Designing successful experiments with primary human cells requires more than the right reagents - it demands careful attention to the variables that drive reproducibility and biological relevance. At Cell Systems, we frequently receive inquiries from researchers about how to achieve optimal results with our primary human endothelial cells and specialized cell culture media.
Based on extensive customer feedback, here are three of the most common challenges and how to address them:
1. Selecting the Right Cells
Choosing the appropriate cell type is critical to achieving your experimental goals. For example, modeling the blood-brain barrier (BBB) or blood–retinal barrier (BRB) requires endothelial cells that express tight junction proteins such as claudin-5, occludin, ZO-1, and VE-cadherin - all essential for generating high TEER values.
Because cellular aging alters transcriptomic profiles, we recommend using early-passage cells (typically passages 3-8) to preserve barrier-relevant phenotypes.
2. Using Assay-Appropriate Medium and Coatings
The medium and surface coating used for cell expansion may not always support functional assays. For barrier modeling, it is often necessary to switch to a barrier-inducing medium prior to testing.
This may include serum-free or low-serum formulations, with supplements such as ascorbic acid, hydrocortisone, or ROCK inhibitors-depending on your model system. Pairing the right medium with the right surface coating can dramatically improve barrier integrity, permeability assays, and in vitro reproducibility.
3. Optimizing Seeding Density
Between seeding and confluence, cells undergo a progressive transformation-including morphological stabilization, polarity establishment, and junctional maturation. Seeding density plays a pivotal role in this process:
• Under-seeding can delay confluence and increase variability in junction formation, potentially resulting in a leaky or unstable monolayer.
• Over-seeding may lead to compression, loss of polarity, or multilayering, compromising barrier integrity.
Finding the optimal density ensures predictable, physiologically relevant outcomes in TEER measurements and other barrier assays.
Trusted by Scientists Worldwide
For more than 30 years, Cell Systems products have been widely adopted and cited in peer-reviewed publications. Researchers across academia, biotech, and pharma use our cells in applications ranging from BBB and BRB models to angiogenesis and drug transport studies.
Many of these methods and protocols are readily accessible online. For example, searching “ACBRI 376 TEER” or “ACBRI 181 TEER” on Google Scholar reveals studies demonstrating how our Primary Human Brain Microvascular Endothelial Cells (ACBRI 376) and Primary Human Retinal Microvascular Endothelial Cells (ACBRI 181) are used in transendothelial electrical resistance (TEER) assays to evaluate barrier integrity.