Culture method of hematopoietic stem cells

Abstract

The invention discloses a culture method of hematopoietic stem cells. The method comprises the following steps: firstly, providing hematopoietic stem cell sources such as umbilical cord blood, bone marrow or peripheral blood, pretreating, and removing unnecessary cell components by using enzymatic hydrolysate containing specific bacillus subtilis or bacillus licheniformis secreted protease and glycosidase; and inoculating the pretreated hematopoietic stem cells into a culture system containing a basic culture medium and a special composition. Culturing at 36.5-37.5 DEG C in an environment with 4-6% COC, and regularly detecting and regulating the proliferation multiple, the cell activity and the expression condition of the surface marker. And collecting, treating and storing after the culture reaches the standard. According to the method, the hematopoietic stem cells can be efficiently amplified, and the proliferation multiple is improved by 1.5-2 times or above compared with a traditional method; the high cell activity is maintained; the high purity is ensured, and the CD34 expression rate is improved; the culture process is precisely regulated and controlled, and a high-quality sufficient cell source is provided for hematopoietic stem cell transplantation.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical engineering, and in particular to a method for culturing hematopoietic stem cells. Background Art

[0002] Hematopoietic stem cells (HSCs) are "seed" cells in the human body that can differentiate into various blood cells and play an important role in the clinical treatment of blood system diseases. At present, HSCs are mainly derived from bone marrow, peripheral blood and placenta (mainly umbilical cord blood), but the number of HSCs from these sources is limited and they are easily polluted by the environment, affecting their quality and safety. Therefore, how to obtain a large number of safe and effective HSCs is crucial to ensure clinical application.

[0003] Hematopoietic stem cell transplantation plays an important role in the treatment of a variety of blood system diseases, immune system diseases and certain solid tumors. However, the current hematopoietic stem cell culture technology still has many limitations, which seriously restricts the development of its clinical application.

[0004] Traditional hematopoietic stem cell culture methods perform poorly in terms of cell expansion efficiency. Due to the lack of effective pretreatment methods and optimized culture systems, the proliferation multiples of hematopoietic stem cells are relatively low, which makes it difficult to meet the clinical demand for a large number of high-quality stem cells. For example, in previous routine cultures, the proliferation multiples of hematopoietic stem cells can often only be maintained at a low level, and cannot provide a sufficient number of cells for transplantation. This makes many patients unable to receive effective transplantation treatment in time due to insufficient cell numbers, or have to face the pain and risk of multiple stem cell collections. Maintenance of cell viability is also a major problem faced by traditional culture methods. During the culture process, hematopoietic stem cells are easily affected by various factors, resulting in decreased cell viability. Under normal circumstances, the cell viability of hematopoietic stem cells obtained by traditional culture methods can only reach 70%-82%, which not only affects the growth state of stem cells during in vitro culture, but also after transplantation, the survival and function of low-viability stem cells in the body will be significantly affected, thereby reducing the success rate of transplantation and the treatment effect, and increasing the risk of recurrence and the incidence of complications in patients.

[0005] In addition, traditional culture methods cannot guarantee the purity of hematopoietic stem cells. The expression rate of the cell surface marker CD34⁺ is not high, and it is easily contaminated by impurity cells, which may introduce unnecessary cell components during the transplantation process, triggering adverse reactions such as immune responses, seriously affecting the safety and effectiveness of the transplantation. At the same time, traditional culture methods lack effective means to accurately control the culture process, and are often unable to make timely and reasonable adjustments to culture conditions according to the real-time growth status of hematopoietic stem cells, resulting in poor success rate and stability of the culture, further limiting the widespread application of hematopoietic stem cell transplantation technology.

[0006] In summary, these problems existing in the existing hematopoietic stem cell culture technology urgently require a new and efficient culture method to solve them, so as to improve the proliferation multiples, cell viability, purity and precise regulation ability of hematopoietic stem cells, thereby providing a more reliable and high-quality cell source for hematopoietic stem cell transplantation and promoting its widespread application and development in clinical treatment. Summary of the invention

[0007] The object of the present invention is to provide a method for culturing hematopoietic stem cells to solve the problems raised in the above background technology.

