Formulations and methods for culturing stem cells

A stem cell, cell technology, applied in the field of preparations without xenogeneic substances, can solve problems such as tendency to be abnormal, insufficient reproducibility, and inability to maintain long-term hESCs

Inactive Publication Date: 2013-01-30
K・拉亚拉 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, these methods suffer from insufficient reproducibility and are currently unavailable for long-term maintenance of undifferentiated hESCs with a stable and normal karyotype
Feeder-free culture with enzymatic passaging can also be harsh for hESCs so that they become more prone to abnormalities

Method used

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  • Formulations and methods for culturing stem cells
  • Formulations and methods for culturing stem cells
  • Formulations and methods for culturing stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1. Effect of medium osmolarity on hESCs.

[0086]To improve the performance of xeno-free formulations, we optimized the osmolarity of the medium according to the invention. The osmolarity was adjusted with 5M NaC. We tested various osmolality in cultures of 5 passages of human embryonic stem cells (hESCs) and examined cell morphology after each passage. Use an osmolarity of 320mOsm for best performance ( Figure 1C ). Using 260mOsm osmolality ( Figure 1A ) formed small heterogeneous colonies and despite an osmolarity of 290mOsm ( Figure 1B ) improved the morphology of the colonies, but the size of the colonies was small. The osmolarity of 350mOsm obviously limited the colony growth ( Figure 1D ).

Embodiment 2

[0087] Example 2. Specific Lipids and Lipid Derivatives Enhance Undifferentiated Growth of hESCs.

[0088] Various lipids and lipid derivatives were tested in the medium according to the invention (RegES) and in conventional hES medium containing KO-SR (Knockout Serum Replacement, Invitrogen). Undifferentiated colonies were evaluated for general morphology, as well as size and thickness, prior to each passage based on visual perception (Table 3). According to the results, conjugated linoleic acid, eicosapentaenoic acid, palmitoleic acid, linoleic acid, linoleic-oil-arachidonic acid mixture and especially retinol improved the expression in hES medium and according to the present invention. Morphology of undifferentiated colonies in both media (Table 3). Furthermore, stearic acid, lysophosphatidylcholine, phosphatidylethanolamine, prostaglandin F2 and DL-isoproterenol lead to poor morphology and / or excessive differentiation in hES medium, whereas in the medium according to the ...

Embodiment 3

[0094] Example 3. Retinol increases proliferation and expression of stem cell markers.

[0095] Retinol was selected for further evaluation in the maintenance of undifferentiated hESCs. Preliminary studies indicated that retinol at concentrations of 0.1-0.5 [mu]M was ineffective and no improvement in morphology or number of undifferentiated colonies was seen. However, further evaluation showed that retinol at a concentration of 2.0 μM or above improved the proliferation of hESCs and induced the expression of hESC-specific markers (Fig. 3). In the presence of 2.0 μM retinol, the growth of the colonies started earlier and the size of the colonies was already larger at day 3 ( Figure 3A-3B ). Proliferation assays showed that hESCs cultured in the presence of 2.0 μM or 3.5 μM retinol had almost 2-fold greater Proliferation rate ( Figure 3E ). Immunocytochemical staining of hESCs cultured in the presence of retinol revealed the expression of stem cell markers Nanog and TRA-1...

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Abstract

The present invention relates to a serum replacement formulation and to a culture medium suitable for the derivation, maintenance and differentiation of stem cells, comprising conjugated linoleic acid, eicosapentaenoic acid, activin A and/or stearic acid.

Description

technical field [0001] The present invention relates to xeno-free formulations for the derivation, maintenance and differentiation of stem cells, such as human embryonic stem cells. Background technique [0002] Human embryonic stem cells (hESCs) are pluripotent cells, which have the potential to differentiate into all cell types of the human body. Human ESCs are of great therapeutic interest because of their ability to proliferate indefinitely in culture and thus provide cells and tissues to replace failed or defective human tissues. In the future, the expectation is high that human ESCs will proliferate and directionally differentiate into specific cell types that can be transplanted into humans for therapeutic purposes or used as cell models in drug discovery and toxicology studies . [0003] Embryonic stem cells are difficult to maintain in culture because they tend to follow their natural cell fate and differentiate spontaneously. Most culture conditions result in so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/0735C12N5/077
CPCC12N2500/25C12N5/0667C12N2501/115C12N2500/99C12N2500/32C12N2500/38C12N2501/16C12N2500/60C12N2500/20C12N5/0606C12N2500/36C12N2500/90C12N2500/98C12N5/00C12N5/0602
Inventor K·拉亚拉R·M·塞佩宁O·霍瓦塔H·斯科特曼
Owner K・拉亚拉
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