阅读说明：本技术 含聚合物微纳小球的造血干细胞体外培养体系及应用 (Hematopoietic stem cell in-vitro culture system containing polymer micro-nano spheres and application ) 是由 钱鹏旭 王琪炜 韩颖丽 于 2019-11-07 设计创作，主要内容包括：本发明提供一种含聚合物微纳小球的造血干细胞体外培养体系及应用。所述体系包含聚苯乙烯微纳小球(C<Sub>8</Sub>H<Sub>8</Sub>)n、组合物和StemSpan<Sup>TM</Sup>无血清培养基。所述聚合物微纳小球是粒径范围在25nm-10μm内的聚苯乙烯小球,该小球以一定剂量添加于小鼠造血干细胞培养体系中,能够实现在10天内对LSK(Lin<Sup>-</Sup>Sca1<Sup>+</Sup>c-Kit<Sup>+</Sup>)和SLAM HSC(Lin<Sup>-</Sup>Sca1<Sup>+</Sup>c-Kit<Sup>+</Sup>CD150<Sup>+</Sup>CD48<Sup>-</Sup>)的大量扩增,且无毒副作用。本发明聚合物微纳小球对造血干细胞体外培养、扩增及功能的促进作用,对于造血干细胞移植、体外扩增等临床及科研领域具有极大的应用潜力。(The invention provides an in vitro hematopoietic stem cell culture system containing polymer micro-nano spheres and application thereof. The system comprises polystyrene micro-nano spheres (C) 8 H 8 ) n, compositions and StemBan TM And (3) serum-free culture medium. The polymer micro-nano spheres are polystyrene spheres with the particle size range of 25nm-10 mu m, and the spheres are added into a mouse hematopoietic stem cell culture system in a certain dose, so that the LSK (Lin) can be treated within 10 days ‑ Sca1 + c‑Kit + ) And SLAM HSC (Lin) ‑ Sca1 + c‑Kit + CD150 + CD48 ‑ ) The amplification is carried out in a large amount, and no toxic or side effect exists. The polymer micro-nano spheres have the promotion effects on the in-vitro culture, the amplification and the functions of hematopoietic stem cells, and have great application potential in the clinical and scientific research fields of hematopoietic stem cell transplantation, in-vitro amplification and the like.)

1. The hematopoietic stem cell in-vitro culture system containing polymer micro-nano spheres is characterized by comprising polystyrene micro-nano spheres, a composition and StemSpan^TMThe serum-free culture medium consists of polystyrene micro-nano spheres with the particle size range of 50nm to 10 mu m, and the composition consists of the following components: stem cell growth factor, thrombopoietin, heparin.

2. The system for in vitro culture of hematopoietic stem cells containing polymer micro-nano beads according to claim 1, wherein the polystyrene micro-nano bead dispersion medium is ultrapure water, the storage condition is room temperature drying, the particle size range is 50nm-10 μm, and the Zeta potential range is-0.064 mV to-0.306 mV.

3. The system for in vitro culture of hematopoietic stem cells containing polymer micro-nano beads according to claim 1, wherein the composition comprises the following components in addition concentration: 10ng/mL stem cell growth factor, 20ng/mL thrombopoietin, 10. mu.g/mL heparin.

4. The system for in vitro culture of hematopoietic stem cells containing polymer micro-nano beads according to claim 1, wherein the molecular formula of the polystyrene micro-nano beads is C₈H₈) n, wherein the polymerization degree n is 500-Is 0.5-5 mug/mL.

5. The system for in vitro culture of hematopoietic stem cells comprising polymeric micro-nano-beads according to claim 1, wherein the composition is mixed with StemSpan^TMSerum-free medium was separated in different containers and stored at-80 ℃ and polystyrene microspheres were prepared in IMDM medium and used at 4 ℃ within a week.

6. The in vitro culture system of claim 1, wherein the in vitro culture system is applied to the ex vivo high-efficiency amplification culture of mouse hematopoietic stem cells.

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an in vitro hematopoietic stem cell culture system containing polymer micro-nano microspheres and a method for efficiently amplifying hematopoietic stem cells in vitro by using the polymer micro-nano microspheres.

Background

Stem cells (Stem cells) are a class of cells with self-renewal capacity and multiple differentiation potential, a property that makes them an indispensable source of seed cells in the fields of regenerative medicine and tissue function. Stem cells are also the best candidates for cell therapy in a number of diseases, such as hematological diseases, neurological injuries, tumors, heart diseases, etc., and thus are of great significance in the clinical field for stem cell research.

