Design of Experiment Based Optimization of a Direct Contact Blood Brain Barrier in Vitro Model for Neuroactivity Screening

Kelsey E Lubin, Purdue University

Abstract

Neurotherapeutics are an essential drug class that is often forgotten or neglected due to the difficulties associated with pharmaceutical development and approval. These compounds face high rates of attrition in clinical trials and late stage development predominantly due to the restrictiveness of the blood brain barrier (BBB). The inherent role of the BBB is to protect and maintain the homeostatic environment around the neuronal cells in the brain parenchyma. This is accomplished by the BBB posing not only as a physical barrier through its restrictive tight junctions that prevent paracellular permeation, but also through the high expression levels of efflux transporters and drug metabolizing enzymes that prevent transcellular permeation of potential drug compounds. In attempting to deliver compounds to the brain the intended outcome is often over-shot to the point of causing neurotoxic implications. One way to mitigate the difficulties associated with drug delivery to the brain and early evaluation of potential toxic compounds is to develop in vitro cell-based models that mimic the in vivo BBB and neurovascular unit (NVU). The mainstays of the BBB phenotype are presented in the brain microvessel endothelial cells (BMECs) and are regulated and influenced by the close contacts of supporting cells of the NVU such as astrocytes, pericytes, and neurons. An in vitromodel that can mimic the close contacts between these four cell types and is capable of being implemented in pharmaceutical development for BBB permeability and neuroactivity screening could lead to better selection of hit and lead candidates, and ultimately reduce the attrition rates of neurotherapeutics.Direct contact coculture and triculture models have been developed in our laboratory that mimic the in vivo cell-cell contacts between the different cell types of the NVU and provide increased barrier properties in comparison to other models utilizing indirect contact between cell types. Early development and optimization of these models was accomplished using the human cerebral endothelial cell line hCMEC/D3. Although this cell line proved useful in early validation stages, it was decided that a different endothelial cell source would be sought out. Work was done using iPSC derived endothelial cells (iCell® endothelial cells) and an alternative immortalized human brain endothelial cell line (HBEC-5i). Both cell lines proved to be amenable to the direct contact coculture and triculture models, with the iCell® models showing greater barrier properties in comparison to those using the HBEC-5i cell line. However, drawbacks of the iCell®model were observed in extending culturing of the cells causing the cells to “roll” to the middle of the filter and proving to be cost prohibitive for extensive optimization. Ultimately, the HBEC-5i cell line was chosen for continued development and optimization due to its immortalized origin and potential for replacing the hCMEC/D3 cell line in the direct contact models.Optimization of the direct contact triculture using the HBEC-5i cell line was required as all of the previous development was performed using the hCMEC/D3 cells. Typically, optimization of in vitrosystems is performed in a one factor at a time manner or not at all. Given the large number of factors that can influence the outcome of this model, a design of experiments (DOE) based optimization approach was taken. DOEs are traditionally used in process optimization of non-biologically based systems; however, the production of the direct contact triculture is a process that could greatly benefit from extensive optimization.

Degree

Ph.D.

Advisors

Knipp, Purdue University.

Subject Area

Physiology|Aging|Genetics|Morphology|Neurosciences|Pharmaceutical sciences

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