Helping to End Addiction Long-term (HEAL): 3D Bioprinted Tissue Models

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The work on this program is focusing on the development of six assay platform projects: 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 for neurological diseases. Many of the compounds being developed for opioid use disorder or pain have targets in the brain, but 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. A bioprinted neurovascular unit model: An alternative model of the BBB is based on the formation of a network of blood capillary vessels in a brain like tissue environment, the so-called neurovascular unit (NVU). We have developed a bioprinted model of the NVU on a 96-well plate 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. 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. 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 and have been able to demonstrate a opioid-induced, withdrawal-like calcium activity in the spheroids mimicking the ventral tegmental area (VTA), and area of the brain involved in addiction behavior. Additional biosensors to detect action potential and neurotransmitter release are being develop to further explore the physiology of these neural spheroids. 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. We have continued to implement experiments to show that iPSC-derived dopamine and GABA neurons can be transduced with optogenetic sensors using AVV viruses. Further, transduced cells exressing these different biosensors can be bioprinted using hydrogels 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. Addiction-in-a-Scaffold System to Identify and Screen Therapeutics (ASSIST): a 3D bioengineering approach to treating opioid use disorder: We are collaborating with Drs. Nieland and Lovett at Tufts University to use their 3-dimensional neural tissue model composed of neuronal progenitor stem cells-derived neurons and support cells (glutamatergic and GABAergic neurons and astrocytes) in a biocompatible 3D silk-based scaffold matrix enveloped in a hydrogel environment, to recapitulates the acute, chronic and/or repeated opioid exposure, and implement drug screens. NCATS is currently adapting the ASSIST technology in-house. 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.