Three dimensional tissues for high-throughput assays
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Publication Number
US-10732174-B2
Publication Date
2020-08-04
Expiration Date
Abstract
Provided herein are methods of detecting responses of bio-artificial tissues to agents by performing assays using three-dimensional bio-artificial tissues. The methods are adaptable to high-throughput platforms.
Core Innovation
The invention relates to an in vitro method for screening agents for a physiological response of cells of a three-dimensional bio-artificial tissue. The method suspends a three-dimensional bio-artificial tissue comprising living cells from a support without a fastener to facilitate tissue adhesion, with the support positioned in a well. The tissue is contacted with an agent and a cell-based assay is performed on the bio-artificial tissue to determine a non-mechanical physiological response of the cells, the assay producing an indicator of that non-mechanical physiologic response.
A key aspect is that the tissue is not removed from the well, while a signal from the indicator is measured. The measured signal is indicative of the level of response of the cells of the tissue to the agent. The three-dimensional bio-artificial tissue comprises at least 5 layers, including multi-layer constructs with at least 5 layers.
In a second independent method, the invention applies the same general approach to screening the effects of a pharmaceutical agent on tissue using a plurality of three-dimensional bio-artificial tissues. Each tissue is suspended from a support without a fastener, and the supports are positioned within a well of a multi-well plate without any mesh covering the bottom of the well to provide an array of locations. A plurality of agents is screened by contacting each bio-artificial tissue in the wells, performing the cell-based assay to generate an indicator of a non-mechanical physiologic response, and measuring indicator signals without removing the tissues from the wells.
Claims Coverage
The document provides two independent claims, each centered on in-well, non-mechanical physiological response screening using a three-dimensional bio-artificial tissue with at least 5 layers and measuring an indicator signal without removing the tissue from the well.
In-well, non-mechanical physiological response screening without removing tissue
Suspending a three-dimensional bio-artificial tissue comprising living cells from a support without a fastener; contacting the bio-artificial tissue with an agent; performing a cell-based assay to determine a non-mechanical physiological response producing an indicator; and, without removing the tissue from the well, measuring a signal from the indicator indicative of the level of response.
Three-dimensional multi-layer bio-artificial tissue (at least 5 layers)
The three-dimensional bio-artificial tissue comprises at least 5 layers.
Multi-well array in-well screening without mesh covering the well bottom
Suspending a plurality of three-dimensional bio-artificial tissues comprising living cells from supports without a fastener; positioning each support within a well of a multi-well plate without any mesh covering the bottom of the well to provide an array of locations; contacting each bio-artificial tissue with same or different agents; performing a cell-based assay in the well producing an indicator of a non-mechanical physiologic response; and, without removing the tissues from the wells, measuring a signal from the indicator from each well indicative of the level of response.
Screening multiple agents across the plurality of tissues
A plurality of agents is screened, each contacting a different bio-artificial tissue, while indicator signals are measured from each well without removing the bio-artificial tissues from the wells.
Overall, the claims cover in-well screening workflows where three-dimensional bio-artificial tissues with at least 5 layers generate an indicator of a non-mechanical physiological response upon agent contact, and indicator signals are measured without removing the tissue, including a multi-well, no-mesh-covering array format for screening multiple agents.
Stated Advantages
Enables determining a non-mechanical physiological response of cells of a three-dimensional bio-artificial tissue to an agent using an indicator-based, cell-based assay.
Allows measurement of an indicator signal without removing the tissue from the well.
Uses three-dimensional bio-artificial tissue comprising at least 5 layers.
Provides an array of locations in a multi-well plate by positioning supports within wells without any mesh covering the bottom of the well, enabling screening of a plurality of agents.
Documented Applications
High-throughput in-well screening of agents using three-dimensional bio-artificial tissues formed on scaffold supports positioned above the well bottom without fasteners, with indicator-based readouts measured in the well using plate readers.
Phenotypic profiling and hit triage using decision tree logic for assay window robustness (Z-factor) and response profiles from tissue indicator readouts after compound exposure.
Quantifying dose-dependent effects of compounds on tissue contractile force, mitochondrial potential, actin/F-actin disruption, and viability in a miniaturized hydrogel tissue construct system with multi-layer tissue constructs.
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