Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets

Abstract

We report the design and fabrication of a robust fluidic platform built out of inert plastic materials and micromachined features that promote optimized convective fluid transport. The platform is tested for perfusion interrogation of rodent and human pancreatic islets, dynamic secretion of hormones, concomitant live-cell imaging, and optogenetic stimulation of genetically engineered islets. A coupled quantitative fluid dynamics computational model of glucose stimulated insulin secretion and fluid dynamics was first utilized to design device geometries that are optimal for complete perfusion of three-dimensional islets, effective collection of secreted insulin, and minimization of system volumes and associated delays. Fluidic devices were then fabricated through rapid prototyping techniques, such as micromilling and laser engraving, as two interlocking parts from materials that are non-absorbent and inert. Finally, the assembly was tested for performance using both rodent and human islets with multiple assays conducted in parallel, such as dynamic perfusion, staining and optogenetics on standard microscopes, as well as for integration with commercial perfusion machines. The optimized design of convective fluid flows, use of bio-inert and non-absorbent materials, reversible assembly, manual access for loading and unloading of islets, and straightforward integration with commercial imaging and fluid handling systems proved to be critical for perfusion assay, and particularly suited for time-resolved optogenetics studies.

In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution

“…Therefore, they provide ideal means for miniaturizing islet perifusion systems. Previously reported microfluidic perifusion systems, developed to study dynamic insulin release from isolated pancreatic islets, rely on calcium imaging to measure the intracellular Ca 2+ response,13,14 on the collection of medium samples for biochemical analysis of secreted insulin15–17 or glucagon,18 or on a combination of both, optical imaging and biochemical readouts 19–22. However, the temporal resolution of the corresponding measurements is limited by the relatively large volumes in the majority of these devices, which preclude rapid liquid exchange and/or require pooling of multiple islets to achieve quantifiable insulin concentrations.…”

Islet–immune interactions in type 1 diabetes: the nexus of beta cell destruction

“…Single cell transcriptomics/ proteomics Assess expression of candidate genes and their protein products for paired cell genotype/ phenotype data, as well as adaptive immune cell repertoire analysis [104] Single cell ATAC-sequencing Investigate the contribution of dysregulated gene networks, particularly in candidate loci, to immune or β-cell function [106,107] CyTOF Following labeling with metal tagged antibodies, deep phenotyping of single cell suspensions can be performed [110] IMC By applying metal tagged antibodies to fixed tissue sections, deep phenotyping of cells can be performed in situ [111,112] Laser capture microdissection Identify differentially expressed genes and proteins to develop disease-predictive biomarkers or targeted therapeutics [108] nanoPOTS Identify novel post-translational modifications within a single islet and the proteomic basis for islet heterogeneity [109] CODEX Examine disease-related tissue and cell subset reorganization, as well as perform multiplexed deep phenotyping [113] Tissue clearing (e.g. X-CLARITY, PARS) Visualization of intact morphology, vasculature, innervation, and extracellular matrix [114] RNA single-molecule FISH Identify the dynamics of candidate gene and checkpoint molecule expression in all subsets present in the tissue microenvironment, examine how they influence cell phenotype and function [115] Microphysiological Systems Introduce targeted therapeutics or innate and adaptive immune cells to assess their contribution to disease development/treatment [116] Investigate the underlying cause of the islet anti-viral immune signature Examine the role of hybrid insulin peptides (HIPs), defective ribosomal products (DRiPs) and neoantigens in β-cell stress or destruction Tissue Slice Profile live immune and endocrine cells across the pancreas in the native tissue environment [24] Assess the role of chemokines and adhesion molecules Determine which cells are directly pathogenic vs bystander or tissue resident cells *nanoPOTS = Nanodroplet processing in one pot for trace samples; PARS = Perfusion-assisted agent release in situ; ATAC = Assay for Transposase Accessible Chromatin; CyTOF = Cytometry time of flight; IMC = Imaging mass cytometry.…”

“…Novel platforms for ex vivo studies of live human islets have been developed to mimic the human islet microenvironment and model islet-immune cell interactions (Table 3). One such technology is the recently developed isleton-a-chip microphysiological system (MPS), which allows laminar flow of immunotherapeutic agents or specific immune cells over human pancreatic islets with immunofluorescent imaging in real time, providing a way to monitor β-cell function and phenotype in response to any number of putatively diabetogenic or protective stimuli [116]. Indeed, in a recent study, Lenguito et al were able to maintain viable islets and reliably detect insulin secretion in response to glucose and KCl stimulation [116], illustrating the capacity to further leverage this MPS to interrogate the etiological role of host-pathogen interactions; specific antigen targets, lymphocyte activation state, as well as function of autoreactive T and B cells at the islet-immune interface within an isogenic system; and the potential for regulatory T cells (T regs ) to inhibit β-cell destruction.…”

Human pseudoislet system enables detection of differences in G-protein-coupled-receptor signaling pathways between α and β cells

“…To overcome this challenge, we developed an integrated microperifusion system consisting of pseudoislets and a microfluidic device that enables studies of islet intracellular signaling using genetically-encoded biosensors in conjunction with hormone secretion (Figures 5A and S3A-S3C). The microfluidic device ( Figure S3A) (Lenguito et al, 2017) is made of bio-inert and non-absorbent materials with optimized design for nutrient delivery, synchronous islet imaging by confocal microscopy, and collection of effluent fractions for analysis of insulin and glucagon secretion. The microperifusion system uses smaller volumes, slower flow rates, and fewer islets than our conventional macroperifusion system ( Figures S3D-S3F).…”

“…The microperifusion platform ( Figures 5 and S3) is based on a previously published microfluidic device with modifications (Lenguito et al, 2017). Design modifications were incorporated using SolidWorks 2018 3D computer-aided design (CAD) software.…”