Effects of growth regulation on conditionally-transformed alginate-entrapped insulin secreting cell lines in vitro.

The ability to control cell growth is an issue of critical importance for the use of transformed beta-cell lines within a bioartificial pancreas. Such control can be achieved either by entrapping the cells in a biomaterial that can inhibit cell proliferation or by genetically modifying the cells to regulate growth. Integrating tetracycline-off or -on operon systems into murine insulinoma cell lines (betaTC-tet and R7T1, respectively) allows cell growth regulation upon exposure to tetracycline (TC) or its derivative doxycycline (Dox), respectively. However, the effects of this regulatory approach on the long-term phenotypic metabolic and secretory stability of alginate-entrapped cells have yet to be thoroughly investigated. In this study, cultures of betaTC-tet and R7T1 cells entrapped in alginate beads were allowed to grow freely, or were growth-regulated, either at the onset, or after 20 days of growth. The data show that growth regulation of alginate-entrapped cells is achievable with chronic administration of the regulatory compound in a concentration-dependent manner. However, as these cultures age, the amount of insulin released does not always reflect the metabolic and histological characteristics of the cultures. This change, coupled with a loss of glucose stimulated insulin secretion in the Dox treated R7T1 cell line, indicate a phenotypic shift of cells with an activated tet-operon. These observations have implications on the selection and long-term function of three-dimensional bioartificial pancreatic constructs that include conditionally transformed beta-cell lines.

Magnetically labeled insulin-secreting cells.

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.

Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II.

Recently, the concept of local renin-angiotensin systems (RAS) capable of generating angiotensin II apart from the circulation has received considerable attention. To investigate this, we generated ACE 1/3 mice in which one allele of ACE is null and the second allele was engineered to express ACE on the surface of hepatocytes. ACE 1/3 mice express no endothelial ACE and lack ACE within the lungs. Their kidneys contain <7.8% the enzyme levels present in control mice. Plasma conversion of angiotensin I to angiotensin II was 43.3% normal. The baseline blood pressure and renal function of the ACE 1/3 mice were normal, probably as a function of a marked increase of both plasma angiotensin I and angiotensin II. When exposed to 2 weeks of a salt-free diet (a stress diet stimulating the RAS), blood pressure in ACE 1/3 mice decreased to 92.3+/-2.0 mm Hg, a level significantly lower than that of wild-type control mice. The ACE 1/3 mice demonstrate the plasticity of the RAS and show that significant compensation is required to maintain normal, basal blood pressure in a mouse with an impaired local vascular and renal RAS.