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Formula of serum free medium for human pluripotent stem cells

A technology of pluripotent stem cells and serum-free medium, applied in the direction of non-embryo pluripotent stem cells, artificially induced pluripotent cells, cell culture active agents, etc., to achieve the effect of wide application and large market

Active Publication Date: 2017-07-18
杨涛
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the average yield of 2-3g / L supported by the current foreign medium, there is still a gap of 10 times

Method used

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  • Formula of serum free medium for human pluripotent stem cells
  • Formula of serum free medium for human pluripotent stem cells
  • Formula of serum free medium for human pluripotent stem cells

Examples

Experimental program
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Embodiment 1

[0035] A formula for serum-free medium for human pluripotent stem cells, including the following raw materials: inorganic salt components, organic components, amino acids and amino acid salts, energy substances and metabolic intermediates, vitamins and antioxidants, proteins and polypeptides, trace elements and chromogenic substances; human pluripotent stem cells were expanded and cultured according to the above method.

[0036] Expansion culture of human pluripotent stem cells: add freshly prepared bFGF (ie: basic fibroblast growth factor, bFGF 2 mg per liter of medium), β-mercaptoethanol (per liter of culture medium) to the prepared medium solution Add β-mercaptoethanol 7μl); maintain the undifferentiated state of human pluripotent stem cells, and make pluripotent stem cells self-renew and proliferate through expansion culture, so as to increase the number of cells, expand the reserve, and carry out subsequent subpackaging and preservation The purpose; with the current marke...

Embodiment 2

[0038] see Figure 2-3 : A formula for serum-free medium for human pluripotent stem cells, including the following raw materials: inorganic salt components, organic components, amino acids and amino acid salts, energy substances and metabolic intermediates, vitamins and antioxidants, proteins and peptides, trace amounts Elements and chromogenic substances; myocardial induction culture was carried out according to the above method, and the culture process was divided into: hanging drop culture for 3 days + suspension culture for 2 days + adherent culture for 5 days, and vitamin C was added at the same time (128.05 mg of ascorbicacid was added to each liter of culture solution) ).

[0039] Cardiac induction culture: Human pluripotent stem cell line IMR90-1 (Wicell Research Institute, Madison, WI, USA) was used to induce the reporter gene MHC-eGFP (myosin heavy chain promoter-driven After the initiation of the induction culture, once the stem cells differentiate into the myocard...

Embodiment 3

[0041] see Figure 4-5 : A formula for serum-free medium for human pluripotent stem cells, including the following raw materials: inorganic salt components, organic components, amino acids and amino acid salts, energy substances and metabolic intermediates, vitamins and antioxidants, proteins and peptides, trace amounts Elements and chromogenic substances; Neural induction was carried out according to the above method: HS-5 cell line was used as a feeder layer in the prepared medium solution, and NT-3 was added at the same time (1342 mg of NT-3 was added per liter of medium).

[0042] Neural induction culture: Human pluripotent stem cell line iPS-1 (Wicell Research Institute, Madison, WI, USA) was co-cultured with human bone marrow stromal cell line HS-5 ( Figure 4 ), to achieve the purpose of inducing and culturing pluripotent stem cells toward nerve cells; the current market similar products to be compared include DMEM / F12+10% fetal bovine serum, Knockout DMEM+10% SR (Invit...

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Abstract

The invention discloses a formula of a serum free medium for human pluripotent stem cells. The serum free medium comprises the following raw materials: inorganic salt, organic matter, amino acid and amino acid salt, energy matter and metabolic intermediates, vitamins and antioxidant, protein and polypeptide, trace elements and color development matter, wherein the culture flow comprises selection of a basic formula, combined screening, result identification and assessment and culture with a new formula. The serum free medium is prepared by the following method: adding injection water to the raw materials to 950 milliliters, slightly stirring till dissolving, adding 2.438 g of sodium hydrogen carbonate, slightly stirring till dissolving, adding injection water to 1 liter, regulating the pH to the required value using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, finally performing positive pressure filtration and sterilization using a filter membrane having the diameter of 0.1 mum, and storing the medium solution in dark at 2-8 DEG C. The formula solves the problem of high cost of domestic imported serum free medium.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a formulation of a serum-free medium for human pluripotent stem cells. Background technique [0002] Biopharmaceuticals are one of the key projects for national development. It is unique in that biopharmaceuticals can only be expressed using active vectors such as cells. Traditional chemical synthesis methods cannot be used in the production of biological drugs and vaccines. As a necessary pillar raw material for biopharmaceuticals, the grade of cell culture medium and the enlargement of the corresponding production process directly reflect the development level of the national biopharmaceutical industry and the level of drug costs. At present, the development and production technology of high-grade serum-free medium and the global cell culture medium market are basically monopolized by American companies such as Sigma, Life, and HYCLONE. One of the main factors for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2501/115C12N2501/11C12N2500/90C12N2500/32C12N2500/35C12N2500/34C12N2500/38C12N2500/40C12N2500/05C12N2500/25C12N2500/30C12N2501/30C12N2501/998C12N2502/1394C12N2500/14C12N2500/16C12N2500/22C12N2500/24C12N2500/36C12N2500/60
Inventor 杨涛隋昳
Owner 杨涛
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