is washed for 2 times by 15-20 mL of DPBS, 6mL of recombinant cell digestive solution TrypLE TM Express preheated at 37 ℃ is added for digestion for 3-5 min at room temperature, when the rounded cell part is separated from a culture dish, 10mL of complete culture medium is added, and 30mL of DPBS is added for stopping digestion.
(3) The cell suspension was transferred to a 50mL centrifuge tube and centrifuged at 1000rpm for 5min.
(4) The supernatant was discarded, and the cells were resuspended with DPBS and centrifuged at 1000rpm for 5min.
(5) The supernatant was discarded, the cells were resuspended with DPBS, a small count was taken, and the remaining cells were centrifuged at 1000rpm for 5min.
(6) The supernatant was discarded and the cells were resuspended in cell cryopreservation solution CryoStor CS 10.
(7) The cell density is adjusted to 5X 10 6 cells/mL, the cells are transferred into a cell freezing bag, the volume of each bag is 10mL, the cells are put into an aluminum box, the temperature is reduced in the program temperature reduction of the preset program, and then the cells are transferred into a liquid nitrogen tank for long-term storage.
Experimental example 1 clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cell (CD 146 High MSC) Main cell Bank and working cell Bank cell basic biological Property detection
1. Cell morphology identification
Cell morphology was observed under a microscope and recorded by photographing. As a result, as shown in FIG. 1D, cells all grew on the wall and were in the shape of a shuttle.
2. Cell viability assay
Cells were stained with AO/PI fluorochromes and counted in a Countstar-Rigel-S2 cytometer and analyzed for cell size distribution. As a result, as shown in FIG. 1E, the cell diameters were concentrated and distributed between 13 μm and 18. Mu.m.
3. Cell STR profile identification
20 Human alleles such as D3S1358, TH01, D21S11, D18S51, penta E and the like and AMEL sex genes were detected by PCR-capillary electrophoresis. The results are shown in FIG. 1B, where there is a single peak or two allele peaks per locus, indicating that the cells are of single individual origin.
4. Chromosome nuclear detection
The working cell bank cell chromosomes were analyzed by the culture method G banding. As a result, the number of chromosomes was 46, XX, and the chromosome structure was not deleted, repeated, inverted, or translocated, as shown in FIG. 1C.
Experimental example 2 clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cells (CD 146 High MSC) adipogenesis, osteogenesis and chondrogenic induced differentiation detection
1. Osteogenic differentiation
P5 generation cells are inoculated into a 24-well plate, cultured by a complete culture medium, when the cells reach 70% fusion, the complete culture medium is changed into an osteogenesis differentiation reagent (goods number: 6114541, DAKEWE), osteogenesis differentiation induction culture is carried out according to the specification, and after 21d of induction culture, the osteogenesis cells are subjected to staining analysis by alizarin red dye liquor (goods number: 6063211, DAKEWE).
2. Adipogenic differentiation
P5 generation cells were inoculated into 24-well plates, cultured in complete medium, and when the cells reached 100% confluence, the complete medium was changed to adipogenic differentiation reagent (accession No. 6063531, DAKEWE), adipogenic differentiation-induced culture was performed according to the instructions, and after 21d of induction culture, the adipogenic cells were stained with oil red O dye (accession No. 6063221, DAKEWE).
3. Chondrogenic differentiation
P5 generation cells are inoculated into a 25 culture flask and cultured by using a complete culture medium, after the cells reach 80% fusion, the cells are digested, transferred into a 15mL centrifuge tube and centrifuged, the supernatant is discarded to retain cell sediment, the complete culture medium is changed into a chondrogenic differentiation reagent (product number: 6114551, DAKEWE), chondrogenic differentiation induction culture is carried out according to the specification, and after 21d of induction culture, chondrogenic cell slices are stained with an alissine blue dye solution (product number: 6063231, DAKEWE).
As shown in fig. 2A, the results demonstrate that CD146 High MSC can be stained with alizarin red (40×), oil red O (400×), and alixin Lan Ranse (100×) after osteogenic differentiation, and has osteogenic, adipogenic, and chondrogenic cell differentiation potential.
