阅读说明：本技术 一种脐带血间充质干细胞无血清培养基、培养方法及其应用 (Serum-free medium for umbilical cord blood mesenchymal stem cells, culture method and application thereof ) 是由 孙博 于 2021-08-11 设计创作，主要内容包括：本发明公开了CDCM培养基及其应用,所述CDCM培养基包括所述CDCM培养基包括α-MEM培养基、胰岛素、硒元素、L-抗坏血酸、b-FGF、EGF、TGF-β、氢化可的松、IGF、肝素、NaHCO-(3)和转运蛋白。本发明还公开了脐带血间充质干细胞的培养方法。本发明使用了化学成分确定的培养基对细胞进行培养。该化学成份确定的培养基能够稳定的维持干细胞生长,实现脐带血间充质干细胞的大量扩增。本发明优化培养皿包被蛋白基质材料,有效支持了脐带血间充质干细胞在CDCM中的生长。本发明最显著的优点是,解决了常规用一定浓度的胎牛血清培养带来的各种隐患或自体血浆带来的规模化扩增受限等问题,对细胞治疗领域意义重大。(The CDCM culture medium comprises an alpha-MEM culture medium, insulin, selenium element, L-ascorbic acid, b-FGF, EGF, TGF-beta, hydrocortisone, IGF, heparin and NaHCO 3 And a transporter protein. The invention also discloses a culture method of the umbilical cord blood mesenchymal stem cells. The present invention uses chemically defined media to culture cells. The culture medium with determined chemical components can stably maintain the growth of stem cells and realize the large-scale amplification of the umbilical cord blood mesenchymal stem cells. The invention optimizes the protein matrix material coated on the culture dish, and effectively supports the growth of the umbilical cord blood mesenchymal stem cells in the CDCM. The most obvious advantage of the invention is that various problems caused by conventional culture with fetal calf serum with certain concentration are solvedHidden troubles or the problem of limited scale amplification brought by autologous plasma and the like, and has great significance in the field of cell therapy.)

1. A CDCM culture medium, characterized in that the CDCM culture medium comprises an alpha-MEM culture medium and further comprises15~25mg L⁻¹Insulin, 10-20 mu g L⁻¹Selenium element, 55-75 mg L⁻¹L-ascorbic acid, 5 to 25 mu g L^{− 1}b-FGF、5~25μg L⁻¹EGF、1~5μg L⁻¹TGF-β、200~500μg L⁻¹Hydrocortisone and 10-30 mu g L⁻¹IGF, 15-30 mg/L heparin, 450-500 mg L⁻¹ NaHCO₃And 250-400 mg L⁻¹A transporter protein.

2. Use of the CDCM medium of claim 1 in the culture of umbilical cord blood mesenchymal stem cells.

3. A culture method of umbilical cord blood mesenchymal stem cells is characterized by comprising the following steps:

1) collecting an umbilical cord blood sample;

2) isolation of mononuclear cell components in cord blood: subjecting the umbilical cord blood samples obtained in the step 1) to heparin anticoagulation, hydroxymethyl cellulose cracking red blood cells, obtaining mononuclear cell components through density gradient centrifugation, and then inoculating the mononuclear cell components to a culture dish coated with protein matrix components;

3) separating the mesenchymal stem cells: adding the CDCM culture medium of claim 1 into the culture dish of step 2) for culture, removing the non-adherent cells in time and replacing the culture medium;

4) culturing and amplifying the mesenchymal stem cells: continuing to culture the CDCM medium of claim 1 with every 5-8 days of fluid change until the cells are 80-95% confluent; the cells are digested with pancreatin, inoculated on a common culture plate, continuously cultured by using the CDCM culture medium of claim 1, and changed once every 2-4 days until fusion, and then subcultured next, so that the mesenchymal stem cells are expanded.

4. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 3, wherein the umbilical cord blood sample is a hospital collected umbilical cord blood sample or a human umbilical cord blood sample obtained from an umbilical cord blood bank, or an umbilical cord blood sample of a normal or premature fetus obtained under aseptic conditions.

5. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 3, wherein the protein matrix component in step 2) comprises gelatin, laminin, fibronectin in a volume ratio of 1-5: 1-5: 0.5-2: 0.5-2 mixing.

