CN111235100B - Culture method of human umbilical cord blood mesenchymal stem cells - Google Patents

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

A method for culturing human umbilical cord blood mesenchymal stem cells

technical field

The invention belongs to the field of cell biology, in particular to a method for culturing human umbilical cord blood mesenchymal stem cells.

Background technique

Mesenchymal stem cells (MSCs) have the potential of self-renewal and multi-directional differentiation, and have now become a cell source of great practical value in the field of gene therapy. MSCs are initially isolated from bone marrow and are the most widely used. MSCs can also be isolated from other tissues and organs. At present, MSCs derived from different tissues have been used in the treatment of clinical diseases, such as umbilical cord blood, menstrual blood, and adipose tissue. Among them, human umbilical cord blood is a good source of cells, which is not from the blood of the mother, but belongs to the blood of the fetus, which is not confused with the blood of the mother. However, umbilical cord blood is usually discarded as "garbage" during childbirth, and its useful value needs to be developed urgently.

Umbilical cord blood has the advantages of abundant sources, easy collection, low immunogenicity, strong proliferation ability, and no ethical restrictions. The role of umbilical cord blood mesenchymal stem cells is becoming more and more prominent, and they can be used as materials for cell transplantation therapy of related diseases. Worldwide, cord blood can be used for the treatment of more than 80 diseases, including leukemia, aplastic anemia, systemic lupus erythematosus, myelodysplastic syndrome, malignant lymphoma, malignancy, severe immunodeficiency and metabolic diseases disease, etc. The content of mesenchymal stem cells in premature infants is higher than that of ordinary fetuses. If the umbilical cord blood of premature infants can be isolated, cultured and preserved, it can provide excellent materials for the treatment of diseases related to premature infants at the cellular level.

However, the proportion of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) in mononuclear cells is only 0.001-0.01%, and the success rate of isolating mesenchymal stem cells from umbilical cord blood is relatively high. Low. Up to now, there are different methods for in vitro isolation and culture of human umbilical cord blood mesenchymal stem cells, and it is still difficult to obtain a large number of MSCs. Therefore, how to choose a simple, practical and low-cost method to isolate and culture the largest proportion of MSCs from human umbilical cord blood and directly apply it to clinical treatment is a key problem that needs to be solved urgently.

The medium is the key to the successful culture of hUCB-MSCs. Most of the prior art adopts DMEM (low sugar or high sugar) medium and adds 5%～20% fetal bovine serum, and the success rate is low (Laitinen A, Lampinen M, Liedtke S, et al. The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells 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 Mesenscult ^TM medium, and obtained a relatively ideal culture effect (Bieback K, Kern S, Klüter H, et al. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood[J]. Stem Cells, 2004, 22(4):625-634). Mesencult ^TM medium is usually an acidic medium, including basal medium and individual supplements, which can promote the efficient proliferation of hUCB-MSCs and maintain their undifferentiated state, thereby inhibiting the growth of other adherent cells, and is considered to be a suitable medium for hUCB. - The best medium for the growth of MSCs. However, the price of Mesenscult ^TM medium is expensive, which leads to a significant increase in the cost of separation and culture, and is especially unsuitable for large-scale production.

The Chinese invention patent with the announcement number CN102559590B discloses a method for sequentially culturing human umbilical cord blood mesenchymal stem cells using two culture media. DMEM/F12 medium was cultured from primary culture to the second passage, and from the third passage, the cells were subcultured to Oricell human umbilical cord mesenchymal stem cell medium to the eighth passage, and the cultured mesenchymal stem cells were able to maintain hUCB - MSCs have good biological characteristics, but the above-mentioned method of sequentially culturing human umbilical cord blood mesenchymal stem cells with two media still unavoidable the use of a special medium for mesenchymal stem cells, which still has the problem of high cost, which is not conducive to obtaining A large amount of blood mesenchymal stem cells can meet the large demand for the treatment of related diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for culturing human umbilical cord blood mesenchymal stem cells, which can obtain higher-purity mesenchymal stem cells in the initial subculture, and use a common medium in the subsequent subculture to satisfy cell stability The need for proliferation significantly reduces the cost of culture.

