CN111235100A - Culture method of human umbilical cord blood mesenchymal stem cells - Google Patents
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Abstract
The invention belongs to the field of cell biology, and particularly relates to a culture method of human umbilical cord blood mesenchymal stem cells. The method comprises the following steps: culturing umbilical cord blood mononuclear cells by using a first culture medium until the growth state of the cells is stable, and then culturing by using a second culture medium, wherein the first culture medium mainly comprises a basic culture medium, fetal calf serum, antibiotics and mesenchymal stem cell growth additives, the volume of the fetal calf serum is 8-12%, and the volume of the mesenchymal stem cell growth additives is 0.1-0.2%; the second culture medium mainly comprises a basic culture medium, fetal calf serum and antibiotics, wherein the fetal calf serum accounts for 8-12% of the volume; the basic culture medium is DMEM/F12 culture medium. The culture method of the human umbilical cord blood mesenchymal stem cells has the advantages of simple steps, low economic cost and high culture success rate, and is suitable for obtaining a large amount of mesenchymal stem cells to meet the requirement of large use amount in treatment of related diseases.
Description
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a culture method of human umbilical cord blood mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) have the potential of self-renewal and multi-directional differentiation, and have become a cell source with great practical value in the field of gene therapy. MSCs were originally isolated from bone marrow and most widely used, and other tissues and organs can be isolated as MSCs. Currently, MSCs of different tissue origin have been applied for the treatment of clinical diseases, such as umbilical cord blood, menstrual blood, adipose tissue, and the like. Among them, human umbilical cord blood is a good source of cells, which is not maternal blood, but is fetal blood, and is not confused with maternal blood. However, cord blood is generally discarded as "garbage" at the time of birth, and its useful value is urgently needed to be developed.
The umbilical cord blood has the advantages of rich source, simple and convenient collection, low immunogenicity, strong proliferation capacity, no ethical limitation and the like, and the mesenchymal stem cells in the umbilical cord blood are more original and have stronger amplification capacity than the mesenchymal stem cells in the bone marrow, so the mesenchymal stem cells in the umbilical cord blood have more and more prominent functions and can be used as materials for cell transplantation treatment of related diseases. Worldwide, umbilical cord blood can be used for treating more than 80 diseases, including leukemia, aplastic anemia, systemic lupus erythematosus, myelodysplastic syndrome, malignant lymphoma, malignant tumor, severe immunodeficiency diseases, metabolic diseases and the like. The content of the mesenchymal stem cells of the premature infant is higher than that of the ordinary fetus, and if the separation, culture and preservation of the umbilical cord blood of the premature infant can be realized, the excellent material can be provided for treating diseases related to the premature infant at a cellular level.
However, the ratio of Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in mononuclear cells is only 0.001-0.01%, and the success rate of separating mesenchymal stem cells from umbilical cord blood is relatively low. At present, the in vitro isolation and culture methods of human umbilical cord blood mesenchymal stem cells are different, and obtaining a large amount of MSCs is still difficult. Therefore, how to select a simple, practical and low-cost method for obtaining the MSCs with the maximum proportion from the human umbilical cord blood by separation and culture is a key problem which needs to be solved urgently and is directly applied to clinical treatment.
The culture medium is the key for successfully culturing the hUCB-MSCs. Most of the prior art adopts DMEM (low sugar or high sugar) culture medium and adds 5% -20% fetal bovine serum, the success rate is low (Laitinen A, Lampinen M, Liedtke S, et. the effects of culture conditions on the functional nature of effective cultured media from human cord blood [ J].Cytotherapy,2016,18(3):423-437；Fujii S,Miura Y,Iwasa M,et al.Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells[J]J Clin ExpHematop,2017,57(1): 1-8); bieback et al used MesenscultTMThe culture medium can provide a desirable culturing effect (Bieback K, Kern S, Kl ü ter H, et al].Stem Cells,2004,22(4):625-634)。MesencultTMThe culture medium is usually an acidic culture medium, including a basal medium and individual additives, which can promote the effective proliferation of the hUCB-MSCs and maintain the undifferentiated state of the hUCB-MSCs, thereby inhibiting the growth of other adherent cells, and is considered to be an optimal culture medium suitable for the growth of the hUCB-MSCs. However, MesenscultTMThe price of the culture medium is expensive, which causes the separation and culture cost to be obviously increased, and the method is particularly not suitable for large-scale production.
