CN110804591B - Hematopoietic stem cell in-vitro culture system containing polymer micro-nano spheres and application - Google Patents

Hematopoietic stem cell in-vitro culture system containing polymer micro-nano spheres and application Download PDF

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CN110804591B
CN110804591B CN201911080272.6A CN201911080272A CN110804591B CN 110804591 B CN110804591 B CN 110804591B CN 201911080272 A CN201911080272 A CN 201911080272A CN 110804591 B CN110804591 B CN 110804591B
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钱鹏旭
王琪炜
韩颖丽
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Zhejiang University ZJU
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Abstract

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)8H8) n, compositions and StemBanTMAnd (3) serum-free culture medium. The polymer is micro-nanoThe pellet is polystyrene pellet with particle size of 25nm-10 μm, and can be added into mouse hematopoietic stem cell culture system at a certain dosage to 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 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.

Description

Hematopoietic stem cell in-vitro culture system containing polymer micro-nano spheres and application
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, StemSpanTMSerum-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, and the Zeta potential range is-0.064 to-0.306 mV.
Compositions and StemSpanTMSerum-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) polystyrene micro-nano small ball pair mouseCulture system for hematopoietic stem cell in vitro expansion: 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. Wherein the polystyrene is micro-nanoThe pellet was used at a concentration of 1. mu.g/mL for a 10-day incubation period, and each group was replicated 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.
Example 1
Detecting the relevant properties of the polystyrene micro-nano spheres: including bulk particle size (scanning electron microscope), hydrodynamic size and Zeta potential (particle size potential analyzer). The results are shown in FIG. 1 and Table 1.
TABLE 1 characterization results of polystyrene micro-nano spheres
Figure BDA0002263745390000031
Figure BDA0002263745390000041
Example 2
SCF (purchased from Shanghai Puxin Biotechnology Co., Ltd., product No. 122-01), TPO (purchased from Shanghai Puxin Biotechnology Co., Ltd., product No. 122-06) and heparin (purchased from Sigma, product No. H3149) were prepared according to 1000 times of the use concentration, and were dispensed into centrifuge tubes, each tube was 40. mu.L, and stored at-80 ℃ for further use.
StemSpanTMSerum-free medium (purchased from StemCell, cat # 09650) was dispensed into centrifuge tubes, 40mL per tube and stored at-80 ℃ until use.
Split-charged StemBan before media preparationTMSerum-free medium was thawed overnight at 4 ℃ and the composition was added to StemBan at 1/1000 volumesTMAnd (5) serum-free culture medium, and uniformly mixing for later use.
Example 3
Taking a C57BL/6 mouse of 6-8 weeks old, killing the neck after carbon dioxide asphyxiation anesthesia, and soaking the whole body in 75% alcohol for 5min for disinfection. The skin of the leg is cut from the side of the lower leg, and the skin is cut at the joint of the femur and the hip bone and the ankle respectively to expose the two ends of the leg bone. The ligaments are cut at the knee joint and femur joint, the femur and tibia are separated, and the muscle and connective tissue on the bone are cleaned. The two ends of the bone were cut, the bone marrow was flushed out with a 3mL syringe that was aspirated with pre-cooled buffer (PBS + 2% fetal bovine serum), repeatedly blown into a single cell suspension, the cells were filtered with a 300 mesh cell screen into a centrifuge tube, and centrifuged at 4 ℃ at 1500rpm for 5 min. The supernatant was aspirated off, 1mL of sterile red blood lysate (8.3g ammonium chloride, 1g sodium bicarbonate, 200. mu. L0.5M EDTA, 1L ultrapure water, 0.22 μm filter sterilized) was added to each leg (femur + tibia), red blood cells were lysed for 3min at room temperature, and 2 volumes of pre-cooled buffer were added to stop lysis. The cells were filtered through a 300 mesh cell screen in a centrifuge tube and centrifuged at 1500rpm at 4 ℃ for 5 min. Adding 1mL of the culture system prepared in example 1, resuspending and blowing to obtain a single cell suspension, counting, and adjusting the cell number to 1-2X 106PermL, 100. mu.L/well of a U-bottom 96-well plate (purchased from BD falcon, cat. No. 353077) was inoculated and cultured in a cell culture chamber at 37 ℃ for 48 hours.
Example 4
When the cells are gathered in a 96-well plate to form macroscopic beads, the polystyrene micro-nano beads with different particle sizes and different concentrations are added, and after incubation for different time, the cell activity is measured by adopting a CCK-8 method, and the result is shown in figure 2.
Grouping experiments:
particle size: 50nm, 100nm, 500nm, 2 μm, 5 μm and 10 μm;
concentration: negative control (0), 0.5, 1, 2, 5, 10, 20. mu.g/mL;
co-incubation time: 3. 7, 14 d;
and taking the average value of each group of 3 multiple wells as the final result.
The polystyrene micro-nano spheres are uniformly mixed with the culture system prepared in the example 1 according to twice of the corresponding working concentration, 100 mu L of mixed liquid is added into each hole, the cell mass is gently blown away, and the polystyrene micro-nano spheres are the working concentration.
Half the change of culture solution every 3 days, namely, sucking 100 mu L of culture solution in each hole, adding 100 mu L of mixed solution containing the working concentration of the polystyrene micro-nano spheres, and gently blowing away cell clusters.