To achieve the above object, the specific technical scheme of the method for culturing human umbilical cord blood mesenchymal stem cells of the present invention is:

A method for culturing human umbilical cord blood mesenchymal stem cells, comprising the steps of: culturing umbilical cord blood mononuclear cells with a first medium until the cells grow in a stable state, and then using a second medium for culturing, wherein the first medium is used for culturing. A medium is mainly composed of basal medium, fetal bovine serum, antibiotics and mesenchymal stem cell growth supplements, of which the volume of fetal bovine serum accounts for 8-12%, and the volume of mesenchymal stem cell growth supplements accounts for 0.1-12%. 0.2%; the second medium is mainly composed of basal medium, fetal bovine serum, and antibiotics, wherein the volume of fetal bovine serum accounts for 8-12%; the basal medium is DMEM/F12 medium.

In the method for culturing human umbilical cord blood mesenchymal stem cells of the present invention, in the initial culture process, the addition of mesenchymal stem cell growth supplements in the medium can significantly accelerate the growth rate of hUCB-MSCs and improve their growth state. The addition of mesenchymal stem cell growth supplements can promote the growth of mesenchymal stem cells, while the acidic environment of the basal medium can inhibit the growth of other heterogeneous cells, such as osteoclast-like cells that are difficult to separate from mesenchymal stem cells. The content of the heterogeneous cell population in the cells obtained from the initial culture is greatly reduced and the purity of the mesenchymal stem cells is significantly improved, amplifying the differential adherence effect between the mesenchymal stem cells and the heterogeneous cells, and can make the obtained mesenchymal stem cells. Cell morphology, proliferation rate and other growth states remained stable. In the subsequent subculture with ordinary medium, the obtained mesenchymal stem cells can well retain various characteristics of the original mesenchymal stem cells, such as high cell proliferation rate or viability, good adipogenic differentiation ability Wait. The method for culturing human umbilical cord blood mesenchymal stem cells of the present invention avoids the use of commercial special culture medium in the subsequent subculture, and significantly reduces the culture cost.

In the prior art, such as the culturing method disclosed in the Chinese invention patent of CN102559590B, if the third generation starts to adopt a common medium, the cell body will be significantly increased when cultured to the fourth and fifth generations, the number of particles in the cytoplasm is increased, and the cells The proliferation rate is significantly reduced, and subsequent passages require the use of high-cost mesenchymal stem cell-specific media to maintain the growth characteristics of the original cells. The reason for this analysis is mainly that the cells were only purified by changing the medium, enzyme digestion, differential adhesion, etc. in the initial culture, but the obtained cells were still mixed with relatively more heterogeneous cells, which led to the subsequent culture. Normal mesenchymal stem cells cannot be obtained by inducing and purifying with a special medium for mesenchymal stem cells.

Compared with the prior art, the culture method of the present invention can maintain a higher purity of mesenchymal stem cells during initial culture, avoid the use of commercial special medium during subsequent subculture, and significantly reduce the culture cost.

The method for culturing human umbilical cord blood mesenchymal stem cells of the present invention has simple steps and low economic cost, and is suitable for obtaining a large amount of mesenchymal stem cells to meet the needs of larger usage in the treatment of related diseases.

Generally, when mononuclear cells are primary cultured and subcultured to passages 2-3 in the above-mentioned first medium, the content of the heterogeneous cell population in the obtained cells is low enough to meet the requirement of sufficient subsequent passages. After culturing, the mesenchymal stem cells with stable growth state can still be obtained, and the second medium is used for subsequent subculture.

When the primary cultured cells are exposed to the in vitro environment for the first time, they will interact with each other, and some active substances that promote growth can be produced between the cells, so that the cells can promote the survival and growth of each other. This growth-promoting effect is small, and the cells have poor adaptability to the environment. If the cell inoculation density is high, the supply of nutrients will be insufficient, and the medium needs to be changed frequently. Generally, the process of primary culture is to use the first medium to make the mononuclear cells into a single cell suspension, and then inoculate the mononuclear cells on the cells at a cell density of 5×10 ⁶ -2×10 ⁷ cells/mL in culture flasks. Generally, the culture flask is placed in a 37°C, 5% _CO2 -saturated humidity incubator for cultivation.

Generally, in primary culture, subculture is carried out when the confluence of cells in the cell culture flask reaches 70-80%. At this time, the vigorous cells in the cell culture flask account for a large proportion, and the success rate of subculture is high.

In primary culture, after mononuclear cells are inoculated in cell culture flasks, half of the medium is changed after 2 days, and the first full amount of medium is changed after 5-7 days. The medium change can not only renew the nutrients in the medium, reduce or remove the metabolites produced, but also continuously dilute the heterogeneous cells in the medium, thereby further improving the purity of the mesenchymal stem cells in the culture flask.