[0008] In order to solve the above technical problems, the technical solution provided by the present invention is: a method for culturing hematopoietic stem cells, which specifically comprises the following steps: 1) Collecting hematopoietic stem cells, wherein the source of the hematopoietic stem cells is umbilical cord blood, bone marrow or peripheral blood containing nucleic acid; 2) pre-treating the hematopoietic stem cell source, including treating with a microbial enzymatic hydrolysate to remove unnecessary cell components, wherein the microbial enzymatic hydrolysate contains proteases and glycosidases secreted by microorganisms, wherein the protease activity is 10-50 U / ml, and the glycosidase activity is 5-30 U / ml; 3) inoculating the pretreated hematopoietic stem cells into a culture system comprising a basal culture medium and a special composition, wherein the basal culture medium comprises essential amino acids, vitamins, inorganic salts and a carbon source; The special composition comprises a pro-proliferation factor, an anti-apoptotic factor and a cell adhesion regulatory factor; the pro-proliferation factor is a combination of platelet-derived growth factor, fibroblast growth factor and insulin-like growth factor, and the concentrations thereof in the culture system are 2-10 ng / ml, 3-15 ng / ml and 1-8 ng / ml respectively; the anti-apoptotic factor is a Bcl-2 protein family agonist and a heat shock protein 70 agonist, and the concentrations thereof are 0.5-3 μM and 1-5 μM respectively; the cell adhesion regulatory factor is an integrin agonist and a selectin antagonist, and the concentrations thereof are 2-8 μM and 1-6 μM respectively; 4) Cultivate at 36.5-37.5°C and 4-6% CO2, replace the culture medium every 3-5 days, and supplement with fresh special composition; 5) Regularly test the proliferation multiples, cell viability and cell surface marker expression of hematopoietic stem cells during the culture process; 6) Adjusting the culture conditions or the ratio of ingredients in the special composition according to the test results; 7) When hematopoietic stem cells reach the predetermined culture indicators, they are collected, processed and preserved for hematopoietic stem cell transplantation.

[0009] As a preferred embodiment, the microorganism is Bacillus subtilis or Bacillus licheniformis.

[0010] As a preferred embodiment, the protease activity is 30 U / ml, and the glycosidase activity is 20 U / ml.

[0011] As a preferred embodiment, the concentration of platelet-derived growth factor is 6 ng / ml, the concentration of fibroblast growth factor is 10 ng / ml and the concentration of insulin-like growth factor is 5 ng / ml.

[0012] As a preferred embodiment, the concentration of the Bcl-2 protein family agonist is 2 μM and the concentration of the heat shock protein 70 agonist is 3 μM.

[0013] As a preferred embodiment, the concentration of the integrin agonist is 5 μM and the concentration of the selectin antagonist is 4 μM.

[0014] As a preferred embodiment, the cell viability detection adopts the trypan blue exclusion method, and the cell surface marker detection adopts flow cytometry.

[0015] As a preferred solution, the storage is carried out by low-temperature freezing, and the cryoprotectant is a mixture of dimethyl sulfoxide and hydroxyethyl starch, and the volume ratio of the two is 1:3-1:5.

[0016] As a preferred embodiment, the iron concentration is 0.5 μM, the zinc concentration is 0.3 μM and the selenium concentration is 0.05 μM.

[0017] The advantages of the present invention are: efficient expansion of hematopoietic stem cells: through specific microbial enzymatic pretreatment and carefully designed special compositions, the present invention can significantly increase the proliferation multiple of hematopoietic stem cells. In many embodiments, compared with the comparative examples, the proliferation multiple can be increased by more than 1.5-2 times, effectively increasing the number of hematopoietic stem cells that can be used for transplantation.

[0018] Maintaining high cell viability: Throughout the culture process, the cell viability of the hematopoietic stem cells cultured in the present invention is always maintained at a high level. As shown in the examples, the cell viability is generally between 85% and 93%, which is significantly improved compared to the 70% to 82% of the traditional method. This is beneficial to ensure the survival and function of the hematopoietic stem cells in the body after transplantation.