The Chinese patent with publication number CN102559590B discloses a method for culturing human umbilical cord blood mesenchymal stem cells through two culture motifs, which uses DMEM/F12 culture medium containing 10% FBS (fetal bovine serum) with the pH value of 6.5-6.8 from primary culture to passage 2, and uses Oricell human umbilical cord mesenchymal stem cell culture medium from passage to passage eight from the third generation, so that the cultured mesenchymal stem cells can keep the good biological characteristics of hUCB-MSCs.
Disclosure of Invention
The invention aims to provide a culture method of human umbilical cord blood mesenchymal stem cells, which can obtain mesenchymal stem cells with higher purity during initial subculture, can meet the requirement of stable cell proliferation by using a common culture medium during subsequent subculture, and obviously reduces the culture cost.
In order to realize the aim, the specific technical scheme of the culture method of the human umbilical cord blood mesenchymal stem cells is as follows:
a culture method of human umbilical cord blood mesenchymal stem cells comprises the following steps: culturing umbilical cord blood mononuclear cells by using a first culture medium until the growth state of the cells is stable, and then culturing by using a second culture medium, wherein the first culture medium mainly comprises a basic culture medium, fetal calf serum, antibiotics and mesenchymal stem cell growth additives, wherein the volume of the fetal calf serum is 8-12%, and the volume of the mesenchymal stem cell growth additives is 0.1-0.2%; the second culture medium mainly comprises a basic culture medium, fetal calf serum and antibiotics, wherein the fetal calf serum accounts for 8-12% of the volume; the basic culture medium is DMEM/F12 culture medium.
In the culture method of the human umbilical cord blood mesenchymal stem cells, the addition of the mesenchymal stem cell growth additive in the culture medium can obviously accelerate the growth speed of the hUCB-MSCs and improve the growth state of the hUCB-MSCs in the initial culture process, and relatively, the addition of the mesenchymal stem cell growth additive can promote the growth of the mesenchymal stem cells, and the meta-acid environment of the basic culture medium can inhibit the growth of other heterogeneous cells, such as osteoclast-like cells which are difficult to separate from the mesenchymal stem cells, so that the content of heterogeneous cell groups in the cells obtained by initial culture is greatly reduced, the purity of the mesenchymal stem cells is obviously improved, the differential adherence effect of the mesenchymal stem cells and the heterogeneous cells is amplified, and the growth states of the obtained mesenchymal stem cells, such as cell morphology, proliferation speed and the like, can be kept stable. When the mesenchymal stem cells are subjected to subculture by using a common culture medium, the obtained mesenchymal stem cells can well retain various characteristics of original mesenchymal stem cells, such as high cell proliferation speed or activity, good adipogenic differentiation capacity and the like. The culture method of the human umbilical cord blood mesenchymal stem cells avoids using a commercial special culture medium during subsequent subculture, and obviously reduces the culture cost.
In the prior art, for example, in the culture method disclosed in the chinese patent application CN102559590B, if a common culture medium is used from the third generation to the fourth and fifth generations, the cell body is significantly increased, the number of particles in the cytoplasm is increased, the cell proliferation rate is significantly reduced, and the growth characteristics of the original cell can be maintained only by using a special culture medium for mesenchymal stem cells with higher cost for subsequent passages. The reason for analyzing the above is mainly that the cells are purified only by liquid exchange, enzyme digestion, differential adherence and the like during initial culture, but relatively more heterogeneous cells are still mixed in the obtained cells, so that normal mesenchymal stem cells cannot be obtained if the culture medium special for the mesenchymal stem cells is not used for induced purification during subsequent culture.
