I use human bone fragments from surgical waste to isolate human primary osteoblast like cells. I use explant method without collagenase treatment and cells migrate to TCPS in 1-2 week period and become confluent around a month. After first tripsinization there are two different populations with different proliferation rates in the flask. One population (probably osteoblasts) is made of large cells with prominent nuclei with extended cell processes and proliferate very slowly. Between these cells there are small colonies of fast proliferating small spindle like cells with small dense nuclei which I assume are fibroblasts. In the literature selective scraping is recommended, but it is lacks delicacy and I am not sure I will be able remove all fibroblasts by this method. It is also suggested to decrease serum concentration to 2.5%. I do not know what to do. Does anyone has any experience with fibroblast contamination?
What we are doing in our lab in such cases is to:
1- Discad the medium.
2- 2x washing with PBS
3- Add Accutase enzyme (about 2 ml per 25 cm2 flask)
4- Incubate at 37 C for 2-3 minutes
5- Add medium (5 ml per 25 cm2 flask) to decativate the enzyme
6- Discard the medium containing floaing fibroblasts
7- 2x PBS washing
8- Add fresh medium.
- This method depend on the fact that fibroblasts detached more faster by action of accutase than othe types of cells.
- We do this as long as it required.
- Honestly, it does not remove the fibroblasts by 100%
Q: Do you use Accutase with dilution or just as packaged concentration?
A: Package conc and also in this case I recommend you to start the incubation time with accutase to be at first 1 minute and see what you get.
Q: How often should we do that to decrease fibroblast population to a minimum ?
A: It is depend, but usually during the following up, if you found a lot of fibroblasts leaving no or litle space for your cells to propagate. And also may be at the step of medium changing, no problem if you add the accutase step if there were many fibroblasts.
Thank you Mohammed. Do you use Accutase with dilution or just as packaged concentration? And lastly, how often should we do that to decrease fibroblast population to a minimum?
Hi,
you can try the things you already suggested. Those are standards.
In addition to selectively use Accutase, you can try to selectively centrifuge (large cells will reach the bottom faster). This works well for hepathocyte isolation.
You could pick colonies of interest like one does in stable clone generation. Not very handy, but works.
You could try fibroblast inhibitors like Human Bone FibrOut™: Fibroblast Inhibitory System (http://www.chiscientific.com). But it´s quite expensive...
What about Facs? Are there markers for osteoblasts?
Good luck
Accutase is a good approach. Your other point about serum free or low serum media is also useful as a number of them are commercially available.
Unfortunately non of the above worked. We are considering magnetic activated cell sorter.
Thank you all for the help.
Hi,
what kind of explant do you use? In the past I used the femor head and never observed contamination with fibroblast. I did not use any enzymatic step, since it was not necessary. For serum concentration I would keep 10% . This is my past experience and it might be different with other type of explants.
T
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