Photosynthetic oxygen generator for bioartificial pancreas

Immunoisolation of pancreatic islets interrupts their vascular connections and results in severe cell hypoxia and dysfunction. This process is believed to be the major obstacle to a successful cure of diabetes by implantation of bioartificial pancreas. Here we describe a new technology for microalga-based, photosynthetic oxygen supply to encapsulated islets, in which a thermophylic strain of the unicellular alga Chlorella was used as a natural photosynthetic oxygen generator. Following determinations of the optimal number of alga cells required for compensation of islet respiration, an appropriate number of islets and algae were co-encapsulated in alginate and perifused with oxygen-free medium at increasing glucose concentrations. No insulin response to glucose was obtained in islets alone, or upon inactivation of photosynthesis by darkness. However, under illumination, photosynthetic- dependent oxygen generation induced higher glucose-stimulated insulin response when compared to normoxic perifusion. Such photosynthetic oxygen generation may have a potential application in development of various bioartificial tissues, in particular the endocrine pancreas.     

PVA hydrogel sheet macroencapsulation for the bioartificial pancreas

We newly developed a sheet-type macroencapsulation device entrapping rat islets from 3% polyvinyl alcohol (PVA) dissolved in Euro-Collins solution containing 10% fetal bovine serum and 5% dimethyl sulfoxide (PVA + EC) using a freezing/thawing technique. The same encapsulation technique but with 3% PVA dissolved only in double-distilled water (PVA) and a culture of free islets were served as controls. After 14-day culture in the CMRL-1066 medium, the islet recovery rate, morphological changes, insulin content, and insulin secretion were evaluated in vitro to prove the feasibility of this method of encapsulation. We also xenotransplanted the device into the peritoneal cavity of diabetic C57BL/6 mice to check its function in vivo. After 1-day culture, the islet recovery rate and insulin content in the PVA group were significantly lower than that in the PVA + EC and free islet groups. After 14-day culture, only the islets in the PVA+EC group maintained a normal morphology and effective insulin secretory response to high glucose while the response was not observed in the PVA group after 1-day culture and no longer observed in the free islets after 7-day culture. After transplantation of rat islets encapsulated in the PVA + EC device to diabetic C57BL/6 mice, nonfasting blood glucose levels showed a rapid decrease from high glucose levels of pre-transplantation, maintaining significantly lower glucose levels during the whole course of study in comparison with the sham-operated group. Our results indicated that this freezing/thawing macroencapsulation technique using 3% PVA + EC was effective for xenotransplantation of islet cells.     

Kinetic modelling of blood glucose variation in a bioartificial pancreas

Methods are presented for assessing the performance of biomaterials for a bioartificial pancreas using a kinetic model of blood glucose variation. The model is composed of simultaneous differential equations that simulate variation of blood glucose concentration of rats outside the biomaterial and decrease of islet number inside the biomaterial. The resulting calculations agree well with the in vivo experimental data and indicate that the characteristics of bioartificial pancreas can be expressed as the actually working number of islets from the viewpoint of rats, regardless of the functioning of the bioartificial pancreas. The contributions of the capability of a biomaterial permeable to insulin and against immune rejection, and other factors that may induce cell death were involved in the actually working number of islets, i.e., the model used a lumped-parameter expression for assessment of the performance of biomaterials for a bioartificial pancreas. This model would be useful as a research tool for analysis of clinical investigation of bioartificial pancreas and physiological significance of blood glucose variation dynamics.     

Permeability of a sol-gel synthesized aminopropyl-silicate-titanate hybrid membrane for the microcapsule-shaped bioartificial pancreas

The aminopropyl-silicate-titanate hybrid membrane was successfully synthesized onto a Ca-alginate gel bead by the sol-gel method, from two kinds of organometallic compound precursors, tetraethyl orthotitanate (TEOTi) [Ti(OC₂H₅)₄] and 3-aminopropyltrimethyl orthosilicate (APTrMOS) [(CH₃O)₃Si(CH₂)₃NH₂]. APTrMOS acted as a binding agent between inorganic titanate and alginate polymer via electrostatic interaction between amino groups of APTrMOS and carboxyl groups of alginate. From the measurements of the diffusion of ovalbumin and γ-globulin through the aminopropyl-silicate-titanate hybrid membrane, it was found that the molecular permeability of the membrane could be controlled by changing the amount of each precursor. The molecular weight cutoff point required for immunoisolation, less than 150 kDa, was achieved under the condition that the amount of APTrMOS was 3.40 mmol/(10 ml of Ca-alginate) and TEOTi was more than 1.42 mmol. The islets encapsulated in the membrane showed almost the same insulin secretion viability as the free islets. Received: September 28, 2000 / Accepted: April 4, 2001     