[0019] High-purity hematopoietic stem cells: From the perspective of the expression rate of the cell surface marker CD34⁺, the culture method of the present invention can maintain a relatively high purity of hematopoietic stem cells. In each embodiment, the expression rate of the CD34⁺ cell surface marker was increased by 10%-20% compared with the control group, thereby reducing the contamination of impurity cells and improving the safety and effectiveness of transplantation.

[0020] Precisely control the culture process: By regularly testing the cell proliferation multiples, vitality and surface marker expression, and adjusting the culture conditions or the proportion of ingredients in the special composition based on the results, it is possible to better adapt to the growth needs of hematopoietic stem cells from different sources and improve the success rate and stability of the culture.

[0021] In summary, the hematopoietic stem cell culture method of the present invention exhibits significant superiority in many aspects, provides a better quality and more sufficient cell source for hematopoietic stem cell transplantation, and has broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a bar graph of the proliferation multiples of Examples 1-8 and the comparative example.

[0023] Figure 2 It is a bar graph of cell viability of Examples 1-8 and Comparative Example.

[0024] Figure 3 It is a bar graph of CD34⁺ expression rate of Examples 1-8 and Comparative Example. DETAILED DESCRIPTION

[0025] For the purpose of the following detailed description, it should be understood that the present invention may adopt various alternative changes and step sequences, unless expressly specified to the contrary. In addition, except in any operating examples, or otherwise indicated, all numbers representing the amount of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the attached claims are approximate values ​​that vary according to the desired performance to be obtained by the present invention. At least it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0026] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0027] In addition, it should be understood that any numerical range described herein is intended to include all subranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the stated minimum value of 1 and the stated maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0028] Example 1: In this example, hematopoietic stem cells are derived from umbilical cord blood with nucleic acid. Enzyme hydrolysate secreted by Bacillus subtilis is used for pretreatment, wherein the protease activity is 10U / ml and the glycosidase activity is 5U / ml. Inoculated in a culture system of basal culture medium and special composition, the concentration of platelet-derived growth factor is 2ng / ml, the concentration of fibroblast growth factor is 3ng / ml and the concentration of insulin-like growth factor is 1ng / ml; the concentration of Bcl-2 protein family agonist is 0.5μM and the concentration of heat shock protein 70 agonist is 1μM; the concentration of integrin agonist is 2μM and the concentration of selectin antagonist is 1μM. Cultured at 36.5°C and 4% CO2, the culture medium is replaced every 3 days and fresh composition is supplemented.

[0029] After 14 days of culture, the test results showed that the proliferation rate of hematopoietic stem cells was 5 times, the cell viability was 85% (detected by trypan blue exclusion method), and the expression rate of CD34⁺ cell surface marker was 80% (detected by flow cytometry).

[0030] Example 2: Hematopoietic stem cells are taken from bone marrow with nucleic acid. The microorganism is Bacillus licheniformis, the protease activity is 30U / ml, and the glycosidase activity is 20U / ml. The concentration of platelet-derived growth factor in the proliferation-promoting factor is 6ng / ml, the concentration of fibroblast growth factor is 10ng / ml, and the concentration of insulin-like growth factor is 5ng / ml; the concentration of Bcl-2 protein family agonist in the anti-apoptotic factor is 2μM and the concentration of heat shock protein 70 agonist is 3μM; the concentration of integrin agonist in cell adhesion regulatory factor is 5μM and the concentration of selectin antagonist is 4μM. The culture environment is 37°C, 5% CO2, and the culture medium and supplement composition are replaced every 4 days.

[0031] After 16 days of culture, the proliferation multiple of hematopoietic stem cells reached 8 times, the cell viability was 90%, and the expression rate of CD34⁺ cell surface marker was 85%.

