Cell Transplantation

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of −1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26—56-month-old sows, we report here that the presence of large (>100 μm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

One impediment for a wider application of islet transplantation is the limited number of donor pancreata for islet isolation. A more efficient utilization of available organs could in part alleviate this problem. Perfluorocarbons (PFCs) have a high oxygen solubility coefficient and maintain high oxygen partial pressures for extended time. They serve also as oxygen “reservoirs” for harvested organs in pancreas organ transplantation. The aim of this study was to test whether the use of PFCs could also be beneficial for the secretory activity and overall viability of cultured purified islets before transplantation. Purified rat islets were cultured in static conditions with or without oxygen-saturated PFCs for 1 or 7 days. Cell death and apoptosis were assessed by trypan blue staining, DNA strand breaks, and caspase 3/7 activity. mRNA levels of insulin and ICA512/IA-2, a membrane marker of secretory granules (SGs), were quantitated by real-time PCR, whereas insulin content and secretion were measured by RIA. Polypyrimidine tract binding protein (PTB), which promotes SG biogenesis, was assessed by Western blotting. The number of SGs and the ultrastructural appearance of β-cells were analyzed by cryoimmunoelectronmicroscopy for insulin. Various parameters, including caspase activity, insulin and ICA512/IA-2 mRNA levels, PTB expression, number of secretory granules, and ultrastructural appearance did not significantly differ between control and PFC-cultured islets. On the other hand, PFC culture islets showed significantly increased DNA fragmentation and a reduced insulin stimulation index at both time points compared to control islets. While advantageous for the transport of human harvested organs, the use of PFH in the culture may be comparable to and/or not provide advantage over conventional protocols for culture of islets for transplantation.

The use of encapsulated insulin-secreting cells constitutes a promising approach towards the treatment of insulin-dependent diabetes. However, long- term storage for off-the-shelf availability still remains an issue, which can be addressed by cryopreservation. This study investigated cryopreservation of a model tissue-engineered pancreatic substitute by two ice-free cryopreservation (vitrification) solutions (designated VS55 and PEG400) in comparison to a conventional freezing protocol. The model substitute consisted of insulin-secreting mouse insulinoma βTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cell viability and static insulin secretion from the thawed cryopreserved groups were characterized and compared against fresh controls. Cell viability tests using alamarBlue® showed that, compared to the fresh groups, the VS55 had the highest viability (p < 0.05), followed by both the PEG400 (p < 0.001) and the frozen groups (p < 0.001). In response to a square wave of glucose, the static insulin secretion data showed that the VS55 and PEG400 groups had similar induction levels against the fresh group, whereas the frozen group had the poorest secretion rate. Cryosubstitution of capsules showed ice formation in the frozen group but no ice in the vitrified groups. Microscopic observations revealed holes and/or tears within beads subjected to freezing, whereas no such abnormalities were detected in the vitrified samples. Overall, vitrification was found to be a promising preservation procedure for this encapsulated cell system.

The development of an optimized animal model for the in vivo analysis of human muscle cells remains an important goal in the search of therapy for muscular dystrophy. Here we examined the efficiency of human myoblast xenografts in three distinct immunodeficient mouse models. We found that different conditioning regimes used to provoke host muscle regeneration (i.e., cardiotoxin versus cryodamage) had a marked impact on xenograft success. Tibialis anterior muscle of Rag2-, Rag-/γc-, and Rag-/γc-/C5- mice was treated by cardiotoxin or cryodamage, submitted to enzymatic digestion, and analyzed by cytofluorometry to quantitate inflammatory cells. Human myoblasts were injected into pretreated muscles from immunodeficient recipients and the cell engraftment evaluated by immunocytochemistry, 4—8 weeks after transplantation. Donor cell differentiation and dispersion within the host muscles was also investigated. Host regeneration in cardiotoxin-treated mice was accompanied by a higher inflammatory cell infiltration when compared to that induced by cryodamage. Accordingly, when compared to the cardiotoxin group, more human myogenic cells were found after cryodamage. When the distinct immunodeficient mice were compared, we found that the alymphoid strain lacking the complement component C5 (Rag-/γc-/C5- mice) was the most efficient host for human muscle xenografts, when compared with C5+Rag-/γc- mice or Rag- mice. Our results demonstrate that cryolesion-conditioned muscles of Rag-/γc-/C5- mice provide the best environment for long-term in vivo human myoblast differentiation, opening the way for a novel approach to study the pathophysiology of human muscle disorders.

