I´m having some trouble growing CRC organoids from biopsies (maybe with low tumor cells present).
Is there any trick that could improve the growth of CRC organoids from biopsies?
Material and methods I´m currently using:
Biopsy digestion medium:
1.58 mg/ml collagenase type II ,
10 ug/mL of hyaluronidase Type IV
100 ug/mL primocin
10 µM RhoKi
Basal expansion medium for CRC organoids (WNT is omitted):
Extracellular matrix:
1. Cultrex BME (R&D)
Passage:
TrypLE + Rhoki
Refresh medium every 2-3 days
Passage every 7 days.
Hi Paolo,
Organoid culture requires 3D cell culture conditions. One good material for 3D cell culture is nanofibrillar cellulose hydrogel, GrowDex. It is clean and animal-free material which is highly biocompatible and tunable. You can embed the colorectal cancer cells in GrowDex and grow them to organoids on 96-well plate. You can use 100ul volume of GrowDex+cells, and then put 100ul of culture medium on top, and replace the medium when needed.
Dear Lauri, thank you for your answer.
Definitely, the matrix is important, but I double that this is the problem in my culture, but if this GrowDex is GMP prove, I´d like to try as I have a project in hand for GMP organoid production. Thanks
Dear Paolo,
Thanks for your reply. At the moment GrowDex is for research use only but I'll let you know when GMP grade hydrogel is available.