Enzymatically Degradable Hydrogel Substrates for Multi-Layered Cell Constructs
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Solution Overview
Problem
Current tissue engineering methods face challenges in recreating the complex structural characteristics of native tissues, particularly in cardiovascular applications, due to limitations in controlling substrate physiochemical properties and damaging cell layers during harvest and transfer processes.
Innovation Solution
A method involving multi-layered cell sheet stacks using enzyme-digestible hydrogel substrates, where cell sheets are stacked and patterned to mimic native tissue structures, allowing for precise control of substrate modulus and preservation of cell viability through enzymatic digestion of scaffold materials.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional scaffold tissue engineering approaches are used to grow engineered tissue, then tissue production is enabled, but the ability to control substrate physiochemical properties (modulus, topology, surface chemistry) simultaneously is limited
Solution Approach 1:
The invention divides the substrate into multiple independent cell sheet layers, each grown on separate temperature-responsive surfaces. This segmentation allows independent control of physiochemical properties for each layer while maintaining overall system functionality, resolving the contradiction between adaptability and complexity.
Solution Approach 2:
The temperature-responsive culture dish surface serves multiple functions: it provides mechanical support during culture, enables controlled cell sheet release through temperature change, and allows stacking to form 3D structures. This multi-functionality reduces the need for multiple specialized substrates, improving adaptability without proportionally increasing system complexity.
2Ease of manufacture
If temperature-responsive culture dishes coated with poly(N-isopropylacrylamide) are used to manufacture cell sheets, then cell sheet production is achieved, but the substrate cost increases and the temperature used to displace cells can damage the cells
Solution Approach 1:
The invention changes the temperature parameter dynamically during the manufacturing process. Cells are cultured at physiological temperature (37°C) for optimal growth, then the temperature is lowered to below the LCST to trigger controlled cell sheet release. This parameter change enables easy manufacture while minimizing cell damage compared to harsh chemical or mechanical detachment methods.
3Manufacturing precision
If multiple myocardial cell sheets are overlaid to develop myocardial tissue masses, then three-dimensional tissue structures are achieved, but the process time and complexity increase
Solution Approach 1:
The invention performs preliminary actions by pre-growing complete cell sheets on temperature-responsive surfaces before stacking. Each cell sheet is fully differentiated and structurally organized before being transferred and stacked with others. This preliminary organization of cells into ready-to-stack units significantly reduces the time and complexity compared to attempting to build 3D structures layer-by-layer in situ.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the rapid fabrication of functionalized three-dimensional thick tissues with high cell viability, capable of mimicking native tissue structures and functions, suitable for vascular and cardiac tissue engineering applications.
Implementation Method 1
scaffold materials that can be digested by specific enzymes
Implementation Method 2
A first cell sheet layer is flipped on to a receiving cell sheet layer to form a sandwich... Pressure is applied to the sandwich to encourage adhesion of the layers
Data Source
AI summary
The present disclosure relates generally to the fields of tissue engineering and regenerative medicine. More particularly, the present disclosure generally relates to systems, methods, compositions and kits to rapidly fabricate functionalized three-dimensional tissues from multiple stacks of cell sheets using enzyme-digestible hydrogel substrates as supports for the cell sheets. Methods to generate the multi-layered cell constructs comprise contacting a cell-sheet on one digestible substrate with another cell-sheet on a different digestible substrate, enzymatically digesting with a first enzyme to remove the first substrate and subsequently adding repeating the steps to add another cell-sheet on same digestible substrate to form a multi-layered cell construct as disclosed herein. Additional aspects relate to using the multi-layered cell constructs for therapeutic use, research and in screening assays.