Microfabricated polymeric vessel mimetics
Inventors
Das, Chandan • Jaeger, Ashley A. • Pohida, Thomas J. • Gottesman, Michael M. • Pursley, Randall H. • McQueen, Philip G. • Morgan, Nicole Y.
Assignees
US Department of Health and Human Services
Publication Number
US-9670447-B2
Publication Date
2017-06-06
Expiration Date
2034-01-28
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Abstract
The present disclosure is directed to embodiments of microstructured membranes, methods of fabricating microstructured membranes, bioreactors housing microstructured membranes, and methods of using bioreactors and microstructured membranes. In some embodiments, the present disclosure allows culturing of cellular tissues in an environment which more accurately resembles a native environment. In some more specific embodiments, the present disclosure allows culturing of tumor cells on a membrane having a microfabricated pattern which mimics a native vasculature system.
Core Innovation
The invention disclosed relates to microstructured oxygen permeable membranes, methods of fabricating these membranes, bioreactors housing them, and methods of using these bioreactors and membranes. The disclosed devices and materials facilitate culturing of cellular tissues in a three-dimensional environment that more closely simulates native in vivo conditions. Specifically, the membrane surface comprises an array of microfabricated pillars that mimic native tissue microvasculature, enabling culture of tumor cells in a way that resembles in vivo tumor cell growth.
The invention addresses the challenge in current bioreactors where culturing thick cell clusters is difficult due to inadequate nutrient transport, especially oxygen, leading to hypoxia and necrosis in the culture. It improves nutrient delivery and waste removal by creating oxygen gradients similar to those in living tissues supported by vasculature. The membrane pillars serve as microfabricated blood vessel mimetics permitting diffusion of oxygen through solid, oxygen-permeable material, thereby enhancing three-dimensional oxygen transport into cell cultures.
Claims Coverage
The patent's independent claims cover inventive features of microfabricated oxygen permeable membranes, bioreactors incorporating such membranes, and methods of cell culturing using these structures. The main inventive features are summarized as follows.
Microfabricated oxygen permeable membrane with blood vessel mimetics
A membrane comprising a base member with a cell culture support surface featuring solid, oxygen-permeable pillars that mimic microvasculature in three-dimensional cell culture. The pillars have heights between greater than 1 μm and less than 1 mm and allow three-dimensional oxygen diffusion through their top and side surfaces. The membrane includes a barrier between pillars that reduces oxygen diffusion through the flat surface to preferentially direct oxygen flow through the pillars.
Microstructured array of cylindrical pillars with specific dimensions and mechanical properties
The pillars are arranged in a microstructured array, substantially cylindrical, with diameters between about 10 μm and 100 μm, heights between about 100 μm and 500 μm, spaced approximately 200 μm apart. Pillars have a shear modulus sufficient to withstand stresses from fabrication and cell culturing and exhibit aspect ratios between 2 and 10, exemplified by 7.5 times height-to-diameter ratio.
Oxygen permeable material comprising organosilicon polymers and hydrogels
The membrane and pillars comprise oxygen-permeable materials such as organosilicon polymers including hydrophilicized polydimethylsiloxane (PDMS), hydrogels, crosslinkers, and photoinitiators, or combinations thereof, enabling appropriate oxygen permeability and biocompatibility.
Bioreactor housing the membrane with oxygen source and sink chambers
A bioreactor structure comprising sealed input and output chambers separated by the membrane. The input chamber connects to an oxygen source, and the output chamber acts as an oxygen sink. Oxygen preferentially flows through the pillars to oxygenate a cell culture on the membrane’s surface, which is hydrophilic and supports cell adherence. The bioreactor maintains an oxygen gradient inducing diffusion from the input to output.
Method of culturing cells with oxygen perfusion through pillars
A method of culturing cells involves providing cells in a culture substrate on the membrane surface and maintaining an oxygen gradient across the membrane such that oxygen perfuses through the solid, oxygen-permeable pillars into the culture substrate, facilitating physiologically relevant oxygen delivery to cells.
The inventive features collectively provide a microfabricated oxygen permeable membrane with structured pillars mimicking vasculature, integrated into a bioreactor that facilitates three-dimensional oxygen diffusion through the pillars. This system supports improved cell culture environments by creating oxygen gradients that resemble in vivo conditions, enabling advanced cell growth and testing methods.
Stated Advantages
Permits culturing of cellular tissues in three-dimensional environments that closely simulate native in vivo conditions.
Improves oxygen distribution and nutrient delivery to cell cultures, reducing hypoxia and necrosis in thick cell clusters.
Facilitates growth and study of tumor cells with oxygenation conditions more accurately mimicking physiological vasculature, enhancing relevance for drug testing and tumor modeling.
Allows formation of larger, viable multicellular tumor spheroids compared to traditional culture techniques.
Enables multiplexed experiments in multiwell configurations for increased throughput and comparative studies.
Documented Applications
Culturing tumor cells in vitro on membranes that mimic native vasculature for research into tumor growth, angiogenesis, metastasis, and drug resistance.
Use as a platform for drug discovery and therapeutic candidate testing by providing more physiologically relevant oxygenation conditions.
Culturing of animal tissues of increased thickness, such as skin tissues, for improved skin and graft tissue development.
Three-dimensional cell culture systems that promote natural cell morphology, metabolism, migration, signaling, gene expression, and differentiation.
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