CN110628718A - Stem cell amplification culture medium and stem cell culture method - Google Patents
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Abstract
The invention relates to a stem cell amplification culture medium and a stem cell culture method, which comprises a basal culture medium, stem cell factors, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6, wherein the final concentration of the stem cell factors is 90 ng/mL-110 ng/mL, the final concentration of the thrombopoietin is 10 ng/mL-30 ng/mL, the final concentration of the FMS-like tyrosine kinase 3 ligand is 90 ng/mL-110 ng/mL, and the final concentration of the interleukin-6 is 10 ng/mL-30 ng/mL. The umbilical cord blood hematopoietic stem cells cultured by the culture medium for the expanded stem cells can show obvious cell expansion effect, the CD34+ surface on the cell surface is also obviously improved, the cell state is more stable during cell expansion, the multidirectional differentiation capability of the cells is maintained, and the culture medium is an economic and efficient culture system.
Description
Technical Field
The invention belongs to the technical field of bioengineering and biomedicine, and particularly relates to a stem cell amplification culture medium and a stem cell culture method.
Background
Hematopoietic stem cells are a group of cells having self-renewal and multipotential differentiation potential, which can proliferate and differentiate into various lineages of cells in the hematopoietic system while maintaining the relative stability of stem cells, and play a significant role in treating blood diseases and immunodeficiency diseases because they have the function of reconstituting the hematopoietic system and immune system, and hematopoietic stem cell transplantation is currently the most effective method for treating the above-mentioned diseases. The cord blood is another important hematopoietic stem cell source except bone marrow and peripheral blood stem cells, and has the advantages of sufficient source, high proliferation and amplification potential, no influence on donor collection, small pollution chance of cytomegalovirus and EB virus, lower immune rejection rate after transplantation than that of bone marrow transplantation of adult donors, and capacity of providing HLA matched stem cells required for transplantation through establishing cord blood bank at any time. Although cord blood hematopoietic stem cells have the above advantages, the limited number of cells in one cord blood greatly limits the clinical application of cord blood hematopoietic stem cells. The in vitro expansion of the hematopoietic stem cells is an important method for solving the defect of the cord blood, so the establishment and optimization of the in vitro expansion scheme of the hematopoietic stem cells of the cord blood are particularly important, and the foundation is expected to be laid for the clinical wide application.
In the previous studies, a large number of attempts have been made to expand hematopoietic stem cells in cord blood, but these have not produced satisfactory results. The traditional method adopts the cell factors in blood to culture cord blood stem cells, but the cells are seriously differentiated, the purity is obviously reduced, and the stem cell transplantation can not be clinically carried out. Later studies were slowly shifted to study some signaling molecules, ligands, etc. in the microenvironment of bone marrow hematopoietic stem cells to effectively expand CD34+ hematopoietic stem/progenitor cells, and to avoid the stem cell differentiation aging and self-renewal inhibition problems caused by exogenous factors. There are also studies to moderately expand hematopoietic stem cells by adding protein molecules such as DLL1, DSL1, etc. to stimulate signal molecules in the hematopoietic stem cell microenvironment. However, the effect of the technology is only slight improvement, and the problem of efficiently amplifying cord blood CD34+ cells is still faced.
Recently, there have been related studies to introduce various exogenous cytokines into the culture medium, which provide a suitable environment for the proliferation and growth of stem cells through synergistic effects, provide targeted stem cell signaling stimulation, and maintain the corresponding cell phenotype. The cytokines commonly used in the research include stem cell factor, monocyte chemotactic factor and interleukin-1 beta, but the expansion multiple of the stem cells is low, the used amount is not uniform, and the purity of the hematopoietic stem cells is not high.
Disclosure of Invention
Based on the method, the culture medium for expanding the stem cells is provided, and the culture medium can obviously improve the expansion multiple of CD34+ hematopoietic stem cells and maintain the stability of the CD34+ hematopoietic stem cells.
