How to limit fibroblast cell contamination in epithelial cell culture - (Nov/02/2006 )

Hello,everyone:
I do epithelial cell primary culture,but when subcultured ,most of the cells transformed to fibroblastic morphology.I digest primary cells with 0.25%trypsin+0.02%EDTA.I tried several times,but can not limit the fibroblat cell.I am very puzzled and wondered how to solve this problem?
Thanks for any suggestion!

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there are two possibilities:

your fibroblasts are contamination in your epithelial cell enrichment; we solve the problem by pre-culturing enriched epithelial cells in a 75 ml bottel coated with collagen where fibroblast should better attach than epithelial cells; we collect the medium after 30 min where we lose some epithelial cells but we lose many more fibroblast


the second possibility is that you are faced with EMT which means there is anything in your handling that induces epithelial cells to fibroblast-like cells; to evaluate this we need more information of your type of cells and your protocol


may be there is a mixture of both possibilities

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how many passages does it take for this to happen? if you are seeing your happy epithelial cells, and then the morphology shifts from the typical cobblestone appearance and they get 'stringy' like fibroblasts, your cells are sick. how confluent are they before passaging, too? I have seen that morphology shift when the cells become senescent, and also when they're sick from other reasons

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[quote name='kosmodrom' date='Nov 3 2006, 08:52 AM' post='75633']

[/quote]

there are two possibilities:

your fibroblasts are contamination in your epithelial cell enrichment; we solve the problem by pre-culturing enriched epithelial cells in a 75 ml bottel coated with collagen where fibroblast should better attach than epithelial cells; we collect the medium after 30 min where we lose some epithelial cells but we lose many more fibroblast


the second possibility is that you are faced with EMT which means there is anything in your handling that induces epithelial cells to fibroblast-like cells; to evaluate this we need more information of your type of cells and your protocol
y be there is a mixture of both possibilitie
[/quote]

Dear Kosmodrom,

For you first possibility,did you mean when do primary epithelial cell culture from tissue fragment,after use collagen coated bottle , other passages culture need not collagen coated?

For second,the cell I cultued is renal tubular epithelial cell. I use renal tubular fragment for primary culture.When most of them were confluent,I spilled the medium,then added prewarmed Ca++,Mg++ free Hanks to rinse twice. Then added prewarmed 0.25%trypsin+0.05%EDTA,37 degree digest 30 minute(otherwise the cell could not detach from the tissue plate).Add complete

medium(DMEM/F12+10F BS) to terminate trypsin,then centrifuge ,suspend the pellet with complete medium ,put the tissue culture plate in 37degree,5%CO2 incubator.But unluckly found the most cells were fibroblast modality,but before digested,most of them were epithelial modality.

Thanks!

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when the cells were grown from the tissue fragment ,they were epithelial morphology.Isubcultured when most of them grew to almost 80% confluent ,but after the day they were digested, most of them were stringy like.I wondered why this happened?How to avoid this shift? And what did you mean "the cell are sick"?
Thanks!

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[quote name='wing111' date='Nov 5 2006, 01:25 PM' post='75821']
[quote name='kosmodrom' date='Nov 3 2006, 08:52 AM' post='75633']

[/quote]

there are two possibilities:

your fibroblasts are contamination in your epithelial cell enrichment; we solve the problem by pre-culturing enriched epithelial cells in a 75 ml bottel coated with collagen where fibroblast should better attach than epithelial cells; we collect the medium after 30 min where we lose some epithelial cells but we lose many more fibroblast


the second possibility is that you are faced with EMT which means there is anything in your handling that induces epithelial cells to fibroblast-like cells; to evaluate this we need more information of your type of cells and your protocol
y be there is a mixture of both possibilitie
[/quote]

Dear Kosmodrom,

For you first possibility,did you mean when do primary epithelial cell culture from tissue fragment,after use collagen coated bottle , other passages culture need not collagen coated?

For second,the cell I cultued is renal tubular epithelial cell. I use renal tubular fragment for primary culture.When most of them were confluent,I spilled the medium,then added prewarmed Ca++,Mg++ free Hanks to rinse twice. Then added prewarmed 0.25%trypsin+0.05%EDTA,37 degree digest 30 minute(otherwise the cell could not detach from the tissue plate).Add complete

medium(DMEM/F12+10F BS) to terminate trypsin,then centrifuge ,suspend the pellet with complete medium ,put the tissue culture plate in 37degree,5%CO2 incubator.But unluckly found the most cells were fibroblast modality,but before digested,most of them were epithelial modality.

Thanks!
[/quote]

pre-culturing on collagen prefers the settling of fibroblast but after your reply I assume that you are predominantly faced with EMT (epithelial mesenchymal transition); EMT of renal tubular cells is induced, among others, by HGF/SF (hepatocyte growth factor/scattering factor), it is part of FBS and you use relatively high amount of FBS; EMT is necessary for tubulogenesis;

so, on the basis of your report, I suggest to avoid FBS but supplement with EGF, insulin, transferrin, Na-selenit, hydrocortison, pyruvate; may be you find similar protocols in literature but the crucial point is to control components for proliferation and to avoid induction of EMT

I would be pleased if you will tell us anytime, if and how you will have succeeded to solve your problem with EMT; good luck

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Thanks for your advice,Iwill have a try.

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I agree with Kosmodron regarding the changing of your supplements

when I say the cells are sick...you are not getting contamination by fibroblasts. if you have epithelial cells, then you passage them, and they pick up a new morphology...this does not mean they have shifted to another cell type. this means they are sick. perhaps it is simply the matter of different supplements. I would also look very carefully at your passaging protocol. perhaps you are not being gentle enough with the cells, and they are harmed by your 'digestion'?

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