Low-density-tolerant feeder-layer-free human pluripotency stem cell culture medium

A human pluripotent stem cell, feeder-free technology, applied in the field of feeder-free human pluripotent stem cell culture medium, can solve the problems of complex composition, high concentration of cytokines, poor use effect, etc.

Active Publication Date: 2016-10-19
FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the birth of mTeSR1 has led to the rapid development of the hiPSC field, mTeSR1 still contains a large amount of BSA, which is not suitable for clinical research and is expensive. Therefore, StemCell uses human serum albumin (HSA) to replace the BSA of mTeSR1, and uses human-derived cytokines , developed TeSR TM 2. The medium does not contain any animal-derived substances and is suitable for clinical research related work. Many other companies have also developed their own unique IPSCs medium accordingly.
However, TeSR2 and other similar media still contain a large amount of cytokines and proteins, with complex components, and the use effect is worse than mTeSR1, which cannot widely meet the needs of basic scientific research and clinical research, and also increases the uncertainty of research development and differentiation. In 2011, the Thomson laboratory identified eight mediums that are the main components of mTeSR1, that is, the E8 medium, which can stably maintain the pluripotency and self-renewal a

Method used

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  • Low-density-tolerant feeder-layer-free human pluripotency stem cell culture medium
  • Low-density-tolerant feeder-layer-free human pluripotency stem cell culture medium
  • Low-density-tolerant feeder-layer-free human pluripotency stem cell culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 (BioC Ⅰ)

[0078] One of the schemes of the feeder-free human pluripotent stem cell culture medium provided by the present invention, the formula of each culture medium is shown in Table 1. The survival rate of single-cell human iPSCs in each medium was detected by MTT method and AP method.

[0079] After culturing human iPSCs for 50 passages using the following groups of media, the karyotype identification and flow cytometry detection of pluripotency markers were performed on human iPSCs. The test results of the fourth group of media are as follows: figure 1 and figure 2 shown.

[0080] Table 1 No feeder layer human pluripotent stem cell culture medium formula

[0081]

[0082]

[0083] The above experimental data show that adding sodium heparin to the culture medium can promote the proliferation rate and single cell survival rate of human iPSCs (including ESCs and iPSCs).

Embodiment 2

[0084] Example 2 (BioC II)

[0085] One of the schemes of the feeder-free human pluripotent stem cell culture medium provided by the present invention, the formula of each culture medium is shown in Table 2. The survival rate of single-cell human iPSCs in each medium was detected by MTT method and AP method.

[0086] After culturing human iPSCs for 50 passages using the following groups of media, the human iPSCs were identified by karyotype and flow cytometric detection of pluripotency markers. The detection results of the fourth group of media are as follows: image 3 and Figure 4 shown.

[0087] Table 2 No feeder layer human pluripotent stem cell culture medium formula

[0088]

[0089]

[0090] The above experimental data show that adding lithium heparin to the culture medium can promote the proliferation rate and single cell survival rate of human iPSCs (including ESCs and iPSCs).

Embodiment 3

[0091] Embodiment 3 (BIOC Ⅰ / BIOC Ⅱ)

[0092] One of the schemes of the feeder-free human pluripotent stem cell culture medium provided by the present invention, the formula of each culture medium is shown in Table 3. The survival rate of single-cell human iPSCs in each medium was detected by MTT method and AP method.

[0093] Table 3 feeder-free human pluripotent stem cell culture medium formula

[0094]

[0095]

[0096] The above experimental data show that the combined use of sodium heparin and lithium heparin can also promote the proliferation rate and single cell survival rate of human iPSCs (including ESCs and iPSCs).

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Abstract

The present invention discloses a feeder-layer-free human pluripotency stem cell culture medium, which contains L-ascorbate-2 magnesium phosphate, sodium selenite, recombinant human insulin, human apotransferrin, basic fibroblast growth factor, transforming growth factor beta 1, heparin lithium and/or heparin sodium, a DMEM/F-12 base culture medium and an osmotic pressure regulator. According to the present invention, with the culture medium, when the hiPSC culture is performed in low density and normal density, the proliferation rate is high, and the cell morphology and the pluripotency are well maintained; the use of the expensive heparitin sulfate is not required so as to substantially reduce the cost; and almost all of the hiPSCs maintaining cultures at the current stage can be met, and various small molecule compounds are added to perform induction reprogramming culture (removal of TGFB1 and DM), such the culture medium is suitable for extensive researches of various basic scientific researches and clinical scientific research experiments.

Description

technical field [0001] The invention relates to a low-density culture medium for human pluripotent stem cells without a feeder layer. Background technique [0002] Pluripotent stem cells (Pluripotency stem cells, iPSC) have the ability of self-renewal and pluripotency, and they can differentiate into any tissue and cell of the three embryonic germ layers (ectoderm, mesoderm, endoderm). Developmental biology and other fields play an important role. iPSCs include embryonic stem cells (Embryonic stem cells, ESCs) and induced pluripotent stem cells (induced pluripotency stem cells, iPSCs). Both ESCs and iPSCs have similar gene expression profiles, self-renewal capabilities, pluripotency, and epigenetic Spectrum, the former comes from the inner cell mass of the blastocyst stage, and the latter comes from the pluripotent stem cells induced and purified by introducing reprogramming factors into adult cells. The most studied IPSC species at this stage include mice and humans, wher...

Claims

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Application Information

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IPC IPC(8): C12N5/074
Inventor 王淋立陈月花宋立兵
Owner FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR)
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