I am working with HepG2 cells, and I want to pre-coat my flask/plate to keep the cells in single layer.
Can I use gelatin to coat the dish or I need to use collagen? I was wondering if any of you knows a safe method to pre-coat the plate for HepG2?
This could help me a lot! Thank you!
Dear Cy!
Please You look at this article:
Article Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Ce...
4.3. Cell Cultures
The human hepatocarcinoma cell line HepG2 (ECACC, Sigma-Aldrich)
4.1. Preparation of PVA/G Hydrogels
PVA/G hydrogels were prepared modifying a procedure described in a previous study [25] in order to obtain sterile scaffolds ready to use. Briefly, a 10% (w/v) solution of PVA (99% hydrolyzed, Mw 85,000–146,000; Sigma-Aldrich, Milan, Italy) in distilled water was prepared using an autoclave cycle, set at 120 °C and 20 min. The PVA solution was cooled down to 60 °C under magnetic stirring, opened inside a cell culture flow cabinet and blended with a 3% (w/v) sterile filtered solution of gelatin (G; from skin bovine type B, 75 Bloom; Sigma-Aldrich) in distilled water to obtain a bioartificial mixture with a PVA/G ratio of 80/20 (w/w).
https://www.abcam.com/ps/pdf/protocols/cell_culture.pdf
3. Creating the correct culturing environment
For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be coated with 1% fibronectin.
Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin by diluting with distilled water followed by filtration. This is efficient to coat about 5 flasks. 2. Pipette coating solution into flask. Rock back and forth to evenly distribute the bottom of the flask. Let sit in incubator for 15-30 minutes. 3. Completely remove coating solution by aspirating before seeding.
In my opinion there should be not much of a difference between collagen and gelatin - use sterile gelatin solution (0,2%) and coat your dishes, leave fo approx. 30 minutes, remove the solution and let the dishes air dry in the hood. Use immediately or store in the fridge (wrapped with parafilm).
Another coating that gives a very homogeneous distribution of HepG2 cells during culture is Polyethyleneimine (PEI - organic polymer with a high density of amino groups also used for transfection - for coating use approx. 2µg/ml; both the branched and the linear forms work for me).
For detailed PEI protocols see also:
doi: 10.1186/1472-6750-4-23
doi: 10.1002/jemt.20742
https://www.sigmaaldrich.com/technical-documents/articles/biofiles/gelatin-solution-product.html
gelatin coating protocol
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