TRANSFECTION OF BRAIN CAPILLARY ENDOTHELIAL CELLS IN PRIMARY CULTURE WITH DEFINED BLOOD–BRAIN BARRIER PROPERTIES

Introduction: The blood-brain barrier (BBB) is crucial for maintaining the central nervous system's (CNS) environment, formed by non-fenestrated brain capillary endothelial cells (BCECs). These cells regulate the passage of substances between the blood and the brain, ensuring CNS protection. A promising strategy for drug delivery to the brain involves transforming BCECs into protein factories through genetic modifications. However, it is challenging to achieve this transfection in non-mitotic BCECs without disrupting their integrity. This study explores the feasibility of non-viral gene therapy in primary BCECs cultured with defined BBB properties, aiming to revolutionize therapeutic approaches for neurological conditions.

Key Findings: The study utilized primary cultures of BCECs, pericytes, and astrocytes isolated from rat brains to construct three different in vitro BBB models. These models were meticulously characterized by their expression of tight junction proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol, reflecting essential BBB characteristics. Among these models, co-culturing BCECs with astrocytes proved the most effective for transfection studies. The BCECs were transfected using Turbofect™, a non-viral transfection agent, to express the red fluorescent protein HcRed1-C1. Notably, transfection efficacy was independent of cell division, showing equal success in mitotic and non-mitotic BCECs without compromising BBB integrity.

Innovative Tools: Several innovative methodologies were employed to ensure the success and accuracy of the study. TEER measurements were used to evaluate the integrity and tightness of the BBB models. High TEER values indicated the effective formation of tight junctions, which is crucial for maintaining BBB properties. Fluorescence-activated cell sorting (FACS) analysis was utilized to monitor cell division and transfection efficiency, ensuring precise evaluation of transfection success in both dividing and non-dividing cells. Immunocytochemistry confirmed the expression of tight junction proteins in transfected BCECs, verifying that the cells retained their BBB characteristics post-transfection. Non-metric dimensional scaling (NMDS) was used to analyze the compositional heterogeneity of the microbial communities, ensuring robust statistical analysis of microbiome data.

TEER and Permeability Analysis: The TEER values were crucial indicators of BBB integrity. The study demonstrated that co-culturing BCECs with astrocytes and pericytes significantly enhanced BBB properties compared to monocultures. The maximal TEER values were 128 ± 9 Ω cm² for monocultures, 299 ± 17 Ω cm² for co-cultures, and 331 ± 28 Ω cm² for triple cultures. This indicated the importance of co-culturing BCECs with other cell types to enhance BBB integrity. The study also highlighted that TEER values around 130–150 Ω cm² were sufficient to obtain low permeability to mannitol, indicating effective barrier properties.

Gene Expression and Barrier Integrity: Gene expression analysis revealed that BCECs in triple culture expressed significantly higher tight junction proteins like claudin-5 and PECAM-1-1 than monocultures. This correlated with the higher TEER values observed, demonstrating the enhanced BBB properties in co- and triple-culture conditions. The study also showed that the expression of these proteins was maintained even after transfection, indicating that the transfection process did not compromise the BBB integrity.

Transfection Efficiency: The transfection efficiency of BCECs was assessed using RT-qPCR and flow cytometry. The results indicated that approximately 4% of BCECs were successfully transfected with the HcRed1-C1 plasmid in mitotic and non-mitotic states. Significantly, the transfection did not disrupt the BBB integrity, as TEER values remained above 130 Ω cm² throughout the observation period. This demonstrated that non-viral gene therapy could effectively apply to non-dividing BCECs without compromising their barrier properties.

Conclusion: This study demonstrated the feasibility of non-viral gene therapy in primary BCECs cultured with defined BBB properties. The transfection successfully divided and non-divided cells, maintaining BBB integrity and providing a foundation for future therapeutic approaches targeting the BBB. The findings suggest that genetically modifying BCECs in vivo might be a feasible strategy for drug delivery to the CNS, offering new possibilities for treating neurological disorders.

Join the Discussion: We invite you to share your thoughts on this innovative approach to CNS drug delivery. Do you believe non-viral gene therapy could revolutionize treatments for neurological conditions? Join the discussion in the comments below.

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Original Research: The original research, "Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties," can be found on PubMed here.