[0032] Example 3: Peripheral blood with nucleic acid is used as the source of hematopoietic stem cells. The protease activity in the Bacillus subtilis enzymatic hydrolysate is 50U / ml, and the glycosidase activity is 30U / ml. The concentration of platelet-derived growth factor, fibroblast growth factor, and insulin-like growth factor in the special composition is 10ng / ml, 15ng / ml, and 8ng / ml; the concentration of Bcl-2 protein family agonist is 3μM, and the concentration of heat shock protein 70 agonist is 5μM; the concentration of integrin agonist is 8μM, and the concentration of selectin antagonist is 6μM. Culture at 37.5℃, 6% CO2, and replace the culture medium and supplement the composition every 5 days.

[0033] After 18 days of culture, the proliferation multiple of hematopoietic stem cells was 10 times, the cell viability was 92%, and the expression rate of CD34⁺ cell surface marker was 88%.

[0034] Example 4: Umbilical cord blood with nucleic acid was used, the microorganism was Bacillus subtilis, the protease activity was 20U / ml, and the glycosidase activity was 10U / ml. The concentrations of the proliferative factors were 4ng / ml of platelet-derived growth factor, 6ng / ml of fibroblast growth factor, and 3ng / ml of insulin-like growth factor; the anti-apoptotic factors were 1μM of Bcl-2 protein family agonist and 2μM of heat shock protein 70 agonist; the cell adhesion regulatory factors were 4μM of integrin agonist and 2μM of selectin antagonist. The culture conditions were 37°C, 5% CO2, and the culture medium and supplement composition were replaced every 3.5 days.

[0035] After 15 days of culture, the proliferation multiple was 6 times, the cell viability was 88%, and the expression rate of CD34⁺ cell surface marker was 82%.

[0036] Example 5: Bone marrow-derived hematopoietic stem cells with nucleic acid, Bacillus licheniformis enzymatic solution with protease activity of 40 U / ml and glycosidase activity of 25 U / ml. The concentrations of the proliferative factors are 8 ng / ml of platelet-derived growth factor, 12 ng / ml of fibroblast growth factor and 6 ng / ml of insulin-like growth factor; the concentrations of the anti-apoptotic factors are 2.5 μM of Bcl-2 protein family agonist and 4 μM of heat shock protein 70 agonist; the concentrations of the cell adhesion regulatory factors are 6 μM of integrin agonist and 5 μM of selectin antagonist. The cells were cultured at 36.8°C and 4.5% CO2, and the relevant operations were performed every 4.5 days.

[0037] After 17 days of culture, the hematopoietic stem cells proliferated 9 times, the cell viability was 91%, and the expression rate of CD34⁺ cell surface marker was 86%.

[0038] Example 6: Peripheral blood hematopoietic stem cells with nucleic acid, protease activity of 35U / ml and glycosidase activity of 15U / ml in Bacillus subtilis enzymatic hydrolysate. Platelet-derived growth factor 7ng / ml, fibroblast growth factor 9ng / ml and insulin-like growth factor 4ng / ml in the special composition; Bcl-2 protein family agonist 1.5μM and heat shock protein 70 agonist 3.5μM; integrin agonist 5μM and selectin antagonist 3μM. The culture temperature was 37.2℃, CO2 concentration was 5.5%, and the culture medium was replaced and supplemented every 4 days.

[0039] After 16 days of culture, the proliferation rate was 7 times, the cell viability was 89%, and the expression rate of CD34⁺ cell surface marker was 83%.

[0040] Example 7: Umbilical cord blood with nucleic acid, Bacillus licheniformis enzymatic solution with protease activity of 15U / ml and glycosidase activity of 8U / ml. Proliferation factor concentrations: platelet-derived growth factor 3ng / ml, fibroblast growth factor 5ng / ml and insulin-like growth factor 2ng / ml; anti-apoptotic factor concentrations: Bcl-2 protein family agonist 0.8μM and heat shock protein 70 agonist 1.5μM; cell adhesion regulatory factor concentrations: integrin agonist 3μM and selectin antagonist 1.5μM. Cultured at 36.6°C, 4.2% CO2, with culture medium and supplement composition replaced every 3.2 days.