The specific technical scheme is as follows:
a stem cell expansion medium comprising a basal medium and cytokines added in the basal medium, the cytokines comprising stem cell factors, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6, wherein,
the final concentration of the stem cell factor is 90 ng/mL-110 ng/mL;
the final concentration of the thrombopoietin is 10 ng/mL-30 ng/mL;
the final concentration of the FMS-like tyrosine kinase 3 ligand is 90 ng/mL-110 ng/mL;
the final concentration of the interleukin-6 is 10 ng/mL-30 ng/mL.
In one embodiment, the basal medium is serum-free IMDM or StemSpan SFEMII medium.
In one embodiment, the method further comprises adding an aromatic hydrocarbon receptor antagonist in the basal medium.
In one embodiment, the aromatic hydrocarbon receptor antagonist is a small molecule compound StemRegenin1, and the final concentration of StemRegenin1 is 0.5-2 μ M.
In one of the embodiments, wherein,
the final concentration of the stem cell factor is 100 ng/mL;
the final concentration of thrombopoietin is 20 ng/mL;
the final concentration of the FMS-like tyrosine kinase 3 ligand is 100 ng/mL;
the final concentration of the interleukin-6 is 20 ng/mL.
Meanwhile, the invention also provides a method for culturing stem cells by using the stem cell amplification culture medium, which comprises the following steps:
(1) centrifuging the blood to obtain mononuclear cells;
(2) sorting the mononuclear cells obtained in the step (1) by magnetic beads to obtain stem cells;
(3) and (3) culturing the stem cells in the step (2) by using the stem cell expansion medium.
In one embodiment, the stem cells are CD34+ hematopoietic stem cells.
In one embodiment, the blood is derived from cord blood, bone marrow, or peripheral blood.
In one embodiment, the centrifugation operation in step (1) is Ficoll lymph separation liquid density gradient centrifugation.
In one embodiment, the concentration of the stem cells cultured in the step (3) is 1X 105Individual cell/mL-2X 105Individual cells/mL.
The stem cell amplification culture medium and the stem cell culture method have the following advantages and beneficial effects:
the inventor of the invention discovers through a large amount of experimental researches that cord blood hematopoietic stem cells can be obviously amplified by reasonably compounding stem cell factors, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6 and other cytokines into a basic culture medium, and synergistic effects are generated among the cytokines. Compared with the traditional stem cell culture medium, the use concentration of thrombopoietin and interleukin-6 is lower, but the umbilical cord blood hematopoietic stem cells can present more obvious cell amplification effect, the expression rate of CD34+ is also obviously improved, the aging caused by obvious cell differentiation does not occur during the amplification, and the prepared stem cell amplification culture medium is an economic and efficient culture system.
Drawings
FIG. 1 is a graph of (A) cell number and (B) amplification fold at different time points in example 7;
FIG. 2 is a graph of the morphology of the cells of example 7 at various time points;
FIG. 3 is a graph showing the expression of CD34+ on the cell surface in example 7.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a stem cell amplification culture medium, which comprises a basic culture medium and cytokines added in the basic culture medium, wherein the cytokines comprise stem cell factors, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6, wherein,
the final concentration of the stem cell factor is 90 ng/mL-110 ng/mL;
the final concentration of thrombopoietin is 10 ng/mL-30 ng/mL;
the final concentration of the FMS-like tyrosine kinase 3 ligand is 90 ng/mL-110 ng/mL;
the final concentration of interleukin-6 is 10 ng/mL-30 ng/mL.
In a specific example, the basal medium is serum-free IMDM or StemSpan sfemi medium, preferably serum-free StemSpan sfemi medium. The serum-free StemBan SFEMII culture medium can provide good growth conditions for the stem cells, and the basic culture medium can further improve the expansion efficiency of the stem cells to a certain extent.
In one particular example, the stem cell expansion medium further comprises an aromatic hydrocarbon receptor antagonist added to the basal medium.
Further, the aromatic hydrocarbon receptor antagonist is a small molecular compound StemRegenin 1. The addition of the small molecule StemRegenin1 can promote the mass amplification and self-renewal of the human hematopoietic stem cells.
