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Methods, surface modified plates and compositions for cell attachment, cultivation and detachment

A technology of cells and compounds, applied in the field of mammalian cell culture, can solve problems such as effect of results

Inactive Publication Date: 2011-05-04
CENTOCOR ORTHO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0040] As another example, Buzzard et al. describe, "We have detected only one karyotype-altering event...Given that our method is quite different from that used by most other groups, the culture method used may have Turns out...has some impact

Method used

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  • Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
  • Methods, surface modified plates and compositions for cell attachment, cultivation and detachment
  • Methods, surface modified plates and compositions for cell attachment, cultivation and detachment

Examples

Experimental program
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Effect test

example 1

[0195] Passaging and maintenance of human embryonic stem cells in the form of cell populations

[0196] The human ES cell lines H1 and H9 were initially maintained on primary mouse embryonic fibroblasts (MEF) inactivated with mitomycin C. Human ES cells are transferred from the MEF feeder layer to Matrigel during repeated passages TM (Becton-Dickinson (Bedford, MA)).

[0197] Use Matrigel TM Surface treatment: Matrigel with low growth factor TM After thawing at 4°C, it was diluted in cold DMEM / F12 (Invitrogen (Carlsbad, CA)) at a ratio of 1:30. Add a volume sufficient to cover the surface into each well (1ml) of each 6cm petri dish (2ml) or 6-well plate, and incubate for 1 hour at room temperature. The treated surface can be used within a few hours or stored at 4°C for up to two weeks.

[0198] Human ES cell culture: by incubating in DMEM / F12 containing 1 mg / ml collagenase IV (Sigma-Aldrich (St. Louis, MO)) for 10 minutes, and then scraping with a pipette to obtain the feeder laye...

example 2

[0201] Passaging of human embryonic stem cells in single cell form

[0202] According to the method disclosed in the US patent application LFS5163USPSP assigned to LifeScan Inc, the human ES cells of the cell line H9 were cultured as single cells. Use TrypLE TM Express cells are subcultured by treating them at 37°C for five minutes, and at 10,000 cells / cm 2 The density of the substrate surface is inoculated.

example 3

[0204] People who use surface modified culture plates without extracellular matrix proteins / components and feeder cells Adhesion, culture and maintenance of pluripotency of embryonic stem cells

[0205] Human ES cells of cell line H1, maintained in Nunclon Delta at passage 49 TM In the MEF conditioned medium on the culture plate, the culture plate was treated with low growth factor Matrigel before the study TM The 1:30 dilution liquid treatment. The cells are separated from the surface for passage by 1mg / ml collagenase separation or manual scraping.

[0206] These cells were then seeded on two untreated wells of a surface-modified culture plate (6-well format). In addition, one well of each culture plate was treated with 0.1% non-heterogeneous human gelatin as a control. Also inoculate the cells directly in Costar TM (Catalog Number 3516; Corning (Corning, NY)), Falcon TM (Catalog No. 351146; Becton Dickinson (Franklin Lakes, NJ)) and Nunclon Delta TM (Catalog No. 140675; Thermo...

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PUM

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Abstract

The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity.

Description

Technical field [0001] This patent application claims the priority of provisional application No. 61 / 030,544 filed on February 21, 2008. [0002] The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell adhesion to, culture on, and separation from the surface of a solid substrate, the surface containing at least about 0.5% N , The total number of O and N is greater than or equal to 17.2%, the contact angle is at least about 13.9 degrees, and the surface does not contain a feeder cell layer and an adsorption layer. In one embodiment of the present invention, cells are treated with a compound capable of inhibiting the activity of Rho kinase. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity. Background technique [0003] Mammalian cell culture is one of many processes in life and health sciences. Containers for mammalian cell culture and analysis involving anchorage-dependent ...

Claims

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Application Information

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IPC IPC(8): C12N5/07C12N5/0735
CPCC12N5/0606C12N2509/00C12N2533/00C12N2501/70C12N5/00
Inventor B·弗里耶S·纳尔逊V·尼尔森T·布雷维T·K·马伍德
Owner CENTOCOR ORTHO BIOTECH
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