[0041] After 14 days of culture, the proliferation rate was 5 times, the cell viability was 86%, and the expression rate of CD34⁺ cell surface marker was 81%.

[0042] Example 8: Bone marrow hematopoietic stem cells with nucleic acid, Bacillus subtilis enzymatic solution protease activity 45U / ml, glycosidase activity 28U / ml. Proliferation factor concentrations platelet-derived growth factor 9ng / ml, fibroblast growth factor 14ng / ml and insulin-like growth factor 7ng / ml; anti-apoptotic factor concentrations Bcl-2 protein family agonist 2.8μM and heat shock protein 70 agonist 4.5μM; cell adhesion regulatory factor concentrations integrin agonist 7μM and selectin antagonist 5.5μM. Cultured at 37.4℃, 5.8% CO2 environment, operated every 4.8 days.

[0043] After 18 days of culture, the hematopoietic stem cells proliferated 11 times, the cell viability was 93%, and the expression rate of CD34⁺ cell surface marker was 90%.

[0044] Comparative Example: The source of hematopoietic stem cells is bone marrow (partially the same as the source in Examples 2, 5, and 8 for comparison purposes), and no enzymatic hydrolysate secreted by Bacillus subtilis or Bacillus licheniformis is used for pretreatment.

[0045] No special composition is added to the basic culture medium, that is, it does not contain platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor, Bcl-2 protein family agonist, heat shock protein 70 agonist, integrin agonist and selectin antagonist.

[0046] The culture environment was set at 37°C and 5% CO2, and the basal medium was replaced every 4 days (without special composition supplementation).

[0047] After 16 days of culture (the same culture time as in Examples 2 and 6 for comparison), the test results showed that the proliferation multiple of hematopoietic stem cells was 4 times, the cell viability was 65% (detected by trypan blue exclusion method), and the expression rate of CD34⁺ cell surface marker was 55% (detected by flow cytometry).

[0048] The comparison table of the embodiment of the present invention and the prior art is shown in Table 1 Table 1 Grouping Stem cell source Microbial species Protease activity (U / ml) Glycosidase activity (U / ml) Multiplication times Cell viability% CD34⁺ expression rate% Example 1 Umbilical cord blood Bacillus subtilis 10 5 5 85 80 Example 2 marrow Bacillus licheniformis 30 20 8 90 85 Example 3 peripheral blood Bacillus subtilis 50 30 10 92 88 Example 4 Umbilical cord blood Bacillus subtilis 20 10 6 88 82 Example 5 marrow Bacillus licheniformis 40 25 9 91 86 Example 6 peripheral blood Bacillus subtilis 35 15 7 89 83 Example 7 Umbilical cord blood Bacillus licheniformis 15 8 5 86 81 Example 8 marrow Bacillus subtilis 45 28 11 93 90 Comparative Example marrow none — — 4 65 55

[0049] Advantages: Increased proliferation multiples: Compared with the comparative examples, the proliferation multiples of hematopoietic stem cells in all the examples were significantly increased, up to 11 times (Example 8), while the comparative example was only 4 times.

[0050] Enhanced cell viability: The cell viability in the examples is generally higher than that in the comparative examples, up to 93% (Example 8), while that in the comparative example is 65%.

[0051] Improving CD34⁺ expression rate: The expression rate of CD34⁺ cell surface markers in the embodiments is generally higher than that in the comparison examples, and can reach up to 90% (Example 8), while the comparison example is 55%.

[0052] Optimizing culture conditions: The proliferation efficiency and quality of hematopoietic stem cells can be significantly improved by adjusting the type of microorganisms, the activity of proteases and glycosidases in the enzymatic hydrolysate, and the culture conditions (such as temperature and CO2 concentration).

[0053] Application of special compositions: The special compositions added in the examples (including pro-proliferation factors, anti-apoptosis factors and cell adhesion regulatory factors) play an important role in improving cell proliferation and maintaining cell characteristics.

