Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

OBJECTIVE: Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). MATERIALS AND METHODS: Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. RESULTS: Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. CONCLUSIONS: The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.

Alginate encapsulant incorporating CXCL12 supports long-term allo- and xenoislet transplantation without systemic immune suppression.

Islet transplantation represents a potentially curative approach for individuals with Type I Diabetes. The requirement for systemic immune suppression to control immune-mediated rejection of transplanted islets and the limited human islet supply represent significant roadblocks to progress for this approach. Islet microencapsulation in alginate offers limited protection in the absence of systemic immunosuppression, but does not support long-term islet survival. The chemokine, CXCL12, can repel effector T cells while recruiting immune-suppressive regulatory T cells (Tregs) to an anatomic site while providing a prosurvival signal for beta-cells. We proposed that coating or encapsulating donor islets with CXCL12 would induce local immune-isolation and protect and support the function of an allo- or xenograft without systemic immune suppression. This study investigated the effect of alginate microcapsules incorporating CXCL12 on islet function. Islet transplantation was performed in murine models of insulin-dependent diabetes. Coating of islets with CXCL12 or microencapsulation of islets with alginate incorporating the chemokine, resulted in long-term allo- and xenoislet survival and function, as well as a selective increase in intragraft Tregs. These data support the use of CXCL12 as a coating or a component of an alginate encapsulant to induce sustained local immune-isolation for allo- or xenoislet transplantation without systemic immunosuppression.

Cryoprotectant transport through articular cartilage for long-term storage: experimental and modeling studies.

OBJECTIVE: Long-term storage of articular cartilage (AC) remains challenging due to poor post-thaw viability. An initial step towards addressing this issue is characterizing cryoprotectant (CPA) transport, since ensuring adequate CPA equilibration throughout the tissue offers protection during cooling. This study takes a systematic approach in determining CPA transport rates through bovine AC and uses that information in mathematical models to determine CPA equilibration times. DESIGN: Diffusion of high concentration single (6.9 M dimethyl sulfoxide (DMSO)) and multi-component CPA solutions (VS55, 3.1 M DMSO+2.2 M 1,2-propanediol (PD)+3.1 M formamide (FM)) was measured through AC using (1)H nuclear magnetic resonance (NMR) imaging and localized spectroscopy, respectively. Using experimentally calculated effective diffusivities, diffusion models describing CPA transport through the tissue matrix and across chondrocyte membranes were combined to design a CPA addition and removal scheme for a cartilage plug of clinically relevant dimensions. RESULTS: (1)H NMR imaging and localized spectroscopy experiments suggested that the permeation of CPAs through AC (5 mm diameter, 5-10 mm in thickness) took on the order of 4 h for full equilibration at 22 degrees C. Imaging clearly showed the permeation of DMSO into cartilage over time and localized spectroscopy was able to distinguish the permeation rates of the individual VS55 components and water. Experimentally measured diffusivity values were used in CPA addition/removal simulations with a cartilage plug of clinically relevant dimensions (5 mm diameter, 2 mm in thickness). Results suggested a multi-step approach for adding and removing high concentration CPAs, with the addition and removal each taking approximately 2 h to complete. CONCLUSIONS: This study provides a foundation for designing CPA addition and removal protocols for effective long-term storage of cartilage tissue using a novel approach to measure CPA permeation.

Efficient transmicrocatheter delivery of functional fibroblasts with a bioengineered collagen gel-platinum microcoil complex: toward the development of endovascular cell therapy for cerebral aneurysms.

