Co-culture model consisting of human brain microvascular endothelial and peripheral blood mononuclear cells

Abstract

Background Numerous systems exist to model the blood-brain barrier (BBB) with the goal of understanding the regulation of passage into the central nervous system (CNS) and the potential impact of selected insults on BBB function. These models typically focus on the intrinsic cellular properties of the BBB, yet studies of peripheral cell migration are often excluded due to technical restraints. New Method This method allows for the study of in vitro cellular transmigration following exposure to any treatment of interest through optimization of co-culture conditions for the human brain microvascular endothelial cells (BMEC) cell line, hCMEC/D3, and primary human peripheral blood mononuclear cells (PBMCs). Results hCMEC/D3 cells form functionally confluent monolayers on collagen coated polytetrafluoroethylene (PTFE) transwell inserts, as assessed by microscopy and tracer molecule (FITC-dextran (FITC-D)) exclusion. Two components of complete hCMEC/D3 media, EBM-2 base-media and hydrocortisone (HC), were determined to be cytotoxic to PBMCs. By combining the remaining components of complete hCMEC/D3 media with complete PBMC media a resulting co-culture media was established for use in hCMEC/D3 – PBMC co-culture functional assays. Comparison with existing methods Through this method, issues of extensive differences in culture media conditions are resolved allowing for treatments and functional assays to be conducted on the two cell populations co-cultured simultaneously. Conclusion Described here is an in vitro co-culture model of the BBB, consisting of the hCMEC/D3 cell line and primary human PBMCs. The co-culture media will now allow for the study of exposure to potential insults to BBB function over prolonged time courses.

New experimental models of the blood-brain barrier for CNS drug discovery

“…On the other hand, the limited scalability, phenotypic drift, and high costs of isolated primary human brain endothelial cells (BMEC) hamper the development of widely usable human BBB in vitro models [89, 90, 91]. As an alternative to primary cultures, immortalized human BBB endothelial cell lines such as hCMEC/D3 [92, 93, 94, 95, 96] exhibit suboptimal monolayer integrity and lower expression of tight junction proteins [97, 98, 99]. Therefore, development of robust and physiologically representative in vitro human BBB model of high fidelity remains a critical objective in the field of neurovascular research.…”

Size-dependent isolation of primordial germ cells from avian species

“…The microporous membrane insert system was originally developed to assess cell migration and invasion (Marsh et al, 2008;Moutasim, Nystrom, & Thomas, 2011), and proved particularly useful for the investigation of in vitro cellular transmigration using cocultures of heterogeneous cell types (Strazza, Maubert, Pirrone, Wigdahl, & Nonnemacher, 2016). In the present study, a microporous membrane insert of 8-μm pore size was used to isolate blood PGCs FIGURE 2 Yield of cells isolated from White Leghorn chicken embryonic blood using MACS versus SDI.…”

Bacteria elevate extracellular adenosine to exploit host signaling for blood-brain barrier disruption

“…Briefly, S. suis and GBS were grown in THY and THB medium (Bacto), respectively, at 37°C under an atmosphere containing 5% CO 2 without shaking. The immortalized human brain microvascular endothelial cell line (HCMEC/D3) was obtained under license from INSERM, France, and cultured in EBM-2 (Lonza) or ECM (Sciencell) endothelial cell media containing 5% FBS (fetal bovine serum; Gibco), and supplemented as previously described [ 27 ].…”