Hematopoietic stem cell in-vitro amplification culture system and hematopoietic stem cell in-vitro amplification method thereof

Abstract

The invention relates to the technical field of cell culture, in particular to a hematopoietic stem cell in-vitro amplification culture system and an in-vitro amplification method of hematopoietic stem cells. According to the method, step-by-step culture is carried out through different culture media, so that more precise regulation and control on the time sequence of the in-vitro life cycle of the HSCs is realized: the first-step activation starting culture is carried out through the first culture medium, the second-step multiplication culture is carried out through the second culture medium, and the natural process of in-vivo HSCs behaviors is simulated through dynamic regulation and control; and the problem that amplification is easy to differentiate is fundamentally solved. According to the invention, a plurality of active components are added into the second culture medium and have a synergistic effect, so that a multi-target and synergistic stemness maintenance network is constructed, amplification differentiation is reduced, and stemness is maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell culture technology, in particular to a hematopoietic stem cell in vitro expansion culture system and a hematopoietic stem cell in vitro expansion method thereof. BACKGROUND

[0002] Hematopoietic stem cells (HSCs) are adult stem cells in the blood system, which are the starting cells of the hematopoietic and immune system. One of the remarkable features of HSCs is the expression of CD34 antigen, which is very small in normal bone marrow, accounting for about 1% to 2% of nucleated cells, and even less in peripheral blood, accounting for about 0.1% of mononuclear cells. HSCs have long-term self-renewal ability and the potential to differentiate into various mature blood cells, and have characteristics such as self-renewal, multi-directional differentiation and homing. At present, HSCs have been widely used in the treatment of clinical blood diseases, and are the most promising target cells for gene therapy. However, the limited source of HSCs (such as the number of HSCs in a single portion of umbilical cord blood is not enough for adult patient transplantation) and the easy differentiation and loss of stemness during in vitro expansion are the main bottlenecks that limit its wide application.

[0003] At present, the in vitro expansion technology of HSCs mainly faces the following challenges:

[0004] ①The traditional expansion scheme mostly uses continuous culture in a single medium containing high concentrations of cytokines (such as SCF, TPO, FLT3-L). Although this method can effectively drive HSCs proliferation and obtain a large number of total cells and CD34+ cells, the strong proliferation signal will force HSCs to rapidly differentiate, resulting in a significant decrease in the proportion of primitive stem cells with long-term reconstitution ability.

[0005] ②In vivo HSCs have processes such as resting, activation, and proliferation, for example, from the perspective of biological characteristics, most of the hematopoietic stem cells are in the resting state (Quiescence) in G0 phase under normal physiological conditions, which helps to protect the integrity of their genomes and prevent excessive proliferation and functional exhaustion. When the body needs, these resting HSCs will be activated to enter the cell cycle for proliferation and differentiation. Although some studies believe that compared with adult bone marrow HSCs, umbilical cord blood HSCs may contain a higher proportion of HSCs in the cell cycle or be more easily activated. But this does not mean that they are completely non-quiescent, and there is still a considerable part of HSCs in the resting state to maintain the stability of the stem cell pool. However, the existing technology mostly uses a single culture environment, without considering that part of the hematopoietic stem cells separated from the umbilical cord or bone marrow is not in the cell cycle, and thus it is difficult to provide the best signal combination required by HSCs in different stages (resting period and cell cycle) during the expansion period.

[0006] ③Although some small molecule compounds (such as SR1, UM171) have been used to inhibit the differentiation of HSCs, single differentiation inhibitor targets and mechanisms are limited, and if a large concentration is used, it may be accompanied by other unknown cytotoxicity. The prior art lacks a comprehensive protection strategy that can synergistically act from multiple targets and multiple pathways to effectively resist multiple stressors (such as oxidative stress, inflammatory stress) in the expansion process.

[0007] Therefore, there is an urgent need in the art for a new type of in vitro expansion culture system and method that takes into account the different process states of HSCs, can effectively suppress the differentiation of HSCs and maintain their primitive stemness through the synergistic action of time-series regulation and multiple active ingredients, while achieving efficient expansion of HSCs. SUMMARY

[0008] Therefore, based on the above background, the present application provides a hematopoietic stem cell in vitro expansion culture system and a hematopoietic stem cell in vitro expansion method thereof.

