I have been trying to grow the Medulloblastoma cell line D341 for weeks now but they are extremely slow growing! I used the exact media and supplementation suggested by the ATCC.
These cells grow in suspension; I tried growing them in a T25/75 flask (no good) and a 6 well plate (better). They start clustering into spheres/aggregates when becoming confluent, however there are such few clusters even after growing for 2-3 weeks. The longer I leave them to grow into more clusters, the old clusters go brown/black and die- I think (see pics attached).
Spinning at 200g for 3 mins gets me a pellet however because the dead stuff is also a cluster/aggregate it is also a part of the pellet. When I resuspend in fresh media there are a lot of dead cells so I think some healthy cells die in the process.
I want to extract DNA from these cells however because they are so little in quantity, I cannot get much DNA. I am using the Qiagen DNeasy extraction kit and in the last step of the protocol it says if you repeat the elution step you can get a higher yield. I have tried this and consequently gotten poor 260/230 and 260/280 values so I would much rather prefer starting off with a high amount.
Has anyone grown these cells? Could you share any advice/tips to get them to grow without dying? As you can see in the pic, some clusters are white aggregates and look healthy. Ideally I want to have many clusters like this so I can extract DNA.
Thanks in advance.
Yan Yan Zhao , seeing your question, I have some suggestions for upgrading and optimizing the culture of CD341.
1. First, optimize the basic culture medium formula and ingredients:
- Replacing the culture medium (such as DMEM/F12) with a neural stem cell-specific culture medium (such as MCE can provide Bi-27 Supplement (50×), Sterile (HY-K3013)) can significantly improve the efficiency of neurosphere formation.
- Because you are extracting DNA instead of doing pharmacodynamic experiments, you can add recombinant proteins rhEGF (20 ng/mL) and rhFGF-basic (10 ng/mL) that promote cell proliferation. EGF can activate the PI3K/Akt pathway to promote proliferation.
- If the cell state is poor, Noggin (100 ng/mL) can be temporarily added to inhibit differentiation and maintain stem cell characteristics.
Reference:
CD341 culture; EGF
https://pubmed.ncbi.nlm.nih.gov/29632646/
CD341 culture; basic-FGF
https://pubmed.ncbi.nlm.nih.gov/20511190/
2. If you have high requirements for the density of CD341, you can try to optimize the metabolic pattern of CD341 to obtain higher purity living cells.
- It is necessary to change the medium by half every 2 days (to avoid the loss of growth factors caused by full-volume medium change) to avoid cell growth retardation.
- When changing the medium, add 1× Cell Detachment Solution and gently blow to dissociate the dead cell clusters, and then purify the living cells by centrifugation at 300 g for 5 minutes.
3. Special passaging method for CD341 cells
Optimal passaging time:
- Passage when the diameter of the sphere reaches 150–200 μm (about 5–7 days of culture), when the cells are in the logarithmic growth phase and have the highest activity. Avoid waiting until the sphere turns black or the diameter is greater than 300 μm.
- It is recommended to split the cells at a ratio of 1:3–1:4 to maintain a moderate density to stimulate sphere formation. If the cell viability is poor, 10% conditioned medium (old medium from the same cell line, containing autocrine factors) can be added during passaging.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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