Synthetic biomaterials designed for the generation of mouse neural organoids for in vitro toxicity screening.

ABSTRACT There is a well recognized need for more accurate and cost effective toxicology screening methods for therapeutic and environmental chemicalsAdvances in the field of stem cell derived brain organoids have led to exploration of these models not only in the study of neural developmentbut also for drug and toxicity screeningHoweverbecause of their costcomplexityand workflow requirementsthese methods have yet to be effectively implemented beyond basic research settingsSuch neural organoids are usually formed on a complex extracellular matrix derived from tumors cultured in rodentscommonly known as MatrigelThe process is cumbersomemakes interrogation difficultand lacks reproducibilityMore recentlyinvestigatorsincluding a Stem Pharm co founderhave demonstrated that brain organoids can be formedculturedand assayed reproducibly in a plate based system on engineered hydrogel substrates with human embryonic stem cell derived precursor cellsThe goal of this project is to apply Stem Pharm s hydrogel platform to the development and validation of an in vitro mouse neural organoid system for toxicity screeningIn specific aimculture conditions will be optimized for the generation of three dimensional brain organoids derived from mouse embryonic stem cells inwell andwell plates by adjusting hydrogel characteristicscell seeding densitiesand media replenishmentReproducibility of the determined method will be assessed via organoid sizeneural maturationand RNA sequencingSpecific aiminvolves subjecting optimized organoids to a small scale screen against known developmental neurotoxinsOrganoids will be assessed by examining their transcriptome alterations in response to toxin treatmentsFinallythe in vitro model will be validated by comparing gene expression patterns to previously generated in vivo toxicant response dataCompletion of this study will result in a hydrogel composition and culturing protocols that support the formation of reproducible mouse brain organoids for medium throughput toxicology screening applicationsPhase II studies will utilize data from Phase I transcriptome analyses to identify potential biomarkerscreate quantitative molecular panels to assess toxicityand develop phenotypic screens that will be used to further validate these assays with additional known developmental neurotoxins and non toxic controlsThis work has the potential to drastically reduce the necessity for and costs associated with the use of live animals for toxicity testingUltimatelyif the responses of these in vitro systems are sufficiently equivalent to or more sensitive than previously generated in vivo animal toxicity datait would represent critical evidence for the validity of the approach and support the legitimacy of analogous organoid models built with human cells PROJECT NARRATIVE Stem Pharm intends to utilize the I Corps program to perform hypothesis driven customer discovery to test product market fit and inform our product development and commercialization strategyThe goal of the parent project is to develop mouse brain organoid models that are amenable to high throughput neuro toxicology testing inwell formatsThis approach will address a longstanding need for alternative models to animal testing and will lead to reduced time and cost for chemical screensThe study will utilize Stem Pharm s proprietary hydrogel material for the formation of brain organoids from mouse embryonic stem cellsValidation of this model and development of biomarkers associated with toxicity will lead to commercially available predictive assays for screening chemicals and drug candidates for neurotoxicity