Experimental example 3 clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cell (CD 146 High MSC) surface marker detection
Detecting the expression condition of the surface marker, adjusting the cell number of each tube to be 5×10 5 cells, and adding a flow antibody: PE-anti-human CD73 (cat No.: 344003, biolegend), perCP/Cy5.5-anti-human CD90 (cat# 328117, biolegend), FITC-anti-human CD105 (cat# 800505, biolegend), PE-anti-human CD166 (cat# 343903, biolegend), PE-anti-human HLA-ABC (cat# 311405, biolegend), alexaFluor-anti-human CD29 (cat# 303015, biolegend), FITC-anti-CD44 (cat# 338803, biolegend), FITC-anti-CD146 (cat# 361011, biolegend), PE-anti-human CD34 (cat# 343605, biolegend), FITC-anti-CD45 (cat# 3049, biolegend), FITC-anti-CD14 (cat# 311405, biolegend), alexaFluor-anti-human CD29 (cat# 303015, biolegend), FITC-anti-CD44 (cat# 338803, biolegend), FITC-anti-CD146 (cat# 300, biolegend), PE-anti-human CD31, 37 mL (cat# 300, 37 mL, 37 mL), and centrifugation.
As shown in FIG. 2B, the results indicated that CD73 was 100.0%, CD90 was 99.9%, CD105 was 99.6%, CD29 was 100.0%, CD44 was 99.8%, CD166 was 99.9%, HLA-ABC was 99.9%; negative markers are not expressed: CD34 of 0.14%, CD45 of 0.10%, CD31 of 0.046%, CD14 of 0.12%, CD19 of 0.56%, HLA-DR of 0.13%
Experimental example 4 clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cell (CD 146 High MSC) immunological response test
1. Lymphocyte proliferation inhibition assay
(1) The post-resuscitated 1-generation CD146 High MSC were inoculated into 12-well plates, 2X 10 5 cells/well, and cultured overnight.
(2) PBMC were isolated, stained with CFSE (cat# 423801, biolegend) at a final concentration of 2. Mu.M for 20min in the absence of light, incubated with PRMI1640 (cat# C1187500BT, gibco) cell culture medium containing 10% FBS (cat# 10099141C, gibco) and washed 2 times after staining.
(3) The supernatant was discarded from overnight cultured CD146 High MSCs and CFSE-labeled PBMC resuspended in PRMI1640 complete medium containing 10% FBS, 1X 10 6 cells/well, 1mL complete medium/well was added.
(4) Blank PBMC were set for culture alone without the addition of activator, control PBMC were cultured alone and PHA (cat No. 11249738001, sigma) was added at a final concentration of 2-5. Mu.g/mL, experimental MSCs were co-cultured with PBMC and activator PHA was added, 3 parallel wells were set per group, and the cells were placed in a 5% CO 2 incubator at 37℃for co-culture for 5 days.
(5) Cell staining: after the end of the incubation, PBMC cells were collected into 1.5mL centrifuge tubes, washed 2 times with 1mL CELL STAINING Buffer (cat# 420201, biolegend), incubated with Pacific Blue anti-human CD45 (cat# 368540, biolegend) for 20min, washed 2 times with 1mL CELL STAINING Buffer, and then resuspended with 300. Mu. L CELL STAINING Buffer.
(6) And (3) flow detection: CD45+ cell populations were circled, CFSE+ parental cell populations were circled on the basis of CD45+ cell populations with a blank, and other sets of daughter cell populations were circled according to the parental cell population location.
As shown in fig. 3A, 3D, the results demonstrate that MSC co-culture with PBMCs can significantly inhibit lymphocyte proliferation p=0.0007.
2. Lymphocyte subpopulation Th1, th17 proliferation inhibition assay
(1) The post-resuscitated 1-generation CD146 High MSC were inoculated into 12-well plates, 2X 10 5 cells/well, and cultured overnight.
(2) PBMCs were prepared separately and cells were resuspended in PRMI1640 cells complete medium containing 10% fbs.
(3) The supernatant of overnight cultured CD146 High MSC was discarded, and PBMC resuspended in PRMI1640 complete medium containing 10% FBS, 1X 10 6 cells/well, 1mL complete medium/well, a blank group, a control group, an experimental group, 3 parallel wells each, were added and incubated in a 5% CO 2, 37℃incubator for 24 hours.
(4) After 24h co-cultivation, the blank PBMC were cultivated without CellActivation Cocktail (PMA+Ionomycin+ BrefeldinA) (cat. No. 423303, biolegend) and the control PBMC were cultivated alone and CellActivation Cocktai and the experimental group MSC were co-cultivated with PBMC and CellActivation Cocktai were added, 3 parallel wells were placed in each group and cultivation was continued for 6h in a 37℃incubator with 5% CO 2.