BACKGROUND AND PURPOSE: Endoaneurysmal implantation of fibroblasts may promote healing of aneurysms and reduce recanalization after therapeutic embolization. The purpose of our study was to develop a device for delivery of fibroblasts with use of current microcoil technology. MATERIALS AND METHODS: Cell carrier devices and cell-free devices were fabricated by associating collagen gels (with or without fibroblasts) with platinum microcoils. During the propagation of control cell carrier devices for 1 week in culture, cell-mediated gel contraction (CMGC) occurred. Modified cell carrier devices created by glutaraldehyde cross-linking, ascorbate coculture, or extended CMGC were also characterized in vitro. Devices were deployed through microcatheters (533 microm lumen, 160 cm length). Gel retention, cell retention, cell death, and the ability to support local cell migration were analyzed in vitro. RESULTS: Cell viability was reduced by glutaraldehyde cross-linking but not by microcatheter transit. During microcatheter transit, cell carrier devices liberated minimal particulate matter and cellular DNA. Liberated particulate matter was reduced by glutaraldehyde cross-linking (P < .05) and extended CMGC (P < .04). Only cell carrier devices treated with glutaraldehyde cross-linking did not exhibit cell migration after microcatheter transit. Passage of cell-free devices through microcatheters sheared off most of their collagen gel. CONCLUSION: Collagen gel-platinum microcoil complexes can mediate efficient transmicrocatheter delivery of viable, migration-capable fibroblasts. CMGC is a necessary component of the process of gel stabilization that enables successful microcatheter transit. Although extended CMGC and glutaraldehyde cross-linking enhance gel stabilization, glutaraldehyde cross-linking decreases cell viability and migratory potential.

Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute.

This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo.

Modeling of encapsulated cell systems.

Tissue engineered substitutes consisting of cells in biocompatible materials undergo remodeling with time as a result of cell growth and death processes. With inert matrices that do not directly influence cell growth, remodeling is driven mainly by the concentration of dissolved oxygen (DO). Insulin-secreting cell lines encapsulated in alginate-based beads and used as a pancreatic substitute represent such a case. Beads undergo remodeling with time so that an initially homogeneous distribution of cells is eventually replaced by a dense peripheral ring of primarily viable cells, whereas inner cells are mostly necrotic. This paper develops and analyzes a mathematical model of an encapsulated cell system of spherical geometry that tracks the viable and dead cell densities and the concentration of DO within the construct as functions of radial position and time. Model simulations are compared with experimental histology data on cell distribution. Correlations are then developed between the average intrabead DO concentration (AIDO) and the total viable cell number, as well as between AIDO and the radial cell and DO distributions in beads. As AIDO can be measured experimentally by incorporating a perfluorocarbon emulsion in the beads and acquiring (19)F nuclear magnetic resonance (NMR) spectroscopic data, these correlations can be used to track the remodeling that occurs in the construct in vitro and potentially in vivo. The usefulness of mathematical models in describing the dynamic changes that occur in tissue constructs with time, and the value of these models at obtaining additional information on the system when used interactively with experimental measurements, are discussed.

Use of glucose-responsive material to regulate insulin release from constitutively secreting cells.

Genetically-engineered cells offer a solution to the cell availability problem in tissue engineering a pancreatic substitute for the treatment of insulin-dependent diabetes. These cells can be non-beta cells, such as hepatocytes or myoblasts, retrieved as a biopsy from the same patient and genetically engineered to secrete recombinant insulin constitutively or under transcriptional regulation. However, the continuous or slowly responsive insulin secretion dynamics from these cells cannot provide physiologic glucose regulation in patients. Our objective consists of using such cells as an insulin source and of regulating insulin release by incorporating a glucose-responsive material, which acts as a control barrier for insulin in a cell-material hybrid device. Experiments were performed with insulinoma betaTC3 cells, HepG2 hepatomas, and C2C12 myoblasts, the latter two genetically-modified to constitutively secrete insulin. The control barrier consisted of concanavalin A (con A)-based glucose-responsive material, which forms a gel at low and a sol at high glucose concentrations. Results demonstrated that the device released insulin at a higher rate in response to glucose challenges. In contrast, a device containing an inert hydrogel instead of glucose-responsive material released insulin at an essentially constant rate, irrespective of the surrounding glucose concentration. Necessary material improvements include increased sensitivity to glucose, so that the material responds to physiologically relevant glucose concentrations, and increased stability. The prospects of developing a properly functional, implantable substitute based on engineered non-beta cells and glucose-responsive material, and the material and device improvements that need to be made prior to in vivo experiments, are discussed.

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

In vivo noninvasive monitoring of a tissue engineered construct using 1H NMR spectroscopy.