6. The method for culturing mesenchymal stem cells of umbilical cord blood according to claim 3, wherein the step 2) comprises the following steps: firstly, diluting anticoagulated umbilical cord blood by using an alpha-MEM culture medium with the same volume, mixing with 4-6g/L methylcellulose, standing for 20-40 minutes, settling erythrocytes, sucking supernatant, centrifuging, preparing single cell suspension by using PBS (phosphate buffer solution), superposing the single cell suspension on lymphocyte separation liquid with the relative density of 1-1.1, centrifuging for 15-30 minutes at 3000 r/min at 2000-1 r/min, taking an interface layer, adding PBS to prepare mononuclear cell suspension, and centrifuging and washing.

7. Use of the culture medium of claim 1, umbilical cord blood mesenchymal stem cells cultured by the method of any one of claims 3-6 in the preparation of blood glucose lowering medicines.

8. The use of the culture medium according to claim 1 and umbilical cord blood mesenchymal stem cells obtained by the culture according to any one of claims 3 to 6 in the preparation of drugs for treating diabetes.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a serum-free culture medium for umbilical cord blood mesenchymal stem cells, a culture method and application thereof.

Background

Stem cell therapy has evolved dramatically in recent years and has gained widespread acceptance as an alternative medical solution. However, most experiments show that the homing capacity and the differentiation capacity of the MSCs are weak, and the MSCs have the problems of unobvious effect on diseases, large difference of individual curative effect and the like. The reasons for the above results are mainly: first, insufficient numbers of stem cells are transplanted; second, even if a sufficient amount of stem cells are obtained, the stem cells may not function normally when loaded with an intragenic mutation or an extragenic modification during the in vitro culture amplification process; third, the in vivo environment of the recipient is unable to support homing and engraftment of the transplanted stem cells, impairing their function, or accelerating their senescence. Therefore, to achieve the desired therapeutic effect of stem cell transplantation, two conditions need to be satisfied: a sufficient number of well-functioning adult stem cells and a recipient in vivo microenvironment sufficient to support the function of the transplanted stem cells. In both of the above conditions, it is crucial to obtain high quality adult stem cells in a young state in a large amount.

The collection of somatic stem cells should be done from the youngest body tissues as much as possible. This is the basic guarantee for separating and obtaining high quality stem cells in young state. The youngest body is, of course, the fetus that has just been delivered. At present, there are well-established techniques for extracting mesenchymal stem cells from tissues of fetus, such as umbilical cord, cord blood, placenta, amnion, etc. Wherein the umbilical blood mesenchymal stem cells have special advantages. The concrete points are as follows: 1) compared with mesenchymal stem cells from other sources, the umbilical blood mesenchymal stem cells are the youngest cells, so that the umbilical blood mesenchymal stem cells have the strongest dryness, stronger homogeneity and higher quality controllability, and are more suitable for being used as cell medicines. 2) The umbilical cord blood mesenchymal stem cells are derived from neonatal tissues, and compared with other mesenchymal stem cells derived from adult tissues, the umbilical cord blood mesenchymal stem cells have weaker immune prototypes, stronger adaptability and better stem cell activity and immunoregulation capability. 3) The umbilical cord blood mesenchymal stem cells can be widely applied to hyperimmune diseases such as dermatitis, arthritis and cytokine storm (such as inflammatory storm caused by disease deterioration of severe patients with new coronary pneumonia) and vascular necrosis diseases such as diabetic foot and chilblain 4). Our own research team has conducted more intensive research from both the point of view of deep sequencing of small RNAs and secretion profiles of extracellular factors.

However, the process of separating and culturing umbilical cord blood mesenchymal stem cells still needs to use fetal bovine serum (CN201210437524.8 a method for efficiently separating and amplifying human umbilical cord blood mesenchymal stem cells) or autologous umbilical cord blood platelet-rich plasma (202011578876.6 a method for culturing umbilical cord blood mesenchymal stem cells), which brings a potential safety hazard to the final application of umbilical cord blood mesenchymal stem cells, and is limited by unstable batch quality of fetal bovine serum or limited collection volume of autologous plasma, and mass preparation with controllable quality is difficult to perform.

The culture medium limited by chemical components can be produced and prepared in a standardized way, for example, the culture medium which is limited by the chemical components of a special formula and is suitable for the umbilical cord blood mesenchymal stem cells can be used, so that large-scale amplification is realized, the problems that serum quality batches are unstable, external risk factors are introduced and the like in the traditional method can be solved, the umbilical cord blood mesenchymal stem cells with controllable quality, uniformity and stability can be produced, the advantages of the umbilical cord blood mesenchymal stem cells are fully applied, the treatment of intractable diseases which are the most main factors influencing the human life can be greatly promoted, and great social and economic values are generated.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a serum-free culture medium (CDCM) for umbilical cord blood mesenchymal stem cells.