(5) Cell staining: after the end of the incubation, PBMC cells were collected into 1.5mL centrifuge tubes, washed 2 times with 1mL CELL STAINING Buffer (cat# 420201, bioleged), and washed 2 times with Pacific Blueanti-human CD45 (cat# 368540, bioleged), PE/Cyanine7 anti-human CD3 (cat# 317334, bioleged), PE anti-human CD8 (cat# 344706, bioleged), perCP/Cyanine5.5 anti-human IFN-gamma (cat# 502526, bioleged), alexa647Anti-human IL-17A (cat# 512310, biolegend), cyto-Fast TM Fix/Perm Buffer Set (cat# 426803, biolegend) stained cell surfaces and intracellular, washed 2 times with 1mL CELL STAINING Buffer after staining, and resuspended in 300. Mu. L CELL STAINING Buffer.
(6) And (3) flow detection: CD45+CD3+CD8-cell populations are circled, and Th1 (CD3+CD8-IFN-. Gamma. +) and Th17 (CD3+CD8-IL-17 A+) are circled on the basis of the CD45+CD3+CD8-cell populations, respectively.
As shown in fig. 3B, 3E, 3C, and 3F, the results show that the MSC co-culture with PBMCs can significantly inhibit the lymphocyte subpopulation Th1 proliferation p=0.0011, and the MSC co-culture with PBMCs can significantly inhibit the lymphocyte subpopulation Th17 proliferation p=0.0039.
3. Lymphocyte subpopulation Treg proliferation promoting assay
(1) The post-resuscitated 1-generation CD146 High MSC were inoculated into 12-well plates, 2X 10 5 cells/well, and cultured overnight.
(2) PBMCs were prepared separately and cells were resuspended in PRMI1640 cells complete medium containing 10% fbs. The supernatant was discarded from overnight cultured CD146 High MSC and PBMC resuspended in PRMI1640 complete medium containing 10% FBS, 1X 10 6 cells/well, 1mL complete medium/well was added.
(3) Control PBMC were set for single culture, experimental MSCs were co-cultured with PBMC, 3 parallel wells were set for each group, and 5% CO 2 was placed in a 37℃incubator for 5 days.
(4) Cell staining: after the end of the incubation, PBMC cells were collected into 1.5mL centrifuge tubes, washed 2 times with 1mL CELL STAINING Buffer (cat# 420201, biolegend), and washed 2 times with Pacific Blueanti-human CD45 (cat# 368540, biolegend), PE/Cyanine7 anti-human CD3 (cat# 317334, biolegend), FITC anti-human CD4 (cat# 300506, biolegend), PE anti-human CD25 (cat# 356104, biolegend), APC anti-human CD127 (cat# 351315, biolegend), washed 2 times with 1mL CELL STAINING Buffer after the end of the staining, and resuspended cells with 300. Mu. L CELL STAINING Buffer.
(5) And (3) flow detection: the CD45+CD3+CD4+ cell population was encircled, and tregs (CD3+CD4+CD25+CD127-/low) were encircled based on the CD45+CD3+CD4+ cell population, respectively.
As shown in fig. 3G, 3H, the results demonstrate that MSC co-culture with PBMCs can significantly promote Treg proliferation p= 0.0202 for lymphocyte subpopulations.
Experimental example 5 clinical-grade human umbilical cord perivascular CD146 High mesenchymal Stem cell (CD 146 High MSC) Oncogenesis test
Balb/c nude mice were inoculated subcutaneously for tumorigenicity detection
(1) Balb/c nude mice were randomly divided into 3 groups, including vehicle control group, hela cell control group, CD146 High MSC group, 10 each.
(2) The vehicle control group was subcutaneously inoculated with DPBS, the Hela cell control group was subcutaneously inoculated with Hela cell line 1×10 6 cells/mouse, the CD146 High MSC group was inoculated with mesenchymal stem cells 1×10 7 cells/mouse, and the tumor formation at the injection site of nude mice was observed, measured and recorded.
(3) At the end of the observation period, all mice were sacrificed, the major organs and injection sites were dissected and observed, and histopathological examination was performed after tissue fixation, sectioning, HE staining.
As shown in fig. 5D, both the vehicle control group and the CD146 High MSC group had no nodule formation and the Hela cell group had progressive nodule formation.
2. Soft agar clone formation assay
(1) 1.2% Low melting agarose was mixed with 2 Xcell culture medium at a volume ratio of 1:1 to prepare 0.6% bottom agarose, and 1.5mL greenhouse solidification was added to each well of the 6-well plate.
