Right now I am using HT29 cells, but I will also use HCT116 and probably breast cancer cell lines.
hi,
Have you tried to culture CSCs spheroids without agarose coating?
Regards
Saurabh
Saurabh Mandal Without low attachment plates, it doesn't work. I have tried to use the hanging drop methods and then transferred the spheroids into a normal 96 well plates, but I didn't get satisfactory results.
Regards
Hello, Enrica!
Please You look at this protocol:
Article A Reliable Tool to Determine Cell Viability in Complex 3-D C...
Cell line and spheroid culturing Experiments were carried out with the human colon carcinoma cell lines HT29 and HCT-116 (ATCC, Manassas, VA). Cells were routinely thawed from frozen stocks and were subcultured for < 25 passages (cumulative population doublings [CPD] < 100). Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1 g/L glucose, 1% sodium pyruvate, 1% L-glutamine, and 3.7% NaHCO3, supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum (FCS), was used as “standard medium” for routine culturing (medium and all ingredients from PAN Biotech GmbH, Aidenbach, Germany). All cultures were kept in a humidified atmosphere with 5% CO2 in air at 37 °C. Cell transfer and preparation of single-cell suspensions were performed by mild enzymatic dissociation using a 0.05% trypsin and 0.02% EDTA solution in phosphate-buffered saline (PBS; PAN Biotech GmbH). Stock cultures were passaged every 3 to 4 days by seeding 0.5 to 1 × 106 cells into T75 culture flasks. Spheroids were initiated in liquid overlay11 by seeding 1.5 × 103 HT29 cells/well and 0.75 × 103 HCT-116 cells/well in a 200-µL medium using agarose-coated 96-well plates (50 µL 1.5% agarose/well).11,12 After an initiation interval of 4 days, 50% of the supernatant was replaced by fresh, standard medium and every 48 h thereafter except for the 72-h drug treatment setup described in the Drug Treatment section (see below)
Thank you Alexandr Chernov ! It seems a good article even if they didn't centrifuge them, I will have a better look and try.
hi,
Why I'm saying, once I was having a shortage of low adherent plates, then, I cultured CSCs spheroids in normal culture dishes, sterile Petri dishes as well as in agarose coated plate. I got good CSCs spheres of nice round shape & size in both normal culture dishes and sterile Petri dishes.
While agarose didn't give satisfactory spheroid. What I understood even trying best, agarose gel coating was thick but not robust. Spheroid gets a somewhat unnecessary sticky attachment with the agarose layer. Agarose being a soft kind of material, CSCs never achieved a good round shape. I was culturing breast CSCs.
I wish you will get good culture with agarose.
Regards
Saurabh
Hi,
I use agarose coated 96 well flat-bottom plates. I pour in 55ul of agarose. I seed the necessary amount of cells, keep the plate in the incubator for an hour and then centrifuge at 2000rpm for 5 minutes. This method always gives me spheroids.
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