| This study examines tailored cells and alginate microcapsules useful for structural and metabolic tissue engineering. Major drawbacks for tissue engineering therapies are the limited source of cells and use of immunosuppressants. In this study, embryonic stem, engineered fibroblasts, and Ca2+-alginate microcapsules were utilized to resolve these constrains.; Embryonic stem cells were coaxed to differentiate into islet-like cell clusters, potentially valuable for cell replacement therapy for diabetes, by manipulating culture conditions. Upon completion of derivation, glucose-stimulated insulin secretion was tested in vitro. Presence of intracellular C-peptide, a by-product of insulin synthesis, was determined by immunostaining and immunogold labeling. Later, exogenous bovine insulin used in media for the traditional protocol was replaced by either human insulin or IGF-I to examine the capability of insulin synthesis by ILCCs. ILCCs cultured without either insulin or IGF-I were also tested. The results showed that insulin secreted by ILCCs was almost entirely sequestered exogenous insulin from media although insulin secretion was glucose-responsive. Uptake of 35S-labeled cysteine also confirmed the lack of significant de-novo insulin synthesis by ILCCs. However, ILCCs possess cellular machinery for insulin synthesis based on presence of intracellular pancreatic markers manifested by immunostaining.; Synovial cells are capable of chondrogenic differentiation, which can be induced by hTGF-beta1. Genetically-modified human and murine fibroblasts to overexpress hTGF-beta1 were encapsulated in Ca2+-alginate microcapsules and tested in vitro and in vivo. In in-vitro study, cell viability was determined following encapsulation and release study to examine the effects of encapsulation process and in-vitro release study. Overexpression of hTGF-beta1 was determined by in-vitro release study. In in-vivo study, microencapsulated genetically-modified cells were implanted into either subcutaneous or intraperitoneal cavity for either 1 or 3 weeks. Therapeutic relevance was demonstrated by survival of encapsulated cells and persistence of hTGF-beta1 overexpression following 1-week implantation, which is equivalent to the duration required for chondrogenic differentiation of synovial cells in the presence of hTGF-beta1. Upon completion of implantation, encapsulated cells survived for at least 3 weeks in mice with a marginal decrease in viability. Enhanced hTGF-beta1 was maintained following 1- and 3-week implant, for at least 72 hours as well. |
