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A feeder cell-free culture medium and system

A technology of feeding cells and culture medium, applied in the field of cell culture, can solve the problem of not understanding the function of MEF

Inactive Publication Date: 2011-02-09
QUEENSLAND UNIVERSITY OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] It is currently unknown what function MEFs have in hES cell cultures

Method used

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  • A feeder cell-free culture medium and system
  • A feeder cell-free culture medium and system
  • A feeder cell-free culture medium and system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0191] Example 1: Analysis of the in vitro microenvironment of human embryonic stem cells

[0192] Materials and Method

[0193] Mouse embryo fibroblast cell culture

[0194] MEF (SCRC-1046 cell line, Cryosite, Lane Cove, Sydney, NSW, AUS) at 80cm 2 Amplify 6 generations in culture flask (Nalge Nunc International, Rochester, NY, USA), use 85% Dulbecco's Modification of Eagle's medium (DMEM) (Invitrogen, MountWaverley, VIC, Australia), add 10% fetus Bovine Serum (FBS) (Invitrogen), 2x10 -3 M L-glutamine (Invitrogen) and 1000IU / mL penicillin / streptomycin (Invitrogen), 5% CO 2 , 37°C. Subsequently, mitomycin-C (Sigma-Aldrich, CastleHill, NSW, AUS) was added to the flask containing MEF, and the cells were incubated at 37°C, 5% CO 2 Incubate for 2.5 to 3 hours. Give petri dish (10cm 2 ) (Nalge Nunc International) cover 0.1% gelatin (Sigma-Aldrich), add MEF at least 1 hour later. MEF cells at 20,000 cells / cm 2 Inoculate 10cm covered with 0.1% gelatin (Sigma-Aldrich) 2 (Nalge Nunc Intern...

Embodiment 2

[0241] Example 2: Proteomics analysis of keratinocyte conditioned medium cultured in vitro

[0242] The purpose of this study is to comprehensively examine the in vitro microenvironment of keratinocytes. Specifically, proteomics methods were used to identify the main factors required for the growth of keratinocytes produced by feeder cells. In addition, using the above-mentioned serum-free medium, the medium is fully defined and has a very low protein content. The significant advantage of the extremely low protein content of the serum-free medium is that it does not "mask" the main factors secreted by feeder cells that can support the growth of keratinocytes. In addition, serum-containing media usually require pre-processing steps before performing proteomic analysis, such as "Multiple Affinity Removal System" (MARS) (Agilent Technologies). The MARS immune depletion technology involves the removal of high-abundance proteins in serum-containing media, which may result in the los...

Embodiment 3

[0277] Example 3: Growth of hES cells without feeder cells and serum

[0278] The hES cells were grown and tested in the following medium formulation: 1ug / mL IGF-I / 1-64VN chimeric protein, 0.1ug / mL bFGF, 35ng / mL activin-A and 40μg / mL laminin.

[0279] Immunofluorescence (IF) was performed using antibodies against Oct4, TRA1-60, SSEA-4, SSEA-1 antigens. IF Research ( Figure 8 ) Confirmed the expression of Oct4, TRA1-60 and SSEA-4, but only low expression of SSEA-1. hES cells also showed a large nuclear-to-cytoplasmic ratio, representing the hES phenotype.

[0280] Rex1 is abnormal, and this result confirms that it is significantly down-regulated in our culture system. However, when examining Oct4 and Nanog, these amplicons in our culture system showed an almost 2-fold increase in expression ( Picture 9 ).

[0281] Together, these data suggest that the system can indeed maintain these cells in an "undifferentiated state."

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Abstract

A cell culture medium and system are provided which eliminates or at least reduces the need for feeder cells. The cell culture medium comprises one or more factors that are normally secreted and / or produced by a feeder cell and a synthetic chimeric protein comprising IGF-I and a portion of vitronectin. The cell culture medium is particularly suitable for propagating human embryonic stem cells and keratinocytes. This invention also relates to compositions and methods which utilize the cells cultured in the cell culture medium of the invention.

Description

Technical field [0001] The present invention relates to cell culture. More specifically, the present invention relates to media, systems and methods for feeder cell-independent cell culture systems. Background technique [0002] Human embryonic stem (hES) cells are derived from the inner cell mass (ICM) of embryo sacs (early embryos that are 4-5 days old). hES cells are pluripotent cell types, which can produce three main germ layers, namely ectoderm, endoderm and mesoderm [1, 2]. In other words, when the necessary differentiation stimulation is given, these cells can develop into more than 200 cell types in adults. Or, when no differentiation stimulation is given, these cells will renew themselves and produce pluripotent progeny cells. [0003] In view of this, it is believed that the pluripotent behavior of hES cells can be manipulated to more effectively produce cells and tissues for therapeutic indications: for example, for the treatment of Parkinson’s disease [3], diabetes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
CPCC12N2502/094C12N2502/1323C12N2500/99C12N2501/115C12N2500/44C12N2501/58C12N2501/235C12N2501/105C12N2501/16C12N5/0629C12N5/0606C12N2500/90A61P17/00A61P17/02
Inventor Z·厄普顿D·利弗斯利S·D·理查兹L·B·科马克D·哈金
Owner QUEENSLAND UNIVERSITY OF TECH
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