Chemical and Biological Engineering ETDs

Publication Date

7-12-2014

Abstract

With the increase in life expectancy and with growing numbers of an aging population, there is a rising need worldwide for replacement tissues and organs. One way to address this growing need is through engineered tissues, such as those generated from stimulus-responsive polymers. Stimulus-responsive polymers undergo a physical or chemical change when a stimulus is applied. One such material is poly(N-isopropyl acrylamide), (pNIPAM), which undergoes a conformation change in a physiologically relevant temperature range to release intact mammalian cell monolayers capable of being used to engineer tissues. Two factors currently limit the use of cell sheets for this purpose: 1) although the NIPAM monomer is toxic, it is unclear (and highly contested) whether its polymerized form is toxic as well; 2) there is little understanding of the mechanism of how cells detach from pNIPAM, and whether the (possibly) cytotoxic polymer would be transferred to implanted engineered tissues. In this work, we present an investigation of the cytotoxicity of pNIPAM-grafted surfaces, as well as an investigation of the mechanism of cell detachment from pNIPAM. The cytotoxicity of substrates prepared using several polymerization and deposition techniques are evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Endothelial, epithelial, smooth muscle, and fibroblast cells were used for the cytotoxicity testing. The mechanism of cell detachment from pNIPAM was investigated using endothelial cells and surfaces synthesized via surface-initiated atom transfer radical polymerization. The detachment experiments were performed at various temperatures with and without an ATP inhibitor. In addition, fluorescent pNIPAM surfaces were generated to determine if any pNIPAM is removed with the detached cells. We find that cell sheets obtained by detachment from pNIPAM films will be suitable for use in engineered tissues, provided that the pNIPAM films that the cells were obtained from are themselves robust (i.e., grafted, covalently linked, or similar). We also find that the cell detachment from pNIPAM is mostly a passive process, and that no pNIPAM is removed from the surfaces during the detachment. Our results therefore provide an important step to clearing the hurdles presently obstructing the generation of engineering tissues from pNIPAM films.

Keywords

cell sheet engineering, stimuli-responsive polymer, thermoresponse, cell detachment, mechanism, cytotoxicity

Document Type

Dissertation

Language

English

Degree Name

Chemical Engineering

Level of Degree

Doctoral

Department Name

Chemical and Biological Engineering

First Advisor

Canavan, Heather

First Committee Member (Chair)

Chi, Eva

Second Committee Member

Diane, Lidke

Third Committee Member

Petsev, Dimiter

Fourth Committee Member

Canavan, Heather

Share

COinS