I harvested fibroblasts from an adult 3 mm punch biopsy 10 days ago. I am culturing them at 37C 5% CO2 and changing medium every 2 day (DMEM, 10% FBS, 1X Pen/Strep). Here is their appearance today, which has been stagnant for a few days now. Do they look healthy? Why are they not dividing?
What type of culture are you using: separation of the epidermis and dermis with enzymes or whole explant culture? Are the images from subcultures or the initial culture?
Separation of epidermis and dermis:
Article Epidermal/Dermal Separation Techniques and Analysis of Cell ...
Example of whole explant culture:
Article Simple, Fast, and Efficient Method for Derivation of Dermal ...
We removed the epidermis after dispase digestion and only cultured dermal cells. This is the initial culture. We have not passaged yet.
The 2 steps with the greatest potential for cell are the Dispase step separating epidermis and dermis, and dissociation of the dermis. The epidermal basal lamina tends to stay attached to dermal matrix, giving the impression more enzyme treatment is needed. This may temp you to keep on digesting. Try reducing time in Dispase. Overdigestion will also lead to cells popping when you centrifuge dispersed dermal cells. Reducing digestion time and/or centrifugation time help here.
I think the density of cells are too low. Fibroblasts are happy to grow at a high density condition which provides ECM for cell expansion. You may try culture cells using a coating plate.
Thank you both for the advice.
Dispase II digestion time was overnight at 4C (~13 hrs) and collagenase IV digestion time was 1.5 hrs at 37C. Do either sound too long?
We are using Thermofisher T75 nunclon coated easy flask. Does this sound like an appropriate model? Agreed that density is low and I will plan to plate in a smaller dish in the future.
The damage done during dissociation is always a combination of the digest time and physical handling. However, before you try changing any dissociation parameters, 1 X 3mm punch biopsy per T 75 is too little material. Start with one well of a 6 well plate or 1 x35 mm dish. Perhaps 2-3 explants per dish. I missed how little tissue you were using.
As I remembered, at our lab，we treat adult skin with 3 x antibiotics in PBS for 15 minutes (3 x 5 minutes), and then cut them into small pieces, seed them onto a dish or 6 well plate( dermis face down and the epidermis face up) add a drop of FBS, put back to CO2 incubator. After 24 hours, add a small volume complete culture medium to the dish or plate. After 5-10 days, fibroblasts will expand from the skin and keratinocytes will be death due to FBS(you can see them under the microscope). After removing all pieces of skin, the attached fibroblasts can be harvested by trypsin digestion. Using this method, you can easily get some divided fibroblasts.
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