Compared with the prior art, the culture method disclosed by the invention can keep higher purity of the mesenchymal stem cells during initial culture, avoids the use of a commercial special culture medium during subsequent subculture, and obviously reduces the culture cost.
The culture method of the human umbilical cord blood mesenchymal stem cells has simple steps and low economic cost, and is suitable for obtaining a large amount of mesenchymal stem cells to meet the requirement of large use amount in the treatment of related diseases.
Generally, when the first culture medium is used for primary culture and subculture of the mononuclear cells to the 2 nd to 3 rd generation, the content of the heterogeneous cell population in the obtained cells is low enough to meet the requirement that the mesenchymal stem cells with stable growth state can still be obtained after sufficient subculture is carried out subsequently, and the second culture medium is used for subsequent subculture.
The primary cultured cells will interact with each other when they are in the first contact with the external environment, and some growth promoting substances will be produced between the cellsThe active substance promotes survival and growth of cells, and if the seeding density of the cells is too low, the growth promoting effect is small, the adaptability of the cells to the environment is poor, and if the seeding density of the cells is high, the nutrient supply is insufficient, and the solution needs to be changed frequently. In general, the primary culture process is to make the mononuclear cell into a single cell suspension by using a first culture medium, and then to make the mononuclear cell into a single cell suspension according to the cell density of 5 × 106-2×107Individual monocytes/mL were seeded into cell culture flasks. The flask is typically selected to be placed at 37 ℃ with a volume fraction of 5% CO2Culturing in a saturated humidity incubator.
Generally, during primary culture, when the cell fusion degree in a cell culture bottle reaches 70-80%, subculture is performed, at the moment, the ratio of the vigorous cells in the cell culture bottle is large, and the success rate of subculture is high.
During primary culture, after the mononuclear cells are inoculated in a cell culture bottle, half of the liquid is changed after 2 days, and the first full liquid is changed after 5-7 days. The liquid change can not only update the nutrient substances in the culture medium and reduce or remove the generated metabolites, but also continuously dilute the heterogeneous cells in the culture medium, thereby further improving the purity of the mesenchymal stem cells in the culture bottle.
During subculture, methods such as enzyme digestion passage and ion chelation digestion passage can be selected, in order to prevent the mesenchymal stem cells from being damaged as far as possible, pancreatin with the mass fraction of 0.25% is used for digestion during subculture, and the treatment time is 2-5 min.
The cell culture flasks were coated with gelatin. The adherent cells can grow faster by adopting the cell culture bottle coated by the gelatin for culture, and various kinds of nucleated cells with the maximum quantity are reserved, so that the mesenchymal stem cells with a small proportion can be preserved to the maximum extent, and the purity of the mesenchymal stem cells in the obtained cells is further improved.
In view of the higher content of mesenchymal stem cells in the umbilical cord blood of a premature infant compared to a normal newborn, it is preferable that the umbilical cord blood is taken from the premature infant in order to satisfy the purity requirement of the cells obtained in the initial culture.
In general, penicillin, streptomycin, and the like are added to the basal medium to prevent bacterial invasion, and the first medium and the second medium contain 100-120U/mL penicillin, 100-120. mu.g/mL streptomycin, and 0.25-0.5. mu.g/mL amphotericin B.
Separating out the cord blood mononuclear cells by adopting a lymphocyte separation liquid method. The method is convenient and quick, simplifies the experimental steps, can avoid the damage to the cells as much as possible, and reduces the loss of the mesenchymal stem cells.