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without loosing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.

Tissue engineering of blood vessels offers a potential new therapy for patients with vascular occlusive disease. In addition, tissue engineering technologies offer the opportunity to study the biology of vascular cells in a biomimetic, three-dimensional environment. A model for vascular tissue engineering was used to study the effects of vascular cell age on extracellular matrix (ECM) deposition, cellular mitosis, and protein synthesis under controlled conditions in vitro. Blood vessels were grown using a three-dimensional polyglycolic acid (PGA) mesh that was seeded with either infant or adult porcine vascular smooth muscle cells. Mechanical forces in the form of pulsatile radial distension were applied for the duration of the 7-week growth period. Overall, infant cells exhibited higher levels of cellular proliferation, ECM deposition, and remodeling activity than cells derived from adult animals. In addition, vessels cultured from infant cells had enhanced physical properties compared to vessels cultured from adult cells. The differentiation state of the smooth muscle cells in the infant and adult constructs was unchanged from the native state. However, the levels of immature pro-collagen, although undetectable in the vessels grown from adult cells, were similar in native vessels and in vessels grown with infant cells. These studies have important implications for the study of aging and vascular disease and remodeling, as well as for the field of tissue engineering.

Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.

Although ex vivo culture of hepatocytes is known to impair functionality, it may still be considered as desirable to propagate or manipulate them in culture prior to transplantation into the host liver. The aim of this study was to clarify whether rat hepatocytes cultured over different periods of time proliferate and retain their hepatocyte-specific functions following transplantation into the recipient liver. Rat hepatocytes were cultured under serum-free conditions in the presence of hepatocyte and epidermal growth factors. Cells derived from wild-type donor livers were transplanted into the livers of CD26-deficient rats. Cell proliferation and the expression of hepatocyte-specific markers were determined before and after transplantation. Cell number increased threefold over a culture period of 10 days. The expression of connexin 32 and phosphoenolpyruvate carboxykinase declined over time, indicating the loss of hepatocyte-specific functions. Hepatocytes cultured over 4 or 7 days and then transplanted proliferated in the host parenchyma. The transplanted cells expressed connexin 32, cytokeratin 18, and phosphoenolpyruvate carboxykinase, indicating the differentiated phenotype. The loss of hepatocyte-specific functions during culture may be restored after transplantation, suggesting that the proper physiological environment is required to maintain the differentiated phenotype.

Conditionally immortalized hepatocytes (CIH) established with a gene for the temperature-sensitive mutant of the T antigen (tsT) have characteristics to stop proliferating and to differentiate at nonpermissive temperatures (37—39°C) due to inactivation of the T antigen. Therefore, they may be a good alternative to primary hepatocytes for experimental investigations or clinical applications. Deinduction of the T antigen results in a transient increase of p53 in these cells, leading to reexpression of normal senescence because of the telomere attrition occurring during the early stages of immortalization. To determine this T antigen dependency for the maintenance of immortality, a type of rat CIH was cultured continuously at 39°C. The frequency of occurrence of T-antigen-independent clones ranged from 0.053% to 0.093%. These clones maintained the temperature-sensitive property of the T antigen; nevertheless, they were able to progress to the S phase and proliferate without undergoing apoptosis at 39°C as at 33°C, a permissive temperature. The temperature-sensitive point mutation of tsT was not affected in these clones and the T antigen was functioning properly. The integrity of the p53 pathway was also maintained from the point of Western blot analysis of p21. Although the telomerase continued to be expressed and the telomere length was maintained, marked chromosomal damage could not be avoided in these cells. It is a plausible explanation that this escape phenomenon from conditional immortalization may be related to the change of other genes involved in cell cycles, which have yet to be elucidated. In conclusion, CIH could lose their temperature-sensitive characteristics without the change of tsT, itself, and the T antigen is not always necessary to maintain their immortality. Therefore, the results obtained from experimental investigations using these cells should be interpreted carefully, and unpredictable phenotypic changes should also be taken into consideration when using them in clinical applications.