An in vitro expansion method of CD34+ hematopoietic stem cells
By combining specific chemical agonists and synthetic polymers, a method for expanding CD34+ hematopoietic stem cells without relying on exogenous biological products was constructed, solving the problems of high cost and unclear composition in existing technologies, and achieving safe and standardized cell expansion results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BOKANG BIOENGINEERING (SHANDONG) CO LTD
- Filing Date
- 2026-05-11
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256257A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, specifically to an in vitro expansion method for CD34+ hematopoietic stem cells. Background Technology
[0002] Hematopoietic stem cells (HSCs), as core seed cells capable of self-renewal and differentiation into all blood cell lineages, have been shown to have radical potential in treating various hematologic disorders, including leukemia, lymphoma, and aplastic anemia, as well as immunodeficiency. Umbilical cord blood is an important source of HSCs due to its relatively convenient availability and low incidence of graft-versus-host disease after transplantation. However, the proportion of HSCs with long-term regenerative hematopoietic function, whether from bone marrow, mobilized peripheral blood, or umbilical cord blood, is extremely low, and they are often mixed with short-term regenerative hematopoietic progenitor cells. This presents a significant bottleneck in obtaining sufficient quantities of functional HSCs for adult transplantation, gene editing, or basic research. Therefore, achieving safe and effective in vitro expansion of HSCs has become crucial for their clinical application. For a long time, strategies for in vitro expansion of HSCs have heavily relied on adding various recombinant cytokines and animal-derived albumin to provide carrier protein functions. While strong mitotic signals promote cell number growth, they also accelerate stem cell differentiation depletion. In addition, this model has significant limitations: First, it is costly, as many high-purity recombinant cytokines are expensive; second, the composition is complex and uncertain, with animal-derived albumin exhibiting batch-to-batch variability, potential pathogen risks, and immunogenicity; third, the chemical composition of the culture system is unclear, which is not conducive to mechanism research and standardized production.
[0003] Based on this, this application provides a method for in vitro expansion of CD34+ hematopoietic stem cells. Summary of the Invention
[0004] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides an in vitro expansion method for CD34+ hematopoietic stem cells, offering a novel culture method that is free from dependence on exogenous complex biological products, has clearly defined chemical components, and specifically supports the self-renewal of functional HSCs rather than their differentiation expansion.
[0005] Technical solution To achieve the above objectives, the present invention provides the following technical solution: An in vitro expansion method for CD34+ hematopoietic stem cells involves seeding isolated CD34+ cells into an expansion medium completely free of exogenous recombinant cytokines and animal-derived albumin for culture. The amplification culture medium comprises: a) Basic culture medium; b) A combination of chemokine agonists, including phosphoinosine 3-kinase (PI3K) activators, thrombopoietin receptor (TPO-R) agonists, and pyrimidine indole derivative UM171; c) Synthetic polymers.
[0006] Furthermore, the basal culture medium is a serum-free culture medium.
[0007] Further, the basal culture medium is at least one of X-VIVO™ 10, X-VIVO™ 15, X-VIVO™ 20, and StemSpan™ SFEM II culture medium.
[0008] Further preferably, the basal culture medium is StemSpan™ SFEM II medium.
[0009] Furthermore, the PI3K activator is 740Y-P, and the TPO-R agonist is butyramide.
[0010] Furthermore, the concentration of 740Y-P used in the amplification medium is 0.1-2 μM, the concentration of butyramide used in the amplification medium is 0.01-0.2 μM, and the concentration of UM171 used in the amplification medium is 50-100 nM.
[0011] Furthermore, the synthetic polymer is polyvinyl alcohol.
[0012] Furthermore, the concentration of polyvinyl alcohol used in the amplification medium is 0.01-0.2 wt%.
[0013] Furthermore, the CD34+ cells are derived from umbilical cord blood.
[0014] Furthermore, the inoculation involves a cell density of 2 × 10⁻⁶ cells. 4 -1×10 5 Cells / mL.
[0015] Furthermore, the specific steps of the in vitro expansion method for CD34+ hematopoietic stem cells are as follows: S1. Cell seeding: After isolation or resuscitation, CD34+ cells are washed and resuspended with amplification medium, and then seeded into culture containers. S2. Culture Maintenance: Collect cell culture medium from wells using a pipette, centrifuge, remove supernatant, resuspend cells in fresh amplification medium, transfer to well plate, change the medium, and return to the incubator; prepare fresh and preheated culture medium for each use, change the culture medium regularly, manually remove old culture medium and replace with new culture medium, and perform cell counting, viability and flow cytometry detection each time.