Hematopoietic Stem Cells (HSC) are predominantly present in the bone marrow and have the ability to self-renew and give rise to all Hematopoietic adult Cell lineages. Therefore, hematopoietic stem cell transplantation has wide application prospects in the treatment of various hematological malignancies, such as leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome and the like. Clinical approaches to hematopoietic stem cells include bone marrow, cord blood and mobilized peripheral blood, however the limited source of hematopoietic stem cells limits their widespread use in clinical therapy. For example, CD34 in a single cord blood aliquot⁺An absolute insufficient number of hematopoietic progenitor cells (HSPCs) makes it difficult to meet the cell mass required for transplantation in adult or heavier child patients; bone marrow collection has been essentially eliminated due to the great trauma, and the bone marrow and mobilized peripheral blood-derived hematopoietic stem cells must search for HLA-matched donors, which also adds difficulty and burden to clinical treatment.

If the number of hematopoietic stem cells in the graft can be increased to promote the reconstruction of the hematopoietic system after the transplantation, the success rate and the application of the hematopoietic stem cell transplantation are inevitably increased. Therefore, there is a great need to search for and develop new methods for the in vitro expansion of hematopoietic stem cells, and there is a need for improvement of the current methods for promoting the in vitro expansion of hematopoietic stem cells.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an in vitro hematopoietic stem cell culture system containing polymer micro-nano microspheres, which comprises polystyrene micro-nano microspheres (C)₈H₈) n (degree of polymerization n is 500-^TMThe serum-free culture medium is characterized in that the polystyrene micro-nano spheres are polymer spheres synthesized by taking styrene as a monomer, the particle size range of the polystyrene micro-nano spheres is between 50nm and 10 mu m, and the composition consists of the following components: stem Cell growth Factor (SCF), Thrombopoietin (TPO), heparin, StemSpan^TMSerum-free medium was purchased from stem cell Technologies Inc, under # 09650. The polystyrene micro-nano small sphere dispersion medium is ultrapure water, the storage condition is room temperature drying, the particle size range is 50nm-10 mu m, the Zeta potential range is-0.064-0.306mV。

Compositions and StemSpan^TMSerum-free medium was separated into different containers and stored at-80 ℃. The polystyrene microspheres were prepared in IMDM medium and placed at 4 ℃ for use within a week.

The addition concentration of each component in the composition is as follows: SCF (10ng/mL), TPO (20ng/mL), heparin (10. mu.g/mL).

The addition concentration of the polystyrene micro-nano spheres is 0.5-20 mu g/mL. The addition concentration is preferably 1. mu.g/mL.

The invention also aims to provide the application of the in vitro culture system in the in vitro efficient amplification culture of the mouse hematopoietic stem cells. The method is an in vitro culture method for efficiently amplifying the mouse bone marrow hematopoietic stem cells, and the promotion effect of adding polystyrene micro-nano microspheres with different concentrations and particle sizes on the number and functions of the hematopoietic stem cells is tested by adopting a CCK-8 method, a flow cytometry analysis technology and a clone formation experiment.

The in vitro amplification culture is realized by the following method:

(1) the culture system of the polystyrene micro-nano spheres for the in-vitro amplification of the mouse hematopoietic stem cells comprises the following steps: isolation of mouse bone marrow Primary cells to StemBan supplemented with composition SCF (10ng/mL), TPO (20ng/mL), heparin (10. mu.g/mL)^TMCulturing in serum-free culture medium, adding polystyrene micro-nano spheres with the concentration of 0.5-20 μ g/mL and the particle size range of 50nm-10 μm, and testing the expansion effect of the hematopoietic stem cells after continuously culturing for 7-14 days;

(2) the CCK-8 method is used for determining the proliferation promoting effect of the polystyrene micro-nano spheres with different concentrations on the bone marrow cells of the mouse: adding the polystyrene micro-nano spheres with the concentration of 0.5-20 mug/mL, and testing the proliferation effect after continuously culturing, wherein the result shows that the addition concentration of the polystyrene micro-nano spheres with the concentration of 0.5-5 mug/mL can obviously promote the proliferation of the bone marrow cells of the mouse;