[0054] Improved clinical application potential: Due to the increased proliferation multiples, viability, and surface marker expression rate of the hematopoietic stem cells in the examples, the potential of these cells in clinical applications also increases accordingly, which may help improve treatment efficiency and reduce side effects.

[0055] The following is a detailed introduction to the advantages of this hematopoietic stem cell culture method: 1. Wide sources and efficient pretreatment: The source of hematopoietic stem cells can be umbilical cord blood, bone marrow or peripheral blood, which provides multiple ways to obtain hematopoietic stem cells of sufficient quantity and quality, increases the availability of raw materials, and can adapt to different clinical needs and donor conditions.

[0056] Pretreatment with a microbial enzymatic solution composed of proteases and glycosidases secreted by microorganisms (such as Bacillus subtilis or Bacillus licheniformis) can accurately remove unnecessary cell components. The protease activity is 10-50U / ml (preferably 30U / ml) and the glycosidase activity is 5-30U / ml (preferably 20U / ml), which can effectively improve the purity and efficiency of subsequent culture, reduce the interference of impurities on hematopoietic stem cell culture, and lay a good foundation for subsequent efficient proliferation.

[0057] 2. The culture system is optimized and the ingredients are precise: The basal culture medium contains essential amino acids, vitamins, inorganic salts and carbon sources, which provide basic nutritional support for hematopoietic stem cells and ensure the normal metabolism and growth needs of cells.

[0058] The proliferation-promoting factors (platelet-derived growth factor, fibroblast growth factor and insulin-like growth factor) in the special composition act synergistically within a specific concentration range (2-10ng / ml, 3-15ng / ml and 1-8ng / ml, preferably 6ng / ml, 10ng / ml and 5ng / ml, respectively), which can significantly promote the proliferation of hematopoietic stem cells, accelerate the expansion of cell number, and meet the cell number requirements for clinical transplantation.

[0059] Anti-apoptotic factors (Bcl-2 protein family agonists and heat shock protein 70 agonists) can effectively reduce cell apoptosis, increase cell survival rate, ensure stable growth of cell number during culture and maintain cell activity when the concentrations are 0.5-3μM and 1-5μM (preferably 2μM and 3μM), respectively.

[0060] The concentrations of cell adhesion regulatory factors (integrin agonists and selectin antagonists) are 2-8μM and 1-6μM (preferably 5μM and 4μM), respectively, which can accurately regulate the adhesion properties of cells, so that hematopoietic stem cells are in a suitable adhesion state in the culture system, which is beneficial to cell proliferation, differentiation and material exchange with the surrounding environment.

[0061] 3. The culture conditions are appropriate and the monitoring and adjustment are scientific: cultured in an environment of 36.5-37.5℃ and 4-6% CO2, which simulates the physiological environment of the human body and is conducive to the normal growth and function maintenance of hematopoietic stem cells.

[0062] Replacing the culture medium and supplementing it with fresh special compositions every 3-5 days can not only replenish the nutrients and regulatory factors consumed by the cells in a timely manner, but also avoid stress damage to the cells caused by a one-time replacement of the culture medium, thereby ensuring the stability and continuity of the culture process.

[0063] During the culture process, trypan blue exclusion method is used regularly to detect cell viability and flow cytometry is used to detect the expression of cell surface markers, which can accurately and timely understand the key information such as the growth status, proliferation ability and differentiation potential of hematopoietic stem cells. According to the test results, the culture conditions or the proportion of the components of the special composition are adjusted to achieve dynamic optimization and precise regulation of the culture process, ensuring that the hematopoietic stem cells are always in the best culture state, and improving the success rate of culture and cell quality.

[0064] 4. The preservation method is reliable: low-temperature freezing is used, and the cryoprotectant is a mixture of dimethyl sulfoxide and hydroxyethyl starch (volume ratio of 1:3-1:5). This preservation method can effectively protect the activity and function of hematopoietic stem cells in a low-temperature environment, reduce the damage of ice crystals to cells, and ensure that the cells can still be used for hematopoietic stem cell transplantation after long-term storage, thereby improving the utilization efficiency of cell resources and storage safety, and providing convenience and guarantee for clinical applications.

