I have been trying to isolate neural stem cells from periventricular tissue (taken from an adult patient). But I do not get any attached cells after 2-3 weeks of culture in NB medium supplemented with glutamine, B27, bFGF, EGF and heparin on PDL/Laminin coated flask. The protocol was carried out as described in a paper on JOVE with slight modification.
1) During tissue dissociation, the tissue (of 10 pieces 1mm size from endoscopic needle sampling) was minced briefly and digested in 1 mL 0.05% Trypsin EDTA with DNase I at 37C for 5 min.(No trituration at this step)
2) The tube was swirl several time during incubation followed by addition of trypsin inhibitor.
3) The pellet was spun down at 1500 rpm, 5 min and supernatant was discarded.
4) The cell pellet was resuspended with growth medium (as mentioned above) and filter through 40 um cell strainer.
5) The cell suspension was spun down again and the pellet was resuspended in growth medium.
6) The cells were seeded at density of 250000 cells in a PDL/Laminin coated T25 flask.
However, we only observed formation on cell clumps after several days of culture and those clumps were not neurosphere.
Is there any step that I need to improve or revise for better result?
what is that paper in JOVE?
minced briefly? Is the other protocol recommend to mince the tissue?
I have successfully isolated the following primary cells from aorta and artery just by enzyme digestion.
Please refer to our JBC paper (http://www.jbc.org/content/270/52/30949.long)
for how we isolated primary smooth muscle cells from the rat aorta.
and novel neuronal type of cells (ANNIES) from mesenteric artery
Somasundaram C et al, 2006
(http://www.karger.com/Article/Abstract/92765)
What do the cells look like right after seeding in the flask? Are there clumps then? What is the count/cm2?
1. Those clumps (if they look dull under microscope) - possibly enzyme digestion was too harsh then you would have very low yield and viability OR enzyme digestion was too mild and you need to triturate the tissue blocks a lot to get single cells and then you would still have very low yield and viability.
2. Myelin contamination (as you are working with adult brain tissues) - Did you see a lot of myelin in the culture? Myelin can significantly affect viability of your culture and possibly affect attachment of the cells. If you see myelin floating in the medium or forming clusters, do a removal step before plating.
3. Batch to batch variation of B27 - Based on my own experience with mouse adult NSC, a certain batch of B27 we purchased always favored neurosphere formation (and we didn't know why).
In short, if you see your clusters/clumps (which have defined edge and are phase bright under microscope) of cells grow in size (which will take at least one week as you are at the early derivation stage), your culture condition may just favor sphere formation. If you see random stuff cluster together in the medium and it looks dull and dark, you may just have too much debris or dead cells. If that's the case, you will need to work on the isolation procedure.
I never cultured neural stem cells isolated from perivascular tissue but neural stem cells derived from human induced pluripotent stem cells. I experienced the similar obstacles.I replaced the PDL/laminin with Matrigel and got a better results. NSCs attacehed and grew nicely.
I referred to this paper http://www.ncbi.nlm.nih.gov/pubmed/24637893
The paper said the tissue is minced to provide more tissue surface for enzymatic digestion.
It was single cell (phase bright) in the medium after seeding. The cell clumps of uneven/elongated shape appeared after 1 day. The cells were seeded 10000/cm2.
Will I be able to collect the NSCs attached on Matrigel for further expansion?
I am curious why there is no explant culture established for neural stem cells? So far the explant culture was used for migration assay but not to isolate NSCs. It seems like the cell derived from explant will differentiate into neuron. I was reading many papers describing cardiosphere isolation using cardiac explant culture.
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