[0009] The technical scheme provided by the present application is:

[0010] A hematopoietic stem cell in vitro expansion culture system, comprising a first culture medium and a second culture medium, wherein:

[0011] The first culture medium is a StemSpan SFEM II-based culture medium containing stem cell factor (50-110) ng / mL, TPO (50-110) ng / mL, FLT3-L (50-110) ng / mL, Astragaloside IV (1-8) μM, heparin (0-5) U / ml;

[0012] The second culture medium is a StemSpan SFEM II-based culture medium containing stem cell factor (50-110) ng / mL, TPO (50-110) ng / mL, FLT3-L (10-30) ng / mL, UM171 (20-40) nM, active peptide (20-50) μM, β-1, 3-glucan (1-6) μg / mL, Radix Notoginseng Cyclopeptide B (1-3) μg / mL, recombinant human insulin (5-12) μg / mL.

[0013] Further, the first culture medium is a StemSpan SFEM II-based culture medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragaloside IV 3 μM, heparin 2 U / ml;

[0014] The second culture medium is a StemSpan SFEM II-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, active peptide 40 μM, β-1,3-glucan 5 μg / mL, Radix Notoginseng cyclic peptide B 2 μg / mL, and recombinant human insulin 10 μg / mL.

[0015] Further, the amino acid sequence of the active peptide is shown as LLPHH.

[0016] Based on the same inventive concept, the application further provides an application of the hematopoietic stem cell in-vitro expansion culture system in hematopoietic stem cell in-vitro expansion culture.

[0017] Based on the same inventive concept, the application further provides a hematopoietic stem cell in-vitro expansion method based on the hematopoietic stem cell in-vitro expansion culture system, comprising the following steps:

[0018] 1) Day 0-2

[0019] The hematopoietic stem cells with CD34+ phenotype derived from umbilical cord or bone marrow are inoculated in the first culture medium for suspension culture;

[0020] 2) Day 3-10

[0021] The hematopoietic stem cells cultured in step 1) are collected, and after the first culture medium is removed, the hematopoietic stem cells are resuspended in the second culture medium for culture, and the medium is replaced every 2-3 days, and the density is maintained at (1-5)×10 5 cells / mL;

[0022] The culture conditions of step 1) and step 2) are as follows:

[0023] The temperature is (36-37.5)℃;

[0024] The culture atmosphere is (4-6)% CO2, (1%-5)% O2.

[0025] Further, the inoculation density of the hematopoietic stem cells in step 1) is 1×10 3-4 cells / mL.

[0026] Further, the hematopoietic stem cells of step 1) are resuspended in the second culture medium at a cell density of (2-5)×10 4 cells / mL in step 2).

[0027] Based on the same inventive concept, the application further provides an application of the hematopoietic stem cells obtained by the hematopoietic stem cell in-vitro expansion method in preparing a cell therapy product for treating blood diseases.

[0028] Further, the blood disease includes leukemia, aplastic anemia, and thalassemia.

[0029] Further, the present application also provides a cell preparation comprising an effective amount of hematopoietic stem cells obtained by the in-vitro expansion method and a medically acceptable auxiliary agent.

[0030] The present application has the following beneficial effects:

[0031] ①The present application realizes more precise timing control of the in-vitro life cycle of HSCs by step-by-step culture with different culture media: the first step of activation start culture is carried out with the first culture medium, which uses a combination of high concentrations of SCF, TPO, and FLT3-L, and is supplemented with Astragaloside IV, to strongly and synchronously activate the resting HSCs from G0 phase (resting phase) into the proliferation cycle, thereby establishing a synchronized starting cell pool for subsequent expansion; the second step of expansion culture is carried out with the second culture medium, which significantly reduces the intensity of differentiation-promoting signals such as FLT3-L, and introduces UM171 to inhibit differentiation, thereby turning the culture towards proliferation and stemness maintenance, and simulating the natural process of HSCs behavior in vivo through dynamic regulation, thereby fundamentally solving the problem of easy differentiation during expansion.