(2) The logarithmic phase CD146 High MSC cells and Hela cells were taken, digested and blown off into a single cell suspension, counted and cell concentration was adjusted to 1X 10 5 cells/mL.
(3) Mixing 0.7% low-melting agarose with 2×cell culture medium at a volume ratio of 1:1, preparing 0.35% upper agar, adding 1mL of upper agar and 100 μL of single cell suspension (CD 146 High MSC about 10000 cells/well, heLa cells about 1000 cells/well) into each well, mixing well, and solidifying in a refrigerator at 2-8deg.C;
(4) The 6-well plate was placed in a cell incubator at 37℃with 5% CO 2 and cultured for 2 to 3 weeks, and after staining with crystal violet, it was observed under a microscope.
As shown in FIG. 5C, CD146 High MSC was not cloned, and HeLa cells were formed with clones of unequal sizes.
Comparative example 1 comparison of CD146 Positive Rate of clinical-grade human umbilical cord vascular Zhou Huatong's jelly and vascular distal Wharton's jelly mesenchymal stem cells
(1) 6 Healthy umbilical cords were selected, umbilical cord blood vessels (vein endothelium was removed) and surrounding Wharton's jelly and vascular distal Wharton's jelly tissue were isolated, primary cells were isolated and cultured, and primary cell banks (P2) and working cell banks (P5) were established by passaging, respectively, as described above.
(2) And (3) detecting the CD146 positive rate of the cells of the main cell bank (P2) and the working cell bank (P5) in a flow mode.
(3) And (3) result statistics:
(4) Analysis of results: as shown in fig. 4A and fig. 4B, the CD146 positive rate in perivascular and distal huamain tissues of human umbilical cord is detected by flow, the blood vessel Zhou Huatong in the main cell bank has a positive rate of 59.07±7.86%, the distal huamain MSC has a positive rate of 44.85±7.59%, and p=0.0099; the positive rate of the vascular Zhou Huatong's collagen MSC in the working cell bank is 53.1+/-4.83%, the positive rate of the vascular distal Walker's collagen MSC is 40.97 +/-6.98%, and p=0.0057.
Comparative example 2 clinical grade human umbilical cord perivascular CD146 High mesenchymal Stem cells and vascular distal Wharton's jelly CD146 Low MSC isolated culture primary cell quantity comparison
(1) 6 Healthy umbilical cords were selected, umbilical cord blood vessels (vein endothelial removal) and surrounding Wharton's jelly and vascular distal Wharton's jelly tissue were isolated separately as described previously and inoculated into 150mm cell culture dishes.
(2) Primary cells were harvested and the average number of cells per dish per cord was counted using a cytometer.
(3) And (3) result statistics:
(4) Analysis of results: as shown in fig. 4F, umbilical cord vessel Zhou Huatong gel CD146 High MSC primary cell number (cells/dish) is 1445000.0 ± 260518.7cells/dis, vascular distal huamai gel CD146 Low MSC is 1160000.0 ± 148996.6cells/dis, p=0.0423, and each primary cell morphology is shown in fig. 4D.
Comparative example 3 clinical grade human umbilical perivascular CD146 High MSC to vascular distal Wharton's jelly CD146 Low MSC Population Doubling Time (PDT)
(1) Perivascular CD146 High mesenchymal stem cells and vascular distal huperzian CD146 Low MSCs in a working cell pool of 3 different individual sources were selected and inoculated at a density of 1 x 10 3cells/cm2 into T25 flasks, 2 replicates per group.
(2) Cells were digested and counted daily, and the number of d1 to d10 cells was counted to draw a growth curve.
(3) Calculate Population Doubling Time (PDT): PDT= (T 2-T1) x lg 2/(lgNt-lg N0)
Wherein PDT-population doubling time;
t 1 -logarithmic phase time 1;
t 2 -logarithmic phase time 2;
Cell number after Nt-t time;
N0-theoretical initial value in logarithmic growth phase.
(4) Analysis of results: as shown in fig. 4C, 4E, the working cell pool was CD146 High MSC population doubling time PDT (CD 146 HighMSC)＝21.57±1.49h,CD146Low MSC population doubling time PDT (CD 146 Low MSC) = 24.84 ±1.03h, p=0.0349).