Direct, noninvasive monitoring of tissue engineered substitutes containing live, functional cells would provide valuable information on dynamic changes that occur postimplantation. Such changes include remodeling both within the construct and at the interface of the implant with the surrounding host tissue, and may result in changes in the number of viable cells in the construct. This study investigated the use of 1H NMR spectroscopy in noninvasively monitoring the viable cell number within a tissue engineered construct in vivo. The construct consisted of mouse betaTC3 insulinomas in a disk-shaped agarose gel, surrounded by a cell-free agarose gel layer. Localized 1H NMR spectra were acquired from within implanted constructs, and the total choline resonance was measured. Critical issues that had to be addressed in accurately quantifying total choline from the implanted cells included avoiding signal from host tissue and correcting for interfering signal from diffusing solutes. In vivo NMR measurements were correlated with MTT assays and NMR measurements performed in vitro on explanted constructs. Total choline measurements accurately and noninvasively quantified viable betaTC3 cell numbers in vivo, in the range of 1 x 10(6) to more than 14 x 10(6) cells, and monitored changes in viable cell number that occurred in the same construct over time. This is the first study using NMR techniques to monitor viable cell numbers in an implanted tissue substitute. It established architectural characteristics that a construct should have to be amenable to NMR monitoring, and it set the foundation for future in vivo investigations with other tissue engineered implants.

Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Hybrid pancreatic tissue substitute consisting of recombinant insulin-secreting cells and glucose-responsive material.

Insulin-dependent diabetes is a serious pathological condition, currently treated by blood glucose monitoring and daily insulin injections, which, however, do not prevent long-term complications. A tissue-engineered pancreatic substitute has the potential to provide a more physiologic, less invasive, and potentially less costly treatment of the disease. A major issue in developing such a substitute is the cells being used. Nonpancreatic cells, retrieved from the same patient and genetically engineered to secrete insulin constitutively or with some glucose responsiveness, offer the significant advantages of being immune-acceptable and relaxing the tissue availability limitations, which exist with islets from cadaveric donors. These cells, however, do not have insulin secretion dynamics appropriate for restoration of euglycemia in higher animals and, eventually, humans. In this study, we present the concept of a hybrid pancreatic substitute consisting of such cells sequestered in a material exhibiting glucose-dependent changes of its permeability to insulin. A Concanavalin A-glycogen material sandwiched between two polycarbonate membranes and exhibiting glucose-dependent sol-gel transformations was used. Rates of insulin transport through this material in gel and sol forms were characterized for both FITC-labeled insulin in solution and insulin secreted by betaTC3 mouse insulinoma cells. Effective diffusivities through sol were found to be up to 3.5-fold higher than through the gel state of the material. A mathematical model of a hybrid construct was formulated and analyzed to simulate the secretory behavior in response to step ups and downs in the surrounding glucose concentration. The experimental and modeling studies indicate that a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

Effect of oxygen tension on chondrocyte extracellular matrix accumulation.

Since cartilage is mainly an avascular tissue, chondrocytes exist in a low-level oxygen environment in vivo. In the present study, we investigated the effect of oxygen tension (20%, 5% and 1% gas phase oxygen concentrations) over a 20-day period on the extracellular matrix accumulation of bovine articular chondrocytes in confluent surface cultures. Matrix accumulation was assessed by the amount of glycosaminoglycan and collagen deposited in the matrix. From initially confluent monolayers, the chondrocytes became distributed throughout a thick layer of extracellular matrix, thus forming a multicell-layer of tissue. Cells maintained their normal rounded shape, indicative of the differentiated phenotype, throughout the 20-day culture period. On a per culture and a per cell basis, the amount of collagen and glycosaminoglycan accumulation in the matrix was lower at the reduced oxygen tensions. Specifically, in 1% oxygen, matrix GAG content reached a steady-state level, with no net increase in GAG levels after two weeks, whereas in 20% oxygen, matrix GAG increased with time. It is concluded that oxygen has a significant effect on the amount of macromolecules accumulated in the extracellular matrix. The implications of these findings in growing cartilage constructs in vitro are discussed.

In vitro effects of transcatheter injection on structure, cell viability, and cell metabolism in fibroblast-impregnated alginate microspheres.