[0032] ②The present application constructs a stemness maintenance network with multiple targets and synergistic effects by the synergistic effect of various active ingredients added in the second culture medium, thereby reducing expansion differentiation and maintaining stemness: UM171 as the core differentiation inhibitor directly acts on epigenetic regulation to prevent HSCs from differentiating too early; active peptide (LLPHH), β-1,3-glucan, and Radix Pseudostellariae cyclic peptide B form a synergistic protection: LLPHH can provide antioxidant stress protection, β-1,3-glucan can regulate the immune microenvironment to reduce inflammatory stress, and Radix Pseudostellariae cyclic peptide B can help alleviate cell replication stress, which together create a low-stress, stemness-maintaining microenvironment for HSCs from different angles, complementing the action of UM171 and producing a synergistic effect; recombinant human insulin provides stable metabolic support to promote cell energy metabolism and ensure the smooth progress of the expansion process.

[0033] ③The method of the present application has a short expansion time, and can achieve efficient expansion in terms of cell number (total cells and CD34+ cells can be expanded by more than 100 times) within 10 days, with high cell survival rate.

[0034] ④All components of the culture medium of the present application are chemically defined substances, completely avoiding the use of animal serum and other uncertain components, thereby ensuring batch consistency and process stability, and laying a solid foundation for standardization and regulatory approval of the production process as a cell therapy product in the future. DETAILED DESCRIPTION

[0035] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0036] The test reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; and the experimental methods are all conventional methods unless otherwise specified.

[0037] The base medium StemSpan SFEMII used in the following embodiments is purchased from Stemcell Technologies;

[0038] Stem cell factor (recombinant human stem cell factor) SCF is purchased from ScienCell;

[0039] Human thrombopoietin TPO is purchased from LMAI Bio;

[0040] FLT3-L (recombinant human FLT3-L protein) is purchased from Mlbio;

[0041] Astragalus membranaceus IV CAS No. 84687-43-4 is purchased from Solabio;

[0042] UM171 CAS No. 1448724-09-1 is purchased from Aladdin, with product number U418551;

[0043] The active peptide with amino acid sequence LLPHH (Leu-Leu-Pro-His-His) is derived from soybean protein, specifically, it is an antioxidant peptide obtained by protein hydrolysis of soybean protein, and the active peptide used in the following embodiments is synthesized by solid phase in the laboratory.

[0044] β-1,3-glucan CAS No. 9051-97-2 is purchased from TargetMol;

[0045] Radix notoginseng cyclic peptide B CAS No. 145459-19-4 is purchased from Solabio;

[0046] Recombinant human insulin is purchased from anrate.

[0047] The CD34+ hematopoietic stem cells derived from umbilical cord and used in the embodiments are self-made in the laboratory, and the preparation steps are as follows:

[0048] Collect 85ml of umbilical cord blood, dilute with equal volume of normal saline, mix gently. Carefully overlay the diluted blood on 0.5 volume of lymphocyte separation medium (Ficoll-Paque PLUS). Centrifuge at 1500-2000 rpm / min for 15 minutes at room temperature (note: set lower acceleration and deceleration to keep the interface clear), the liquid in the centrifuge tube is divided into plasma layer, annular milky white PBMC layer, separation medium layer, red blood cell layer from top to bottom; Carefully transfer the annular milky white PBMC layer to another centrifuge tube with a Pasteur pipette, resuspend in enough normal saline, centrifuge again at 1500-2000 rpm / min for 15 minutes, discard the supernatant, resuspend the precipitate in normal saline again, centrifuge again at 1500-2000 rpm / min for 15 minutes, discard the supernatant, and the cell precipitate is the preliminary separation of PBMC.

[0049] Resuspend the PBMC precipitate with staining buffer containing fluorescently labeled anti-human CD34 antibody, incubate on ice for a specified time in the dark. After staining, centrifuge twice with washing buffer to remove unbound antibodies. Resuspend the cells in buffer containing appropriate serum, filter through a cell strainer, and use a flow cytometer to sort CD34 positive cells. The sorted CD34 + The purity of the cells is greater than 95% as detected by flow cytometry.

