Large-scale culture reagent and culture method of hematopoietic stem cells and their application

Abstract

The present invention provides a large-scale culture reagent, a culture method and an application of hematopoietic stem cells, and belongs to the field of cell culture technology. The large-scale culture reagent of hematopoietic stem cells provided by the present invention includes 100-150ng / mL SCF, 50-75ng / mL FLT3-L, 50-100ng / mL TPO, 25-30ng / mL transferrin, 150-350μg / mL nicotinamide, 90-210μg / mL valproic acid and 400-800ng / mL UM171. Using the large-scale culture reagent and culture method provided by the present invention, the cell viability of CD34+ umbilical cord blood hematopoietic stem cells can still reach more than 95% on the 10th day of culture, and the cell proliferation multiples are more than 240 times. After 10 days of amplification, the positive ratio of CD34+ cells is still greater than 79%. It has good application prospects in the preparation of cell therapy products for the treatment of blood diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a large-scale culture reagent and culture method of hematopoietic stem cells and their application. Background Art

[0002] Hematopoietic stem cells (HSC) are adult stem cells in the blood system and are the starting cells of the hematopoietic and immune systems. Hematopoietic stem cells have the ability to self-renew for a long time and the potential to differentiate into various mature blood cells. They have the characteristics of self-renewal, multidirectional differentiation and homing. Therefore, HSC has been widely used in clinical HSC transplantation. Hematopoietic stem cell transplantation technology is a common and effective treatment for a variety of blood diseases and immune system diseases such as leukemia, lymphoma, aplastic anemia, thalassemia, and is the most promising target cell for gene therapy.

[0003] However, in the current treatment of blood-related diseases, adult hematopoietic stem cell transplantation is limited by extremely low matching probability and graft-versus-host disease, which makes it impossible to cure bone marrow and malignant hematopoietic diseases; even if umbilical cord blood hematopoietic stem cells are used, they are still limited by insufficient number and poor quality, and cannot be used in adults and children with larger body weights. Therefore, increasing the number of cells obtained by in vitro culture and increasing the proportion of functional hematopoietic stem cells among them is the primary solution to improve the bottleneck of clinical application of hematopoietic stem cells.

[0004] Therefore, effective large-scale hematopoietic stem cell culture technology is crucial to fully utilize hematopoietic stem cell resources. The existing hematopoietic stem cell proliferation and preparation methods have low proliferation efficiency and slow proliferation rate, which cannot meet the actual needs.

[0005] The study of hematopoietic stem cell expansion has been going on for more than 30 years. In the early days, people used cytokines in the blood to culture hematopoietic stem cells, which resulted in cell differentiation and weakened transplantation function. Later, people found that Wnt signaling molecules, Notch ligands, retinoic acid antagonists, etc. in the microenvironment of bone marrow hematopoietic stem cells can effectively expand CD34+ hematopoietic stem / progenitor cells. Using CHIR99021 or BIO to activate the Wnt signaling pathway maintains the transplantation ability of hematopoietic stem cells cultured in vitro; and adding DLL1, DSL1, etc. to the culture system of hematopoietic stem cells can moderately expand hematopoietic stem cells by activating Notch signals. Other studies have found that PTN secreted by bone marrow endothelial stromal cells can also slightly expand hematopoietic stem cells. Under physiological conditions, hematopoietic stem cells are under hypoxic conditions, and the oxygen stress generated by in vitro culture will damage the self-renewal and transplantation function of hematopoietic stem cells by increasing ROS levels; people have found that the addition of antioxidants and the inhibition of mTOR can offset these damages. However, the above technologies have not been able to significantly expand hematopoietic stem cells. In general, there is no clear consensus on the optimal in vitro expansion conditions for HSC. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a large-scale culture reagent, a culture method and an application of hematopoietic stem cells. The large-scale culture reagent of hematopoietic stem cells provided by the present invention includes 100-150ng / mL SCF, 50-75ng / mL FLT3-L, 50-100ng / mL TPO, 25-30ng / mL transferrin, 150-350μg / mL nicotinamide, 90-210μg / mL valproic acid and 400-800ng / mL UM171. Using the large-scale culture reagent provided by the present invention, the cell viability of CD34+ umbilical cord blood hematopoietic stem cells can still reach more than 95% on the 10th day of culture, and the cell proliferation multiples are more than 240 times. After 10 days of amplification, the positive proportion of CD34+ cells is still greater than 79%.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In one aspect, the present invention provides a large-scale culture reagent for hematopoietic stem cells, wherein the large-scale culture reagent comprises SCF, FLT3-L, TPO, transferrin, nicotinamide, valproic acid and UM171.