The technical problem to be solved by the invention is to provide a culture method of umbilical cord blood mesenchymal stem cells.

The invention finally solves the technical problem of providing the culture medium and the application of the umbilical cord blood mesenchymal stem cells obtained by the culture of the method in preparing the medicines for reducing blood sugar.

The technical scheme is as follows: the CDCM culture medium comprises an alpha-MEM culture medium and 15-25 mg L of the CDCM culture medium calculated by taking the alpha-MEM culture medium as a reference^-1Insulin, 10-20 mu g L^-1Selenium element, 55-75 mg L^-1L-ascorbic acid, 5 to 25 mu g L^-1b-FGF、5～25μg L^-1EGF、1～5μg L^-1TGF-β、200～500μg L^-1Hydrocortisone, 10～30μg L^-1IGF, 15-30 mg/L heparin, 450-500 mg L^-1NaHCO₃And 250-400 mg L^-1A transporter protein.

The invention also provides a culture method of the umbilical cord blood mesenchymal stem cells, which comprises the following steps:

1) collecting an umbilical cord blood sample;

2) isolation of mononuclear cell components in cord blood: subjecting the umbilical cord blood samples obtained in the step 1) to heparin anticoagulation, hydroxymethyl cellulose cracking red blood cells, obtaining mononuclear cell components through density gradient centrifugation, and then inoculating the mononuclear cell components to a culture dish coated with protein matrix components;

3) separating the mesenchymal stem cells: adding the CDCM culture medium into the culture dish in the step 2) for culture, removing nonadherent cells in time and replacing the culture medium;

4) culturing and amplifying the mesenchymal stem cells: continuing to culture with the CDCM medium, and changing the culture medium every 5-8 days until the cells are 80-95% fused; digesting the cells by pancreatin, inoculating the cells to a common culture plate, continuously culturing by using the CDCM culture medium, changing the culture solution once in 2-4 days until the cells are fused, and carrying out next passage to realize the amplification of the mesenchymal stem cells.

Wherein, the cord blood sample is a cord blood sample collected in a hospital or a human cord blood sample obtained from a cord blood bank, or a cord blood sample of a normal or premature fetus obtained under an aseptic condition.

Wherein the protein matrix component in the step 2) comprises gelatin protein, laminin, accessory fibronectin and fibronectin which are mixed according to the optimal volume ratio of 1-5: 0.5-2.

Wherein, the step 2) specifically comprises the following processing steps: firstly, diluting anticoagulated umbilical cord blood by using an alpha-MEM culture medium with the same volume, mixing with 4-6g/L methylcellulose, standing for 20-40 minutes, settling erythrocytes, sucking supernatant, centrifuging, preparing single cell suspension by using PBS (phosphate buffer solution), superposing the single cell suspension on lymphocyte separation liquid with the relative density of 1-1.1, centrifuging for 15-30 minutes at 3000 r/min at 2000-1 r/min, taking an interface layer, adding PBS to prepare mononuclear cell suspension, and centrifuging and washing.

The invention also comprises the application of the culture medium and the umbilical cord blood mesenchymal stem cells obtained by the culture method in the preparation of the blood sugar reducing medicine.

The invention also discloses the application of the culture medium and the umbilical cord blood mesenchymal stem cells obtained by the culture of the method in the preparation of drugs for treating diabetes.

Has the advantages that: compared with the prior art, the invention has the following advantages: the present invention uses chemically defined media to culture cells. The culture with determined chemical components can stably maintain the growth of stem cells and realize the large-scale amplification of the umbilical cord blood mesenchymal stem cells. Meanwhile, the invention further optimizes the culture dish coating protein matrix material on the basis of the previous patent (CN201210437524.8, a method for efficiently separating and amplifying the umbilical cord blood mesenchymal stem cells) and effectively supports the growth of the umbilical cord blood mesenchymal stem cells in CDCM. The invention has the most obvious beneficial effect of solving the problems of various hidden troubles caused by the conventional culture of fetal calf serum with certain concentration or the limitation of large-scale amplification caused by autologous plasma and the like in the prior art, and has great significance in the field of cell therapy.

Drawings

FIG. 1 is a result of culturing mesenchymal stem cells of umbilical cord blood for 20 days according to the present invention;

FIG. 2 shows the cell morphology obtained in three culture systems;

FIG. 3 is the blood glucose concentration change of diabetic mice after the injection of umbilical cord blood mesenchymal stem cells.