[0050] Example 1: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0051] 1) Days 0-2

[0052] Hematopoietic stem cells derived from umbilical cord with CD34+ phenotype are seeded at a density of 1 x 105cells / mL in the first culture medium (fibronectin (10 μg / mL) coated culture plate) for suspension culture; 4

[0053] The first culture medium is StemSpan SFEMII-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragalus membranaceus IV 3 μM, heparin 2 U / mL;

[0054] The culture conditions are:

[0055] The temperature is 37°C;

[0056] The culture atmosphere is 5% CO2, 5% O2.

[0057] 2) Days 3-10

[0058] ​After centrifugation of the culture medium of step 1), the hematopoietic stem cell cell pellet was collected, and the hematopoietic stem cells were cultured at a density of 5x10 4 resuspended in a second culture medium, half-volume exchange every 2 days, maintaining a density of 1x10 5 cells / mL.

[0059] The second culture medium is a StemSpan SFEMII-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, active peptide 40 μM, β-1,3-glucan 5 μg / mL, Radix Notoginseng Cyclopeptide B 2 μg / mL, recombinant human insulin 10 μg / mL.

[0060] The amino acid sequence of the active peptide is shown as LLPHH.

[0061] The culture conditions are as follows:

[0062] The temperature is 37°C.

[0063] The culture atmosphere is 5% CO2, 5% O2.

[0064] Comparative Example 1: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0065] Hematopoietic stem cells derived from umbilical cord and having a phenotype of CD34+ were inoculated at a density of 1x10 4 suspended culture in a first culture medium (fibronectin (10 μg / mL)-coated culture plate);

[0066] Days 0-10

[0067] The first culture medium is a StemSpan SFEMII-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragalus membranaceus Glycoside IV 3 μM, heparin 2 U / mL.

[0068] Half-volume exchange every 2 days, maintaining a density of 1x10 5 cells / mL.

[0069] This comparative example selects a one-step culture with the first culture medium compared to Example 1.

[0070] Comparative Example 2: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0071] Days 0-10

[0072] The CD34+ hematopoietic stem cells derived from umbilical cord were inoculated in the first culture medium at a density of 1 x 10 4 The cells were cultured in suspension (on a fibronectin (10 μg / mL)-coated culture plate) in the second culture medium.

[0073] The medium was half-changed every 2 days to maintain the density at 1 x 10 5 cells / mL.

[0074] The second culture medium was selected for one-step culture in the comparative example 1.

[0075] Comparative example 3: An in-vitro hematopoietic stem cell expansion method of an in-vitro hematopoietic stem cell expansion system, comprising the following steps:

[0076] 1) On day 0-2

[0077] The CD34+ hematopoietic stem cells derived from umbilical cord were inoculated in the first culture medium at a density of 1 x 10 4 The cells were cultured in suspension (on a fibronectin (10 μg / mL)-coated culture plate) in the first culture medium.

[0078] The first culture medium was a StemSpan SFEMII-based culture medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, heparin 2 U / mL.

[0079] The culture conditions were as follows:

[0080] The temperature was 37°C.

[0081] The culture atmosphere was 5% CO2, 5% O2.

[0082] 2) On day 3-10

[0083] After centrifugation of the culture medium of step 1), the hematopoietic stem cell cell pellet was collected, and the hematopoietic stem cells were resuspended in the second culture medium at a density of 5 x 10 4 The cells were cultured, and the medium was half-changed every 2 days to maintain the density at 1 x 10 5 cells / mL.

[0084] The second culture medium was a StemSpan SFEMII-based culture medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, active peptide 40 μM, β-1, 3-glucan 5 μg / mL, Radix Notoginseng cyclic peptide B 2 μg / mL, recombinant human insulin 10 μg / mL.

[0085] The amino acid sequence of the active peptide is shown as LLPHH.

[0086] The culture conditions are as follows:

[0087] The temperature is 37℃;

[0088] The culture atmosphere is 5% CO2, 5% O2.

[0089] The present comparative example is compared with Example 1, and the first culture medium does not contain Astragaloside IV.

[0090] Comparative Example 4: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0091] 1) Days 0-2

[0092] Hematopoietic stem cells derived from umbilical cord and having a phenotype of CD34+ are inoculated in the first culture medium at a density of 1×10 4 The hematopoietic stem cells are inoculated in the first culture medium for suspension culture (on a fibronectin (10 μg / mL)-coated culture plate);

[0093] The first culture medium is a StemSpan SFEMII-based culture medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragaloside IV 3 μM, heparin 2 U / mL;

[0094] The culture conditions are as follows:

[0095] The temperature is 37℃;

[0096] The culture atmosphere is 5% CO2, 5% O2.

