Keratinocyte SFM (1X)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the matrix gel in advance on ice or in fridge	Change the half of the media every 2–3 days

Upstream tips
Defrost the matrix gel in advance on ice or in fridge
Protocol tips
Change the half of the media every 2–3 days

Publication protocol

Seeding of Epithelial Cells into Matrix Gel and Changing Media: Collect necessary materials: human primary prostate epithelial cells (PrE), matrix gel on ice, 96-well microplate, keratinocyte serum-free media (KSFM) on ice, charcoal stripped fetal bovine serum (FBS), and dihydrotestosterone (DHT). Prepare KSFM-based organoid media by combining 47.5 mL of KSFM with 2.5 mL of charcoal stripped FBS and dihydrotestosterone (DHT) to a final concentration of 10 nM DHT, then reserve on ice. Plate cells into a 3D matrix in a cell culture hood using sterile technique. Gently mix cold KSFM-based organoid media with ice-cold matrix gel to obtain a 50% matrix gel mixture by slowly pipetting up and down, avoiding bubbles. NOTE: Matrix gel should be thawed overnight at 4 °C and kept on ice whenever possible. It solidifies quickly at room temperature (RT). Pre-wet a pipette tip in cold media and transfer 40–50 μL of 50% matrix gel onto the bottom of each well to be used on a 96-well plate. Coat the bottom of the well to form a base layer. NOTE: Do not fully dispense to the second stop on pipette, as this can create bubbles. Place the 96-well plate into 37 °C incubator for 30–45 min to solidify the base layer. NOTE: Do not plate epithelial cells onto the base layer until it has solidified, or cells may fall through the matrix onto the bottom of the plate and grow as a monolayer. Mix epithelial cells suspended in KSFM-based organoid media with matrix gel to achieve a final concentration of 100–1,000 cells/100 μL and 33% matrix gel. Plate epithelial cells on top of base layers using 100 μL of 33% matrix gel/cell mixture per well. NOTE: Previous experiments have shown that seeding epithelial cells too densely in 3D will restrict the size of organoid growth, while seeding too sparsely can result in very low yield13. It is important to optimize seeding conditions for the cell type of interest, based on the patient-specific organoid formation efficiency. Place the 96-well plate in a 37 °C incubator for 30–45 min to allow the matrix gel to solidify, then gently add 100 μL of KSFM-based organoid media on top of cells by pipetting slowly against the edge of the well. Change the media every 2–3 days. Pipetting against the side of the well with space between the media and matrix gel, carefully remove 50 μL of media and replace with 50 μL of fresh media. NOTE: For a long-term culture, the matrix gel needs to be changed every 2–3 weeks. If the matrix gel culture appears unstable and displays translucent wrinkles under the microscope, then the matrix gel has broken down and needs to be replaced.

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Manufacturer protocol

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