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-10851344-B2
Publication Date
2020-12-01
Expiration Date
2035-08-07
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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 are accurate models of human tissues including heart, skeletal muscle, and neuronal tissues. The core innovation lies in using a device comprising a cell adhesion substrate and a removable elastomeric stencil overlay with patterned microwells. These microwells, having shapes such as circular, oval, rectangular, square, V-shaped, or triangular holes joined by canals, force mammalian cells to align and self-assemble into three-dimensional micro-tissues exhibiting authentic tissue properties.
The problem addressed by the invention pertains to the limitations inherent in current animal-based and in vitro tissue models for toxicity testing and drug efficacy studies. Animal-based tests are ethically questionable, costly, time-consuming, and often poor predictors of human responses. Existing in vitro models, particularly for cardiac tissues, require large cell numbers, take long to prepare, have short functional lifespans, and do not readily permit single-cell analyses. There is a need for scalable, reproducible, physiologically relevant, and accessible in vitro tissue models that employ fewer cells and allow high-throughput testing while maintaining mature tissue-like characteristics.
The described devices overcome these problems by confining cells within deep microwells that provide sufficient surface area for cell adhesion and interconnectivity through narrow canals to induce uniaxial alignment and mechanical stress gradients. This geometry promotes self-assembly into functional micro-tissues without requiring exogenous extracellular matrices, enabling rapid generation of tissues with physiological electrical and contractile behaviors. The micro-tissues respond synchronously to drugs, manifest mature phenotypes, and are compatible with automated handling and analytical methods, thus offering clinically relevant platforms for drug screening and disease modeling.
Claims Coverage
The patent includes one independent claim describing a method for generating micro-tissues using a specifically designed device comprising a cell adhesion substrate and an elastomeric stencil with patterned microwells. The claim focuses on the use of geometrically defined microwells to induce alignment and self-assembly of mammalian cells into dogbone-shaped micro-tissues. There are 24 main inventive features extracted from the independent claim.
Device with patterned microwells
A device comprising a cell adhesion substrate and a removable elastomeric stencil overlay having cut-out patterned microwells formed by two or more holes joined by a canal, enabling confining of cells.
Dogbone-shaped micro-tissue formation
Method of seeding mammalian cells into microwells comprising circular, oval, rectangular, square, V-shaped or triangular holes joined by canals, culturing them to self-assemble into dogbone-shaped micro-tissues aligned along canals.
Seeding cell number range
Seeding about 2000 to about 9500 cells per microwell in the device to form micro-tissues.
Microwell depth specification
Microwells with depths of at least 250 μm or at least 500 μm to prevent tissue bridging and promote distinct micro-tissues.
Microwell geometric dimensions
Microwells with at least two square holes of side length 250 μm to 1000 μm connected by a canal with a length of 250-1000 μm and width 50-200 μm; a ratio of hole size to canal width of at least five.
Use of mixed mammalian cell types
Seeding a mixture of mammalian cell types typically present in organs such as heart, muscle, or neuronal tissue into microwells.
Genetic modification of cells
Cells can comprise heterologous marker genes, reporter genes including calcium indicators, mutant genes relevant to disease models, such as mutations in genes linked to cardiac function or other tissue functions.
Cell loading and culture methods
Cells can be seeded by settling via gravity or fluid flow through membranes; culturing includes introducing test compounds and evaluation of contractility, morphology, and gene expression.
Micro-tissue analytical assessment
Determination of cell alignment, three-dimensional structure formation, contractile activity along the longitudinal axis, and response to drugs with increased synchronicity compared to 2D cultures.
Stencil removal and downstream analysis
Methods include reversible bonding allowing stencil removal to yield intact micro-tissues for further analysis including mRNA and protein expression and histological evaluation.
The independent claim covers a method of using a specifically designed device with an elastomeric stencil overlay featuring dogbone-shaped microwells to induce alignment and self-assembly of mammalian cells into functional three-dimensional micro-tissues. Key inventive features include the microwell geometry, cell seeding density, depth of wells to prevent bridging, use of mixed cell types including genetically modified cells, capability for high-throughput culture and drug testing, and reversible stencil bonding enabling tissue retrieval and analysis.
Stated Advantages
Enables generation of clinically relevant human tissue models that accurately mimic in vivo tissue properties.
Requires fewer cells than conventional tissue constructs, facilitating rapid and high-throughput testing.
Micro-tissues exhibit physiologically relevant synchronous contractility and drug responsiveness superior to two-dimensional monolayer cultures.
Eliminates the need for exogenous extracellular matrix gels, simplifying tissue fabrication and enabling automation.
Allows reversible stencil removal for intact tissue retrieval, supporting detailed molecular and histological analyses.
Supports formation of micro-tissues reflecting genetic disease states, enabling patient-specific and isogenic disease modeling.
Compatible with robotic fluid handling and membrane loading methods for scalable and reproducible tissue generation.
Documented Applications
In vitro generation of three-dimensional micro-tissue models of human heart, skeletal muscle, neuronal, and other tissues for drug efficacy and toxicity testing.
High-throughput screening of drug candidates using human cardiac micro-tissues exhibiting mature electrophysiological and contractile properties.
Modeling genetic cardiac diseases such as hypertrophic cardiomyopathy, dilated cardiomyopathy, anthracycline-induced cardiotoxicity, arrhythmogenic right ventricular dysplasia, left ventricular non-compaction, double inlet left ventricle defects, and Long QT syndrome using micro-tissues derived from patient-specific or genetically engineered cells.
Modeling muscular and neuronal diseases and conditions including muscular dystrophies, neuropathies, myasthenia gravis, Parkinson’s disease, multiple sclerosis, and others utilizing micro-tissues formed from relevant cell types.
Pharmacological testing of drug responses, including inotropic and chronotropic effects, in physiologically relevant human tissue models.
Use of calcium indicator-expressing cells in micro-tissues for continuous monitoring of intracellular calcium flux and electrical activity.
Embedding and histological analysis of micro-tissues for morphology, biomarker expression, and structural studies.
Formation of micro-muscles that can be mounted on standard muscle testing apparatuses to study contractile force, tension response, and electrical pacing behavior.
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