HEAL: 3D Bioprinted Tissue Models

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Development of blood brain barrier models as a platform for compound testing A physiological relevant in vitro blood brain barrier (BBB) model is needed to establish the brain penetration potential of compounds being developed as drugs. Many of the compounds being developed for opioid use disorder or pain have targets in the brain and, in some cases, it would be beneficial to prevent brain penetration. Therefore, being able to establish brain penetrance during preclinical development is critical. Current in vitro models (e.g. PAMPA) are very simplistic and do not include the relevant endothelial, pericyte and astrocytes composing the BBB. The project team has established a 96-well transwell BBB model using human iPSC-derived human brain endothelial cells (ECs) and have used transepithelial electrical resistance (TEER) measurements to demonstrate tight junction and barrier formation. Using primary brain microvascular endothelial cells (BMEC), the team has also developed a BBB model that includes astrocytes and pericytes in a Mimetas microfluidic plate and have demonstrated low permeability to molecules of different molecular weights. This BBB model will allow evaluation of drug penetration in the brain, as well as to examine the effects of drugs on the physiology of the BBB in the context of pain and addiction. Biofabrication of a neurovascular unit (NVU) tissue model in multiwell format Brain tissue models that include brain microvasculature consisting of brain endothelial cells in a microenvironment of astrocytes, pericytes and neuronal cells is critically needed as an assay platform to assess the toxic effects of drugs in the brain, as well as to study efficacy of compounds on brain relevant pathophysiological disease phenotypes. Bioprinting techniques are being used to create a microvascularized brain tissue models made up of primary brain endothelial cells, pericytes and astrocytes in a 96-well plate format. Vasculogenesis and angiogenesis in these tissues has been shown by cell fluorescence imaging. The project team is also in the process of including neuronal cells and using a new multiwell plate platform that will enable perfusion though the vasculature. Neural spheroids models for opioid addiction screening 3D brain cellular models of relevant physiological complexity and amenable to HTS are needed as preclinical assay platforms to assess the toxicity and efficacy of compounds developed to treat OUD and pain. Spheroids are multi-cell type aggregates with physiologically functional activity and are being utilized as assay platforms for disease modeling and drug screening. Brain spheroids are produced using iPSC-derived neuronal cells and astrocytes, and cell composition is tailored to mimic that of different regions of the brain. Current work is focused on validating designer neural spheroids as a HTS platform for the discovery and validation of new treatments for OUD. The 3DTBL has neural 3D spheroid cultures in 384-well plates to use as a high throughput system to test compounds. The project team has developed a calcium flux (FLIPR) assay using these cortical spheroids and screened a library of 700 compounds targeting brain targets/signaling pathways involved in opioid use disorder. Team members are also further tailoring designer, self-organized brain organoids which are generated using controlled mixtures of iPSC-derived excitatory (glutamatergic), inhibitory (GABAergic) and dopamine neurons and astrocytes. These tailored neural spheroids assay platforms are being used to establish opioid-like activity signatures using detection methods commonly used in HTS, including fluorescence imaging calcium flux. The team has demonstrated that these neural spheroids have synchronized calcium waves depending on the neuronal composition. Developing optogenetic tools to create neuronal circuits in a multiwell plate format Opioids modulate reward circuits in the brain, including the ventral tegmental area (VTA), playing a central role in addiction and withdrawal, medically known as opioid use disorders (OUD). The ability to recreate neuronal circuits of addiction and reward in a test-tube would allow evaluation of the addictive potential of new pain medicines and facilitate the development of therapeutics to treat OUD. The project team is using bioprinting techniques to develope simplified ex vivo reward circuit. For example, the ventral tegmental area, is being constructed by bioprinting with the goal of testing the effects of compounds in reward and addiction neuronal circuits. Proof of principle experiments have shown that iPSC-derived dopamine and GABA neurons can be transduced with optogenetic sensors using AVV viruses. Further, cells can be bioprinted and action potential activities, following light activation, can be detected using microarrayelectrodes (MEA) and calcium binding fluorescence biosensors (GCamp) using confocal and epifluorescence, both in a 2D and 3D format. Innervated 3D skin models for pain sensing Pain drugs are being developed using engineered cell lines and animal models which are not very predictive of activity in humans, thus leading to a high number of failures in clinic in spite of good preliminary genetic evidence. There is a critical need to develop in vitro pain models that are more predictive of drug activity in the clinic. These in vitro cellular models should include human sensory neurons in the context on the tissue were pain is produced and in a format that is amenable to screening to ensure these models make an impact as preclinical assays for drug development. NCATS 3DTBL has established a robust protocol for the biofabrication of skin tissues in a HTS amenable multiwell platform. At the same time, NCATS SCTL has developed robust protocols for iPSC differentiation into sensory neurons. The two labs are working together towards the assembly of a functional innervated skin model that can use to quantitate pain or itch signals from the skin to the sensory neurons. The project team is working to evaluate formation of extensions from the sensory neurons into the skin tissue. Based on published data the team is also testing different approaches, including using DRG spheroids and designing custom wells that will allow DRG extension formation into the skin. Increasing endogenous matrix production in 3D Tissue models for improved physiological properties The extracellular matrix component (ECM) of tissues plays a critical role in tissue morphology and function by providing mechanical and physiological clues to the cells in the tissue. The native ECMs in each tissue are complex and generated by the cells in the tissue. Reproducing the complexity of the native ECMs in the biofabricated tissue is critical to ensure the correct physiological function of the tissue. Macromolecular crowding (MMC) is a technique that has been used to enhanced ECM secretion from cells. It is based on the addition of chemically inert macromolecules that increase ECM deposition by excluded volume effect. NCATS 3DTBL are applying MMC techniques to increase the native ECM in neuronal tissues and improve their neuronal network function. The project team has demonstrated that by treating astrocytes with Ficoll and ascorbic acid, ECM deposition increases, including collagen IV, fibronectin and laminin. Furthermore, when neurons are added to the astrocytes cultures treated with MMC, neuronal activities increase, as measured by calcium waves. We are currently investigating whether the MMC treated cultures induce a higher number of synapsis and more dense neuronal cultures to improve the quality of tissue models that will be used to more accurately model pain, addition, and overdose.