[0097] 2) Days 3-10

[0098] After centrifugation of the culture medium of step 1), the hematopoietic stem cell cell pellet is collected, and the hematopoietic stem cells are inoculated in the second culture medium at a density of 5×10 4 The hematopoietic stem cells are resuspended in the second culture medium for culture, and the medium is replaced every 2 days, and the density is maintained at 1×10 5 cells / mL;

[0099] The second culture medium is a StemSpan SFEMII-based culture medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, and recombinant human insulin 10 μg / mL.

[0100] The present comparative example is compared with Example 1, and the second culture medium does not contain active peptides, β-1,3-glucan, and Radix Notoginseng cyclic peptide B.

[0101] Comparative Example 5: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0102] 1) Day 0-2

[0103] The CD34+ hematopoietic stem cells derived from umbilical cord were resuspended in the first medium at a density of 1 x 10 4 The cells were seeded in the first medium for suspension culture (on fibronectin (10 pg / mL) coated culture plates);

[0104] The first medium was StemSpan SFEM II-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragalus Root Glycoside IV 3 mM, heparin 2 U / mL;

[0105] The culture conditions were as follows:

[0106] The temperature was 37℃;

[0107] The culture atmosphere was 5% CO2, 5% O2.

[0108] 2) Day 3-10

[0109] After centrifugation of the medium of step 1), the hematopoietic stem cell cell pellet was collected, and the hematopoietic stem cells were resuspended in the second medium at a density of 5 x 10 4 The cells were cultured in the second medium, and the medium was replaced every 2 days, and the density was maintained at 1 x 10 5 cells / mL;

[0110] The second medium was StemSpan SFEM II-based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, active peptide 40 mM, beta-1, 3-glucan 7 pg / mL, recombinant human insulin 10 pg / mL.

[0111] The amino acid sequence of the active peptide is shown as LLPHH.

[0112] The culture conditions were as follows:

[0113] The temperature was 37℃;

[0114] The culture atmosphere was 5% CO2, 5% O2.

[0115] In the comparative example, Taizishen cyclic peptide B was replaced by beta-1, 3-glucan compared with example 2.

[0116] Comparative Example 6: A method for in vitro expansion of hematopoietic stem cells in a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0117] 1) Day 0-2

[0118] The CD34+ hematopoietic stem cells derived from umbilical cord were inoculated in the first culture medium at a density of 1 x 10 4 The cells were inoculated in the first culture medium for suspension culture (on fibronectin (10 pg / mL) coated culture plates) ;

[0119] The first culture medium was StemSpan SFEMII based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragalus membranaceus saponin IV 3 mM, heparin 2 U / mL;

[0120] The culture conditions were as follows:

[0121] The temperature was 37°C;

[0122] The culture atmosphere was 5% CO2, 5% O2.

[0123] 2) Days 3-10

[0124] After centrifugation of the culture medium of step 1), the hematopoietic stem cell cell pellet was collected, and the hematopoietic stem cells were inoculated in the second culture medium at a density of 5 x 10 4 The cells were resuspended in the second culture medium every 2 days for half volume exchange, and the density was maintained at 1 x 10 5 cells / mL;

[0125] The second culture medium was StemSpan SFEMII based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, active peptide 40 mM, Radix Notoginseng Cyclopeptide B 7 pg / mL, recombinant human insulin 10 pg / mL.

[0126] The amino acid sequence of the active peptide was as shown in LLPHH.

[0127] The culture conditions were as follows:

[0128] The temperature was 37°C;

[0129] The culture atmosphere was 5% CO2, 5% O2.

[0130] In the comparative example, the beta-1, 3-glucan was replaced by Radix Notoginseng Cyclopeptide B compared to Example 2.

