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.