A Simple Approach To Culture Patient-Specific Fibroblasts
The study of phenotypes specific to diseases can be done using cell lines. For example, induced pluripotent stem cells were generated to express mutations in the gene most commonly affected in Parkinson’s disease called Leucine-Rich Repeat Kinase-2 (LRRK2). The research team led by Nguyen generated neurons from patient fibroblasts that resembled the early stages of the disease to hence allow studies for modelling the disease and test therapeutic agents. Fibroblasts from patients hence allow for multiple studies such as mechanisms behind diseases and designing appropriate and effective therapy.
Traditional approaches to isolate fibroblasts utilize enzymes that however have challenges such as the long process (up to 4 hours), the possibility of over-digestion and lowered cell viability. A research article published in 2013 in the Journal of visualized experiments by Vangipuram and team discussed the establishment of a fibroblast culture from a 4-mm human skin biopsy. The protocol was without enzymatic treatments and was described hence as “technically simple” lasting 15-20 min. It was also found to be reproducible when there was a small amount of few samples (this was observed for elderly patients or those with chronic conditions). Immediately after receiving the biopsy sample, the protocol can be done with high success to hence allow culturing of samples derived from patients.
800 μl of complete DMEM/20% FBS media is added to 6-well plates coated with gelatin. The skin punch biopsy sample is first prepared in medium followed by its dissection into evenly sized pieces. Care must be taken to avoid jagged edges that cause poor cell growth or attachment. The pieces are then placed on the bottom of culture plates followed by incubation. Media is added every 2 days to replenish what is lost via evaporation. Cell monitoring is done every day once and media is changed every 2 days.
48 hours after dissection, keratinocyte growth is seen followed by fibroblast outgrowth over 7-10 days. The growth of the latter can be encouraged over that of keratinocytes using DMEM high glucose media supplemented with 20% FBS. The cells show the morphology defined for fibroblasts: with oval nuclei in spindle-shaped cells. The cultures were found to express a specific marker called SERPINH1 (also called HSP-47) that is involved in collagen synthesis.
While 20% FBS is recommended for initial culture, 10% FBS can be used for later passaging of cells. Dilution of the keratinocytes is seen after 2 passages. As other cells such as keratinocytes need specific growth factors and supplements for active growth, the medium used results in the pure culture of fibroblasts.
Thus, research comes up with newer and simpler approaches for Primary Cultures of pure fibroblasts. This can speed up more studies to especially focus on human diseases.
References:
- Nguyen, H. N., Byers, B., Cord, B., Shcheglovitov, A., Byrne, J., Gujar, P.Pera, R. R. (2011). LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell stem cell, 8(3), 267–280. doi:10.1016/j.stem.2011.01.013
- Vangipuram, M., Ting, D., Kim, S., Diaz, R., &Schüle, B. (2013). Skin punch biopsy explant culture for derivation of primary human fibroblasts. Journal of visualized experiments :JoVE, (77), e3779. doi:10.3791/3779