[0131] Comparative Example 7: A method for hematopoietic stem cell in vitro expansion of a hematopoietic stem cell in vitro expansion culture system, comprising the following steps:

[0132] 1) Days 0-2

[0133] The CD34+ hematopoietic stem cells derived from umbilical cord were inoculated in the first culture medium at a density of 1 x 10 4Inoculate into the first medium for suspension culture (on fibronectin (10 μg / mL) coated culture plate);

[0134] The first medium is StemSpan SFEMII based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, Astragalus membranaceus IV 3 μM, heparin 2 U / ml;

[0135] The culture condition is:

[0136] The temperature is 37°C;

[0137] The culture atmosphere is 5% CO2, 5% O2.

[0138] 2) Days 3-10

[0139] After centrifugation of the medium of step 1), the hematopoietic stem cell cell pellet is collected, and the hematopoietic stem cells are cultured at a density of 5 x 10 4 Resuspend in the second medium for culture, half the amount of liquid is changed every 2 days, and the density is maintained at 1 x 10 5 cells / mL;

[0140] The second medium is StemSpan SFEMII based medium containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM171 35 nM, β-1,3-glucan 5 μg / mL, Radix Notoginseng Cyclopeptide B 2 μg / mL, recombinant human insulin 10 μg / mL.

[0141] The culture condition is:

[0142] The temperature is 37°C;

[0143] The culture atmosphere is 5% CO2, 5% O2.

[0144] The present comparative example removes the active peptide compared to Example 1.

[0145] The following are related characterization experiments:

[0146] ① Cell proliferation and survival

[0147] The samples of Example 1, Comparative Example 1 to Comparative Example 7 were taken at 0 (D0), 2 (D2), 5 (D5) and 10 days (D10) of culture for counting, obtaining the data of cell proliferation fold, and the cell viability was detected by trypan blue exclusion method, and the results are shown in Table 1, each group was treated in triplicate, and the average value was taken. Cell total number expansion fold = Dn cell number * 100% / D0 cell number.

[0148] Table 1: Hematopoietic stem cell proliferation fold and survival rate (%)

[0149]

[0150] From the above table, the final expansion fold of Example 1 (287 folds) is much higher than that of Comparative Example 1 (116 folds) using only the first-step medium and Comparative Example 2 (75 folds) using only the second-step medium, which shows that compared with the one-step method of Comparative Examples 1 and 2, the two-step method of Example 1 can significantly benefit the expansion of hematopoietic stem cells. It is analyzed that because the first step of Example 1 activates hematopoietic stem cells efficiently, it makes them enter the cell proliferation cycle and lay the quantity foundation; the second step can efficiently maintain expansion and avoid differentiation exhaustion. The cell viability of Example 1 at D10 (92%) is also significantly higher than that of Comparative Example 1 (75%) and Comparative Example 2 (81%), which indicates that the two-step culture of the present application can better maintain the healthy state of cells. The expansion fold (193 folds) and D10 viability (87%) of Comparative Example 3 (without Astragaloside IV) are both lower than those of Example 1. This shows that Astragaloside IV in the activation period can enhance the response of cells to cytokines, improve the initial activation efficiency, and have a lasting positive impact on subsequent expansion. Comparative Example 4 has the lowest expansion fold (164 folds) and viability (82%) when compared with Example 1 and Comparative Examples 5 to 7, which highlights the important influence of the addition of active peptides, β-1, 3-glucan and Radix Pseudostellariae cyclic peptide B on maintaining expansion and cell health. The data of Comparative Examples 5, 6 and 7 are all better than those of Comparative Example 4 but worse than those of Example 1, which shows that there is a synergistic effect among the active peptides LLPHH, β-glucan and Radix Pseudostellariae cyclic peptide B. The absence of any single component or the replacement of the function overlap (such as Comparative Examples 5 and 6) cannot completely replace the antioxidant-anti-inflammatory-anti-replication stress comprehensive protection network formed by the combination of the three.

[0151] ②CD34+phenotype cell proportion

[0152] And at 10 (D10) days of culture, the expression of CD34+protein was determined by flow cytometry, and each group was treated in triplicate, and the average value was taken.

[0153] Specifically, about 5x10 5 cells were centrifuged at 2000 rpm for 5 minutes, resuspended with PBS after removing the supernatant, and incubated with fluorescently labeled CD34 antibody and PBS resuspended cells. The incubation conditions were: 4°C, 15 minutes, then washed twice with PBS, discarded the supernatant, and resuspended with 0.5 mL PBS. The treated cells were detected and analyzed by flow cytometry. The expression of CD34 protein is shown in Table 2.

