Deborah J Quick1, Kristi S Anseth. 1. Department of Chemical Engineering, University of Colorado, Boulder, CO 80309, USA.
Abstract
PURPOSE: Toward the ultimate goal of developing an engineered tissue capable of mimicking complex natural healing processes, we have designed a photopolymer platform that enables simultaneous encapsulation of cells and plasmid DNA in degradable hydrogels. Photopolymerization enables spatial and temporal control of gel formation under physiological conditions, but the presence of photoinitiator radicals poses challenges for DNA photoencapsulation. METHODS: The effects of photoinitiating conditions (ultraviolet light and photoinitiator radicals) on plasmid DNA were studied. Protection methods were identified. Plasmid DNA was photoencapsulated in photocrosslinked hydrogels, and the quantity and quality of the released DNA were assessed. Plasmid DNA was simultaneously entrapped (coencapsulated) with cells in hydrogels to assess in situ transfection. RESULTS: Experiments showed that in the absence of other species, plasmid DNA was sensitive to photoinitiator radicals, but the addition of transfection agents and/or antioxidants greatly reduced DNA damage by radicals. Encapsulated plasmid DNA was released from degradable, photocrosslinked hydrogels in active forms (supercoiled and relaxed plasmids) with an overall -60% recovery. Released DNA was capable of transfecting both plated and encapsulated cells. Encapsulated cells expressed the encoded gene of the coencapsulated plasmid as the polymer degraded. CONCLUSIONS: This photopolymerization platform allows for the creation of engineered tissues with enhanced control of cell behavior through the spatially and temporally controlled release of plasmid DNA.
PURPOSE: Toward the ultimate goal of developing an engineered tissue capable of mimicking complex natural healing processes, we have designed a photopolymer platform that enables simultaneous encapsulation of cells and plasmid DNA in degradable hydrogels. Photopolymerization enables spatial and temporal control of gel formation under physiological conditions, but the presence of photoinitiator radicals poses challenges for DNA photoencapsulation. METHODS: The effects of photoinitiating conditions (ultraviolet light and photoinitiator radicals) on plasmid DNA were studied. Protection methods were identified. Plasmid DNA was photoencapsulated in photocrosslinked hydrogels, and the quantity and quality of the released DNA were assessed. Plasmid DNA was simultaneously entrapped (coencapsulated) with cells in hydrogels to assess in situ transfection. RESULTS: Experiments showed that in the absence of other species, plasmid DNA was sensitive to photoinitiator radicals, but the addition of transfection agents and/or antioxidants greatly reduced DNA damage by radicals. Encapsulated plasmid DNA was released from degradable, photocrosslinked hydrogels in active forms (supercoiled and relaxed plasmids) with an overall -60% recovery. Released DNA was capable of transfecting both plated and encapsulated cells. Encapsulated cells expressed the encoded gene of the coencapsulated plasmid as the polymer degraded. CONCLUSIONS: This photopolymerization platform allows for the creation of engineered tissues with enhanced control of cell behavior through the spatially and temporally controlled release of plasmid DNA.
Authors: Joseph C Clarke; Bradley W Tuft; John D Clinger; Rachel Levine; Lucas Sievens Figueroa; C Allan Guymon; Marlan R Hansen Journal: Hear Res Date: 2011-05-18 Impact factor: 3.208