Preferably, the final concentration of StemRegenin1 is 0.5. mu.M to 2. mu.M.
In one particular example, the first and second sensors, among others,
the final concentration of stem cell factor is preferably 100 ng/mL;
the final concentration of thrombopoietin is preferably 20 ng/mL;
the final concentration of FMS-like tyrosine kinase 3 ligand is preferably 100 ng/mL;
the final concentration of interleukin-6 is preferably 20 ng/mL.
Among the above cytokines, stem cell factor can function through c-kit, a tyrosine receptor anchored and expressed on the surface of hematopoietic stem cells, and the expression of c-kit will cause the expansion of hematopoietic stem cells. Thrombopoietin can stimulate the proliferation and differentiation of megakaryocytes, regulate and maintain the proliferation and self-renewal of early hematopoietic stem cells. The FMS-like tyrosine kinase 3 ligand transmits signals to cells through the combination of specific TKRs, so that the proliferation of hematopoietic stem cells is induced, the apoptosis of the hematopoietic stem cells is obviously reduced, and the in-vitro viability of the hematopoietic stem cells is improved and enhanced.
The invention also provides a method for culturing stem cells by using the stem cell amplification culture medium, which comprises the following steps:
(1) centrifuging the blood to obtain mononuclear cells;
(2) sorting the mononuclear cells obtained in the step (1) by magnetic beads to obtain stem cells;
(3) and (3) culturing the stem cells in the step (2) by using the stem cell expansion medium.
In one specific example, the stem cells are CD34+ hematopoietic stem cells.
Preferably, the blood is derived from umbilical cord blood, bone marrow or peripheral blood.
In one particular example, the centrifugation operation is Ficoll lymph separation liquid density gradient centrifugation.
In one specific example, the concentration of stem cells cultured in the stem cell expansion medium is 1X 105Individual cell/mL-2X 105Individual cells/mL.
The stem cell expansion medium and the stem cell culture method of the present invention will be described in further detail below with reference to specific examples.
Serum-free StemBan SFEMII basal medium used in the following examples, Stemcell, manufacturer, shelf number 09655; stem cell factor, PeproTech as manufacturer, 300-07 of shelf number; thrombopoietin, PeproTech, manufacturer, shelf number 300-18; FMS-like tyrosine kinase 3 ligand, PeproTech as manufacturer, 300-19 of shelf number; interleukin-6, PeproTech, Protech, shelf number 200-06; StemRegenin1, Cellagen Tech as a manufacturer, and C7710 as a shelf number; ficoll lymph isolate was produced as Stemcell, cat # 07851.
It is understood that in other embodiments, the basal medium and cytokines used are not limited thereto.
The following examples and comparative examples used the following cell purification procedure:
1. extraction of cord blood mononuclear cells
(1) Mixing freshly obtained umbilical cord blood and PBS in a volume ratio of 1:1, slowly adding the umbilical cord blood and PBS into Ficoll lymph separation liquid containing 0.5 times of volume, and paying attention to not damage a separation interface;
(2) and (3) carrying out gradient centrifugation on the cord blood mixed solution for 30 minutes at the centrifugation speed of 800g/min, after layering, carefully sucking the leucocyte into a new centrifugal tube by using a suction tube, washing the cell for 3 times by using PBS, and cracking red to obtain the mononuclear cell.
2. Purification of cord blood CD34+ hematopoietic Stem cells
Counting the separated mononuclear cells at every 10 th8Adding 300 μ L of magnetic bead sorting buffer, 100 μ L of FcR blocking agent and 100 μ L of CD34 magnetic beads into each cell, mixing the cells, and incubating at 4 deg.C in the dark for 30 minThen adding 10mL of magnetic bead sorting buffer solution for washing, centrifuging for 10 minutes, removing supernatant, adding 500 μ L of magnetic bead sorting buffer solution for resuspending cells to prepare for magnetic bead sorting, wetting a separation column with 500 μ L of magnetic bead sorting buffer solution in advance, placing a separation column with a proper model on a magnetic bead sorter, then slowly adding the cell suspension into the separation column, finally washing the column with buffer solution for 3 times, taking down the separation column, and washing the cells in the separation column into a centrifugal tube with 1mL of buffer solution, namely the CD34+ hematopoietic stem cells.