Specifically, the method for separating the cord blood mononuclear cells by adopting the lymphocyte separation solution method comprises the following specific steps:
1) extracting umbilical cord blood of a newborn under an aseptic condition, and performing heparin anticoagulation to obtain heparin anticoagulated umbilical cord blood;
2) diluting heparin anticoagulation umbilical cord blood in equal proportion by using a D-Hanks balanced salt solution;
3) superposing diluted heparin anticoagulant umbilical cord blood onto lymphocyte separation liquid to obtain mixed liquid, wherein the height ratio of the diluted heparin anticoagulant umbilical cord blood to the lymphocyte separation liquid is 2: 1;
4) centrifuging the mixed solution obtained in the step 3) for 20-25min at 1500-.
The method adopts a lymphocyte separation liquid method to separate the mononuclear cells in the umbilical cord blood, and the used reagents are common commercially available reagents, so the method is low in cost and suitable for large-scale popularization and production.
Drawings
FIG. 1 is a cell morphology map of hUCB-MSCs subcultured to passage 2 using the culture method of the present invention;
FIG. 2 is a cell morphology map of hUCB-MSCs (200X) subcultured to passage 8 using the culture method of the present invention;
FIG. 3 is a graph showing the growth of hUCB-MSCs subcultured to passage 2 and subcultured to passage 8 using the culture method of the present invention;
FIG. 4 is a graph showing the results of the identification of the phenotype of flow cytometry of hUCB-MSCs subcultured to passage 2 using the culture method of the present invention;
FIG. 5 is a graph showing the results of the identification of the phenotype of flow cytometry of hUCB-MSCs subcultured to generation 8 using the culture method of the present invention;
FIG. 6 shows the results of RT-PCR identification of hUCB-MSCs subcultured to passage 2 using the culture method of the present invention;
FIG. 7 shows the results of RT-PCR identification of hUCB-MSCs subcultured to generation 8 using the culture method of the present invention;
FIG. 8 shows the adipogenic differentiation results of hUCB-MSCs subcultured to passage 2 using the culture method of the present invention;
FIG. 9 shows the adipogenic differentiation results of hUCB-MSCs subcultured to generation 8 using the culture method of the present invention.
Detailed Description
The application of the method of the present invention will be specifically described with reference to the following examples. It should be noted that the examples given in this specification are only for the purpose of facilitating understanding of the present invention, and they are not intended to be limiting, i.e., the present invention may be embodied in other forms than those shown in the specification. Therefore, any technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
The reagents or material sources used in the following examples are:
D-Hanks balanced salt solution was purchased from Beijing Solaibao Tech Co., Ltd;
the Ficoll lymphocyte separation was purchased from tianjin tertiary ocean biology corporation;
PBS balanced salt solution was purchased from Beijing Solaibao Tech technologies, Inc.;
DMEM/F12 medium was purchased from Gibco, USA;
CD45-FITC, CD29-PE, and CD44-FITC were purchased from BD Biosciences, USA;
TRIzol Reagent is available from Ambion, USA;
the cDNA reverse transcription kit is purchased from TaKaRa company;
the cord blood mesenchymal stem cell adipogenic induction differentiation medium kit is purchased from Guangzhou Seisai company;
mesenchymal stem cell growth supplements were purchased from ScienCell, usa and contain growth factors, hormones and proteins as major components.
In the following examples, umbilical cord blood of premature infants is used as a raw material for culturing the human umbilical cord blood mesenchymal stem cells, and specific information of the umbilical cord blood of premature infants is as follows: according to the agreement of the lying-in woman and the family members, 30-50 mL of umbilical cord blood of the premature infant is collected in a third subsidiary hospital of the New county medical college, anticoagulant (heparin concentration is 20U/mL) is added for anticoagulation, separation is carried out within 6-8 h, and the umbilical cord blood is stored in a refrigerator at 4 ℃. All parturients were HIV negative, HBsAg negative, and no congenital disease was found in the preterm.
