I isolated primary liver cells from porcine by two step collagenase method. I am using RPMI 1640 medium for culturing the cells on collagen coated plates (collagen coating is done by myself). The cells are not attaching even it is 10 to 14 days old, but they are viable and forming clusters. Please suggest me any method to have cell attachment
Depending on what you want to do with the cells, you could try using Matrigel or a similar product. I found it to be very good for rat and human hepatocytes.
Thank you for the suggestions. The cells are not attached on the scaffolds also, I will try on nanospun mats. Cell isolation is carrried out immediately after taking organ to the lab and enzymes are also completely removed.
Hi Janani,
The success or degree of attachment depends on the initial viability of the cells, and attachment efficiency is in fact a good assay for cell health. What was your initial viability? Also, I suggest RPMI1640 is not recommended for primary porcine hepatocyte (PHHs) cultures [Nelson et al Scientific Reports; 2013] as cells form discrete clumps in culture (I used serum-free medium), with diminished cell attachment efficiency and fucntionality. In fact, I recommend either Sigma William's E or Medium 199 for PHHs culture. I also develped a high viability/ high yield technique for PHHs isolation (for bioartificial liver apps). I can send you these papers if you wish[[email protected]]. PHHs produce there own ECM under the right culture conditions and dont necessarily require added scaffolds, unlesss you want 3D organoid cultures.
Best Wishes,
Lenny Nelson
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