[0016] Furthermore, the specific steps of the in vitro expansion method for CD34+ hematopoietic stem cells are as follows: S1. Cell seeding: Wash the isolated or resuscitated CD34+ cells once with amplification medium, and seed at 2×10⁻⁶. 4 -1×10 5 The concentration was resuspended and 1 mL was transferred to a 24-well plate; S2. Culture Maintenance: Collect cell culture medium from the wells into 15 mL tubes using a pipette, centrifuge at 440 g for 5 minutes at room temperature, remove the supernatant, resuspend the cells in fresh expansion medium, transfer to a 24-well plate, change the medium, and return to a CO2 incubator; prepare fresh medium and preheat to 37°C before each use, change the medium every 2-3 days, manually remove the old medium and replace with preheated new medium, and perform cell counting, viability and flow cytometry detection each time.
[0017] Beneficial technical effects In this invention, a complete replacement of exogenous recombinant cytokines and animal-derived albumin is achieved by employing a specific combination of chemical agonists and synthetic polymers. This not only reduces culture costs but also establishes a culture system with clearly defined chemical composition, high batch stability, and no risk from animal-derived components, thereby improving the safety of clinical applications and the level of production standardization.
[0018] Secondly, the culture system of this invention can balance expansion and stem cell maintenance. As shown in the example data, the proportion of CD34+CD90+ stem cell subsets decreased on day 5 of culture using the conventional cytokine method, indicating rapid depletion of functional stem cells. In the culture system of this invention, the cell expansion fold showed a stable increase, while the proportions of the key stem cell phenotypes CD34+CD90+ and CD34+CD45RA- were effectively maintained for at least 12 days throughout the entire culture cycle. This indicates that the specific signal combination provided by this invention, synergistically with the physicochemical support environment constructed by the synthetic polymer, can preferentially activate and maintain the self-renewal program of HSCs, inhibit their premature differentiation, and thus achieve functional expansion. Finally, the method has a clear and reproducible operation procedure, uses conventional serum-free basal culture medium and a defined additive concentration, and is easy to implement and apply in laboratory and large-scale production environments. In summary, this invention overcomes the shortcomings of traditional techniques, such as low expansion quality, complex composition, and high cost, and provides a novel solution for efficient, economical, safe, and high-quality expansion of functional hematopoietic stem cells, which has significant practical application value for promoting the development of stem cell therapy products. Attached Figure Description
[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is the initial flow cytometry plot of the cells. Figure 2 This is a test image from the fourth day of sample culture in Example 1. Figure 3 This is a test image of the sample in Comparative Example 1 on the fourth day of culture.
[0021] Figure 4 This is a test graph of the sample cultured for seven days in Example 1. Figure 5 This is a test image of the sample in Comparative Example 1 on the seventh day of culture.
[0022] Figure 6 This is a test image from the ninth day of sample culture in Example 1.
[0023] Figure 7 This is a test image of the sample cultured on the twelfth day in Example 1. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0025] The raw materials used in the embodiments of this invention are shown below, and all reagents used are analytical grade.
[0026] Example 1 A method for in vitro expansion of CD34+ hematopoietic stem cells, the specific operation steps of which are as follows: S1. Prepare CD34+ cells. Wash CD34+ cells once with amplification medium at a concentration of 2×10⁻⁶. 4 Resuspend the cells at a concentration of 1 cell / mL, and then transfer 1 mL of culture medium into a 24-well plate; S2. Use a pipette to collect all cell culture medium from the wells into a 15mL tube, centrifuge at 440 g for 5 minutes at room temperature, remove the supernatant, add fresh amplification medium, resuspend the cells and transfer them to a 24-well plate. After changing the culture medium, return the plate to the tissue culture incubator. Prepare fresh culture medium before each use and preheat it to 37°C. Change the culture medium every 2 days by manually removing the conditioned medium by pipetting and replacing it with preheated and freshly prepared culture medium. Perform cell counting, viability and loss detection each time. The amplification medium is StemSpan™ SFEM II medium containing 0.1% PVA, 1 μM 740Y-P, 0.1 μM butyramide and 70 nM UM171.