Fabrication of bioartificial pancreas using decellularized rat testicular tissue

AimsDiabetes is a chronic disease that is associated with a decrease or disfunction of β-cell. In the present study, fabrication of bioartificial pancreas using MIN-6 β-cell line seeded in decellularized rat testicles was investigated.Main methodsIn this experimental study, the whole body of testes were decellularized and after characterization, were seeded by MIN-6 cell line. The expression of insulin-related genes and proteins including PDX-1, Glut2, Insulin, and Neurogenin-3 were evaluated. Insulin secretion was assessed under different concentrations of glucose. Seeded scaffolds with or without MIN-6 cells were transplanted to the rat's mesentery and their blood sugar and body weight were evaluated every three days for 28 days and analyzed with H&E staining.ResultsHistological assessments indicated the cells were completely removed after decellularization. The scaffold had no toxic impacts on the MIN-6 cells (P? 0.02).Insulin release in response to different concentrations of glucose in 3D culture (testis-ECM) was significantly more than the traditional 2D monolayer culture (P < 0.001). Moreover, the relative genes and proteins expression were significantly higher in the 3D culture, compared to the 2D control group. In vivo transplantation of the (testis- Extra Cellular Matrix) testis-ECM scaffolds showed appropriate positions for transplantation with angiogenesis and low infiltration of inflammatory cells. The recellularized scaffolds could drop blood sugar levels and increase the body-weight of STZ-diabetic rats (P < 0.01).SignificanceOur study clearly confirmed that ECM valuable organ scaffolds prepared by decellularization of the testicular tissue is suitable for the fabrication of bioartificial pancreas for transplantation.     

Suitability of cellulose molecular dialysis membrane for bioartificial pancreas: in vitro biocompatibility studies

The success of immunoisolation devices for islet transplantation depends on the properties and biocompatibility of semipermeable immunobarrier membranes. In the present study, we have evaluated the in vitro biocompatibility of the cellulose membrane Spectra/Por 2 (MW no larger than 12- 14,000) for its possible application in islet immunoisolation. The membrane was found to be hydrophilic (octane contact angle: 153.2+/-0.66 degrees) and exhibited decreased protein adsorption. It showed mechanical stability after 1 month of storage in PBS (pH 7.4) with tensile strength, percent elongation, and Young's modulus of 88.88 MPa, 36.22, and 291.8 MPa, respectively. It allowed regulated transport of glucose and insulin in an in vitro diffusion assay. The high viability of NIH3T3 fibroblasts and the inability of lymphocytes to proliferate in vitro on exposure to the membrane leach-out products suggested its noncytotoxic and nonimmunogenic nature. Macrophages, when cultured on membranes, did not show increased expression of inflammatory surface marker such as CD11b/CD18, CD45, CD14, and B 7.2. Image analysis studies showed integrity and intact morphology of mouse islets cultured on and inside the membranes with high viability (91%, 89.7%). These islets also retained their functionality, as judged by insulin secretion. The present study provides sufficient documentation to consider cellulose molecular dialysis membrane Spectra/Por 2 (MW no larger than 12-14,000) as a potential candidate for immunoisolation of islets.     

