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Defined media for expansion and maintenance of pluripotent stem cells

A technology for cell culture, basal medium, applied in the field of defined medium for the expansion and maintenance of pluripotent stem cells, which can solve problems such as inconsistency

Inactive Publication Date: 2014-11-19
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both approaches have the disadvantage of inconsistencies in the conditioned medium or feeder cell batches that continuously support the expansion of pluripotent stem cells

Method used

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  • Defined media for expansion and maintenance of pluripotent stem cells
  • Defined media for expansion and maintenance of pluripotent stem cells
  • Defined media for expansion and maintenance of pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0066] Test multiple culture conditions to identify the optimal culture for proliferation of undifferentiated embryonic stem cells base component

[0067] in MATRIGEL TM (1:30 dilution; BD Biosciences, Franklin Lakes, NJ) coated dishes in 1 culture medium (StemCell Technologies, Vancouver, Canada), and the cells of the human embryonic stem cell line H1 (at the 35th to 40th passage) passaged with EDTA were used as the starting population to test various medium compositions . Cells were passaged as small colonies using 5-10 min EDTA treatment at room temperature. Cultures were routinely split at a ratio of 1:6 to 1:10 at each passage. Table I lists initial media formulations tested for their ability to proliferate H1 cells while maintaining their undifferentiated morphology and markers of pluripotency.

[0068] Table I

[0069] Media preparations evaluated

[0070]

[0071]

[0072]

[0073] *: Trace elements C** (Mediatech, Manassas, VA), HEPES (4-(2-hyd...

example 2

[0092] Recovery of undifferentiated embryonic stem cells by culturing H1 cells in IH-3 medium spiked with ascorbic acid main features of cells

[0093] In order to identify the relationship between H1 cells cultured in IH-3 and in IH-1 and The reasons for the decrease of SSEA-4 and ZPF42 in H1 cells cultured in medium 1 were compared, and a gap analysis was performed to identify the Major reagents present in 1 and IH-1 but not in IH-3 medium. IH-3 medium was supplemented with trace element C, ascorbic acid, lithium chloride or defined lipids as indicated in Table V.

[0094] Table V

[0095] Modifications to IH-3 Medium

[0096] culture medium

Addition to IH-3 medium

IH-3-1

1x trace element C

IH-3-2

0.25mM ascorbic acid

IH-3-3

1mM LiCl

IH-3-4

1:500X defined lipid

[0097] H1 cells cultured in IH-3 for passage 14 were subsequently cultured in the above medium formulations and compared with cells cultu...

example 3

[0104] Long-term culture of H1 cells in IH-3 and IH-1 medium maintains pluripotency and stable karyotype

[0105] As described in Example 1, in MATRIGEL TM (1:30 dilution) in the coated dish 1 medium, and the cells of the human embryonic stem cell line H1 (passage 35 to passage 40) passaged in EDTA were used as the starting population to evaluate the use of IH-1, IH-3-2 and 1 medium for long-term culture. Cells were passaged as small colonies using 5-10 min EDTA treatment at room temperature. The components of the test media are listed in Table VII.

[0106] Table VII

[0107] Components Used in IH-1, IH-3-2, and IH-3RT Medium Preparations .

[0108]

[0109] Such as Figures 11A to 11D As can be seen in , H1 cells cultured in IH-1, IH-3-2 and IH-3RT for 20 passages retained the typical ES morphology. The results of PCR analysis of H1 cells cultured in IH-1, IH-3-2 and IH-3RT for 15 passages are shown in Figure 12A to Figure 12F middle. The results of PCR ...

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Abstract

The present invention provides methods to promote the proliferation of undifferentiated pluripotent stem cells in defined media. Specifically, the invention provides a defined cell culture formulation for the culture, maintenance, and expansion of pluripotent stem cells, wherein culturing stem cells in the defined cell culture formulation maintains the pluripotency and karyotypic stability of the cells for at least 10 passages. Further disclosed is a cell population grown under defined media conditions that express OCT4, SOX2, NANOG, and FOXA2.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application Serial No. 61 / 607,706, filed March 7, 2012, which is hereby incorporated by reference in its entirety for all purposes. technical field [0003] The present invention is in the field of the proliferation and maintenance of pluripotent stem cells under defined media conditions. Background technique [0004] Expansion of undifferentiated pluripotent stem cells has traditionally employed "feeder" cells that provide sufficient factors to support attachment, proliferation, and maintenance of markers of pluripotency. Early methods for the generation and culture of human embryonic stem cells required the use of mouse embryonic fibroblast (MEF) feeder cells. Subsequent techniques include the replacement of feeder cells with "conditioned medium" and extracellular matrix coatings. Conditioned medium is medium that has been modified by feeder cells such as...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/02
CPCC12N2500/36C12N2500/05C12N2501/105C12N2501/115C12N5/0606C12N2500/38C12N2501/15C12N2500/25C12N2500/34
Inventor A.雷扎尼亚
Owner JANSSEN BIOTECH INC
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