Example 1
The method for culturing the human umbilical cord blood mesenchymal stem cells comprises the following steps:
(1) isolation of mononuclear cells:
1) extracting umbilical cord blood of premature infant under aseptic condition, and anticoagulating with heparin;
2) diluting heparin anticoagulation umbilical cord blood in equal proportion by using a D-Hanks balanced salt solution;
3) superposing diluted heparin anticoagulant umbilical cord blood onto Ficoll lymphocyte separation liquid to obtain mixed liquid, wherein the height ratio of the diluted heparin anticoagulant umbilical cord blood to the lymphocyte separation liquid is 2: 1;
4) centrifuging the mixed solution obtained in the step 3) for 20min at 2000r/min, carefully absorbing a white cloud layer at an interface,
the pellet was centrifuged 2 times at 1000r/min in PBS balanced salt solution and washed.
(2) Culturing umbilical cord blood mesenchymal stem cells of premature infants:
1) adding the mononuclear cells separated in the step (1) into a first culture medium to prepare a single cell suspension, and counting the cells;
2) cell density 5X 106Seeding the mononuclear cells obtained in the step 1) into a gelatin-coated T25 cell culture flask per mL;
3) placing the cell culture flask at 37 deg.C with 5% volume fraction of CO2Culturing in saturated humidity incubator, changing liquid half after 2 days, changing liquid for the first time after 5-7 days, changing liquid for the first time every 3-4 days, and observing original liquid every day under inverted phase contrast microscopeGrowth and morphological characteristics of the passage cells;
4) when the cell fusion degree reaches 70-80%, digesting with 0.25% pancreatin, carrying out 1:2 subculture (namely, one bottle of cells is divided into two bottles for subculture), and continuing to subculture to the 2 nd generation;
5) from the 3 rd generation cells, subculture was performed to 10 th generation using the second medium.
The specific preparation process of the first culture medium comprises the following steps: 40mL of FBS (fetal bovine serum), 1mL of mesenchymal stem cell growth supplement were added to 500mL of DMEM/F12 medium, while adding penicillin, streptomycin, amphotericin B, such that the resulting medium contained 100U/mL penicillin, 100. mu.g/mL streptomycin, and 0.25. mu.g/mL amphotericin B.
The specific preparation process of the second culture medium comprises the following steps: 40mL of FBS (fetal bovine serum) was added to 500mL of DMEM/F12 medium together with penicillin, streptomycin and amphotericin B so that the resulting medium contained 100U/mL of penicillin, 100. mu.g/mL of streptomycin and 0.25. mu.g/mL of amphotericin B.
Example 2
The method for culturing the human umbilical cord blood mesenchymal stem cells comprises the following steps:
(1) the isolation of mononuclear cells was performed as in example 1.
(2) Culturing umbilical cord blood mesenchymal stem cells of premature infants:
1) adding the mononuclear cells separated in the step (1) into a first culture medium to prepare a single cell suspension, and counting the cells;
2) 1X 10 in terms of cell density7Seeding the mononuclear cells obtained in the step 1) into a gelatin-coated T25 cell culture flask per mL;
3) placing the cell culture flask at 37 deg.C with 5% volume fraction of CO2Culturing in a saturated humidity incubator, changing liquid half after 2 days, changing liquid for the first time in full after 5-7 days, changing liquid for the full after every 3-4 days, and observing the growth condition and morphological characteristics of the primary cells every day under an inverted phase contrast microscope;
4) when the cell fusion degree reaches 70-80%, digesting with 0.25% pancreatin, carrying out 1:2 subculture, and continuing subculturing to the 2 nd generation;
5) from the 3 rd generation cells, subculture was performed to 10 th generation using the second medium.
The specific preparation process of the first culture medium comprises the following steps: 50mL of FBS and 1mL of mesenchymal stem cell growth supplement were added to 500mL of DMEM/F12 medium, and penicillin, streptomycin and amphotericin B were added simultaneously, so that the resulting medium contained 100U/mL of penicillin, 100. mu.g/mL of streptomycin and 0.25. mu.g/mL of amphotericin B.