Example 1
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a culture medium containing the above-mentioned cytokines at final concentrations of 90ng/mL, 20ng/mL, 100ng/mL, 20ng/mL, 1. mu.M, respectively, of expanded stem cells, which was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Example 2
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the above-mentioned cytokines at final concentrations of 100ng/mL, 20ng/mL, 90ng/mL, 20ng/mL, 1. mu.M, respectively, of expanded stem cells, which was kept at 4 ℃ for future use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under culture conditions500 μ L of fresh expanded stem cell medium was supplemented every 2 days depending on the cell culture status.
Example 3
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the above-mentioned cytokines at final concentrations of 100ng/mL, 30ng/mL, 100ng/mL, 20ng/mL, 1. mu.M, respectively, of expanded stem cells, which was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Example 4
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the above-mentioned cytokines at final concentrations of 100ng/mL, 20ng/mL, 100ng/mL, 30ng/mL, 1. mu.M, respectively, of expanded stem cells, which was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Example 5
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the above-mentioned cytokines at final concentrations of 100ng/mL, 20ng/mL, 0.5. mu.M, respectively, of expanded stem cells, which was left at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Example 6
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a culture medium containing the expanded stem cells at the final cytokine concentrations of 110ng/mL, 30ng/mL, and 2. mu.M, respectively, and the culture medium was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Example 7
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin-6, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the expanded stem cells at the final cytokine concentrations of 100ng/mL, 20ng/mL, and 1. mu.M, respectively, and the medium was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Comparative example 1
1. Preparation of culture medium for expanding stem cells
Adding the cell factor stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6 into a serum-free StemBan SFEMII basal medium to obtain a culture medium containing the amplified stem cells with the final concentrations of the cell factors of 100ng/mL, 20ng/mL, 100ng/mL and 20ng/mL respectively, and placing the culture medium at 4 ℃ for later use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Comparative example 2
1. Preparation of culture medium for expanding stem cells
Cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, StemRegenin1 were added to serum-free StemBan SFEMII basal medium to obtain a medium containing the expanded stem cells at the final cytokine concentrations of 100ng/mL, 20ng/mL, 100ng/mL, and 1. mu.M, respectively, and the medium was kept at 4 ℃ for further use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Comparative example 3
1. Preparation of culture medium for expanding stem cells
Adding cytokine stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6 into IMDM basal medium of 10% FBS to obtain culture medium containing amplified stem cells with final concentrations of the above cytokines of 100ng/mL, 50ng/mL, 100ng/mL and 50ng/mL respectively, and standing at 4 deg.C for use.
2. Cell culture
CD34+ hematopoietic stem cells were seeded at a density of 1X 10 onto 35mm cell culture dishes5cells/mL, 2mL of the above expanded stem cell medium was added, and 5% CO was added at 37 ℃2Culturing under the culture condition, and supplementing 500 mu L of fresh expanded stem cell culture medium every 2 days according to the cell culture state.
Comparative example 4
No cytokine was added to serum-free StemBan SFEMII basal medium and other experimental conditions were the same as in comparative example 3.
Cell expansion evaluation test
Trypan blue staining method counts cells: counting the cell amount and the survival rate of the cells in the examples 1 to 7 and the comparative examples 1 to 4 by using a trypan blue staining method in1 to 7 days respectively, fully mixing 10 mu L of suspension and 10 mu L of trypan blue after the cells are mixed uniformly, adding 10 mu L of the suspension into a counter, and performing machine detection to obtain the cell amount and the survival rate. Meanwhile, the morphological change of the cells is recorded by photographing for 1-7 days of cell culture.