[0009] Specifically, the large-scale culture reagents include 100-150 ng / mL SCF, 50-75 ng / mL FLT3-L, 50-100 ng / mL TPO, 25-30 ng / mL transferrin, 150-350 μg / mL nicotinamide, 90-210 μg / mL valproic acid and 400-800 ng / mL UM171.

[0010] Preferably, the large-scale culture reagents include 125 ng / mL SCF, 60 ng / mL FLT3-L, 75 ng / mL LTPO, 25 ng / mL transferrin, 250 μg / mL nicotinamide, 150 μg / mL valproic acid and 800 ng / mL UM171.

[0011] Preferably, the mass ratio of nicotinamide to valproic acid is 5:3.

[0012] Specifically, the total mass of nicotinamide and valproic acid: the mass of UM171 = 500-1000:1.

[0013] Preferably, the total mass of nicotinamide and valproic acid: the mass of UM171=500:1.

[0014] Specifically, the hematopoietic stem cells include bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, placental hematopoietic stem cells or umbilical cord blood hematopoietic stem cells.

[0015] Preferably, the hematopoietic stem cells are umbilical cord blood hematopoietic stem cells.

[0016] Specifically, the preparation method of the large-scale culture reagent includes: adding SCF, FLT3-L, TPO, nicotinamide, transferrin, valproic acid, and UM171 to the basic culture medium.

[0017] Preferably, the basal culture medium includes RPMI1640, DEME, StemPro, IMDM, α-MEM or StemSpanSFEMII.

[0018] Further preferably, the basal culture medium is StemSpan SFEMII.

[0019] On the other hand, the present invention provides a large-scale culture method for hematopoietic stem cells, the large-scale culture method comprising: inoculating hematopoietic stem cells into the above-mentioned large-scale culture reagent for culture.

[0020] Specifically, the seeding density of the hematopoietic stem cells is 1×10 5 -1×106cells / mL.

[0021] Preferably, the seeding density of the hematopoietic stem cells is 5×10 5 cells / mL.

[0022] Specifically, the culture conditions are 36-38° C. and 3-7% CO 2 for 5-10 days.

[0023] Preferably, the culture conditions are 37° C., 5% CO 2 for 10 days.

[0024] In another aspect, the present invention provides use of the large-scale culture reagent or large-scale culture method in preparing a cell therapy product for treating blood diseases.

[0025] The technical effects achieved by the present invention are:

[0026] By using the large-scale culture reagent and method provided by the present invention, the cell viability of CD34+ umbilical cord blood hematopoietic stem cells can still reach more than 95% on the 10th day of culture, and the cell proliferation multiples are more than 240 times. After 10 days of expansion, the CD34+ cell positive ratio is still greater than 79%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the cell proliferation multiple.

[0028] Figure 2 To detect the in vitro clone formation ability. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below by way of examples. The following examples are only a part of the present invention and are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are not specifically stated, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0030] The experimental materials used in the present invention are shown in Table 1:

[0031] Table 1 Experimental materials

[0032]

[0033]

[0034] Basic Experimental Example 1 Obtaining CD34+ Umbilical Cord Blood Hematopoietic Stem Cells

[0035] 1. Obtaining umbilical cord blood mononuclear cells (PBMC):

[0036] (1) Collect 80-120 ml of umbilical cord blood using a disposable blood bag (containing anticoagulants such as sodium heparin), transfer the umbilical cord blood from the blood bag to a 500 ml culture bottle, dilute it 2-3 times with physiological saline, mix it well, and then add it dropwise to 0.4 times the volume of lymphocyte separation solution, taking care not to damage the interface;

[0037] (2) Centrifuge at 1500-2000 rpm for 20 min. The centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, the second layer is the circular milky white PBMC layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer;

[0038] (3) Carefully pipette the second ring-shaped milky white PBMC layer into another 50 mL centrifuge tube, add physiological saline, and centrifuge at 1500-2000 rpm / min for 5-10 minutes;

[0039] (4) Discard the supernatant, resuspend with physiological saline, centrifuge at 1500-2000 rpm / min for 5-10 minutes, and discard the supernatant to obtain PBMC cell pellets.

