The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices(ECM)for cell micro-environmental modulation.Matrigel embedding is problematic...The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices(ECM)for cell micro-environmental modulation.Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations.In this study,we developed a novel microwell culture platform based on 3D printing.This platform,without using Matrigel or external signaling molecules(i.e.,SMAD and Wnt inhibitors),successfully generated matured human cerebral organoids with robust formation of high-level features(i.e.,wrinkling/folding,lumens,neuronal layers).The formation and timing were comparable or superior to the current Matrigel methods,yet with improved consistency.The effect of microwell geometries(curvature and resolution)and coating materials(i.e.,mPEG,Lipidure,BSA)was studied,showing that mPEG outperformed all other coating materials,while curved-bottom microwells outperformed flat-bottom ones.In addition,high-resolution printing outperformed low-resolution printing by creating faithful,isotropically-shaped microwells.The trend of these effects was consistent across all developmental characteristics,including EB formation efficiency and sphericity,organoid size,wrinkling index,lumen size and thickness,and neuronal layer thickness.Overall,the microwell device that was mPEG-coated,high-resolution printed,and bottom curved demonstrated the highest efficacy in promoting organoid development.This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.展开更多
基金We thank Hemdeep Patel from the ResinWorks3D company for providing us the high-resolution 3D printed mold devices as a gift.We also thank Angela Clyde for the initial discussion on device design.We thank Utah State University’s College of Engineering Undergraduate Research Program(EURP)for supporting Andrew Kjar.We also thank Utah State University Research Catalyst(RC)program for supporting Cheng Chen.
文摘The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices(ECM)for cell micro-environmental modulation.Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations.In this study,we developed a novel microwell culture platform based on 3D printing.This platform,without using Matrigel or external signaling molecules(i.e.,SMAD and Wnt inhibitors),successfully generated matured human cerebral organoids with robust formation of high-level features(i.e.,wrinkling/folding,lumens,neuronal layers).The formation and timing were comparable or superior to the current Matrigel methods,yet with improved consistency.The effect of microwell geometries(curvature and resolution)and coating materials(i.e.,mPEG,Lipidure,BSA)was studied,showing that mPEG outperformed all other coating materials,while curved-bottom microwells outperformed flat-bottom ones.In addition,high-resolution printing outperformed low-resolution printing by creating faithful,isotropically-shaped microwells.The trend of these effects was consistent across all developmental characteristics,including EB formation efficiency and sphericity,organoid size,wrinkling index,lumen size and thickness,and neuronal layer thickness.Overall,the microwell device that was mPEG-coated,high-resolution printed,and bottom curved demonstrated the highest efficacy in promoting organoid development.This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.