Assessment and modeling of poly(vinyl alcohol) bioartificial pancreas in vivo

Pancreatic islets surrounded by a semipermeable membrane to prevent an immune response by the host immunosystem is a potential way of treating type I diabetes mellitus. Our previous in vitro studies have demonstrated that poly(vinyl alcohol) (PVA) membranes satisfy the basic requirements for a bioartificial pancreas. This study was designed to evaluate the performance of PVA tubular membrane chambers containing islets in vivo. When the m-2 type of PVA chamber was implanted into streptozotocin-induced diabetic rats, nonfasting blood glucose levels dropped from 500 +/- 35 mg/dl to the lowest value 210 +/- 22 mg/dl. Furthermore, the performance of the bioartificial pancreas can be enhanced by the increased numbers of implanted chambers. If three m(-2) chambers were implanted, nonfasting blood glucose levels in the diabetic rats decreased to 130-160 mg/dl and such a low blood glucose value was maintained for 1 month. This indicates that implanting three m-2 chambers in the diabetic rats could provide improved permeability of insulin to normalize blood glucose levels and improved survival of islets from the immune system of the recipient. For improving the design of the bioartificial pancreas, a mathematical model was developed to account for the changes in blood glucose levels of the diabetic rats. We demonstrated such a mathematical analysis was helpful to understand the characteristics of islet inside an artificial environment.     

Concept and computational design for a bioartificial nephron-on-a-chip

A MEMS-based, (Micro Electro Mechanical System) bioartificial device is proposed for replicating the function of a single nephron. Consistent with the anatomy and physiology of humans, our device has 3 distinct sections, replicating the function of the glomerulus, the proximal tubule, and the loop of Henle. Construction of a bioartificial loop of Henle in particular requires control of diffusion-scale features. The proposed device can be built using existing microfabrication technologies and populated with various renal cell types. A computational model is also developed to analyze the coupled, multiphase mass transport in this system. Using the model, a design is generated with flow and solute transport properties matching those of the human nephron.     

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12–14 days culture at 22°C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.     

Hybrid bioartificial liver: establishing a reversibly immortalized human hepatocyte line and developing a bioartificial liver for practical use

Recently, much attention has been attracted by a novel therapy for liver failure using a hybrid bioartificial liver (BAL) support device that incorporates living liver cells. Researchers in various fields have considered the following cells for potential use in BALs: human embryonic stem (ES) cells; somatic stem cells; differentiated tissue cells; and cells derived from tissues of different animal species, particularly from the pig. With their pluripotency, human ES cells are extremely useful, and many research groups are joining the race to develop BALs. One such effort involves the breeding of transgenic pigs to overcome interspecies barriers. Recent reports suggest, however, that porcine endogenous retrovirus may infect human tissues, and clinical application of transgenic pigs has become a controversial issue. To avoid such risks, we recommend that researchers adopt a reversible immortalization system that uses the Cre-loxP site-specific recombination reaction targeting human hepatocytes in their final differentiated state. This system has allowed us to establish a safe human hepatocyte line that is capable of differentiation at low cost and on a large scale. We are also designing and developing an artificial liver module made of a combination of hollow fibers and nonwoven fabrics. The objective of this review article is to report our therapeutic strategy, which aims at the earliest possible introduction of the treatment of liver failure using BALs.     

Method for measuring in vivo oxygen transport rates in a bioartificial organ.

Oxygen transport is crucial for the proper functioning of a bioartificial organ. In many cases, the immunoisolation membrane used to protect the transplanted cells from the host's immune system can be a significant barrier to oxygen transport. A method is described for measuring the in vitro and in vivo oxygen transport characteristics of a planar immunoisolation membrane. The in vitro oxygen permeability of the membrane was found to equal 9.22 x 10(-4) cm/sec and was essentially the same as the in vivo value of 9.51 x 10(-4) cm/sec. The fact that the in vitro and in vivo membrane permeabilities are identical indicates that any fibrotic tissue adjacent to the immunoisolation membrane did not present a significant resistance to the transport of oxygen. The measured oxygen permeability was also found consistent with the solute permeabilities obtained in a previous study for larger molecules. Based on the oxygen permeability results, theoretical calculations for this particular membrane indicate that about 1,100 islets of Langerhans/cm2 of membrane area can be sustained at high tissue densities and only 660 islets/cm2 can be supported at low tissue densities.     

Agarose mold embedding of cultured cells for tissue microarrays.