Flow cytometry analysis of stem cell CD34+ expression
Collecting cell suspension, washing with PBS 1 time, counting cells, and collecting 5 × 105The cells were used for flow cytometry, the supernatant was discarded after centrifugation of the cells, 100. mu.L of PBS and 5. mu.L of CD34+ flow antibody were added, incubation was carried out at 4 ℃ for 30 minutes, then washing was carried out 2 times with PBS, 500. mu.L of PBS was added to resuspend the cells, and then the cells were tested on the machine.
The number of cells, the amplification factor and the expression rate of CD34+ after one week of culture of the cells of examples 1 to 7 and comparative examples 1 to 4 are shown in Table 1.
TABLE 1
As can be seen from the results of examples 1 to 7 and comparative examples 1 to 4 in Table 1, the cord blood CD34+ hematopoietic stem cells cultured by the expanded stem cell culture medium obtained by the method of the invention can achieve 106-fold expansion, wherein the expression rate of CD34+ is as high as 88.6%, and the dosage of the CD34+ is less than that of the conventional thrombopoietin and interleukin-6, but the expansion fold is obviously improved. Comparing example 7 with comparative example 1, it is clear that StemRegenin1 has a large effect on the cell expansion fold and the expression rate of CD34+ in stem cells, and comparing example 7 with comparative example 2, it is clear that interleukin-6 has a large effect on the proliferation of cells, but has a small effect on the expression rate of CD34 +. The results of comparative example 3 show that the cytokine prepared by the traditional method at high concentration does not significantly improve the expansion fold of the stem cells, and the expression rate of CD34+ is not high. From FIG. 1, it is reflected that the cord blood hematopoietic stem cells cultured by the amplification medium described in example 7 of the present invention exhibited an accelerated amplification process starting on the third day, and the cell density was sparser and denser on days 3, 5 and 7 during the culture process from the scanning electron microscope (FIG. 2) to the first two days, and the morphology of the cells was not changed during the whole process, and quantitative analysis by a flow cytometer showed that the expression rate of CD34+ in the stem cells was 88.606% (FIG. 3).
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A stem cell expansion medium comprising a basal medium and cytokines added to the basal medium, the cytokines comprising stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand and interleukin-6, wherein,
the final concentration of the stem cell factor is 90 ng/mL-110 ng/mL;
the final concentration of the thrombopoietin is 10 ng/mL-30 ng/mL;
the final concentration of the FMS-like tyrosine kinase 3 ligand is 90 ng/mL-110 ng/mL;
the final concentration of the interleukin-6 is 10 ng/mL-30 ng/mL.
2. The stem cell expansion medium of claim 1, wherein the basal medium is serum-free IMDM or StemSpan sfemi medium.
3. The stem cell expansion medium of claim 1, further comprising an aromatic hydrocarbon receptor antagonist added to the basal medium.
4. The stem cell expansion medium according to claim 3, wherein the aromatic hydrocarbon receptor antagonist is compound StemRegen 1, and the final concentration of the compound StemRegen 1 is 0.5 μ M to 2 μ M.
5. The stem cell expansion medium according to claim 1, wherein,
the final concentration of the stem cell factor is 100 ng/mL;
the final concentration of thrombopoietin is 20 ng/mL;
the final concentration of the FMS-like tyrosine kinase 3 ligand is 100 ng/mL;
the final concentration of the interleukin-6 is 20 ng/mL.
6. A method of culturing stem cells in a stem cell expansion medium according to any one of claims 1 to 5, comprising the steps of:
(1) centrifuging the blood to obtain mononuclear cells;
(2) sorting the mononuclear cells obtained in the step (1) by magnetic beads to obtain stem cells;
(3) culturing the stem cells in the step (2) with the stem cell expansion medium.
7. The method for culturing stem cells according to the stem cell expansion medium of claim 6, wherein the stem cells are CD34+ hematopoietic stem cells.
8. The method for culturing stem cells according to the stem cell expansion medium of claim 6, wherein the blood is derived from umbilical cord blood, bone marrow, or peripheral blood.