[0027] Comparative Example 1 The difference between this comparative example and Example 1 is that the culture medium used is a conventional amplification medium supplemented with cytokines, specifically StemSpan™ containing 100 ng / mL SCF, 100 ng / mL FL, 50 ng / mL TPO, 20 ng / mL IL-3, and 150 ng / mL IL-6. StemSpan™ SFEM II is a modified serum-free culture medium specifically designed for the in vitro culture and expansion of hematopoietic stem cells.
[0028] The cell systems in Example 1 and Comparative Example 1 were then tested. Flow cytometry analysis was performed on the culture systems on days 4 and 7, and on days 9 and 12, the culture system in Example 1 was further analyzed by flow cytometry. The test results are as follows: Figure 1 - Figure 7 As shown.
[0029] In the figure, the culture medium of Example 1 is labeled as stem cell culture medium, and the culture medium of Comparative Example 1 is labeled as 3a culture medium.
[0030] Figure 1 This is the initial flow cytometry plot of the cells. Figure 2 This is a test image from the fourth day of sample culture in Example 1. Figure 3 This is a test image of the sample in Comparative Example 1 on the fourth day of culture.
[0031] Figure 4 This is a test graph of the sample cultured for seven days in Example 1. Figure 5 This is a test image of the sample in Comparative Example 1 on the seventh day of culture.
[0032] Figure 6 This is a test image from the ninth day of sample culture in Example 1.
[0033] Figure 7 This is a test image of the sample cultured on the twelfth day in Example 1.
[0034] Flow cytometry results showed that Figure 1 The phenotypic characteristics of CD34+ cells isolated or resuscitated from umbilical cord blood at the start of culture were presented. Among them, the proportion of CD34+CD90+ cells was quite high at 32.30%, indicating that the starting cell population was enriched with a large number of extremely primitive hematopoietic stem cells with strong regenerative potential.
[0035] By comparison Figure 2 and Figure 3 The data shows that the proportion of cells with the CD34+CD90+ phenotype in Example 1 is much higher than that in Comparative Example 1. Figure 4 and Figure 5 As can be seen from the samples, the proportion of cells with the CD34+CD90+ phenotype in Example 1 was still higher than that in Comparative Example 1. Figure 6 and Figure 7 The results showed that the cell system in Example 1 maintained a high level of stemness throughout the at least 12-day culture period.
[0036] It should be noted that the terms "comprising," "including," and any variations thereof used herein indicate non-exclusive inclusion, meaning that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to the process, method, article, or apparatus. Unless otherwise specified, an element defined by "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.
[0037] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
[0038] Those skilled in the art should understand that the above description is only a part of the specific embodiments of the present invention, and not all of the embodiments.
Claims
1. A method for in vitro expansion of CD34+ hematopoietic stem cells, characterized in that, The isolated CD34+ cells were seeded in an amplification medium completely free of exogenous recombinant cytokines and animal-derived albumin and cultured. The amplification culture medium comprises: a) Basic culture medium; b) A combination of chemical agonists, including a phosphoinosine 3-kinase activator and the thrombopoietin receptor agonist pyrimidine indole derivative UM171; c) Synthetic polymers.
2. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The basal culture medium is a serum-free culture medium.
3. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The phosphoinosine 3-kinase activator is 740Y-P; The thrombopoietin receptor agonist is butyramide.
4. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 3, characterized in that, The concentration of 740Y-P used in the amplification medium is 0.1-2 μM, and the concentration of butyramide used in the amplification medium is 0.01-0.2 μM.
5. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The concentration of UM171 used in amplification medium is 50-100 nM.
6. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The synthetic polymer is polyvinyl alcohol.
7. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 6, characterized in that, The concentration of polyvinyl alcohol used in the amplification medium is 0.01-0.2 wt%.
8. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The CD34+ cells were derived from umbilical cord blood.
9. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The inoculation process, in in vitro culture, involves setting the seeding density of CD34+ cells at 2 × 10⁻⁶. 4 -1×10 5 Cells / mL.
10. The method for in vitro expansion of CD34+ hematopoietic stem cells according to claim 1, characterized in that, The specific operating steps are as follows: (1) Cell seeding: After isolation or recovery, CD34+ cells are washed and resuspended with amplification medium and then seeded into culture containers; (2) Culture maintenance: Collect cell culture medium in wells with pipettes, centrifuge and remove supernatant, add fresh expansion culture medium to resuspend cells, transfer to well plates, change culture medium and transfer back to incubator; fresh and preheated culture medium should be prepared for each use, and the culture medium should be changed regularly. Old culture medium should be removed manually and replaced with new culture medium. Counting, viability and flow cytometry detection should be performed each time.