There are several indications for the placement of samples of cultured cells in tissue microarrays (TMAs). To optimize this technique, three embedding procedures were compared: embedding of fixed cells pelleted by centrifugation, embedding of cells dispersed in an agarose matrix, and embedding of pelleted cells packed into the center of hollow agarose molds. TMAs were made from these preparations. The number of cells per tissue spot and the number of histologic sections that could be obtained from the preparations were determined. The agarose matrix and agarose mold techniques resulted in the longest core samples, while the cell pellet and agarose mold methods resulted in the greatest cell density. Thus, the use of cylindrical agarose molds optimizes both the number of cells present on a histologic section of a TMA, and the number of histologic sections that can be obtained from a TMA. This technique results in a paraffin-embedded cell preparation that yields a cell density of approximately 1000 cells per 0.6-mm diameter circular histologic section, and that produces uniform core samples the full thickness of the donor block. Histologic sections of TMAs prepared in this manner were validated in immunohistochemical and in situ hybridization assays.     

A newly developed bioartificial pancreas successfully controls blood glucose in totally pancreatectomized diabetic pigs

Construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treating diabetic patients. Various types of BAP devices have been developed, but most of them involve extravascular implantation of islets in microcapsules or diffusion chambers. These devices have poor diffusive exchange between the islets and blood, and often rupture. To overcome these problems, we developed a new type of BAP composed of polyethylene-vinyl alcohol (EVAL) hollow fibers that are permeable to glucose and insulin and a poly-amino-urethane-coated, non-woven polytetrafluoroethylene (PTFE) fabric that allows cell adhesion. Porcine islets attached to the surface of the PTFE fabric, but not to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between blood flowing inside the fibers and cells outside. We inoculated this BAP with porcine islets and connected it to the circulation of totally pancreatectomized diabetic pigs. We found that blood glucose levels were reduced to a normal range and general health was improved, resulting in longer survival times. In addition, regulation of insulin secretion from the BAP was properly controlled in response to glucose both in vitro and in vivo. These results indicate that our newly developed BAP may be a potential therapy for the treatment of diabetes in humans.     

In vitro and in vivo evaluation of alginate/sol-gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol-gel process. The membrane has identical to Si-O-Si identical to bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499 +/- 32 microns in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly-L-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4 +/- 0.5 (mean +/- SEM)% and 88.0 +/- 1.5% (p < 0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.     

The optimal hepatocyte density for a hollow-fiber bioartificial liver

A bioartificial liver (BAL) based on viable porcine hepatocytes can serve as a bridge to liver transplantation in patients with acute liver failure (ALF). To support liver functions, an adequate mass of hepatocytes is needed, which depends upon the cell density in the BAL device. This study evaluated the optimal density of hepatocytes within BAL devices that were constructed by perfusing porcine hepatocyte suspensions mixed with cytodex-3 into polysulfon hollow-fibers. The BAL devices were prepared with 6 different cell densities. The mass of hepatocytes in each device was evaluated for (a) cell viability, (b) ability to degrade diazepam, (c) ability to synthesize urea, (d) incorporation of [3H]-leucine into protein, (e) glucose-6-phosphatase activity, (f) total RNA content, and (g) p53 gene expression. Hepatocyte viability was about 90% in each device. With increasing hepatocyte density, the diazepam concentration in the medium decreased from 9.26 +/- 0.96 mg/L at 1 x 10(5) cells/ml to a minimum of 5.25 +/- 1.02 mg/L at 5 x 10(6) cells/ml and thereafter remained at low levels. Urea production and [3H]-leucine incorporation into protein increased progressively until the cell density reached 5 x 10(6)/ml and thereafter remained at high levels. Glucose-6-phosphatase activity and total RNA content stayed at high levels until the cell density reached 5 x 10(6)/ml and then progressively decreased. p53 gene expression differed from the other parameters, since it increased only when the cell density reached 5 x 10(7)/ml. In conclusion, the density of 5 x 10(6) cells/ml is a critical inflection point for most of the functional parameters, although p53 gene expression is not elevated at this cell density. These findings suggest that 5 x 10(6) cells/ml is the optimal hepatocyte density in the hollow-fiber BAL device.     

