I have been culturing human microvascular endothelial cells (HMEC-1) on my own-made low-serum media. The cells expand fine in wells and flasks T25, T75, and T180 using 0.2 ml/cm2, so the media is not a problem. I coat 1% gelatin and seed at 20k per cm2. When I try to scale up to multi-layer flasks, I have problems with cells not attaching under the same culture conditions. I have read that cells need time to adapt to low serum conditions, but the population doubles in around two days, indicating that their growth under the current conditions is consistent. I have also read little pieces about how oxygen exchange in multi-layer flasks differs from single-layer, but I have no experience with this. Any advice is appreciated.
One explanation is that the oxygen tension in multi-layer flasks is lower than in single-layer flasks, which may affect the cell attachment and viability. According to a study by Kato et al. (2017), the oxygen tension in the bottom layer of a 5-layer flask was about 2.5% compared to 5% in a single-layer flask, and the cell viability was significantly lower in the multi-layer flask. They suggested that increasing the oxygen concentration in the incubator or using a gas-permeable multi-layer flask could improve the cell growth and attachment.
Another explanation is that the cell density in multi-layer flasks is higher than in single-layer flasks, which may induce cell-cell contact inhibition and reduce the cell proliferation and attachment. According to a study by Lee et al. (2016), the cell density in a 5-layer flask was about 5 times higher than in a single-layer flask, and the cell proliferation rate was significantly lower in the multi-layer flask. They suggested that reducing the cell seeding density or using a medium with higher serum concentration could enhance the cell growth and attachment.
A third explanation is that the surface coating in multi-layer flasks is different from in single-layer flasks, which may affect the cell adhesion and spreading. According to a study by Kim et al. (2014), the surface coating in a 5-layer flask was thinner and less uniform than in a single-layer flask, and the cell adhesion and spreading were significantly lower in the multi-layer flask. They suggested that increasing the coating concentration or using a different coating material such as collagen or fibronectin could improve the cell growth and attachment.
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