3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways.
- Award ID(s):
- 2051065
- NSF-PAR ID:
- 10315195
- Date Published:
- Journal Name:
- Frontiers in Pharmacology
- Volume:
- 12
- ISSN:
- 1663-9812
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract -
Abstract Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin‐like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue‐derived, epithelial‐only intestinal organoids. HELP enables the encapsulation of dissociated patient‐derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal‐derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin‐ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid–matrix interactions and potential patient‐specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (
G’ ≈ 1 kPa), slower stress relaxation rate (t 1/2≈ 18 h), and higher integrin ligand concentration (0.5 × 10−3–1 × 10−3m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal‐derived products or synthetic polyethylene glycol for potential clinical translation. -
Abstract The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co‐culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision‐cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung‐on‐a‐chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.
-
Abstract Engineering physiologically relevant in vitro models of human organs remains a fundamental challenge. Despite significant strides made within the field, many promising organ‐on‐a‐chip models fall short in recapitulating cellular interactions with neighboring cell types, surrounding extracellular matrix (ECM), and exposure to soluble cues due, in part, to the formation of artificial structures that obstruct >50% of the surface area of the ECM. Here, a 3D cell culture platform based upon hydrophobic patterning of hydrogels that is capable of precisely generating a 3D ECM within a microfluidic channel with an interaction area >95% is reported. In this study, for demonstrative purposes, type I collagen (COL1), Matrigel (MAT), COL1/MAT mixture, hyaluronic acid, and cell‐laden MAT are formed in the device. Three potential applications are demonstrated, including creating a 3D endothelium model, studying the interstitial migration of cancer cells, and analyzing stem cell differentiation in a 3D environment. The hydrophobic patterned‐based 3D cell culture device provides the ease‐of‐fabrication and flexibility necessary for broad potential applications in organ‐on‐a‐chip platforms.
-
Human induced pluripotent stem cell (hiPSC)-derived brain organoids can recapitulate the complex cytoarchitecture of the brain as well as the genetic and epigenetic footprint of human brain development. Although the brain organoids are able to mimic the structures and functions of brain in vitro, the 3D models have difficulty in integrating a complex vascular network that can provide the interaction with organoids. Here we report on a microfluidicbased three-dimensional, vascularized cortical organoid tissue construct consisting of 1) a perfused micro-vessel against an extracellular matrix (ECM), dynamic flow and membrane-free culture of the endothelial layer, 2) a sprouted vascular network using a combination of angiogenic factors, and 3) a vascularized hiPSCderived cortical organoid. We report on an optimization of density/stiffness of ECM to induce angiogenic sprouting and effect of angiogenic factors to trigger robust, rapid, and directional angiogenesis for concentration-driven and repetitive sprout formation. Vascularized network in the microfluidic device was further characterized in terms of morphology, directional alignment under perfusion, lumen formation, and permeability. HiPSCderived cortical organoid was generated, placed, and integrated into a vascularized network in the vascularized microfluidic device. We investigate how vascularized micro-vessels interact with cortical organoid. This paper further demonstrates the potential utility of a membrane-free vascularized cortical organoid in perfusion used to model Alzheimer’s disease and for toxicity screening of nerve agents.more » « less