Five recommended points to efficiently culture mice endothelial cells

Endothelial cells (ECs) are natural barriers and platforms for exchanging materials in the body and are considered as gatekeepers. As genetic engineering can be easily done in mice, the study of cardiovascular diseases is often done in this animal. The primary culture of mouse lung ECs (MLECs) serves as common systems to study diseases associated with damaged endothelial cells such as hypertension, diabetes, acute lung injury and atherosclerosis.

A team led by Wang published a protocol in Scientific Reports in 2019 to isolate functional ECs from mice lungs without the need for pooling samples. The system involved collagenase I digestion and sorting using CD31 and CD102 microbeads. Several points were recommended to allow the successful isolation of cells:

1. Collagenase I should be dissolved in serum-free DMEM media rather than PBS for good viability.
2. The cell yield requires the correct dissociation of cells from the tissue. Dissociation of tissue is not enough when underdigestion is done while cell viability is lowered with overdigestion. A correct balance can be achieved when enzymatic digestion is combined with mechanical pressure such as gentle pipetting.
3. The seeding is best done in dishes with small volumes. Senescence was faster when large dishes were used as the time for confluency was more than when smaller dishes are used.
4. The EC yield is lower when neonatal or young mice are used due to faster proliferation. Also, the cell division is lowered when freezing and thawing cycles are involved.
5. As ECs are specialized and heterogeneous, the use of more than 2 antibodies is recommended to confirm the cell identity.
6. When genetically engineered mice are involved, re-genotyping is warranted before isolation. This is because of the probable falling of ear tags.

These primary cell cultures of ECs can aid research in several ways like studying endothelial cell functioning like inflammation or angiogenesis. New approaches such as RNA-sequencing can be performed on cultured cells that can be used to study genes and their regulation. Such generated cultures can be used for knock out studies that can cast light on specific molecules and their role in diseases.

Nowadays, the generation of knockout genes is greatly facilitated by the use of technology such as CRISPR/Cas. An example is a 2014 published article in Circulation by Zhang that discussed the generation of Liver kinase B1 (LKB1) knockout mice. Mouse lung ECs (MLECs) were used to confirm the deletion to show the role of the protein on endothelial cells.

With the European Heart Journal reporting that cardiovascular diseases rank number one to cause morbidity and death, the use of such primary cultures can offer insights into understanding the disease and design suitable treatments.

References:

Wang, J., Niu, N., Xu, S. et al. A simple protocol for isolating mouse lung endothelial cells. Scientific Reports  9, 1458 (2019) doi:10.1038/s41598-018-37130-4.

Zhang, W. et al. Endothelial cell-specific liver kinase B1 deletion causes endothelial dysfunction and hypertension in mice in vivo. Circulation 129, 1428–1439 (2014).

Adam Timmis, Nick Townsend, Chris Gale, Rick Grobbee, Nikos Maniadakis, Marcus Flather, Elizabeth Wilkins, Lucy Wright, Rimke Vos, Jeroen Bax, Maxim Blum, Fausto Pinto, Panos Vardas, ESC Scientific Document Group, European Society of Cardiology: Cardiovascular Disease Statistics 2017, European Heart Journal, Volume 39, Issue 7, 14 February