During subculture, methods such as enzymatic digestion and passage, ion chelation digestion and passage can be selected. In order to minimize damage to the mesenchymal stem cells themselves, trypsin digestion with a mass fraction of 0.25% is used for subculture, and the treatment time is 2-5min. .

The above cell culture flasks were coated with gelatin. The use of gelatin-coated cell culture flasks can make adherent cells grow faster and retain the largest number of nucleated cells, thereby ensuring that a small proportion of mesenchymal stem cells can be preserved to the greatest extent, and further improve the Purity of mesenchymal stem cells obtained.

Considering that the content of mesenchymal stem cells in the umbilical cord blood of premature infants is higher than that of normal newborns, in order to meet the purity requirements of the cells obtained from the initial culture, it is preferable to obtain the umbilical cord blood from premature infants.

Usually, penicillin, streptomycin, etc. are added to the basal medium to avoid bacterial invasion, and the first medium and the second medium above contain 100-120 U/mL penicillin, 100-120 μg/mL streptomycin and 0.25-0.5 μg/mL amphotericin B.

Umbilical cord blood mononuclear cells were isolated by lymphocyte separation medium. The method is convenient and fast, simplifies experimental steps, can avoid damage to cells as much as possible, and reduce the loss of mesenchymal stem cells.

Specifically, the specific steps for separating umbilical cord blood mononuclear cells by the lymphocyte separation solution method are:

1) Under sterile conditions, the umbilical cord blood of the newborn was extracted, and heparin anticoagulation was performed to obtain heparin anticoagulated umbilical cord blood;

2) Use D-Hanks balanced salt solution to dilute heparin anticoagulated cord blood in equal proportions;

3) Superimpose the diluted heparin anticoagulated umbilical cord blood on the lymphocyte separation liquid to obtain a mixture, and the height ratio of the diluted heparin anticoagulated umbilical cord blood to the lymphocyte separation liquid is 2:1;

4) Centrifuge the mixed solution obtained in step 3) at 1500-2000r/min for 20-25min, absorb the white cloud layer at the interface, centrifuge with PBS balanced salt solution at 1000-1500r/min, and wash the precipitate for a total of 2 times.

The invention adopts the lymphocyte separation liquid method to separate the mononuclear cells in the umbilical cord blood, the reagents used are all common commercially available reagents, the cost is low, and it is suitable for large-scale popularization and production.

Description of drawings

Figure 1 is a cell morphology diagram of hUCB-MSCs subcultured to the second generation using the culture method of the present invention;

Figure 2 is a cell morphology diagram of hUCB-MSCs (200×) subcultured to the 8th passage using the culture method of the present invention;

3 is a growth curve diagram of hUCB-MSCs subcultured to the 2nd generation and subcultured to the 8th generation using the culture method of the present invention;

Figure 4 is a flow cytometric phenotype identification result of hUCB-MSCs subcultured to the second generation using the culture method of the present invention;

5 is a flow cytometric phenotype identification result of hUCB-MSCs subcultured to the 8th passage using the culture method of the present invention;

6 is the RT-PCR identification result of hUCB-MSCs subcultured to the second generation using the culture method of the present invention;

Fig. 7 is the RT-PCR identification result of the hUCB-MSCs subcultured to the 8th generation using the culture method of the present invention;

Figure 8 shows the adipogenic differentiation results of hUCB-MSCs subcultured to the second generation using the culture method of the present invention;

Figure 9 shows the results of adipogenic differentiation of hUCB-MSCs subcultured to the 8th passage using the culture method of the present invention.

Detailed ways

The application of the method of the present invention will be specifically described below with reference to specific embodiments. It should be particularly pointed out that the examples exemplified in the description of the present invention are only for helping the understanding of the present invention, and they do not have any limiting effect, that is, the present invention may have other embodiments besides the examples exemplified in the description. Therefore, any technical solutions formed in the form of equivalent replacement or equivalent transformation all fall within the protection scope of the present invention.

The sources of reagents or materials used in the following examples are:

D-Hanks balanced salt solution was purchased from Beijing Soleibao Technology Co., Ltd.;

Ficoll lymphocyte separation solution was purchased from Tianjin Haoyang Biological Company;

PBS balanced salt solution was purchased from Beijing Soleibao Technology Co., Ltd.;

DMEM/F12 medium was purchased from Gibco, USA;

CD45-FITC, CD29-PE and CD44-FITC were purchased from BD Biosciences, USA;

TRIzol Reagent was purchased from Ambion Company in the United States;

cDNA reverse transcription kit was purchased from Dalian TaKaRa Company;

Umbilical cord blood mesenchymal stem cell adipogenic differentiation medium kit was purchased from Guangzhou Saiye Company;

Mesenchymal stem cell growth supplements were purchased from American ScienCell Company, and its main components were growth factors, hormones and proteins.

