Dear all,
I am trying to isolate and culture bovine endometrial stromal cells (stromal fibroblast cells) by using collagenase type II for digestion (conc. 2mg/ml). I tried tow times but the result is not satisfactory. the working protocol as follows (main steps)-
In either case, after 2 days there is no visible cells. there were few very scattered small cells and they died after 7 days of culture.
Thanks in advance for your suggestions.
May be the problem is RBC lysis buffer which destroyed all the cell. İnstead use stem cell lysis buffer or use ficol method with density gradiends methods for RBC isolation.
Best
Dear Hocam,
thanks for suggestions. I may come to you physically to discuss about this issue.
Hi Sohel,
First, did you use the FBS to stop the digestion after collagenase? You'd better to use the culture medium or FBS to stop the digestion.
Second, the bovine endometrial stromal cells are bigger than other cells, you may try the 70 um filter.
In addition, 400 g for 5 minutes only can exclode part of red blood cells. Therefore, I guess most of your cells are still in the supernantants.
Last but not at least, the collagenase is too strong to digest the tissue, you may try the trypsin accoding your experiments.
you may found a trustable protocol to isolate the bovine endometrial stromal cells.
Good luck
Hi,
Do you get a mix of cells that do not attach or is there a problem of getting proper cells to seed?
1) Time and collagenase concentration might be too long (too high), you can check your survival rate here.
2) It is important to get a proper buffer suitable for both enzyme and cells. Easiest
is serum free media or SFM with DPBS 1:7
3) Check your lines for contamination, it is usually an issue with primary lines.
4) You can always try alternative method: cut small pieces (0,2cm x 0,2 cm) and let fibroblasts to esacpe into Petri. It worked for bovine skin fibroblasts.
5) IMO 500g 5min is enough for every cell line in suspension, and 70um pore is way to big (I would expect 8-15um to be more less enough, but I wouldnt recommend this method)
Thanks Sarah and Wojciech Witarski for your valuable suggestions. I will try to make it in combination of your suggestions.
Sarah, can you please give me a complete protocol to isolate endometrial stromal fibroblast using trypsin (including concentration and time). thanks in advance.
You're welcome.
Jividen, K., Movassagh, M. J., Jazaeri, A., Li, H. Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens. J. Vis. Exp. (87), e51513, doi:10.3791/51513 (2014).
Hi Wojciech Witarski,
Thanks again for your suggestion. My university does not have access to this journal. Do you have access to this journal? If so, please send me the article.
Thanks in advance. Have a good day.
https://www.researchgate.net/publication/260337745_Journal_of_Visualized_Experiments_Two_Methods_for_Establishing_Primary_Human_Endometrial_Stromal_Cells_From_Hysterectomy_Specimens
please let me know if there is a problem.
Data Journal of Visualized Experiments Two Methods for Establishi...
No there is not, I am able to download the article. thank you so much Witarski.
We usually use Collagenase Type I and Type IV for human endometrial tissue digestion and we have better results with Collagenase Type I. The digestion procedure takes 30-60 minutes and gives most satisfactory results.
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