[0040] 2. Obtain CD34+ umbilical cord blood hematopoietic stem cells from the above PBMC using MACS

[0041] (1) Each umbilical cord blood PBMC was resuspended in a mixture of 50 μL human CD34+ magnetic beads, 50 μL FcR blocker, and 150 μL 0.5% BSA and incubated at 4°C for 30 min;

[0042] (2) At the same time, place the magnet and magnetic rack in a clean bench and irradiate with ultraviolet light for 30 minutes;

[0043] (3) Add 10 mL of sterile PBS and mix well, then centrifuge at 1500-2000 rpm / min for 5-10 min, discard the supernatant, and retain the PBMC pellet;

[0044] (4) Place the MACS adsorption column into the magnet, add 500 μL 0.5% BSA for rinsing, and collect the outflowing liquid with a 15 mL tube;

[0045] (5) Resuspend the PBMC pellet in step (3) with 500 μL 0.5% BSA, mix well and transfer to a MACS adsorption column until the liquid completely flows out;

[0046] (6) Wash three times with 500 μL 0.5% BSA, remove the adsorption column and place it in a 15 mL tube;

[0047] (7) Add 1 mL of 0.5% BSA and use a piston to push the liquid into a 15 mL tube. The resulting liquid contains CD34+ umbilical cord blood hematopoietic stem cells.

[0048] Example 1

[0049] A large-scale culture reagent for hematopoietic stem cells, wherein the large-scale culture reagent is a StemSpan SFEMII culture medium containing 125 ng / mL SCF, 60 ng / mL FLT3-L, 75 ng / mL TPO, 25 ng / mL transferrin, 250 μg / mL nicotinamide, 150 μg / mL valproic acid and 800 ng / mL UM171.

[0050] Example 2

[0051] A large-scale culture reagent for hematopoietic stem cells, wherein the large-scale culture reagent is a StemSpan SFEMII culture medium containing 100 ng / mL SCF, 50 ng / mL FLT3-L, 50 ng / mL TPO, 30 ng / mL transferrin, 350 μg / mL nicotinamide, 210 μg / mL valproic acid and 460 ng / mL UM171.

[0052] Example 3

[0053] A large-scale culture reagent for hematopoietic stem cells, wherein the large-scale culture reagent is a StemSpan SFEMII culture medium containing 150 ng / mL SCF, 75 ng / mL FLT3-L, 100 ng / mL TPO, 25 ng / mL transferrin, 150 μg / mL nicotinamide, 90 μg / mL valproic acid and 400 ng / mL UM171.

[0054] Comparative Example 1

[0055] The difference between Comparative Example 1 and Example 1 is that 250 μg / mL nicotinamide is not added.

[0056] Comparative Example 2

[0057] The difference between Comparative Example 2 and Example 1 is that 150 μg / mL valproic acid is not added.

[0058] Comparative Example 3

[0059] The only difference between Comparative Example 3 and Example 1 is that 150 μg / mL valproic acid and 250 μg / mL nicotinamide are not added.

[0060] Comparative Example 4

[0061] The only difference between Comparative Example 4 and Example 1 is that UM171 is replaced by SR1.

[0062] Comparative Example 5

[0063] A large-scale culture reagent for hematopoietic stem cells, wherein the large-scale culture reagent is a StemSpan SFEMII culture medium containing 125 ng / mL SCF, 60 ng / mL FLT3-L, 75 ng / mL TPO, 25 ng / mL transferrin, 400 μg / mL nicotinamide, 300 μg / mL valproic acid and 1000 ng / mL UM171.

[0064] Experimental Example 1: Cultivation and determination of CD34+ umbilical cord blood hematopoietic stem cells

[0065] The CD34+ umbilical cord blood hematopoietic stem cells prepared in Basic Experimental Example 1 were suspended and inoculated in the large-scale culture reagents of Examples 1-3 and Comparative Examples 1-5 for culture. The cell inoculation density in the 24-well plate was 5×10 5 cells / mL, and cultured in a 37°C, 5% CO2 incubator. Fresh large-scale culture reagents were added to each group every 2 days to adjust the cell density to 5×10 5 cells / mL, and hematopoietic stem cells were harvested on the 10th day of culture.

[0066] 1. Detection of cell viability and proliferation efficiency of hematopoietic stem cells

[0067] On the 0th, 5th and 10th days of culture (D0, D5 and D10), samples were taken for counting to obtain data on cell viability and cell proliferation times. The cell viability is shown in Table 2, and the cell proliferation times are shown in Table 2. Figure 1 shown.

