Cells dont attach to cross-linked gelatin

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ghunter
ghunter's picture
Cells dont attach to cross-linked gelatin

Hi, guys,

I made some cross-linked gelatin gels and intended to use these for cell culture. To my surprise, cells like NIH3T3 or BHK 21 dont attach/spread on to these gels readily. I thought gelatin is frequently used for coating plastic culture surface to promote cell attachment.

These are glutaldehyde fixed gelatin 5% (w/v), extensively washed after blocking the reactive groups with glycine. Cells are not spreading on it after two days, most remain rounded, while they are very happy on the regular plastic dishes.

Anyone has some comments on whats going on here?

Thanks in advance!

vik

JoshPRoberts
JoshPRoberts's picture
I'm writing an article for

I'm writing an article for The Scientist about just this kind of thing -- challenges in 3D cell culture. If ghunter, or anyone else, has some insights into 3D cell (tissue) culture -- with or without solutions to the challenges -- I'd love to hear about them! Please send me a note with contact information.

Thanks.

ghunter wrote:

Hi, guys,

I made some cross-linked gelatin gels and intended to use these for cell culture. To my surprise, cells like NIH3T3 or BHK 21 dont attach/spread on to these gels readily. I thought gelatin is frequently used for coating plastic culture surface to promote cell attachment.

These are glutaldehyde fixed gelatin 5% (w/v), extensively washed after blocking the reactive groups with glycine. Cells are not spreading on it after two days, most remain rounded, while they are very happy on the regular plastic dishes.

Anyone has some comments on whats going on here?

Thanks in advance!

vik

yusfan84
yusfan84's picture
hye guys..

hye guys..

i also done research on microcarrier focusing on DF-1 cells. One of my partner also used material with gelatin coated based..but he found that the cells also not attach on the microcarrier. he read that this type of microcarrier required serum free media. Hurm..I also planning to develop my own microcarrier. Thus u have any idea to coated gelatin on material surface such as plastic, glass or ceramic...? I still searching on the technique.

Abe
Abe's picture
Also consider that the

Also consider that the multiple washings may not be removing all the glutaraldehyde, which may be persisting at low concentration. See:

J Biomed Mater Res. 2002 Jul;61(1):121-30.

Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis.

Gough JE, Scotchford CA, Downes S.

School of Biomedical Sciences, E Floor, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom. eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%6a%2e%67%6f%75%67%68%40%69%63%2e%61%63%2e%75%6b%22%3e%6a%2e%67%6f%75%67%68%40%69%63%2e%61%63%2e%75%6b%3c%2f%61%3e%27%29%3b'))

Collagen has been investigated as a potential natural biomaterial, because of its occurrence in the extracellular matrix. Collagen requires crosslinking in this context, by reagents that are often cytotoxic. Glutaraldehyde is one such agent that is potentially cytotoxic. The aim of this study was to determine the cause of poor cell attachment and growth on collagen/poly(vinyl alcohol) bioartificial composite films, when crosslinked with glutaraldehyde. Dehydrothermal crosslinking was used as a comparison. Human osteoblasts were observed to undergo apoptosis on glutaraldehyde crosslinked films dependent on concentration of collagen present. Higher collagen content resulted in higher levels of apoptosis with poor cell attachment and spreading of remaining cells. Post-treatment of films with 8% L-glutamic acid prevented the apoptotic response of osteoblasts and allowed attachment and spreading. The addition of 100 nM insulin-like growth factor-1 to the culture medium also prevented apoptosis. Glutaraldehyde toxicity of crosslinked collagen has been demonstrated in this study, the mechanism of which is apoptosis. This study indicates that poor biocompatibility and induction of apoptosis on collagen/poly(vinyl alcohol) films crosslinked by glutaraldehyde are attributed to glutaraldehyde components on the surface of the films (not residual glutaraldehyde), whose effects can be quenched by glutamic acid, and prevented by insulin-like growth factor-1. Copyright 2002 Wiley Periodicals, Inc.

PMID: 12001254 [PubMed - indexed for MEDLINE]

nerve2007
nerve2007's picture
Its basically that your

Its basically that your crosslinking agent still remains on the scaffolds and hence cells dont grow.

alexpennos
alexpennos's picture
First of all, hello to

First of all, hello to everyone here!
Could you be more specific on how you crosslinked the gel? And moreover, is there any possible way to increase the concetration of the gelatin.
P.S. my main goal is to produce a high-concetration, thick and rigid, collagen-gel.

Abe
Abe's picture
Gelatin can be crosslinked

Gelatin can be crosslinked with transglutaminase.  It's non-toxic, available from Sigma, but pretty expensive.   See Yung et al 2007 (Journal of Biomed Materials Research, 83A (4): 1039-1046) for background.