Publication Date

2011-04-19

Availability

Embargoed

Embargo Period

2012-04-19

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biomedical Engineering (Engineering)

Date of Defense

2011-04-08

First Committee Member

Cherie Stabler

Second Committee Member

Weiyong Gu

Third Committee Member

Camillo Ricordi

Fourth Committee Member

Luca Inverardi

Fifth Committee Member

Juan Dominguez-Bendala

Abstract

While clinical transplantation of islets of Langerhans for the treatment of insulin dependent Diabetes Mellitus has shown significant promise in recent years, there remains a need for procedural optimizations to improve cell viability, functionality and ultimately, graft longevity. One of the most critical factors to islet cell survival is the proper oxygenation of these highly metabolic cellular aggregates. In culture, islets experience suboptimal oxygen profiles delimited by steep gradients across culture media. When retransplanted, they are subjected to extremes of hypoxia and anoxia, resulting in pronounced graft dysfunction and cell loss, which is further exacerbated when these cells are immunoisolated in polymer matrices. This study examined the effects of improving both in-vitro culture and immunoisolation of islet cells by optimizing oxygen mass transfer via oxygen carriers in the form of perfluorocarbons. Specifically, new systems for these applications were developed utilizing perfluoromoeities and conventional culture (polydimethylsiloxane) and immunoisolation (sodium alginate) matrices. During in vitro culture of islet cells, the use of perfluoro-impregnated PDMS culture platforms enhanced cell recovery, viability and function over the culture period. Additionally, marginal mass transplants of the islets cultured in these novel platforms functioned better in recipients than relevant controls. In immunoisolation, the optimization of perfluorocarbon emulsions was performed investigating the effects of combinations of surfactants and perfluorocarbons on oxygen mass transfer and cell viability. Emulsions were well characterized using particle size analysis by dynamic light scattering, perfluorocarbon inclusion by gravimetry and oxygen diffusivity measurements utilizing fluorescent optodes. A novel method was developed for the assessment of dissolved oxygen content of these emulsions. Optimal emulsions, as determined by predicted/measured oxygen transfer enhancement over relevant controls, were utilized in alginate matrices for microencapsulation of cell lines, initially, and then, islets of Langehans. The effects of these potential improvements were assessed by in-vitro potency assays, including a novel method for assessing glucose stimulated insulin release, and in transplantation efficacy in rodent marginal mass models. While the improvements in culture were promising in cell line studies, the observed benefit did not translate in islet culture. The cause was found to be related to permeability impediments generated from the surfactant components utilized in emulsion manufacture. In addition to the development of several new methods for the characterization of oxygen containing solutions and the potency assessment of isolated islets of Langerhans, the impact of these studies is important in the field of polymer engineering. We observed that the use of Polyethylene glycol (PEG) based materials may limit transport of nutrients and oxygen critical to cells. Additionally, we developed cell culture platforms that enhance the viability, number and function of cultured islet cells, potentially impacting the clinical realm where cell preservation is critical to transplant outcome.

Keywords

encapsulation; cell culture; islets of Langerhans; oxygen mass transfer

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