[0068] Table 2 Cell viability (%)

[0069]

[0070] The test results show that, using the large-scale culture reagents of Examples 1-3 of the present invention, the cell viability of CD34+ umbilical cord blood hematopoietic stem cells can still reach more than 95% on the 10th day of culture, and the cell proliferation multiples are all more than 240 times.

[0071] 2. Detection of hematopoietic stem cell surface markers

[0072] On the 0th and 10th days (D0 and D10) of culture, the expression of CD34 protein was measured. 5 The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was removed and resuspended in PBS, and the fluorescently labeled CD34 antibody was incubated with the cells resuspended in PBS at 4°C for 15 minutes, and then washed twice with PBS, the supernatant was removed, and the cells were resuspended in 0.5 mL PBS. The treated cells were detected and analyzed by flow cytometry. The expression of CD34 protein is shown in Table 3.

[0073] Table 3 Expression of CD34 protein (%)

[0074]

[0075]

[0076] The results in Table 2 show that the CD34+ umbilical cord blood hematopoietic stem cells cultured with the large-scale culture reagents of Examples 1-3 of the present invention had a CD34+ cell positive ratio greater than 79% after 10 days of expansion.

[0077] Experimental Example 2 In vitro clone formation ability detection

[0078] (1) The CD34+ umbilical cord blood hematopoietic stem cells prepared in Basic Experimental Example 1 were suspended and inoculated in the large-scale culture reagent of Example 1 or StemSpan SFEMII medium (as a control) for culture.

[0079] (2) After CD34+ umbilical cord blood hematopoietic stem cells were cultured to the logarithmic growth phase, they were trypsinized, resuspended in complete culture medium (StemSpan SFEMII+10% fetal bovine serum) into a cell suspension, and counted.

[0080] (3) 700 cells / well were inoculated in each experimental group in a 6-well culture plate.

[0081] (4) Continue culturing until 14 days or until the number of cells in most individual clones is greater than 50. Change the medium every 3 days and observe the cell status.

[0082] (5) After cloning is completed, the cells are photographed under a microscope, then washed once with PBS, 1 mL of 4% paraformaldehyde is added to each well for fixation for 30-60 min, and then washed once with PBS.

[0083] (6) Add 1 mL of crystal violet stain to each well, stain the cells for 10-20 min, and wash once with PBS.

[0084] (7) Calculate the number of clones in each well.

[0085] The results of the test are as follows Figure 2 As shown, the experimental results show that the CD34+ umbilical cord blood hematopoietic stem cells cultured with the large-scale culture reagent provided by the present invention have good in vitro cloning ability.

[0086] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A reagent for large-scale culture of hematopoietic stem cells, characterized in that: The large-scale culture reagents are composed of SCF, FLT3-L, TPO, transferrin, nicotinamide, valproic acid, UM171 and StemSpan SFEM II culture medium; The large-scale culture reagents include 100-150 ng / mL SCF, 50-75 ng / mL FLT3-L, 50-100 ng / mL TPO, 25-30 ng / mL transferrin, 150-350 μg / mL nicotinamide, 90-210 μg / mL valproic acid and 400-800 ng / mL UM171.

2. The large-scale culture reagent according to claim 1, characterized in that The mass ratio of nicotinamide to valproic acid is 5:

3.

3. The large-scale culture reagent according to claim 1, characterized in that The total mass of the nicotinamide and valproic acid: the mass of UM171 = 500-1000:

1.

4. The large-scale culture reagent according to claim 1, characterized in that The hematopoietic stem cells include bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, placental hematopoietic stem cells or umbilical cord blood hematopoietic stem cells.

5. The large-scale culture reagent according to claim 1, characterized in that The preparation method of the large-scale culture reagent comprises: adding SCF, FLT3-L, TPO, nicotinamide, transferrin, valproic acid, and UM171 to StemSpan SFEMII culture medium.

6. A large-scale culture method for hematopoietic stem cells, characterized in that: The large-scale culture method comprises: inoculating hematopoietic stem cells into the large-scale culture reagent described in any one of claims 1 to 5 for culture.

7. The large-scale culture method according to claim 6, characterized in that: The seeding density of the hematopoietic stem cells is 1×10 5 -1×10 6 cells / mL.

8. Use of the large-scale culture reagent according to any one of claims 1 to 5 or the large-scale culture method according to any one of claims 6 to 7 in the preparation of a cell therapy product for treating blood diseases.

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