Reversible stencils for fabricating micro-tissues
Inventors
Huebsch, Nathaniel • Conklin, Bruce • Healy, Kevin E. • Loskill, Peter
Assignees
J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-11898167-B2
Publication Date
2024-02-13
Expiration Date
2035-08-07
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The invention relates to devices, methods, kits, and compositions for in vitro generation of three-dimensional micro-tissues that are accurate models of heart, skeletal muscle, neuronal, and other tissues.
Core Innovation
The invention provides devices, methods, kits, and compositions for in vitro generation of three-dimensional micro-tissues that serve as accurate models of human tissues including heart, skeletal muscle, neuronal, and others. Specifically, the device comprises a cell adhesion substrate and a removable elastomeric stencil overlay with cut-out patterned microwells made up of two or more distinct holes of various shapes (circular, oval, rectangular, square, V-shaped, triangular) joined by a canal. This configuration forces cellular alignment and self-assembly into micro-tissues with dimensions and biomarker expression relevant to in vivo tissues.
The problem addressed is the inadequacy of current animal and ex vivo models for predicting human responses to chemical substances including pharmaceuticals. Animal tests are costly, time-consuming, ethically questionable, and poor human models. Existing in vitro three-dimensional cardiac tissue constructs require large cell numbers, long preparation times, have short functional utility, and are difficult for single cell analyses. There is a need for reproducible, physiologically relevant, human cell-based tissue models that can be generated in large numbers, use reasonable cell quantities, exhibit mature phenotypes and allow functional and molecular analyses.
Claims Coverage
The patent includes 24 independent claims covering a device for cell confinement and micro-tissue formation, detailing physical architecture and functional components, materials, and biological applications.
Device architecture with patterned microwells
A device comprising a cell adhesion substrate and a removable elastomeric stencil overlay with microwells containing two or more holes of specified geometric shapes each joined by a canal (shaft), where the substrate binds cells within the holes and canals.
Material composition of elastomeric stencil
The removable elastomeric stencil comprises materials selected from PDMS, functionalized PDMS, polyimide, polyurethane, SU8, thermoplastics, PMMA, PC, PS, PET, PVC, fibrin, glass, quartz, silicon, and hydrogels including polyacrylamide, polyethylene glycol, alginate, agarose, gelatin, collagen, or combinations thereof.
Microwell dimensions for optimal tissue formation
Microwells have a depth of at least 250 μm; canals about 50 μm to 150 μm wide; and the canal-to-hole width ratio is about 1:3 to 1:10 to promote cell alignment and tissue stability.
Blocking agent for stencil surfaces
Stencil walls including microwell walls are coated with blocking agents such as Pluronics, polyethylene oxide, alginate, poly-N-isopropylacrylamide, bovine serum albumin, bisacrylamide, agarose, polyethylene glycol diacrylate or combinations to inhibit cell adhesion to stencil surfaces.
Substrate material and bonding
The substrate comprises materials such as glass, silicon, polyolefin, polystyrene, poly(meth)acrylates, polyacrylamide, polycarbonate, polyethylene glycol, polyvinyl derivatives, polyamide, polyimide, polyurethane, PVDF, phenolic, amino-epoxy resins, polyesters, polyethers, polyglycolic acid, polyphenyleneterephthalamide, polyphosphazene, polypropylene, or combinations, and may be permanently or reversibly covalently bonded to the stencil.
Integration of porous membrane and microfluidics
The substrate optionally comprises a porous membrane preventing cellular flux but permitting fluid flow, and can include a network of microfluidic channels beneath the membrane operably connected to reservoirs and microwells, optionally with micropumps, to deliver test compounds or reagents precisely to microwells.
Coating with cell adhesion molecules
The substrate may be coated with cell adhesion molecules including fibronectin, alginate, E-selectin, gelatin, laminin, matrigel, collagen, fibrinogen, bisacrylamide, RGD peptides, PHSRN peptides, DGEA peptides, or combinations to facilitate cell attachment.
Inclusion of cells and mixtures for tissue modeling
The device contains mammalian cells in microwells, potentially mixtures of cell types found in mammalian organs such as heart, muscle, or neuronal tissue, including genetically modified cells expressing marker or reporter genes or disease-associated mutant genes.
Cell seeding and micro-tissue self-assembly
The method involves seeding about 1000 to 10,000 cells, including mixtures of differentiated or genetically modified cells, into microwells of specified geometry, culturing them to promote alignment along canals and three-dimensional self-assembly into micro-tissues exhibiting synchronous contractility and mature functional responses.
In summary, the claims cover a device composed of a substrate and removable elastomeric stencil with geometrically patterned microwells and canals for cell alignment and tissue formation, materials suitable for constructing the stencil and substrate, coatings to inhibit or promote adhesion, integration with microfluidic networks, and containing specific mammalian cells or mixtures thereof to form physiologically relevant micro-tissues. Methods include seeding cells and culturing to induce micro-tissue formation with functional properties.
Stated Advantages
Enables rapid, reproducible generation of three-dimensional micro-tissues that accurately model human tissues such as heart, muscle, and neuronal tissues.
Requires fewer cells than existing engineered tissue constructs, facilitating high throughput and statistically significant studies.
Avoids the need for exogenous extracellular matrix gels, simplifying tissue fabrication and enabling automation.
Micro-tissues exhibit mature physiologic properties such as synchronous contractility and appropriate drug responses not observed in two-dimensional cultures.
The patterned microwell geometry promotes cellular alignment and three-dimensional self-assembly enhancing tissue functionality.
Allows for incorporation of mixed cell populations and genetically defined cells for disease modeling and drug testing.
Can be integrated with microfluidic networks for targeted delivery of test compounds and precise experimental control.
Documented Applications
In vitro generation of micro-tissues modeling heart, skeletal muscle, neuronal, and other tissues for pharmacological efficacy, safety, and toxicity testing.
Modeling cardiac diseases such as hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular dysplasia, left ventricular non-compaction, long QT syndrome through patient-derived or genetically engineered cells.
Use in drug screening to identify therapeutic agents alleviating symptoms of cardiac, muscular, or neuronal diseases.
Creation of three-dimensional muscular and neuronal micro-tissue models for studying muscular dystrophies, neuropathies, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, and other conditions.
Studying tissue physiology including contraction force, response to stretch (Frank-Starling behavior), drug-induced chronotropic and inotropic effects.
Application in high-throughput automated systems for loading cells and testing micro-tissues with drugs or other compounds.
Use of genetically encoded calcium indicators (e.g., GCaMP6f) in micro-tissues for non-invasive, real-time functional monitoring of intracellular calcium flux.
Interested in licensing this patent?