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CelesteAvalos CelesteAvalos
wrote...
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9 years ago
Hi everyone!

Hope that someone can help me with this issue:
I have been trying to obtain fibroblast cells from a 3,5 mm skin biopsy from belly skin and from behind the ear with an enzyme digestion protocol.
Even though we can see a cell suspension under the microscope after digestion, there is little or definitely no attachment on the plate after days of culture. We have tried different enzyme concentrations (we are using Liberase TM from Roche) and the digestion time is about 3 to 4 hours. We use conventional cell culturing media, DMEM +SF10%+PenStrep.

Any ideas of how many fibroblasts can I obtain from a 3,5 mm biopsy?

Or can anyone give me some advice on how to improve the yield, viability and attachment of these cells?


Every tip or trick would definitely help.  Slight Smile


Thanks!
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wrote...
9 years ago
I used collagenase to digest the minced tissue it takes about 2 hr to completely dissolve them..than to a 70 um cell strainer..pellet the cells and culture with DMEM with 10% serum..they attach at the next day...it's not difficult really
wrote...
9 years ago
http://www.currentprotocols.com/WileyCDA/CPUnit/refId-cb0201.html Slight Smile

I've used the explants method. We use to mince the skin punch biopsy and place around 5-10 pieces on the botton of a T25 flask. We add 1,5mL of CHANG medium, put the flask in an incubator (5% of CO2 in atmospheric air) then let the skin pieces adhere 3 days. After this time, the medium is completed to 4 mL. After one week of culture, the cells began to appear aroud the skin pieces. the medium is changed to 4/5 (HAM F'10 + 12%SVF, +antibiotics if necessary) + 1/5 CHANG medium until the cell culture become confluent.
Then the cells are trypsinized and reseeded in 1T75 flask in HAM F'10 + 12% SVF. We use to duplicate our cells every weeks.
Biology - The only science where multiplication and division mean the same thing.
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