Detailed Description

The alpha-MEM medium of the present invention is a conventional commercially available product.

EXAMPLE 1 preparation of culture Medium CDCM-1 and coated protein matrix

The invention provides a culture medium CDCM with limited chemical composition and a protein matrix coated culture plate matched with the CDCM. The CDCM medium comprises the following components: alpha-MEM Medium and additional Components, the amount of the additional components added was 19.4mg L based on the volume of the alpha-MEM Medium^-1Insulin, 14 μ g L^-1Selenium element,64mg L^-1L-ascorbic acid, 10. mu. g L^-1b-FGF、10μg L^-1EGF、2μg L^-1TGF- β、362μg L^-1Hydrocortisone, 20 μ g L^-1IGF, 22.5mg/L heparin, 543mg L^-1NaHCO₃And 310.7mg L^-1Transferrin (merck, usa, T3309);

the coated protein matrix is prepared by mixing gelatin protein, laminin, accessory fiber connexin and fibronectin at the same concentration according to the volume ratio of 3: 1: 0.5.

EXAMPLE 2 preparation of the culture Medium CDCM-2 and the corresponding coated protein matrix

The invention provides a culture medium CDCM with limited chemical composition and a protein matrix coated culture plate matched with the CDCM. Within the scope of the present invention, the specific concentration of each component of the culture medium is adjusted to obtain CDCM-2: alpha-MEM Medium and additional Components in an amount of 25mg L based on the amount (by volume) of the alpha-MEM Medium^-1Insulin, 20 μ g L^-1Elemental selenium, 55mg L^-1L-ascorbic acid, 25ug L^-1b-FGF、25μg L^-1EGF、5μg L^-1TGF-β、200μg L^-1Hydrocortisone, 10ug L^-1IGF、30mg L^-1Heparin, 500mg L^-1NaHCO₃And 250L^{- 1}Transferrin; the coated protein matrix is prepared by mixing gelatin protein, laminin, accessory fibronectin and fibronectin at the same concentration according to the preferred volume ratio of 1: 5: 2: 0.5.

EXAMPLE 3 preparation of the culture Medium CDCM-3 and the corresponding coated protein matrix

The invention provides a culture medium CDCM with limited chemical composition and a protein matrix coated culture plate matched with the CDCM. Within the scope of the present invention, the specific concentration of each component of the culture medium is adjusted to obtain CDCM-2: alpha-MEM Medium and additional Components, the amount of the additional components added was 15mg L based on the alpha-MEM Medium^-1Insulin, 10 μ g L^-1Elemental selenium, 75mg L^-1L-ascorbic acid, 5ug L^-1b-FGF、5μg L^-1EGF、1μg L^-1TGF-β、500μg L^-1Hydrocortisone, 30ug L^-1IGF、15mg L^-1Heparin, 450mg L^-1NaHCO3 and 400mg L^{- 1}Transferrin; the coated protein matrix is prepared by mixing gelatin protein, laminin, accessory fibronectin and fibronectin at the same concentration according to the preferred volume ratio of 5: 1: 0.5: 2.

Example 4 preparation of umbilical cord blood mesenchymal stem cells

Obtaining umbilical cord blood of normal or premature fetus under aseptic condition, 25-200ml, heparin anticoagulation. The separation and purification process of the umbilical cord blood mesenchymal stem cells is carried out within 24 hours after delivery.

The anticoagulated cord blood was diluted with the same volume of α -MEM, mixed with 5g/L of methylcellulose at a ratio of 4: 1, allowed to stand for 30 minutes, and the erythrocytes were settled. The supernatant was aspirated, centrifuged, and then single-cell suspension was prepared with PBS, and the supernatant was superimposed on a lymphocyte separation medium having a relative density of 1.077, centrifuged at 2500rpm for 20min, and a single-cell suspension was prepared by adding PBS to the interface layer, and the interface layer was centrifuged and washed to culture the cells in CDCM-1 prepared in example 1, CDCM-2 prepared in example 2, and CDCM-3 prepared in example 3, respectively. Cells were added to the protein matrix coated cell culture plates. A cell. When the cells reached 90% confluence, passages were performed. At the time of passage, a common culture dish was used, and expansion of cells was performed in the above-mentioned CDCM medium.

Coating cell culture plate: gelatin protein, laminin, fibronectin and laminin were diluted to 2mg/ml respectively according to the instructions and mixed according to the volume ratio of examples 1 to 3, and then transferred to a cell culture plate and left at room temperature for 1 hour. Can be stored at 4 deg.C for 3 months in sterile environment.