Comparative example 4 comparison of CD146 Positive Rate of clinical-grade human umbilical vascular Zhou Huatong's gum and umbilical full Wharton's jelly mesenchymal Stem cells
(1) 3 Healthy umbilical cords were selected, umbilical cord blood vessels (vein endothelium removed) and surrounding Wharton's jelly and all Wharton's jelly tissues of the umbilical cord were isolated separately according to the method described previously, primary cells were isolated and cultured, and subcultured.
(2) And detecting the CD146 positive rate of the P5 generation cells in a flow mode.
(3) And (3) result statistics:
(4) Analysis of results: as shown in fig. 4G and fig. 4H, the CD146 positive rate flow test result of the P5 generation mesenchymal stem cells in all the huthrough glue tissues of human umbilical cord blood vessel Zhou Huatong, the blood vessel Zhou Huatong glue MSC positive rate is 56.97±4.26%, the all the huthrough glue MSC positive rate of umbilical cord is 46.7±4.69%, and p= 0.0481.
Comparative example 5 clinical grade human umbilical cord perivascular CD146 High MSC and vascular distal Wharton's jelly CD146 Low MSC comparative value-added migration potential
(1) Human umbilical cord perivascular CD146 High MSC and vascular distal Wharton's jelly CD146 Low MSC were compared for value-added migration capacity by a scratch test.
(2) Cells were seeded into 24-well plates at 8 x10 3cells/cm2, after cells were grown, streaked at the center of the 24-well plates, and the 24-well plates were placed into a zenCELL owl live cell dynamic imaging and analysis system for photography.
(3) The percent cell healing was analyzed for 0h, 6h, 12h, 24h using Image J software.
(4) Analysis of results: as shown in fig. 5A-5B, the working cell bank CD146 High MSC and CD146 Low MSC had no significant difference in cell healing rate at 0h, 6h, 12h, and significant difference in cell healing rate at 24h p= 0.0491.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
Claims (10)
1. A method for isolated culture of cd146+ mesenchymal stem cells, comprising: umbilical cord blood vessels separated from isolated human umbilical cord tissues and the surrounding Wharton's jelly tissues of the umbilical cord blood vessels are used as culture objects to carry out tissue mass adherence culture.
2. The isolated culture method of claim 1, wherein the umbilical vessel is an umbilical artery and an endothelial-stripped umbilical vein;
And/or the Wharton's jelly tissue around the umbilical cord blood vessel is Wharton's jelly tissue within a range of 2mm around the umbilical artery and the umbilical vein.
3. The method according to claim 1, wherein the tissue mass adherence culture comprises: culturing the tissue block in a serum-free complete medium, harvesting P 0 generation cells, inoculating the P 0 generation cells to a new serum-free complete medium, performing gradual expansion culture, and harvesting P 1 -Pn generation cells.
4. The isolated culture method of claim 3, wherein the serum-free complete medium comprises a basal medium and an additional component, the additional component comprising a serum-free human platelet lysate, an alkaline fibroblast growth factor, and an epidermal growth factor;
the concentration of the serum-free human platelet lysis extract is 2% -10% v/v, preferably 5% v/v;
The final concentration of the basic fibroblast growth factor is 1-20 ng/mL, preferably 5ng/mL;
The final concentration of the epidermal growth factor is 1-20 ng/mL, preferably 4ng/mL.
5. The method according to claim 4, wherein the basal medium is αMEM or DMEM/F12, preferably αMEM.
6. The isolated culture method according to claim 3, wherein the step-wise expansion culture is performed until P 5 cells are harvested.
7. The isolated culture method of claim 3 wherein the culture conditions include: culturing in an incubator at 37 ℃ with 5% CO 2; ii) culturing until the cell fusion degree is 80-90%.
8. A method of isolated culture according to claim 3, wherein the inoculation has a density of 5000 to 15000cells/cm 2, preferably 8000 to 10000cells/cm 2.
9. The isolated culture method of claim 3 wherein the method of harvesting comprises: firstly adding a cell digestive juice, when a cell rounding part is separated from a culture vessel, adding a serum-free complete culture medium and a stop solution, centrifuging the obtained cell suspension, and re-suspending and filtering a cell precipitate obtained by centrifugation; the cell digestive juice is recombinant protein digestive enzyme, preferably TrypLE TM Express cell digestive juice.
10. The method according to claim 3, further comprising adjusting the cell frozen stock solution for the harvested cells to a certain cell density and then freezing the cells; the cell freezing solution is serum-free cell freezing solution, preferably any one of CTS TM Synth-a-FreezeTM cell freezing solution and CryoStor CS10 cell freezing solution.
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