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.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. I used the technique of serum-free cell culture to develop a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of defined supplements. SFME cells, cultured in a nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin, have maintained a diploid karyotype with no detectable chromosomal abnormalities for more than 200 generations. The cells did not undergo growth crisis and remain in culture today. SFME cells were dependent on EGF for survival and were reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acid protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts. Following the derivation of SFME cells my research focussed on (1) defining more precisely the growth response of SFME cells to medium supplements, (2) investigating the relationship between the nonsenescent nature of SFME cells and their responses to serum and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture of human embryonic astrocytes. SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum was extracted by charcoal, a procedure that removed steroid and thyroid hormones. However, the effect of serum on untransformed SFME cells could not be prevented by addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are involved. SFME cell death resulting from EGF deprivation exhibited characteristics associated with apoptosis or programmed cell death. Ultrastructural analysis showed cells became small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1- phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length fragments generating a regularly spaced ladder. I applied the serum-free approach used to derive SFME cells to the multipassage culture of human embryonic astrocytes. Cells were cultured in nutrient medium supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth factor and heparin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized GFAP. / Graduation date: 1992

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

Univ., Diss.--Göteborg, 2005. / Literaturverz. S. [289] - 313.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on Feb. 9, 2010. Major professor: Dr. Robert Crockett. "Presented to the faculty of California Polytechnic State University, San Luis Obispo, California." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering." "November 2009." Includes bibliographical references (p. 47-52).

Thesis (M. Phil.)--University of Hong Kong, 1985.

Not available / text

published_or_final_version / Botany / Master / Master of Philosophy

No description available.