Formation of chondrospheres using MicroTissues 3D Petri dishes

The tissue spheroids were formed using MicroTissues 3D Petri dish micro-molds (Sigma Aldrich, cat.# Z764019-6EA, 81 circular wells 800µm x 800µm) according to manufacturing protocol. 

Prepare 2% agarose solution in PBS using microwave oven; 

Allow molten agarose to cool to about 60-70°C;

Pipette 450 µl of molten agarose into micro-molds for casting MicroTissues 3D Petri dishes;

After the agarose had gelled (~ 4 min), carefully flex the micro-molds to remove MicroTissues 3D Petri dishes;

Transfer MicroTissues 3D Petri dishes to a 12-well culture plate;

To equilibrate MicroTissues 3D Petri dishes, add cell culture medium (2.5 mL/well);

Incubate for 20 minutes or longer; 

Trypsinize cells, count and prepare cell suspension with concentration of 3.4x10⁶ per milliliter;

Remove culture medium surrounding outside of MicroTissues 3D Petri dishes;

Seed 190 µl of cell suspensions into MicroTissues 3D Petri dishes;

Incubate for 40 minutes or longer; 

Add an additional culture medium into each well of 12-well plate to the outside of MicroTissues 3D Petri dishes;

Incubate the 12-well plate containing MicroTissues 3D Petri dishes at 37ºC in a humidified atmosphere with 5% CO₂ for 2 days;

To harvest the spheroids from the micro-wells carefully squirt culture medium into the MicroTissues 3D Petri dishes using a micro-pippette;

Draw the spheroids up into the micro-pipette.

Reagents and Materials:

MicroTissues 3D Petri dish micro-molds (Sigma Aldrich, cat.# Z764019-6EA, 81 circular wells 800µm x 800µm);

Agarose (Helicon, cat.# Am-0710-0.1);

Dulbecco's modified Eagle's medium, DMEM (Gibco, cat.# 12491-015);

Fetal bovine serum, FBS (Gibco, cat.# 16000-044);

Dulbecco's phosphate buffered saline, PBS (Gibco, cat.# 18912-014);

Antibiotic-antimycotic (Gibco, cat.# 15240-062);

Trypsin/EDTA (Gibco, cat.# 25200-114);

L-glutamine (Paneco, cat.# F032);

12-well cell culture plates (Corning, cat.# 3512).