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Culture medium for pluripotent stem cells

A sub-totipotent stem cell and culture medium technology, which is applied in the field of kits for proliferation and/or maintenance of sub-totipotent stem cells, and can solve problems such as inapplicability

Inactive Publication Date: 2019-04-26
普乐思尔有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this only seems to be true for selected hPSC lines and not in general

Method used

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  • Culture medium for pluripotent stem cells
  • Culture medium for pluripotent stem cells
  • Culture medium for pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1 - Preparation of a chemically defined medium according to the invention

[0159] The stem cell medium is a chemically defined medium according to the present invention, prepared in the following manner. First, fill the mixing vessel to 90% of the final volume with cell culture grade water. Weighed or appropriate amounts of the stock solutions of the components are then added under permanent stirring. The osmolarity was adjusted to 280 mOSM / kg using NaCl solution. After dissolving all components, adjust the volume and filter-sterilize the medium. The final medium has the following composition: Calcium chloride dihydrate 2.01 x 10 2 mg / L, magnesium chloride hexahydrate 4.90×10 1 mg / L, anhydrous magnesium sulfate 4.40×10 1 mg / L, copper sulfate pentahydrate 7.80×10 -4 mg / L, ferric nitrate nonahydrate 6.00×10 -2 mg / L, ferrous sulfate heptahydrate 3.75×10 -1 mg / L, potassium chloride 1.87×10 2 mg / L, sodium chloride 5.60×10 3 mg / L, anhydrous disodium hydroge...

Embodiment 2

[0160] Example 2 - Initiating hPSC Culture

[0161] 2.1 Extracellular Matrix (ECM) Coating of Culture Vessels

[0162] 0.5 μg / cm for culture vessel 2 Recombinant human vitronectin was coated as ECM. For this purpose, in each cm 2100 μl of diluted vitronectin solution was applied to the culture vessel surface. The coated culture vessels were left at room temperature for 2 hours. Aspirate the vitronectin solution before seeding the cells.

[0163] 2.2 Plating cells (Day 0)

[0164] The cell line used in this experiment was the human foreskin fibroblast derived hiPS cell line SBI#SC101A-1 (available from System Biosciences (SBI), Palo Alto according to the defined reference number).

[0165] Stem cell medium was prepared as described in Example 1. For cryopreserved cells, a ROCK-inhibitor (10 μM Y-27632 or 2 μM Thiazovivin) was added to the stem cell medium. Alternatively, for existing proliferating cultures, clump passage was performed as described in Example 3 below...

Embodiment 3

[0173] Example 3 - Serial passage of hPSC cultures

[0174] Aspirate the stem cell medium with Ca-free 2+ / Mg 2+ Wash hPSCs twice with Duchenne's PBS. Next, add 200 to 300 μl / cm 2 Dissociation buffer, the solution at 37 °C and 5% CO 2 Incubate for 6 to 10 minutes in an incubator. Carefully aspirate the dissociation buffer and add 1 to 5 ml of fresh medium. Use a serological pipette to wash the colonies off the surface. To maintain a sufficient size of cell clumps, avoid more than 4 to 5 washes. Cell pellets were then dispensed into fresh ECM-coated culture vessels with fresh stem cell medium and culture continued according to the cell attachment procedure of Example 2 (step 2.3).

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Abstract

The present invention relates to a chemically defined medium for eukaryotic cell culture, comprising water, at least one carbon source, one or more vitamins, one or more salts, one or more fatty acids, one or more buffer components, selenium, at least one substance of the group of functional inhibitors of acid sphingomyelinase (FIASMAs) and at least one polypeptide of the TGF-beta superfamily withthe ability to inhibit stem cell differentiation, its use in the culture of human pluripotent stem cells, a cell culture system comprising human pluripotent stem cells and the chemically defined medium, as well as a kit for proliferation and / or maintenance of human pluripotent stem cells.

Description

technical field [0001] The present invention relates to a chemically defined medium for eukaryotic cell culture, use of the medium for proliferation and / or maintenance of subtotipotent stem cells, a cell culture system comprising subtotipotent stem cells and the chemically defined medium, As well as kits for the proliferation and / or maintenance of sub-totipotent stem cells. Background technique [0002] The ability to self-renew and differentiate into various terminally mature cell types is a hallmark shared by all types of stem cells. These mechanisms are tightly controlled in normal stem cells, but potential is gradually lost during the stepwise multistage differentiation of stem cells to terminally differentiated mature cells (Sanges et al., 2010). [0003] The potency of a stem cell specifies its potential to differentiate into different cell types. Stem cells can be classified according to their potency, eg totipotent stem cells can differentiate into all cell types i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/00
CPCC12N5/0696C12N2500/14C12N2500/16C12N2500/22C12N2500/24C12N2500/25C12N2500/32C12N2500/34C12N2500/35C12N2500/38C12N2500/40C12N2500/46C12N2500/60C12N2500/98C12N2501/15C12N2501/16C12N2501/727C12N2501/73C12N2533/50C12N2533/90C12N2500/05C12N2500/30C12N2500/36C12N2501/999
Inventor 哈根·维兰德
Owner 普乐思尔有限公司
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