I'm trying to isolate endothelial cells from mouse brains for culture and I've tried a variety of protocols with mixed success and was hoping for some pointers. I've had no issue with the isolation/digestion of the capillary fraction but when it comes to separating the capilliaries from the debris/erythrocytes I've had less luck, this is what I've tried so far:
1. Horizontal pasteur pipette which allows the debris to 'settle' so the endothelial cells can be squirted off. This was the original lab protocol and I've found it very ineffective.
2. Percoll gradient. This was from a JOVE video and is a gradient set up at 3000g for an hour using 10x PBS, 1xPBS, FBS and percoll. The capillary fraction is layered on top and spun for 700g for 10 mins (no accel/brake) then the cells/capillary fragments are supposed to be in the interphase. This resulted in zero cells in the interphase and a lot of debris pelleted right at the bottom of the tube, very much not what was on the video.
3. Optiprep gradient. Pretty much same as above but I used optiprep because the percoll hadn't arrived and I had it in the lab. This time I ended up with a diffuse pink layer (medium), a clear interphase (supposedly cells), a pale red band (erythrocytes/debris?) and a clear layer. I tried the interphase - no cells, then I tried the red band which got me capillary fragments as required but also tons of what I assume are erythrocytes (very small cells...?)
Anyway, I'm wondering whether anyone has any recommendations for getting a cleaner vessel fragment fraction? The one time I have managed to get a very dirty fraction the fragments did grow into endothelial cells, I'm just wondering whether there's a cleaner/better way to do it by modifying my gradients somehow?
Hello Yvonne,
You may use a RBC lysis buffer to get rid of erythrocytes. You can purchase it or make your own. Here is the recipe for the one we use to isolate aortic adventitial cells (including ECs):
10xRBC lysis solution
41.5g ammonium chloride
5.0 g potassium bicarbonate
0.18g disodium EDTA
400mL dH2O
pH 7.2-7.4 with 1M HCl or NaOH
Final volume 500mL with dH2O
As for the debris, I have a few question:
- do they prevent EC growth?
- do they stick to the flask or well?
I'm wondering if the debris could 'simply'be washed out when changing the media on ECs after a couple of days in culture, once the ECs have attached?
Alternatively, you may use CD31+ beads to clean up ECs from debris.
Hi Yvonne ,
maybe I can help. I will send you a detailed protocol on our preparation.
Steffen
Hi Yvonne,
Have you considered using magnetic beads isolation?
https://www.miltenyibiotec.com/_Resources/Persistent/1d34687c38aae836c9b307c2d7c5fdeb2ccc457c/SP_Isolation-endothelial-cells-adult-mouse-brain.pdf
Regards
Palm
Hi Yvonne,
Maybe per fusing the animal to reduce some of the contaminant.
Also, please have a look to Edu´s thesis, he worked in different methods of isolation and you might find some useful information. I think to recall he used CD3+ magnetic beads for the last step http://oro.open.ac.uk/71145/
Hi Tuo,
Here is the brief protocol that we used to isolate mouse brain ECs:
The brains from six mice without cerebella, white matter, and leptomeninges, were minced and digested with 500 U/ml of collagenase II (Worthington Co.) and 0.6 U/ml of Dispase II (Boehringer) in HBSS for 40 min at 37˚C. The digested material was filtered through a 100-um nylon mesh and washed twice in M199 + 10% FCS. Subsequently, the filtered cells were incubated with rat anti-mouse PECAM-1 antibody (MEC 13.1, BD Biosciences) for 30 minutes at room temperature, washed, and co-incubated with the sheep anti-rat IgG-conjugated Dynabeads M450 (Dynal Biotech) for 20 min in M199 supplemented with 1% FCS. MCVEC attached to the microbeads were captured by Dynal magnet and cultured in fibronectin-coated T-25 cell culture dish (Sarstead Inc) in EBM-2 media supplemented with endothelial cell growth factors (Lonza) until confluence. The homogeneity of MCVEC isolated by this procedure was 85- 95% (identified by DiI-Ac-LDL uptake). Passage 1-2 cells were used in the experiments at three days post-confluence.
Hope that answers your questions.
Hi Gediminas,
Thank you so much for sharing your protocol. I still have some details to ask:
- What age are those mice?
- So you don't remove myelin or RBC, right?
- Roughly how many cells can you obtain out of 6 mouse brains?
- How long does it take for the extracted endothelial cells to attach after plating?
Thanks you!