[0154] Table 2: CD34+phenotype cell proportion

[0155] Group Expression rate (%) of CD34+ Example 1 group 91.5 Comparative example 2 group 58.3 Comparative example 3 group 82.5 Comparative example 4 group 68.7 Comparative example 5 group 85.4 Comparative example 6 group 83.6 Comparative example 7 group 80.9

[0156] CD34+ cell expression rate is a key indicator for measuring whether the culture system can maintain the undifferentiated, primitive state of cells. As shown in the above table, the CD34+ expression rate of Example 1 is as high as 91.5%, which is an extremely excellent result. It can be seen that the two-step method of the present application has excellent stemness maintenance ability for expanding hematopoietic stem cells. This result is combined with the highest total cell expansion fold (287-fold) and cell viability (92%) in Table 1. It can be seen that Example 1 can achieve large-scale expansion while effectively inhibiting differentiation, so that most cells maintain the original stem / progenitor cell phenotype. This is directly benefited from the efficient synchronous activation in the first step and the low differentiation stress environment created by the network synergy of UM171, active peptide, beta-glucan and radix notoginseng cyclic peptide B in the second step. At the same time, the CD34+ ratio of Comparative Example 2 using only the second step medium is only 58.3%, which shows that if the first step of strong activation is not used to establish a high-quality starting cell pool, and directly enters the expansion and maintenance mode, not only the expansion efficiency is low, but also the cells are more likely to differentiate. The CD34+ ratio of Comparative Example 3 (82.5%) is significantly lower than that of Example 1 (91.5%), which shows that Astragaloside IV in the activation stage is not only beneficial to the subsequent expansion, but also enhances the synchronization and quality of activation, which has an important influence on maintaining a high proportion of CD34+ cells in the entire expansion cycle. Example 1 is compared with Comparative Examples 4 to 7. The CD34+ ratio of Comparative Example 4 is the lowest (68.7%), which is greatly different from that of Example 1 (91.5%), highlighting that the joint addition of active peptide, beta-glucan and radix notoginseng cyclic peptide B is a decisive factor for maintaining a high CD34+ expression rate. The CD34+ ratios of Comparative Examples 5 to 7 are all significantly better than that of Comparative Example 4, but are still significantly lower than that of Example 1, which proves that there is an irreplaceable synergistic effect among LLPHH, beta-glucan and radix notoginseng cyclic peptide B in maintaining the stemness ability of cells.

[0157] ③Respectively take the cells of Example 1 and its Comparative Examples 1 to 7 cultured to the 10th day and inoculate them in MethoCult TM H4435 medium, add 1 ml of medium to each well of a six-well plate, inoculate at a density of 1000 cells / well, and place in a 37°C, 5% CO2, 5% O2 incubator for 14 days. Calculate the CFU-GEMM, BFU-E, CFU-GM (colony-forming units per 10 3 input cells), and each group is treated in triplicate, and the average value is taken.

[0158] Table 3: Colony situation

[0159] Group CFU-GEMM BFU-E CFU-GM Example 1 group 22 75 95 Comparative example 1 group 7 30 58 Comparative example 2 group 10 38 65 Comparative example 3 group 16 55 78 Comparative example 4 group 12 48 66 Comparative example 5 group 18 60 82 Comparative example 6 group 17 58 80 Comparative example 7 group 15 56 77

[0160] CFU assay is a reliable standard to evaluate the functionality of cells, in which CFU-GEMM (mixed colony) best reflects the multi-differentiation potential of the most primitive hematopoietic stem / progenitor cells. High level of total colony number and CFU-GEMM number is a direct evidence of the strong regenerative capacity of the cells. As can be seen from the above table, Example 1 produced the most total colony number and CFU-GEMM (22), which corresponds to its extremely high CD34+ proportion (91.5%), indicating that the cells expanded by it are not only phenotypically stem cells, but also functionally strong stem cells with extremely high multi-lineage differentiation potential. This provides the most critical predictive indicator for the success rate of its clinical transplantation. While the total cell number of Comparative Example 1 and Comparative Example 2, especially Comparative Example 1, has increased, the proportion of primitive cells with function is extremely low, and the clinical value is limited. In Comparative Example 3, due to the absence of Astragaloside IV in the first step of activation culture, its CFU-GEMM decreased to 16, indicating that Astragaloside IV has an important influence on obtaining a high-quality functional stem cell pool. As can be seen from Comparative Example 4 to Comparative Example 7, the synergy between LLPHH, β-glucan and Taizishen cyclic peptide B is beneficial to obtaining high-quality functional hematopoietic stem cells.