The specific preparation process of the second culture medium comprises the following steps: 50mL of FBS (fetal bovine serum) was added to 500mL of DMEM/F12 medium together with penicillin, streptomycin and amphotericin B so that the resulting medium contained 100U/mL of penicillin, 100. mu.g/mL of streptomycin and 0.25. mu.g/mL of amphotericin B.
Example 3
The method for culturing the human umbilical cord blood mesenchymal stem cells comprises the following steps:
(1) the isolation of mononuclear cells was performed as in example 1.
(2) Culturing umbilical cord blood mesenchymal stem cells of premature infants:
1) adding the mononuclear cells separated in the step (1) into a first culture medium to prepare a single cell suspension, and counting the cells;
2) at a cell density of 2X 107Seeding the mononuclear cells obtained in the step 1) into a gelatin-coated T25 cell culture flask per mL;
3) placing the cell culture flask at 37 deg.C with 5% volume fraction of CO2Culturing in a saturated humidity incubator, changing liquid half after 2 days, changing liquid for the first time in full after 5-7 days, changing liquid for the full after every 3-4 days, and observing the growth condition and morphological characteristics of the primary cells every day under an inverted phase contrast microscope;
4) when the cell fusion degree reaches 70-80%, digesting with 0.25% pancreatin, carrying out 1:2 subculture, and continuing subculturing to the 2 nd generation;
5) from the 3 rd generation cells, subculture was performed to 10 th generation using the second medium.
The specific preparation process of the first culture medium comprises the following steps: 60mL of FBS and 1mL of mesenchymal stem cell growth supplement are added to 500mL of DMEM/F12 culture medium, and penicillin, streptomycin and amphotericin B are simultaneously added, so that the obtained culture medium contains 100U/mL of penicillin, 100 mu g/mL of streptomycin and 0.25 mu g/mL of amphotericin B.
The specific preparation process of the second culture medium comprises the following steps: 60mL of FBS (fetal bovine serum) was added to 500mL of DMEM/F12 medium, together with penicillin, streptomycin and amphotericin B, so that the resulting medium contained 100U/mL of penicillin, 100. mu.g/mL of streptomycin and 0.25. mu.g/mL of amphotericin B.
Comparative example
The method for sequentially culturing human umbilical cord blood mesenchymal stem cells by adopting two culture media disclosed in the Chinese invention patent with publication number CN102559590B is used as a comparison.
Examples of the experiments
35 parts of cord blood are separated from the group, and 29 parts of the cord blood are subjected to primary culture for 22-25 days to obtain uniform hUCB-MSCs, wherein the culture success rate is 82.86%. The series identification and characterization of the mesenchymal stem cells are carried out by observing morphological characteristics of the hUCB-MSCs, drawing an in-vitro growth curve of the hUCB-MSCs, detecting cell surface markers by flow cytometry, detecting specific genes of embryonic stem cells by RT-PCR, detecting adipogenic differentiation capacity and other experiments. The specific experimental procedures are detailed below.
Morphological characteristics of (I) hUCB-MSCs
The morphological characteristics of the cells of the 2 nd generation and the 8 th generation are respectively observed under an inverted phase contrast microscope, the results are respectively shown in figure 1 and figure 2, the cells grow in a vortex shape, and the cell fusion degree can reach 80-90%. The results show that the cell morphology of the hUCB-MSCs at the 8 th generation and the 2 nd generation is almost the same after a plurality of rounds of passage, and the morphological characteristics of the hUCB-MSCs are not obviously changed.