(3) detecting the amplification effect of the polystyrene micro-nano spheres with different particle sizes on the hematopoietic stem cells in the bone marrow cells of the mice by using a flow cytometry analysis technology: adding polystyrene micro-nano spheres with the particle size range of 50nm to 10 mu m, wherein the concentration is 1 mu g/mL; continuously culturing, collecting cells, dyeing by using a hematopoietic stem cell marker logistic antibody, and analyzing the proportion and the number change of the hematopoietic stem cells on a machine, wherein the result shows that the addition concentration of polystyrene micro-nano spheres of 50nm, 100nm, 500nm, 2 mu m, 5 mu m and 10 mu m can obviously improve the proportion and the number of the hematopoietic stem cells in the bone marrow cells of mice;

(4) the cloning formation experiment is applied to detect the effect of the polystyrene micro-nano spheres with different particle sizes on the in vitro functions of the mouse hematopoietic stem cells: adding polystyrene micro-nano spheres with the particle sizes of 50nm, 500nm and 5 mu m, wherein the concentration is 1 mu g/mL; continuously culturing, collecting cells, culturing in a semi-solid culture medium for 14 days, and observing the cloning formation number of the hematopoietic stem cells under a microscope, wherein the result shows that the addition concentration of the polystyrene micro-nano spheres of 50nm, 500nm and 5 mu m can obviously improve the in vitro function of the hematopoietic stem cells of the mice;

the invention discloses a promotion effect of polymer micro-nano spheres on in-vitro culture, amplification and functions of hematopoietic stem cells. The polymer micro-nano spheres are added into a mouse hematopoietic stem cell culture system in a certain dosage, and can realize LSK (Lin) within 10 days^-Sca1⁺c-Kit⁺) And SLAM HSC (Lin)^-Sca1⁺c-Kit⁺CD150⁺CD48^-) The amplification is carried out in a large amount, and no toxic or side effect exists. The invention is added with polystyrene micro-nano spheres and a composition (cytokine) simultaneously for in vitro culture of mouse hematopoietic stem cells, and the expansion effect on the hematopoietic stem cells is larger than that of the composition only added with the cytokine. The invention has great application potential in clinical and scientific research fields of hematopoietic stem cell transplantation, in-vitro amplification and the like.

Drawings

FIG. 1 shows the hydration particle size, the polydispersity index and the Zeta potential of a part of polystyrene micro-nano spheres which are characterized by a Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS);

FIG. 2 is a cell viability experiment used for determining the proliferation effect of polystyrene micro-nano spheres with different concentrations on mouse bone marrow cells; the using concentrations of the polystyrene micro-nano spheres are respectively 0.5, 1, 2, 5, 10 and 20 mu g/mL, and the culture time is respectively A: 3. b: 7. c: and 14 days.

FIG. 3 is a flow cytometry analysis technique for determining the expansion effect of polystyrene micro-nano spheres with different sizes on hematopoietic stem cells in mouse bone marrow cells, wherein the polystyrene micro-nano spheres are used at a concentration of 1 μ g/mL for 10 days, and are polystyrene microspheres with a control group, a particle size of 50nm, a particle size of 500nm and a particle size of 5 μm from left to right.

FIG. 4 is a graph showing the measurement of LSK (Lin) in mouse bone marrow cells by using flow cytometry analysis techniques on control, 50nm, 500nm and 5 μm polystyrene microspheres^-Sca1⁺c-Kit⁺) And SLAM HSC (Lin)^-Sca1⁺c-Kit⁺CD150⁺CD48^-) Statistics of the proportion of total cells. The use concentration of the polystyrene micro-nano spheres is 1 mu g/mL, the culture time is 10 days, and each group is repeated for three times.

FIG. 5 is a graph showing the measurement of LSK (Lin) in mouse bone marrow cells by using flow cytometry analysis techniques on control, 50nm, 500nm and 5 μm polystyrene microspheres^-Sca1⁺c-Kit⁺) And SLAM HSC (Lin)^-Sca1⁺c-Kit⁺CD150⁺CD48^-) Counting statistics of the total number. The use concentration of the polystyrene micro-nano spheres is 1 mu g/mL, the culture time is 10 days, and each group is repeated for three times.

FIG. 6 is a graph showing the effect of polystyrene micro-nano beads with different sizes on the in vitro differentiation function of mouse hematopoietic stem cells measured by a Colony-forming experiment (Colony-Formation Unit), wherein BFU-E, G, M, GM and GEMM respectively represent the clones forming different lineages of blood cells after the hematopoietic stem cells are differentiated.

Detailed Description

The invention is further described with reference to the accompanying drawings and examples.

The invention discloses a polymer micro-nano small sphere and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.