I've been trying to culture human fetal microglia from whole human fetal brain samples. I've been using the protocol by Durafort, et al. (link attached). Of course the first time I tried the protocol it worked great, but every sample thereafter has been resulting in nearly complete cell death.
The first time I did the prep there were clearly lots of cells that stuck to the flask the next day and my total microglia yield was on target for what the authors reported. Now the cells that are there barely stick to the flask and are easily lifted just by gently moving the flask, and they look nothing like the cells that I got the first time I completed the prep.
The one difference is that the first time I used heat-inactivated FBS and since then I've been using charcoal-stripped FBS. Otherwise the media is the same (DMEM, 5% FBS, 1% pen/strep).
As I've only tried the prep a couple of times, I'm wondering if this is something with sample variability or maybe even if there is something going wrong in how the samples are being shipped to us (in media vs. PBS, in which case the cells may just be dying in the PBS). I just completed a prep yesterday and will have another look in a few days (I don't want to touch the flasks just in case any cells may get lifted).
If you happen to know of a method that works or tips or tricks that work for them I would really appreciate it! If you have any questions for me let me know. Thanks!
http://www.ncbi.nlm.nih.gov/pubmed/23813381
I can't offer another protocol but would like to pick up two other things you mentioned:
1. You get your "alive" samples / cells shipped in PBS? How long does the shipping take? I wouldn't leave viable cells in PBS for any longer than absolutely necessary. If this process is taking too long (I don't know how sensitive the cells are but I would assume very) it can for sure have an influence on the cell viability or at least on their well-being and recovery after the transport and isolation. Is it not possible to have them shipped in the future culture medium? Or maybe it's a possibility to add at least some FBS to the PBS to see if it makes a difference?
2. Do you have any possibility of going back to heat-inactivated FBS just to make sure your problems aren't resulting from something as simple as that? Or is there any special reason why you turned to charcoal-stripped FBS? As far as I understood the stripping doesn't only get rid of many undesirable components but also potentially necessary lipophillic compounds like growth factors and hormones which may be crucial for your cells?
Hope you'll get your experiments back on track :)
Thanks for your response, Angela. Let me address your questions:
1) Our samples are shipped fresh, packed on ice, overnight. We receive them the following morning, so we process them within 24hrs of when they were collected. We requested that they were shipped to us in media with some FBS, but we are going to double-check to make sure that they are doing this. I agree, if the samples are being shipped in PBS that this will be detrimental to cell survival.
2) Switching back to heat-inactivated FBS is something I will probably try this week. We decided to try using charcoal-stripped FBS for some studies involving sex differences, but I believe we can do the initial culture with heat-inactivated serum and then switch to charcoal-stripped serum once the microglia have been isolated from the mixed glial culture and plated. However, cultures with primary neonatal mouse mixed glial cultures with charcoal-stripped FBS have not shown any obvious differences in survival or proliferation of microglia.
Considering these two points, I think there may be something with how they are shipped to us or with the initial processing. I am going to double-check with the company today to see how they have been shipping our samples.
Thanks!
Sorry then I have one follow-up question: The first time you did the experiment were the samples also comming from an external supplier already? Or did you do it yourself?
Good luck with the FBS-approach. Otherwise I think you best guess might be to double and triple-check with whoever is supplying you. Maybe something in their procedures changed and they didn't realise it may have such a detrimental effect on your experiments?
Is this tutorial video helpful for you? The procedure is shown for three adult mouse brains but it is the same for human brain tissue.
https://youtu.be/XYg7KSsZZys
- Badges
- Science method