[0065] In summary, this hematopoietic stem cell culture method has been systematically and scientifically designed and optimized from stem cell source, pretreatment, culture system construction, culture condition control, process monitoring to final preservation. It has significant advantages such as high efficiency, stability and controllability. It has broad application prospects and important clinical value in the field of hematopoietic stem cell transplantation.

[0066] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for culturing hematopoietic stem cells, characterized in that: The specific steps include: 1) Collecting hematopoietic stem cells, wherein the source of the hematopoietic stem cells is umbilical cord blood, bone marrow or peripheral blood containing nucleic acid; 2) pre-treating the hematopoietic stem cells, including treating with a microbial enzymatic hydrolysate to remove unnecessary cell components, wherein the microbial enzymatic hydrolysate contains proteases and glycosidases secreted by the microorganisms, wherein the protease activity is 10-50 U / ml, and the glycosidase activity is 5-30 U / ml; 3) Inoculating the pretreated hematopoietic stem cells into a culture system comprising a basal culture medium and a special composition, wherein the basal culture medium comprises essential amino acids, vitamins, inorganic salts and a carbon source; the special composition comprises a pro-proliferation factor, an anti-apoptotic factor and a cell adhesion regulatory factor; the pro-proliferation factor is a combination of platelet-derived growth factor, fibroblast growth factor and insulin-like growth factor, and the concentrations thereof in the culture system are 2-10 ng / ml, 3-15 ng / ml and 1-8 ng / ml, respectively; the anti-apoptotic factor is a Bcl-2 protein family agonist and a heat shock protein 70 agonist, and the concentrations thereof are 0.5-3 μM and 1-5 μM, respectively; the cell adhesion regulatory factor is an integrin agonist and a selectin antagonist, and the concentrations thereof are 2-8 μM and 1-6 μM, respectively; 4) Cultivate at 36.5-37.5°C and 4-6% CO2, replace part of the culture medium every 3-5 days, and supplement with fresh special composition; 5) Regularly test the proliferation multiples, cell viability and cell surface marker expression of hematopoietic stem cells during the culture process; 6) Adjusting the culture conditions or the ratio of ingredients in the special composition according to the test results; 7) When hematopoietic stem cells reach the predetermined culture indicators, they are collected, processed and preserved for hematopoietic stem cell transplantation.

2. The method for culturing hematopoietic stem cells according to claim 1, characterized in that: The microorganism is Bacillus subtilis or Bacillus licheniformis.

3. The method for culturing hematopoietic stem cells according to claim 2, characterized in that: The protease activity is 30 U / ml, and the glycosidase activity is 20 U / ml.

4. The method for culturing hematopoietic stem cells according to claim 3, characterized in that: The concentration of platelet-derived growth factor is 6 ng / ml, the concentration of fibroblast growth factor is 10 ng / ml and the concentration of insulin-like growth factor is 5 ng / ml.

5. The method for culturing hematopoietic stem cells according to claim 4, characterized in that: The concentration of the Bcl-2 protein family agonist was 2 μM and the concentration of the heat shock protein 70 agonist was 3 μM.

6. The method for culturing hematopoietic stem cells according to claim 5, characterized in that: The integrin agonist concentration was 5 μM and the selectin antagonist concentration was 4 μM.

7. The method for culturing hematopoietic stem cells according to claim 6, characterized in that: The cell viability detection adopts the trypan blue exclusion method, and the cell surface marker detection adopts flow cytometry.

8. A method for culturing hematopoietic stem cells according to any one of claims 1 to 7, characterized in that: The storage is carried out by low-temperature freezing, and the cryoprotectant is a mixture of dimethyl sulfoxide and hydroxyethyl starch, and the volume ratio of the two is 1:3-1:

5.

9. A method for culturing hematopoietic stem cells according to any one of claims 1 to 7, characterized in that: The iron concentration was 0.5 μM, the zinc concentration was 0.3 μM, and the selenium concentration was 0.05 μM.

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