PURPOSE: To determine if microsphere-encapsulated cell preparations can be delivered through a microcatheter without compromising microsphere structure, cell viability, or metabolism. MATERIALS AND METHODS: Fibroblast-impregnated microspheres were fabricated by using 1.0% alginate and rabbit synovial fibroblasts. Fibroblast-impregnated alginate microspheres injected through microcatheters were analyzed in parallel with identical noninjected microspheres. The effects of transcatheter injection on structure and cell viability (percentage of viable cells per microsphere) were correlated with microsphere size. Structural effects were analyzed by using light microscopy, and 7-day percentage (ratio of live cells to dead cells) cell viability was assessed with confocal microscopy and fluorescent staining. In a second series of experiments, the metabolism of small microspheres was studied during a course of 7 days by using a spectrophotometric bioanalyzer. RESULTS: Transcatheter injection caused fracturing and/or fragmentation of large (800-1,000 microm) and medium (500-750 microm) microspheres, while small (250-400 microm) microspheres were structurally unaffected by transcatheter injection. Fracturing and fragmentation were associated with cell release from the alginate matrix. Although transcatheter injection reduced cell viability by 17%-23% in all size categories, it did not cause a detectable alteration in the rate of glucose metabolism. CONCLUSION: Transcatheter injection was physiologically well tolerated by fibroblasts encapsulated in alginate microspheres; however, when microsphere diameter exceeded the catheter diameter, fracturing and fragmentation of microspheres compromised the sequestration function of the microsphere vector.

The effects of alginate composition on encapsulated betaTC3 cells.

The effects of alginate composition on the growth of murine insulinoma betaTC3 cells encapsulated in alginate/poly-L-lysine/alginate (APA) beads, and on the overall metabolic and secretory characteristics of the encapsulated cell system, were investigated for four different types of alginate. Two of the alginates used had a high guluronic acid content (73% in guluronic acid residues) with varying molecular weight, while the other two had a high mannuronic acid content (68% in mannuronic acid residues) with varying molecular weight. Each composition was tested using two different polymer concentrations. Our data show that betaTC3 cells encapsulated in alginates with a high guluronic acid content experienced a transient hindrance in their metabolic and secretory activity because of growth inhibition. Conversely, betaTC3 cells encapsulated in alginates with a high mannuronic acid content experienced a rapid increase in metabolic and secretory activity as a result of rapid cell growth. Our data also demonstrate that an increase in either molecular weight or concentration of high mannuronic acid alginates did not alter the behavior of the encapsulated betaTC3 cells. Conversely, an increase in molecular weight and concentration of high guluronic acid alginates prolonged the hindrance of glucose metabolism, insulin secretion and cell growth. These observations can be best interpreted by changes in the microstructure of the alginate matrix, i.e., interaction between the contiguous guluronic acid residues and the Ca2+ ions, as a result of the different compositions.

Non-Invasive monitoring of a bioartificial pancreas in vitro and in vivo.

Monitoring biochemical processes relevant to the function, survival, and longevity of tissue-engineered pancreatic constructs is important for the development of an optimum construct design as well as patient care management after implantation. In this report we demonstrate the ability of nuclear magnetic resonance (NMR) techniques to monitor aspects of intracellular metabolism, overall morphology, and distribution of a microencapsulation based bioartificial pancreas in vitro and in vivo.

Effects of short-term hypoxia on a transformed cell-based bioartificial pancreatic construct.

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma betaTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

In vitro monitoring of total choline levels in a bioartificial pancreas: (1)H NMR spectroscopic studies of the effects of oxygen level.

This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of (1)H NMR detectable metabolites from alginate/poly-l-lysine/alginate (APA)-encapsulated betaTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P<0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed.

Effects of pH on murine insulinoma betaTC3 cells.

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.

Engineering challenges in the development of an encapsulated cell system for treatment of type 1 diabetes.

Implantation of glucose-responsive, insulin-secreting cells is promising in providing a treatment for type I diabetes, which is more effective, less invasive, and potentially less costly than conventional insulin injections. However, in spite of promising results with animal studies, a clinical product or therapeutic procedure based on encapsulated cells does not yet exist. This is because a number of barriers remain to be addressed, which include a source of functional cells, a stable, biocompatible membrane offering immune protection to the implant, a construct architecture ensuring cell viability and construct function, and the engineering of immune acceptance of the construct post-implantation. This article reviews these barriers and the current state-of-the-art, with special emphasis on the engineering challenges involved, and discusses possible ways to tackle the complex problems currently preventing this approach from reaching clinical practice.