Growth curves of (di) hUCB-MSCs
Respectively taking the 2 nd generation and 8 th generation hUCB-MSCs to prepare single cell suspension, adjusting the cell concentration to 5 × 104/mL, inoculating to a 24-well plate according to 200 uL/well, placing at 37 deg.C and 5% CO2And culturing in an incubator with saturated humidity. From day 2, two random wells were selected for digestion and counted each day, and the average value was taken asDay cell numbers, counted for 12 consecutive days, and in vitro growth curves of hUCB-MSCs were plotted, as shown in FIG. 3. The growth curve shows that the proliferation speed and the growth cycle of the 2 nd generation and the 8 th generation of hUCB-MSCs have no obvious change, specifically, the 1 st to 3 rd days are the growth incubation period of the cells, from the 4 th day, the cells enter a logarithmic growth phase, and then begin to proliferate in large quantities, the cell protrusions can be observed under an inverted microscope to continuously extend to the periphery, the cell density is gradually increased, the cells are connected with each other, the peak is reached by the 10 th day, the speed is reduced later, and the cells enter a plateau phase. The exponential growth phase doubling time was about 67 h.
Compared with the scheme of the comparative example, the hUCB-MSCs obtained by the culture method of the human umbilical cord blood mesenchymal stem cells have strong proliferation activity and short doubling time, and the 8 th generation and the 2 nd generation have no obvious difference in proliferation speed, growth cycle and the like.
(III) identification of hUCB-MSCs
The flow cytophenotype identification results of fig. 4 and fig. 5 show that the hUCB-MSCs of generation 2 and generation 8 can stably and uniformly express the stem cell markers CD29 and CD44 with high expression, and hardly express the marker antigen CD45 of hematopoietic precursor cells. The specific experimental process is as follows: taking hUCB-MSCs of 2 nd generation and 8 th generation respectively, digesting by using 0.25% pancreatin, centrifuging for 10min at 1000rpm, discarding supernatant, washing and precipitating for 3 times by using PBS, counting 1 × 106 cells by means of heavy suspension, adding 20 μ L of flow monoclonal antibodies including CD45-FITC, CD29-PE and CD44-FITC respectively, standing for 30min at normal temperature in a dark place, centrifuging for 5min at 1000rpm, adding 400 μ L of PBS for heavy suspension after removing unbound antibodies, detecting by using a flow cytometer, and specifically operating according to the using rules of the instrument.
FIGS. 6 and 7 show the results of detection of cell surface markers by RT-PCR, and it can be seen that all of the hUCB-MSCs of passage 2 and passage 8 exhibited positive mRNA expression of Oct4, Sox2 and Nanog. The specific experimental process is as follows: taking the 2 nd generation and 8 th generation hUCB-MSCs respectively, extracting total RNA of the cells by a Trizol method, and carrying out specific operation according to a kit instruction. 500ng of RNA was taken, and the RNA was reverse-transcribed into cDNA using PrimeScript RT Reagent Kit, and the expression levels of three genes, Oct4, Nanog, and Sox2, were detected by PCR using cDNA as a template, and GAPDH as an internal control. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, and termination at 72 ℃ for 5 min.
(IV) differentiation potential test of hUCB-MSCs
Respectively taking hUCB-MSCs of 2 nd generation and 8 th generation, preparing into single cell suspension, inoculating into 6-well plate, placing at 37 deg.C and 5% CO2And culturing in an incubator with saturated humidity. When the cells are fused by 80-90 percent, the cells are changed into the culture medium A for inducing differentiation by adipogenic differentiation of human umbilical cord blood mesenchymal stem cells, after 3 days of induction, the culture medium A is discarded, the culture medium B is changed into the culture medium B for inducing differentiation by adipogenic differentiation of human umbilical cord blood mesenchymal stem cells, after 24 hours, the culture medium B is discarded, and the culture medium A is changed back again. The solution A and solution B were alternately applied for 3 rounds for about 12 days, and the culture was continued for 3 days with the solution B until the lipid droplets became large and round. After the fatting induction and differentiation of the round is finished, fixing by using a 4% neutral formaldehyde solution, and after the identification by dyeing of 0.3% oil red O dye solution, placing the culture plate under an inverted microscope to observe the fatting dyeing effect. After differentiation culture, the results of oil red O staining are shown in FIGS. 8 and 9, which show that lipid droplets can be formed in the 2 nd and 8 th generation hUCB-MSCs, indicating that the hUCB-MSCs isolated and cultured by the method still have multidirectional differentiation potential after multiple rounds of subculture.
