Is there a reliable protocol to isolate and "purify" only tumorspheres in a sphere culture, that also contains some cell debris, some single cells that have failed to form spheres, and cell clumps that do not look like spheres? If all of the cells are isolated, will they alter the results (such as gene expression)?
As far as I have read, previous publications related to sphere culture did not mention anything related to this issue. If it is ok to include them, what would be the arbitrary acceptable threshold without altering the results? Thank you very much in advance.
Evaluate the spheres' size by microscopy (i.e., 100 um), then use cell strainer, i.e., 50 um, all the 100 um spheres will be collected by the strainer, but individual cells and debris will pass through. Wash the spheres using media to ensure that all debris and individual cells pass through with the flow. Then flip the strainer upside down and pour media from 1 ml pipet on the fliped strainer with some force to return collected spheres back into the tube.
I think the approach by Anton L. should work just fine!
Another possibility would be to use low-g centrifugation to pellet only larger organoids and get rid of small debris / single cells?
Best regards,
Christian
Hello!
Article A simplified and modified procedure to culture brain glioma ...
Culture of primary glioma cells and tumor spheres
Tumor-sphere cultures were performed according to reported procedures (4–6,8), with minor modifications. Following resection, the tumor tissues were washed, minced in phosphate-buffered saline (PBS) and subjected to enzymatic dissociation. The tissues were then mechanically dissociated with a graded series of fire-polished Pasteur pipettes, and passed through a series of cell strainers, and centrifuged at 800 × g for 5 min. Tumor cells were resuspended in DMEM/F12 containing 15% FBS and plated at a density of 2×105 live cells/ml. When tumor cells grew as monolayers in flasks, the medium was changed to committed stem cell medium (serum-free DMEM/F-12, 1:50 of B-27 supplement, 20 ng/ml rhEGF, 20 ng/ml rhFGF-b, 100 IU/ml penicillin G and 100 μg/ml streptomycin). Fresh rhEGF and rhFGF-b were added each week and the medium was changed twice a week. Cells were maintained in a standard tissue culture incubator with 5% CO2 and 100% relative humidity at 37°C.
Proliferation assay and limited dilution assays
In the proliferation assay, when suspending cells emerged and the primary tumor spheres were clear, they were collected and dissociated into single-cell suspensions through a fire-polished Pasteur pipette, and then reseeded in the stem cell media at the same cell density. Each cell line was serially passaged over 4 generations. To evaluate the self-renewal capacity of tumor spheres, tumor spheres were mechanically dissociated into single-cell suspensions and reseeded into 96-well microwell plates by limited dilution at a cell density of 1–2 live cells per well. Each well was fed with fresh stem cell media every 3–4 days. The formation of cell clones was inspected under a phase-contrast microscope after 7–10 days.
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