At the corresponding detection time point, 20 μ L of CCK-8 reagent is added into each well, the cell mass is blown off slightly, the cell is incubated in a cell culture box at 37 ℃ for 1-2h, and the absorbance of each well at 450nm is measured by a microplate reader.
Cell viability calculation method:
cell viability ═ OD450Experimental group-OD450blank)/(OD450Negative control-OD450Blank) x 100%
Supplementary explanation:
blank control (background) is medium plus CCK 8; if the drugs have color interference, the drugs with different concentrations are added as the ground color to be deducted respectively.
Example 5
Primary mouse bone marrow cells were prepared as described in example 2, with an initial hematopoietic stem cell count of 50 per well, and when cells aggregated in a 96-well plate to form macroscopic beads, polystyrene micro-nano beads of different particle sizes were added, and after incubation for 10 days, the ratio and number of LSK and SLAM HSC were determined by flow cytometry. The results are shown in FIGS. 3 to 5.
Grouping experiments:
particle size: 50nm, 100nm, 500nm, 2 μm, 5 μm and 10 μm;
concentration: negative control (0), 1. mu.g/mL;
each group required 6-12 wells, and the experiment was repeated 3 times, averaged to the final result.
Method for adding polystyrene micro-nano beads and replacing culture solution according to embodiment 3, after incubating for 10d, each group of cells is collected, after counting, the cells are resuspended by 100 μ L of precooled PBS + 2% FBS, and then placed on ice for standby.
Every 10 th6The cells mixed the flow antibodies in the following proportions:
Figure BDA0002263745390000051
the antibody mixture is added according to the proportion of 1063.6 μ L of cells was added to the cell suspension and incubated on ice for 30-45min in the dark.
Isotype Control setting (Isotype Control, for drawing door)
ISO1:ISO-PE-Cy5 1μL；
ISO2:Lin-PE-Cy5 1μL、ISO-PE-Cy7 0.5μL、ISO-APC 0.5μL；
ISO3:Lin-PE-Cy5 1μL、Sca1-PE-Cy7 0.5μL、c-Kit-APC 0.5μL、ISO-PE 0.5μL、ISO-eF4500.5μL。
Single-standard pipe: about 50000 cells per tube were added with 0.2. mu.L of Lin-PE-Cy5, Sca1-PE-Cy7, c-Kit-APC, CD150-PE, CD48-eF450 antibodies, respectively, for adjusting color compensation.
After the incubation is finished, the cell is centrifuged at 1500rpm at 4 ℃ for 5min to remove unbound antibodies, and the LSK (Lin) is detected by a flow machine (Beckman, DxFLEX)-Sca1+c-Kit+) And SLAM HSC (Lin)-Sca1+c-Kit+CD150+CD48-) The difference in the ratio and the number.
A flow detection step: the specific software of the flow cytometry analyzers of different manufacturers and models has different use methods, but the detection steps are similar roughly. Taking Beckman DxFLEX as an example, firstly opening a software drawing scatter diagram, adjusting the voltage of each detection channel by an upper blank tube (not combined with an antibody), sequentially loading a single standard tube for adjustment and compensation, loading a same-type control tube for drawing a gate, and finally loading each experimental group for detection.
Example 6
Preparing primary mouse bone marrow cells by the method of example 2, adding polystyrene micro-nano beads with different particle sizes when the cells are aggregated in a 96-well plate to form macroscopic beads, incubating for 7d, and determining the in-vitro differentiation function of hematopoietic stem cells by a CFU method. The results are shown in FIG. 6.
Cell counts were collected and each group of 30000 cells was resuspended in 300. mu.L of the culture system prepared in example 1. A5 mL syringe was fitted with a 16G blunt tip needle, the cell suspension aspirated, and added to 3mL of semi-solid medium (StemCell, MethoCult)TMGF M3434).
To each well of the 6-well plate, 1mL of semi-solid medium mixed with cells, about 10000 cells per well, was added, and the mixture was placed in a wet box and cultured in a cell incubator at 37 ℃. Clone formation was observed under a microscope every 2d, BFU-E was formed (about 10-12d), counted after 2d, counted and stored by photographing.
Different clone count criteria:
BFU-E (Burst-forming unit-erythroid): the cells are small and irregular, the clone has no core and is yellow or red;
CFU-M (Colony-forming unit-macro): cells are large and gray oval;
CFU-G (Colony-developing unit-journal): the cells are round and bright, and are smaller and uniform than CFU-M;
GM: the clone has a core and is formed by mixing G and M cells;
GEMM (granulocyte, erythroid, macrohage, megakaryocyte): consists of four cells, namely granulocytes, erythroblasts, macrophages and megakaryocytes, the cloning is large, the number of the cells is usually more than 500, and the core is black.

Claims (3)

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 StemSpanTMThe 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, liverA peptide; 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, and the Zeta potential range is-0.064 to-0.306 mV;
the addition concentration of each component in the composition is as follows: 10ng/mL stem cell growth factor, 20ng/mL thrombopoietin, 10 μ g/mL heparin;
the molecular formula of the polystyrene micro-nano bead is (C)8H8) n, wherein the polymerization degree n is 500-.
2. 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 StemSpanTMSerum-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.
3. The in vitro culture system of claim 1, wherein the in vitro culture system is used for the in vitro efficient amplification culture of mouse hematopoietic stem cells, and the application is that the in vitro culture system can efficiently amplify the mouse hematopoietic stem cells in the in vitro culture.
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