Based on the experimental results, the human umbilical cord blood mesenchymal stem cells are obtained by separating and culturing according to the culture method of the human umbilical cord blood mesenchymal stem cells, the culture success rate is high by adopting the culture method of the human umbilical cord blood mesenchymal stem cells, a commercial culture medium special for the mesenchymal stem cells is not used in the whole process, the separation and culture costs are greatly reduced, and the method is suitable for large-scale culture of the umbilical cord blood mesenchymal stem cells and is used for treating related diseases.
Claims (10)
1. A culture method of human umbilical cord blood mesenchymal stem cells is characterized by comprising the following steps: culturing umbilical cord blood mononuclear cells by using a first culture medium until the growth state of the cells is stable, and then culturing by using a second culture medium, wherein the first culture medium mainly comprises a basic culture medium, fetal calf serum, antibiotics and mesenchymal stem cell growth additives, wherein the volume of the fetal calf serum is 8-12%, and the volume of the mesenchymal stem cell growth additives is 0.1-0.2%; the second culture medium mainly comprises a basic culture medium, fetal calf serum and antibiotics, wherein the fetal calf serum accounts for 8-12% of the volume; the basic culture medium is DMEM/F12 culture medium.
2. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 1, wherein the stable growth state is obtained by performing primary culture and subculture on the mononuclear cells to 2-3 generations with a first culture medium, and performing subsequent subculture with a second culture medium.
3. A culture method of human umbilical cord blood mesenchymal stem cells according to claim 2, wherein the primary culture comprises preparing the mononuclear cells into a single cell suspension with a first culture medium, and then culturing the mononuclear cells at a cell density of 5 x 106-2×107Individual monocytes/mL were seeded into cell culture flasks.
4. A culture method of human umbilical cord blood mesenchymal stem cells according to claim 2 or 3, wherein during the primary culture, subculture is performed until the cell fusion degree in a cell culture flask reaches 70-80%.
5. A culture method of human umbilical cord blood mesenchymal stem cells according to claim 2 or 3, wherein in the primary culture, after the mononuclear cells are inoculated in a cell culture flask, half of the culture solution is changed after 2 days, and the culture solution is changed for the first time in full after 5-7 days.
6. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 3, wherein the cell culture flask is coated with gelatin.
7. The method for culturing human umbilical cord blood mesenchymal stem cells according to any one of claims 1 to 3, wherein the umbilical cord blood is taken from a premature infant.
8. The method for culturing human umbilical cord blood mesenchymal stem cells according to any one of claims 1 to 3, wherein the content of antibiotics in the first and second culture media is: 100-120U/mL penicillin, 100-120. mu.g/mL streptomycin and 0.25-0.5. mu.g/mL amphotericin B.
9. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 1, wherein umbilical cord blood mononuclear cells are isolated by a lymphocyte separation method.
10. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 9, wherein the specific steps of mononuclear cell isolation are as follows:
1) extracting umbilical cord blood of a newborn under an aseptic condition, and performing heparin anticoagulation to obtain heparin anticoagulated umbilical cord blood;
2) diluting heparin anticoagulation umbilical cord blood in equal proportion by using a D-Hanks balanced salt solution;
3) superposing diluted heparin anticoagulant umbilical cord blood onto lymphocyte separation liquid to obtain mixed liquid, wherein the height ratio of the diluted heparin anticoagulant umbilical cord blood to the lymphocyte separation liquid is 2: 1;
4) centrifuging the mixed solution obtained in the step 3) for 20-25min at 1500-.
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