9. The method for culturing stem cells according to the stem cell expansion medium of any one of claims 6 to 8, wherein the centrifugation operation in step (1) is Ficoll lymph separation liquid density gradient centrifugation.
10. The method for culturing stem cells according to any one of claims 6 to 8, wherein the concentration of the stem cells cultured in the step (3) is 1X 105Individual cell/mL-2X 105Individual cells/mL.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112080469A (en) * | 2020-09-02 | 2020-12-15 | 暨南大学 | Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro |
| CN112553159A (en) * | 2020-12-10 | 2021-03-26 | 上海市东方医院（同济大学附属东方医院） | 3D model for simulating bone marrow environment to culture hematopoietic stem cells and preparation method and application thereof |
| CN113502263A (en) * | 2021-06-09 | 2021-10-15 | 华南理工大学 | Differentiation promoting culture medium and method for promoting CD34 positive cells to differentiate into platelets |
| CN113881633A (en) * | 2021-12-06 | 2022-01-04 | 山东省齐鲁干细胞工程有限公司 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
| CN114196632A (en) * | 2021-11-02 | 2022-03-18 | 济南赛尔生物科技股份有限公司 | Culture medium for efficiently culturing hematopoietic stem cells in vitro |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080065A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof |
| CN105112374A (en) * | 2015-09-13 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Ex-vivo expansion culture medium of umbilical cord blood hematopoietic stem cells and application thereof |
| US20180163177A1 (en) * | 2016-12-11 | 2018-06-14 | Healthbanks Biotech Co. Ltd. | Serum-free culture medium and method for expanding hematopoietic stem cells |
| CN108265028A (en) * | 2016-12-30 | 2018-07-10 | 联亘生物科技（上海）有限公司 | For the cultivating system of amplifying candidate stem cell in vitro |
-
2019
- 2019-09-29 CN CN201910932605.7A patent/CN110628718A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080065A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof |
| CN105112374A (en) * | 2015-09-13 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Ex-vivo expansion culture medium of umbilical cord blood hematopoietic stem cells and application thereof |
| US20180163177A1 (en) * | 2016-12-11 | 2018-06-14 | Healthbanks Biotech Co. Ltd. | Serum-free culture medium and method for expanding hematopoietic stem cells |
| CN108265028A (en) * | 2016-12-30 | 2018-07-10 | 联亘生物科技（上海）有限公司 | For the cultivating system of amplifying candidate stem cell in vitro |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112080469A (en) * | 2020-09-02 | 2020-12-15 | 暨南大学 | Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro |
| CN112080469B (en) * | 2020-09-02 | 2022-03-25 | 暨南大学 | Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro |
| CN112553159A (en) * | 2020-12-10 | 2021-03-26 | 上海市东方医院（同济大学附属东方医院） | 3D model for simulating bone marrow environment to culture hematopoietic stem cells and preparation method and application thereof |
| CN114686432A (en) * | 2020-12-28 | 2022-07-01 | 苏州方舟生物科技有限公司 | Efficient amplification culture system for neutrophils and application thereof |
| CN114686432B (en) * | 2020-12-28 | 2024-02-02 | 苏州方舟生物科技有限公司 | Efficient amplification culture system for neutrophils and application thereof |
| CN113502263A (en) * | 2021-06-09 | 2021-10-15 | 华南理工大学 | Differentiation promoting culture medium and method for promoting CD34 positive cells to differentiate into platelets |
| CN113502263B (en) * | 2021-06-09 | 2023-05-23 | 华南理工大学 | Differentiation-promoting culture medium and method for promoting differentiation of CD34 positive cells into platelets |
| CN114196632A (en) * | 2021-11-02 | 2022-03-18 | 济南赛尔生物科技股份有限公司 | Culture medium for efficiently culturing hematopoietic stem cells in vitro |
| CN113881633A (en) * | 2021-12-06 | 2022-01-04 | 山东省齐鲁干细胞工程有限公司 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
| CN113881633B (en) * | 2021-12-06 | 2022-02-22 | 山东省齐鲁干细胞工程有限公司 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
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