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

National Center For Advancing Translational Sciences

Investigators

Linked publications & trials

Abstract

The work on this program is focusing on the development of six assay platform projects: Development of blood brain barrier models as platforms 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 Team has used brain microvascular endothelial cells (BMEC), human astrocytes and pericytes to develop two models of the BBB on microfluidic tissue plates. In one model endothelial cells create a channel in contact with a hydrogel containing astrocytes and pericytes. We have demonstrated low permeability to molecules of different molecular weights. In another model, a microvasculature is created between two channels of endothelial to assess perfusion and leakiness of microvessels. We have demonstrated that we can create a microvasculature that is perfusable with large macromolecules for at least two weeks. We are currently increasing the physiological relevance of this model by adding neurons, oligodendrocytes, and microglia. These two BBB models 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, and other neuroinflammatory, neurodegenerative diseases. 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 an 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 developed to further explore the physiology of these neural spheroids. The Team has also showed that neural spheroids mimicking different brain regions can be functionally connected to form assembloids, which should enable the possibility of creating neural circuits of addiction and pain in vitro. The Team has also started testing compounds in a chronic mOR agonists treatment model using VTA neural spheroids and testing the effects of mOR antagonists and other compounds being developed to prevent addiction. Biofabrication of functional neuronal circuits of addiction 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 will 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 develop in vitro reward circuits. We are using iPSC-derived dopamine, glutamatergic and GABA neurons transduced with optogenetic or calcium binding fluorescence biosensors (GCamp) biosensors using AVV viruses and mixed with hydrogels to create spatial arrangements to mimic connectivity of different regions of the brain. We are using light to stimulate neural activity at one end of the circuit, and we are measuring calcium fluorescence at the other end using fluorescence confocal microscopy. We are exploring how different circuit shapes affect activation and pharmacological modulation by mu-opioid receptor agonists and antagonists. 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 has reproduced the ASSIST technology in-house and is working on adapting it to high throughput screening 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 despite 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 where 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 be used to quantitate pain or itch signals from the skin to the sensory neurons. The project team is working to evaluate the 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.