Separating and amplifying umbilical cord blood mesenchymal stem cells: mixing umbilical cord blood collected under sterile condition with cells at a volume ratio of 1: 1 in alpha-MEM, diluting, mixing with 5g/L methylcellulose at a ratio of 4: 1, standing for 30 min, and settling erythrocytes. The supernatant was aspirated, centrifuged, and then single-cell suspension was prepared with PBS, and the single-cell suspension was added to a lymphocyte separation medium Ficoll-Hypaque (Sigma, USA) with a relative density of 1.077, centrifuged at 2500rpm for 20min, and the interface layer was removed, and PBS was added to prepare single-cell suspension, and centrifuged and washed.

Since Ficoll-Hypaque has a specific gravity of 1.077g/ml, which is heavier than monocytes but lighter than erythrocytes, monocytes can be separated from residual erythrocytes. The collection boundary layer can collect relatively pure mononuclear cells.

The obtained mononuclear cells were diluted with PBS, centrifuged at 2000rpm for 10min, the supernatant removed, and fresh PBS was added for dispersion and dilution. After another wash under the same conditions, cells settled on the bottom of the centrifuge tube were visualized.

Subsequently, the collected cells were uniformly scattered using the CDCM-1 configured in example 1, the CDCM-2 configured in example 2, or the CDCM-3 configured in example 3. After cells were scattered, they were inoculated on coated cell culture plates and the medium was changed 7 days later. The plates were washed to remove non-adherent cells. The culture was continued using umbilical cord blood mesenchymal stem cell medium supplemented with 10% fetal bovine serum, with a change every 7 days until the cells were nearly 90% confluent.

90% of fused cells were digested with trypsin, inoculated on a common plate, cultured using CDCM-1, CDCM-2 or CDMC-3 as in the case of prozone culture, and the medium was changed once every 3 days. Until confluency and next passage until passage 25. The cell doubling times for the three culture systems are compared in FIG. 1. As can be seen, the cell proliferation reaches 40 striking generations under the CDCM-1 culture environment, and the obvious prolongation of the doubling time appears, while the doubling time reaches 82 hours at the 30 th generation of CDCM-2, and the aging signs appear; the CDCM-3 culture environment grew slowly as the cells passed through passage 25 and aged rapidly. The morphology of the cells obtained in the third generation of the three culture systems is shown in FIG. 2. As shown in the figure, the CDMC-1, CDMC-2 and CDMC-3 systems can maintain the state of cells well when cultured to the third generation.

Example 5 characterization of cell surface antigens from the following Table of cultured mesenchymal Stem

To characterize the mesenchymal stem cell surface antigens obtained in the above three culture systems of example 4, the cell surface markers CD34, CD45, CD3, CD73, CD105, CD9 were analyzed using FACs. The results are shown in Table 1.

TABLE 1

Table 1 demonstrates that, for the stem cells isolated and cultured in the present invention, CD34, CD45, and CD3 are expressed low, and CD73, CD105, and CD90 are expressed high. This result indicates that the cells isolated and expanded by the present invention are mesenchymal stem cells.

Example 6

Male non-obese diabetic (NOD) mice of 20 weeks of age were used, weighing 24-27 g. Feeding in 12 hours of light and 12 hours of dark cycle. In carrying out the experiment, NOD mice were divided into two groups of 6-8 mice each. After the spontaneous increase in blood glucose to 750-⁶Umbilical cord blood-derived mesenchymal stem cells. After 2 weeks a second injection was performed, i.e. the splenic artery was injected with 1X 10 suspended in 0.1ml of physiological saline⁶Umbilical cord blood-derived mesenchymal stem cells. The control group was subjected to the same procedure, but injected with physiological saline only (sham group). After the second injection, blood was collected from the orbit every three days, and the blood glucose level was recorded using a blood glucose meter to observe the blood glucose recovery. Blood glucose changes as shown in fig. 3, the blood glucose level of the cell-treated mouse diabetes model was already significantly lower than that of the control group injected with physiological saline alone at day 21 after the injection of the umbilical cord blood-derived mesenchymal stem cells prepared in example 4. 30 days after cell treatment, blood glucose levels in the diabetic mouse model had decreased from 791mg/dl at day zero to 482 mg/dl; the physiological saline injected control group was increased from 809mg/dl to 880mg/dl on day zero, demonstrating that the umbilical cord blood-derived mesenchymal stem cells prepared in example 4 have the ability to become a cell-like drug that lowers the blood glucose level of diabetes, thus demonstrating the present invention.