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A kind of erythrocyte progenitor cell serum-free medium and method of using the same

A serum-free medium and progenitor cell technology, applied in cell culture active agent, culture process, animal cells, etc., can solve the problem of not involving the reconstruction of erythrocyte progenitor cells, maintaining stemness and limited quantity, and inability to expand erythrocyte progenitor cells, etc. problems, to achieve the effect of high clinical application and scientific research value

Active Publication Date: 2021-02-09
安徽中盛溯源生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] Recent studies have reported the reconstitution of non-T cell populations in peripheral blood, (Chou et al., 2011; Mack et al., 2011; Okita et al., 2013), but not the reconstitution of erythroid progenitors, since under normal conditions , the number of erythrocyte progenitor cells is extremely small, about 500-1000 cells / ml in peripheral blood, and about 3.5 times higher in umbilical cord blood. Quantity is limited

Method used

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  • A kind of erythrocyte progenitor cell serum-free medium and method of using the same
  • A kind of erythrocyte progenitor cell serum-free medium and method of using the same
  • A kind of erythrocyte progenitor cell serum-free medium and method of using the same

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Effect test

Embodiment 1

[0049] Example 1: Erythrocyte progenitor serum-free medium

[0050] Adopt IMDM culture medium (serum-free) as basal medium, add therein the composition that is selected from A, B, C, D, the composition in any column of E in Table 2:

[0051] Table 1 Formulas of different ratios of serum-free medium for erythrocyte progenitor cells

[0052]

[0053]

Embodiment 2

[0054] Example 2. Primary isolation and culture of erythroid progenitor cells

[0055] Collect 10ml of adult peripheral blood, transfer it to a lymphocyte separation tube, centrifuge, take the mononuclear cell layer, wash it twice with DPBS centrifugation, take a sample and count it, and take 0.5×10 cells according to the counting result. 6 The cells / ml were inoculated in 6-well plates and divided into six groups, among which 5 groups were experimental groups and 1 group was the control group. The 5 experimental groups were correspondingly added with a ratio of erythrocyte progenitor cell serum-free medium in Table 1, and the control group Group added Stemspan medium, the amount added was 2ml / well, and the above 6 groups of cells were placed at 37°C, 5% CO 2 cultured in an incubator. The 5 experimental groups added 2ml of fresh erythrocyte progenitor cell serum-free medium on the 4th day of expansion, the control group added Stemspan medium accordingly, and the 5 experimental...

Embodiment 3

[0056] Example 3. Identification of erythroid progenitor cells

[0057] Such as figure 1 As shown, when the erythrocyte progenitor cells were cultured for 1 day, 8 days, and 16 days, the change process of the cell morphology was observed through a microscope, and photographed for preservation. The scale bar of the microscope is 200 μm.

[0058] As shown in Figure 2, on the 10th day of culturing erythrocyte progenitor cells, flow cytometry was used to detect the expression of surface molecules CD71 and CD235a, and the positive rate and average fluorescence intensity indicators were detected respectively.

[0059] Such as image 3 As shown, the total number of cells obtained after the expansion of erythrocyte progenitor cells was counted after 1 day, 8 days, and 16 days of culture. EP-GM means the erythrocyte progenitor cell medium of Formula A in Table 1, and Stemspan is the control group.

[0060] Results: The isolated blood mononuclear cells expanded in the erythrocyte prog...

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Abstract

The invention belongs to the field of culture medium, and in particular relates to a serum-free culture medium for erythrocyte progenitor cells, including basal culture medium and other additive components; the other additive components include ITS additive, GlutaMAX, Lipid Concentrate, L-ascorbic acid 2-phosphorylated hemi Magnesium salt hydrate, inorganic salts, lipoic acid, hydrocortisone, stem cell factor, erythropoietin, and interleukin-3. The beneficial effects of the present invention are: the composition of the whole medium system is clear, the quality is stable, and the erythrocyte progenitor cells in the blood cells are selectively amplified, so that the erythrocyte progenitor cells can effectively express specificity on the basis of maintaining their original stemness characteristics CD71+ / CD235a+ / – is a marker, and the ratio of CD71+ / CD235a+ / – accounts for about 50% to 100%. The culture medium of the present invention is suitable for clinical application and scientific research.

Description

technical field [0001] The invention belongs to the field of medium, and in particular relates to a serum-free medium for erythrocyte progenitor cells and a use method thereof. Background technique [0002] Induced pluripotent stem cells (iPSCs) are an important source of stem cells to replace embryonic stem cells for the treatment of diseases, the study of embryonic developmental stages, and the detection of disease mechanisms. It is especially important when studying human embryonic development, disease mechanisms, and treatments because ethical issues involving the use of human embryos are avoided. [0003] Human iPSCs (hiPSCs) were initially generated by overexpressing several transcription factors in dermal fibroblasts (Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008). Like human embryonic stem cells (hESCs), hiPSCs are capable of differentiating into functional cell lineages and thus hold great potential for disease modeling, drug screening, and future ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789C12N5/0786
CPCC12N5/0641C12N5/0645C12N5/0647C12N2500/24C12N2500/38C12N2501/125C12N2501/14C12N2501/2303
Inventor 俞君英董成友张颖
Owner 安徽中盛溯源生物科技有限公司
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