From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells.

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

In vitro and in vivo proposal of an artificial esophagus.

Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. This study evaluates the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of homologous esophageal acellular matrix and autologous smooth muscle cells (SMCs). Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker and expressed bFGF as protein, showing angiogenic activity in vivo on the chick embryo chorioallantoic membrane (CAM). Moreover, they supported cell adhesion, and inasmuch as just after 24 h from seeding, the scaffold appeared completely covered by SMCs. To verify the biocompatibility of our constructs, defects created in the porcine esophageal wall were covered using homologous acellular matrices with and without cultures of autologous SMCs. At 3 week from surgery, the patches composed of only acellular matrices showed a more severe inflammatory response and were negative for alpha-smooth muscle actin immunostaining. In contrast, the cell-matrix implants presented ingrowth of SMCs, showing an early organization into small fascicules. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous SMCs may represent a promising tissue-engineering approach for the repair of esophageal injuries.

Effects of hyperbaric oxygen on proliferative and apoptotic activities and reactive oxygen species generation in mouse fibroblast 3T3/J2 cell line.

BACKGROUND: Hyperbaric oxygen (HBO) therapy is widely used to treat problem wounds associated with pathologic conditions compromising blood supply and tissue oxygenation because increased tissue oxygen levels enhance collagen synthesis, cell proliferation, and angiogenesis. However, little is known about the dose of hyperoxia needed to achieve optimal therapeutic effects. Moreover, HBO, by enhancing the production of reactive oxygen species (ROS), may also exert cytotoxic effects. In vitro models are simplified systems that may aid the development of treatment protocols with HBO. Hence, we have investigated the effects of HBO on the growth and ROS production of the 3T3/J2 fibroblast cell line in relation to the pressure and the duration of exposure. METHODS: 3T3/J2 fibroblasts were plated (5 x 10(3) cells/cm2) on six-well microtiter plates in phosphate buffered saline (PBS), put in a compression chamber, and exposed to 100% oxygen at a pressure of 1.0 or 2.5 atmosphere absolute (ATA) for 15, 30, 60, or 120 minutes. Then the cells were incubated in Dulbecco's modified minimum essential medium (DMEM) for 24, 48, or 72 hours, and at the end of the post-HBO incubation period, their number was determined. In other experiments, cells were detached just after HBO exposure, seeded on 60 mm Petri dishes, and cultured for 10 days in DMEM, and the colony forming units were counted. The effects of HBO exposure (2.5 ATA) on the apoptotic rate of cultured cells were investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and enzyme-linked immunosorbent assays. To measure ROS production, 60 minutes before HBO exposure, 2',7'-dichlorofluorescin (DCF) diacetate (200 nmol/mL) was added to PBS, and after HBO exposure (2.5 ATA), cells were lysated, and fluorescence-emission intensity was measured and converted to micromol DCF/microg protein. RESULTS: At 1.0 ATA, all HBO exposures increased the proliferation rate of cultured fibroblasts and their clonal growth efficiency. At 2.5 ATA, 15-minute exposure to HBO was ineffective, whereas 30- and 60-minute exposures raised the proliferation rate and clonal growth efficiency. Conversely, a 120-minute exposure significantly decreased these parameters compared with control cultures. The exposure of cells to HBO at 2.5 ATA for 120 minutes raised the apoptotic rate of cultured fibroblasts, whereas shorter exposure times were ineffective. All exposure periods to HBO at 2.5 ATA enhanced ROS production from cultured fibroblasts. CONCLUSIONS: Collectively, our findings allow us to conclude that (1) all of the exposure periods to HBO at 1.0 ATA or 30- and 60-minute periods at 2.5 ATA enhance cell growth, (2) 120-minute exposure to HBO at 2.5 ATA exerts a marked proapoptotic effect, and (3) no evident relationships occur between the effects of HBO on cell growth and ROS production.

Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells.

Several attempts have been performed to achieve a suitable tracheal replacement for the treatment of different conditions characterized by a lack of sufficient tissue for surgical reconstruction. Actually, tracheal homografts can induce long-term stenosis and their growth potential is not known. Thus, in this work porcine tracheal matrices have been obtained by a detergent-enzymatic method. The treatment decreased the antigenicity of matrices which were able to support the in vitro adhesion of both chondrocytes and tracheal epithelial cells. On the contrary, only few cells were observed in tracheal matrices prepared with formalin, Thimerosal, and acetone, suggesting that the long-term stenosis occurring in vivo is probably because of an insufficient cell ingrowth. In summary, our results indicate that the detergent-enzymatic method allows us to obtain tracheal matrices which can function as a promising support to achieve an in vitro tissue-engineered cell-matrix construct.