[0161] The above describes the present application and its embodiments, which is not restrictive, and the examples shown are only one of the embodiments of the present application, and the actual structure is not limited thereto. In general, if a person skilled in the art is inspired thereby, without departing from the purpose of the present application, without creative design, similar structure and embodiments to the technical solution should belong to the protection scope of the present application.

Claims

1. An in vitro expansion and culture system for hematopoietic stem cells, characterized in that, It includes a first culture medium and a second culture medium, wherein: The first culture medium was based on StemSpan SFEMII, containing stem cell factor (50-110) ng / mL, TPO (50-110) ng / mL, FLT3-L (50-110) ng / mL, astragaloside IV (1-8) μM, and heparin (0-5) U / mL. The second culture medium is based on StemSpan SFEMII and contains stem cell factor (50-110) ng / mL, TPO (50-110) ng / mL, FLT3-L (10-30) ng / mL, UM171 (20-40) nM, active peptide (20-50) μM, β-1,3-glucan (1-6) μg / mL, Codonopsis pilosula cyclic peptide B (1-3) μg / mL, and recombinant human insulin (5-12) μg / mL.

2. The in vitro expansion and culture system for hematopoietic stem cells according to claim 1, characterized in that, The first culture medium was based on StemSpan SFEMII, containing stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 65 ng / mL, astragaloside IV 3 μM, and heparin 2 U / ml; The second culture medium was based on StemSpan SFEMII and contained stem cell factor 80 ng / mL, TPO 50 ng / mL, FLT3-L 20 ng / mL, UM17 135 nM, active peptide 40 μM, β-1,3-glucan 5 μg / mL, Codonopsis pilosula cyclic peptide B 2 μg / mL, and recombinant human insulin 10 μg / mL.

3. The in vitro expansion and culture system for hematopoietic stem cells according to claim 1 or 2, characterized in that, The amino acid sequence of the active peptide is shown in LLPHH.

4. The application of the hematopoietic stem cell in vitro expansion culture system according to any one of claims 1 to 3 in the in vitro expansion culture of hematopoietic stem cells.

5. A method for in vitro expansion of hematopoietic stem cells based on the in vitro expansion culture system for hematopoietic stem cells according to any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Day 0-2 CD34+ hematopoietic stem cells derived from the umbilical cord or bone marrow were seeded into the first culture medium for suspension culture. 2) Days 3-10 Collect the hematopoietic stem cells cultured in step 1), remove the first culture medium, and resuspend them in the second culture medium for further culture. Change half of the medium every 2-3 days to maintain a density of (1-5)×10⁻⁶. 5 cells / mL; The culture conditions for steps 1) and 2) are as follows: The temperature is (36-37.5)℃; Cultivation atmosphere: (4-6)% CO2, (1%-5%) O2.

6. The method for in vitro expansion of hematopoietic stem cells according to claim 5, characterized in that, The seeding density of hematopoietic stem cells in step 1) is 1×10⁻⁶. 3-4 cells / mL.

7. The method for in vitro expansion of hematopoietic stem cells according to claim 5, characterized in that, In step 2), the cell density is (2-5)×10 4 The hematopoietic stem cells from step 1) were resuspended in the second culture medium at cells / mL.

8. The use of hematopoietic stem cells amplified and cultured by the in vitro amplification method of claims 4-7 in the preparation of cell therapy products for treating blood diseases.

9. The application according to claim 8, characterized in that, The blood disorders mentioned include leukemia, aplastic anemia, and thalassemia.

10. A cell preparation, characterized in that, Includes an effective amount of hematopoietic stem cells amplified and cultured by the in vitro amplification method of claims 4-7 and medically acceptable adjuvants.

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