Agarose microwell assay for cell-mediated cytotoxicity

A colony-inhibition micromethod for tumour cells is described, which conbines the high cloning efficiency of the dilute agar test (Heppner, 1973) with the quantitation advantages of actylamide microwells (Haskill, 1973), simply by moulding microwells in agarose over a feeder layer. It uses 150 tumour cells per 30 mm dish of 200 wells, with cloning efficiency of 50–80%. At an effector: target cell ratio of 10 : 1 the agarose microwell assay method gives a colony-inhibition of 25–85%.     

Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: application to bioartificial pancreas

To develop a novel type of immunoisolation membrane for a microcapsule-shaped bioartificial pancreas, we attempted to use a sol-gel synthesized silicate. An aminopropyl-silicate membrane derived from 3-aminopropyltrimethoxysilane and tetramethoxysilane was formed on Ca-alginate gel beads via electrostatic interaction. The positively charged amino groups remaining on the surface of the resultant gel beads were neutralized by immersion in an aqueous Na-alginate solution. From measurements of the partition coefficients and effective diffusivities of different substances to the gel beads, it was found that the aminopropyl-silicate membrane prepared under optimized composition of silicon alkoxide precursors successfully rejected gamma-globulin, giving good permeability to substances having a low molecular weight. Islets could be encapsulated within the newly developed microcapsules while retaining their ability to secrete insulin.     

Perifusion model for detoxification of drugs in a bioartificial liver

Detoxification of a drug in a bioartificial liver (BAL) during an in vitro experiment was theoretically carried out based on a perifusion model. The detoxification capacity assay, the rates of disappearance of the chemicals such as flow-limited and enzyme-limited drugs in the BAL system could be defined from models of hepatic perfusion-elimination relationships. When the flow-limited drug administrated under a quasi-equilibrium condition, a two-compartment model for the concentration behavior of the drug was introduced and compared with a one-compartment model. For both models, equations involving hepatic drug clearance and various pharmacokinetic parameters were derived under initial bolus loading and constant-rate infusion plus bolus loading conditions. The concentration of enzyme-limited drug in the BAL decreased linearly with time in contrast with the concentration profile of the flow-limited drug followed by exponential functions. The perifusion model offers a quantitative understanding of the elimination kinetics of chemicals such as flow-limited and enzyme-limited drugs in a bioartificial liver and a comparison between the BAL and human liver.     

Critical issues in bioartificial liver development.

End-stage liver disease accounts for over 30,000 deaths annually in the United States. Orthotopic liver transplantation is the only clinically proven treatment for patients with end-stage liver failure. A limitation of this therapy is a shortage of donor organs available. This donor organ shortage is exacerbated by the fact that the number of patients listed for transplantation has continued to increase. As a result, there has been a continuing increase in the number of patients who die waiting for a donor liver. Extracorporeal bioartificial liver devices consisting of viable hepatocytes have the potential to provide temporary support for patients with fulminant hepatic failure, thereby serving as a "bridge" to transplantation. In some patients, this temporary support would allow the native liver to regenerate function, eliminating the need for transplantation and the resulting life-long immunosuppressive therapy, all of which translates into a cost savings to the health care system. Although the bioartificial liver device is a promising technology for the treatment of liver failure, significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. An overview of the critical issues in the development of bioartificial liver devices is discussed.     

Biodegradable polymeric matrices for bioartificial implants

Biomaterials made of polymers, metals or their alloys, ceramics and their composites, are used as implants to restore or to replace the damaged soft and hard tissue/organ functions for an intended time period. Biomaterials made of synthetic materials are very simple materials compared to their natural counterparts, they only replace very simple functions of the damaged tissue during healing. Natural tissues have been used for both soft and hard repair and replacement, but they do have serious limitations such as: shortage of donor tissue, donor site morbidity, unpredictable resorption characteristics, immunogenic response, risk of disease transmission, and ethical limitations. Tissue engineering is a relatively new approach, in which healthy mammalian cells are used with supporting matrices, usually made of either natural or synthetic polymers as composite bioartificial implants. Primary cells, especially embryonic stem cells, cell lines, hybridomas, genetically modified cells are considered as potential sources for this application. Both closed and open matrices are used as support matrices. Nondegradable and biocompatible microcapsules and hollow fibers are utilized in closed systems, especially for immunoprotection of the transplanted cells. Biodegradable polymers, both natural and synthetic are used in the preparation of bioartificial implants carrying only autogenic cells.