In the following examples, the umbilical cord blood of premature infants is used as the raw material for the culture of human umbilical cord blood mesenchymal stem cells of the present invention. The specific information of the umbilical cord blood of premature infants is as follows: With the consent of the mother and her family members, the premature infants were collected at the Third Affiliated Hospital of Xinxiang Medical College. 30-50 mL of umbilical cord blood was added with anticoagulant (heparin concentration of 20 U/mL) for anticoagulation, and separated within 6-8 hours, and stored in a refrigerator at 4°C. All mothers were HIV-negative, HBsAg-negative, and no congenital disease was found in preterm infants.

Example 1

The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment includes the following steps:

(1) Isolation of mononuclear cells:

1) The umbilical cord blood of premature infants was drawn under sterile conditions, and heparin anticoagulation;

2) Use D-Hanks balanced salt solution to dilute heparin anticoagulated cord blood in equal proportions;

3) Superimpose the diluted heparin anticoagulated umbilical cord blood on the Ficoll lymphocyte separation liquid to obtain a mixture, and the height ratio of the diluted heparin anticoagulated umbilical cord blood to the lymphocyte separation liquid is 2:1;

4) Centrifuge the mixture obtained in step 3) at 2000 r/min for 20 min, carefully absorb the white cloud layer at the interface,

The pellets were centrifuged at 1000 r/min with PBS balanced salt solution and washed for a total of 2 times.

(2) Culture of umbilical cord blood mesenchymal stem cells in premature infants:

1) adding the mononuclear cells isolated in step (1) into the first culture medium to prepare a single cell suspension, and count the cells;

2) At a cell density of 5×10 ⁶ cells/mL, inoculate the mononuclear cells in step 1) into a gelatin-coated T25 cell culture flask;

3) Place the cell culture flask in a 37°C, _CO2 -saturated humidity incubator with a volume fraction of 5%, change the medium in half after 2 days, change the medium in full after 5-7 days, and change the medium in full every 3-4 days thereafter , observe the growth and morphological characteristics of primary cells daily under an inverted phase contrast microscope;

4) When the cell confluence reaches 70-80%, digest with 0.25% trypsin and carry out 1:2 subculture (that is, one bottle of cells is divided into two bottles for subculture), and continue to subculture to the second generation;

5) From the 3rd passage, use the second medium to subculture to the 10th passage.

The specific preparation process of the above-mentioned first culture medium is as follows: 40 mL of FBS (fetal bovine serum) and 1 mL of mesenchymal stem cell growth supplement are added to 500 mL of DMEM/F12 medium, and penicillin, streptomycin, and amphotericin B are added simultaneously. , the resulting medium contains 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B.

The concrete preparation process of above-mentioned second substratum is: add 40mL FBS (fetal bovine serum) in the DMEM/F12 substratum of 500mL, add penicillin, streptomycin, amphotericin B simultaneously, make in gained substratum contain 100U/F12 substratum. mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B.

Example 2

The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment includes the following steps:

(1) The separation method of mononuclear cells is the same as that in Example 1.

(2) Culture of umbilical cord blood mesenchymal stem cells in premature infants:

1) adding the mononuclear cells isolated in step (1) into the first culture medium to prepare a single cell suspension, and count the cells;

2) Inoculate the mononuclear cells in step 1) into a gelatin-coated T25 cell culture flask at a cell density of 1×10 ⁷ cells/mL;

3) Place the cell culture flask in a 37°C, _CO2 -saturated humidity incubator with a volume fraction of 5%, change the medium in half after 2 days, change the medium in full after 5-7 days, and change the medium in full every 3-4 days thereafter , observe the growth and morphological characteristics of primary cells daily under an inverted phase contrast microscope;

4) When the cell confluence reaches 70-80%, digest with 0.25% trypsin and carry out 1:2 subculture, and continue subculture to the second generation;

5) From the 3rd passage, use the second medium to subculture to the 10th passage.

The specific preparation process of the above-mentioned first culture medium is as follows: adding 50 mL of FBS and 1 mL of mesenchymal stem cell growth supplement to 500 mL of DMEM/F12 medium, and simultaneously adding penicillin, streptomycin, and amphotericin B to make the obtained medium. Contains 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B.

