Development of 3D Human Blood Brain Barrier Formation Assay

Abbreviated title: BBB NAM for DNT Statement of Work Development of a blood-brain barrier formation and integrity model for the evaluation of potential developmental neurotoxicity The purpose of this acquisition is to develop an in.vitro.blood-brain barrier formation and integrity new approach method (NAM) for the evaluation of potential developmental neurotoxicity hazard of foods and dietary supplements. This assessment will first require optimization of human-derived brain microvascular endothelial cell, neuron, and astrocyte models, as well as in-plate assay control chemical exposure concentration and duration. Second, four (4) performance compounds with expected activity in assay endpoints will need to be evaluated to demonstrate assay performance. Background The mission of the Division of Toxicology (DT) in the Office of Chemistry and Toxicology (OCT), Office of Laboratory Operations and Applied Science (OLOAS) of the Human Foods Program (HFP) is to develop, evaluate, and integrate modern predictive toxicological new approach methods (NAMs) to support regulatory and public health decision making. As such, DT aims to develop and assess the relevance and predictive capacity of in.vitro. (human-derived, cell-based) NAMs for human safety evaluations of chemicals of interest to HFP and the Agency. To do this, chemicals of immediate interest need to be evaluated for their potential effects on the developing nervous system, a primary research area for HFP. The developing nervous system is exceedingly vulnerable to chemical-induced toxicity, particularly from exposures in pregnancy through the maternal diet, as well as in infancy, childhood, and adolescence. At present, the recommended in.vitro.NAMs for the evaluation of potential developmental neurotoxicity (DNT) applied in regulatory applications do not incorporate or model the blood-brain barrier (BBB). The BBB is imperative for elimination or minimization of toxic insult to the central nervous system; thus, NAMs which recapitulate chemical-induced effects on barrier formation and/or integrity are essential to the interpretation of potential DNT hazard. As such, this acquisition aims to identify a contract organization that can develop a BBB formation and integrity in.vitro.NAM which incorporates three (3) human- derived cell types (i.e., brain microvascular endothelial cells, neurons, astrocytes) in a multi-chip microfluidic plate format (i.e., minimum 40 microfluidic chips per plate). Development of the NAM will be invaluable to the prediction of potential DNT hazard of foods and dietary supplements. -- 1 of 4 -- Abbreviated title: BBB NAM for DNT Objective To develop an in.vitro.blood-brain barrier formation and integrity new approach method for the evaluation of potential developmental neurotoxicity hazard. Development of this NAM will allow DT/OCT/OLOAS/HFP/FDA to predict potential chemical-induced developmental neurotoxicity, which is a vital area of concern for public health decision-making in HFP and the Agency. Scope The scope of this work encompasses the optimization of human-derived brain microvascular endothelial cells, neurons, and astrocytes for a multi-chip microfluidic plate format assay, selection of appropriate in-plate assay control chemicals for each assay endpoint, and evaluation of four (4) performance compounds with expected activity in those endpoints. Tasks 1. The contractor shall obtain three (3) applicable human-derived cell types for the purposes of model development and optimization in consultation with the technical point of contact (TPOC) a. Cell types shall include: i. Brain microvascular endothelial cells (HBMEC acceptable) ii. Neurons (induced pluripotent stem cell (iPSC)- or embryonic stem cell (ESC)-derived acceptable) – no neurotransmitter specific requirement iii. Astrocytes (iPSC- or ESC-derived acceptable) 2. The contractor shall optimize the selected human-derived cell types above for a multi-chip microfluidic plate format (a minimum of 40 microfluidic chips per plate is acceptable) 3. The contractor shall evaluate at a minimum phase contrast images, transendothelial electrical resistance (TEER), fluorescein isothiocyanate-labeled (FITC) dextran permeability, and cell viability to assess barrier formation and integrity 4. The contractor shall evaluate at a minimum quantitative immunofluorescence (IF) of nuclear, neuronal, and astrocyte markers, as well as cell viability to assess developmental neurotoxicity -- 2 of 4 -- Abbreviated title: BBB NAM for DNT 5. The contractor shall evaluate at a minimum two (2) in-plate assay control chemicals at three (3) concentrations to include at a single concentration in subsequent chemical evaluations. Applicable chemical exposure duration and concentration(s) will be selected in consultation with the TPOC. a. Staurosporine recommended as an in-plate assay control for cell viability and quantitative immunofluorescence of neuronal and astrocyte markers b. Tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) recommended as in-plate assay controls for phase contrast images, TEER, and FITC dextran permeability 6. The contractor shall evaluate four (4) performance compounds at eight (8) concentrations. Performance compound selection and concentrations assessed will be determined in consultation with the TPOC. 7. The contractor shall provide raw experimental data for all in-plate assay control chemicals including vehicle controls, as well as for performance compounds across individual multi-chip microfluidic plate. 8. The contractor shall report all experimental protocols and any deviations from standard procedure. Delivery Deliverable # Task # Title Required Format Due Date Recipient 1 7, 8 Raw experimental data, noting any deviations from standard procedure Electronic file (.csv or .RData preferred) shared with TPOC no more than sixty (60) days after completion of experiments TPOC Government-Furnished Property (GFP) None Security Co…