The concrete preparation process of above-mentioned second substratum is: add 50mL FBS (fetal bovine serum) in the DMEM/F12 substratum of 500mL, add penicillin, streptomycin, amphotericin B simultaneously, make the gained substratum contain 100U/ mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B.

Example 3

The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment includes the following steps:

(1) The separation method of mononuclear cells is the same as that in Example 1.

(2) Culture of umbilical cord blood mesenchymal stem cells in premature infants:

1) adding the mononuclear cells isolated in step (1) into the first culture medium to prepare a single cell suspension, and count the cells;

2) Inoculate the mononuclear cells in step 1) into a gelatin-coated T25 cell culture flask at a cell density of 2×10 ⁷ cells/mL;

3) Place the cell culture flask in a 37°C, _CO2 -saturated humidity incubator with a volume fraction of 5%, change the medium in half after 2 days, change the medium in full after 5-7 days, and change the medium in full every 3-4 days thereafter , observe the growth and morphological characteristics of primary cells daily under an inverted phase contrast microscope;

4) When the cell confluence reaches 70-80%, digest with 0.25% trypsin and carry out 1:2 subculture, and continue subculture to the second generation;

5) From the 3rd passage, use the second medium to subculture to the 10th passage.

The specific preparation process of the above-mentioned first culture medium is as follows: adding 60 mL of FBS and 1 mL of mesenchymal stem cell growth supplement to 500 mL of DMEM/F12 medium, and simultaneously adding penicillin, streptomycin, and amphotericin B to make the obtained medium. Contains 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B.

The concrete preparation process of above-mentioned second substratum is: add 60mL FBS (fetal bovine serum) in the DMEM/F12 substratum of 500mL, add penicillin, streptomycin, amphotericin B simultaneously, make in gained substratum contain 100U/F12 substratum. mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B.

Comparative ratio

The method of sequentially culturing human umbilical cord blood mesenchymal stem cells by using two culture media disclosed in the Chinese invention patent with publication number CN102559590B is used as a comparison.

Experimental example

A total of 35 umbilical cord blood were isolated in this group. After 22-25 days of primary culture, 29 of them obtained homogeneous hUCB-MSCs, and the culture success rate was 82.86%. By observing the morphological characteristics of hUCB-MSCs, drawing the growth curve of hUCB-MSCs in vitro, detecting cell surface markers by flow cytometry, detecting embryonic stem cell-specific genes by RT-PCR, and detecting the ability of adipogenic differentiation, the mesenchymal cells were carried out. Serial identification and characterization of stem cells. The specific experimental procedures are described in detail below.

(1) Morphological characteristics of hUCB-MSCs

The morphological characteristics of the cells of the second and eighth passages were observed under an inverted phase contrast microscope, and the results were shown in Figure 1 and Figure 2, respectively. The results showed that after several rounds of passage, the cell morphology of hUCB-MSCs at passage 8 and passage 2 were almost the same, and there was no obvious change in their morphological characteristics.

(2) Growth curve of hUCB-MSCs

Take hUCB-MSCs of passage 2 and passage 8 respectively, prepare single cell suspension, adjust the cell concentration to 5×104/mL, inoculate 200uL/well into 24-well plates, and place at 37°C, 5% CO ₂ . , cultured in a humidified incubator. From the 2nd day, two wells were randomly selected for digestion and count each day, and the average value was taken as the number of cells on that day. The cells were counted for 12 consecutive days, and the in vitro growth curve of hUCB-MSCs was drawn, as shown in Figure 3. The growth curve showed that the proliferation rate and growth cycle of hUCB-MSCs in the second and eighth passages did not change significantly. Specifically, the first to third days were the growth latency of the cells, and from the fourth day, the cells entered the logarithmic growth phase. , and then began to proliferate. Under the inverted microscope, it can be observed that the cell protrusions continue to expand to the surrounding, the cell density gradually increases, and the cells are connected to each other, reaching a peak on the 10th day, and then slowing down and entering a plateau phase. The exponential growth period doubling time is about 67h.

Compared with the scheme of the comparative example, the hUCB-MSCs obtained by using the method for culturing human umbilical cord blood mesenchymal stem cells of the present invention have strong proliferation activity, short doubling time, and the 8th and 2nd generations have a higher proliferation rate and better growth rate. There was no significant difference in growth cycle.

(3) Identification of hUCB-MSCs

The results of flow cytometric phenotyping in Figure 4 and Figure 5 show that hUCB-MSCs at passage 2 and passage 8 can stably and uniformly express high stem cell markers CD29 and CD44, and hardly express the marker antigen CD45 of hematopoietic precursor cells. The specific experimental process is as follows: hUCB-MSCs of passage 2 and passage 8 were taken, digested with 0.25% trypsin, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the pellet was washed three times with PBS, resuspended and counted at 1×106 Cells were added with 20 μL of flow mAbs, including CD45-FITC, CD29-PE and CD44-FITC, placed in the dark for 30 min at room temperature, and then centrifuged at 1000 rpm for 5 min. After removing unbound antibodies, resuspended in 400 μL of PBS. Use a flow cytometer for detection, and the specific operations are carried out in accordance with the rules for the use of the instrument.

Figures 6 and 7 show the results of detecting cell surface markers by RT-PCR. As can be seen from the figures, the mRNA expressions of Oct4, Sox2 and Nanog in hUCB-MSCs of the second and eighth passages were all positive. The specific experimental process is as follows: hUCB-MSCs of passage 2 and passage 8 were taken respectively, and total cell RNA was extracted by Trizol method. The specific operation was carried out according to the kit instructions. 500ng of RNA was taken, and the RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit. Using cDNA as a template, the expression levels of Oct4, Nanog, and Sox2 were detected by PCR, and GAPDH was used as an internal reference. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, a total of 30 cycles, and the reaction was terminated at 72 °C for 5 min.

(4) Differentiation Potential Experiment of hUCB-MSCs

The hUCB-MSCs of passage 2 and passage 8 were taken respectively to prepare single cell suspension, inoculated in 6-well plates, and cultured in an incubator at 37°C, 5% CO ₂ and saturated humidity. When the cells reach 80% to 90% confluence, the cells are replaced with human umbilical cord blood mesenchymal stem cells adipogenic differentiation medium A solution, after 3 days of induction, discard the A solution and replace it with human cord blood mesenchymal stem cells adipogenic differentiation medium Medium B solution, after 24h, discard B solution and replace it with A solution again. Solution A and solution B acted alternately for 3 rounds, about 12 days, and continued to maintain culture with solution B for 3 days until the lipid droplets became larger and rounded. After this round of adipogenic induction and differentiation, the cells were fixed with 4% neutral formaldehyde solution, and identified by 0.3% oil red O staining solution, and then the culture plate was placed under an inverted microscope to observe the staining effect of adipogenicity. After differentiation and culture, the results of Oil Red O staining are shown in Figure 8 and Figure 9. The figures show that the formation of lipid droplets can be seen in the hUCB-MSCs of the second and eighth passages, indicating that the hUCB-MSCs isolated and cultured by this method have undergone multiple rounds of passages. It still has the potential for multi-directional differentiation.

Based on the above experimental results, the human umbilical cord blood mesenchymal stem cells isolated and cultured by the method for culturing human umbilical cord blood mesenchymal stem cells of the present invention are human umbilical cord blood mesenchymal stem cells, and the culture method for human umbilical cord blood mesenchymal stem cells of the present invention is used. High, commercial mesenchymal stem cell-specific medium is not used in the whole process, which greatly reduces the cost of isolation and culture. This method is suitable for large-scale umbilical cord blood mesenchymal stem cell culture and its use in the treatment of related diseases .

Claims (5)

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;

the cord blood is taken from a premature infant; the mesenchymal stem cell growth additive is from ScienCell corporation of America;

the stable growth state is that the first culture medium is used for carrying out primary culture and subculture on the mononuclear cells to the 2 nd generation, and the second culture medium is used for subsequent subculture;

the primary culture comprises the use of a firstPreparing the mononuclear cells into a single cell suspension by using a culture medium, and then, preparing the single cell suspension according to the cell density of 5 x 10⁶-2×10⁷Inoculating single nuclear cells into a cell culture bottle per mL;

during primary culture, after inoculating mononuclear cells into a cell culture bottle, changing liquid for half a day after 2 days, changing liquid for the first time for the full time after 5-7 days, and changing liquid for the full time every 3-4 days later;

the antibiotic contents in the first culture medium and the second culture medium are as follows: 100-120U/mL penicillin, 100-120. mu.g/mL streptomycin and 0.25-0.5. mu.g/mL amphotericin B.

2. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 1, wherein during the primary culture, subculture is performed until the cell fusion degree in a cell culture flask reaches 70-80%.

3. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 1, wherein the cell culture flask is coated with gelatin.

4. 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.

5. The method for culturing human umbilical cord blood mesenchymal stem cells according to claim 4, 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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