Peritoneal dissolved oxygen and function of encapsulated adult porcine islets transplanted in streptozotocin diabetic mice.

BACKGROUND: Alginate-encapsulated islet xenografts have restored normoglycemia in diabetic animals for various periods of time. Plausible mechanisms of graft failure in vivo include immune rejection and hypoxia. We sought to understand the effects of encapsulated adult porcine islet (API) dosage on the peritoneal dissolved oxygen (DO) level in correlation to the achieved glycemic regulation in diabetic mice. METHODS: Adult porcine islets encapsulated in barium alginate were transplanted intraperitoneally in streptozotocin diabetic BALB/c mice at 6000 and 4000 islet equivalents (IEQ) and in normal mice at 500 IEQ; APIs encapsulated in calcium alginate were transplanted at 6000 IEQ in diabetic mice. In all cases, cell-free barium alginate capsules containing a perfluorocarbon emulsion were co-implanted for DO measurements using 19 F NMR spectroscopy. Blood glucose levels and peritoneal DO were measured over 60 days or until graft failure. Explanted capsules were evaluated microscopically and histologically. RESULTS: Both barium and calcium alginate-encapsulated APIs at 6000 IEQ reversed diabetes until day 60; barium alginate-encapsulated APIs at 4000 IEQ also reversed diabetes but with a higher failure rate. Transplanted APIs significantly reduced the peritoneal DO, approximately in a dose-dependent manner. The number of viable islets and the insulin content per capsule decreased over time. Capsules retrieved from normoglycemic mice exhibited minimal host cell adherence. CONCLUSIONS: Transplantation of encapsulated APIs can reduce peritoneal DO to severely hypoxic levels. Although normoglycemia could be maintained within the study period, the DO levels suggest that hypoxia is a factor contributing to loss of islet viability and insulin secretion with time in mice.

Multiple clinically relevant immunotherapies prolong the function of microencapsulated porcine islet xenografts in diabetic NOD mice without the use of anti-CD154 mAb.

BACKGROUND: Our goal was to identify clinically relevant immunotherapies that synergize with microencapsulation to protect adult porcine islet (API) xenografts in diabetic NOD mice. We have shown previously that dual costimulatory blockade (CTLA4-Ig plus anti-CD154 mAb) combined with encapsulation protects APIs long-term in NOD mice. Since no anti-CD154 mAbs currently are approved for use in humans, we tested the efficacy of other targeted immunosuppression regimens that might be used for diabetic patients receiving encapsulated islets. METHODS: Microencapsulated APIs were transplanted i.p. in diabetic NOD mice given either no immunosuppression or combinations immunosuppressive reagents. Graft function was monitored by blood glucose levels, i.p. glucose tolerance tests, and histology. Mechanisms of rejection were investigated by phenotyping host peritoneal cells and measuring graft site cytokine and chemokine levels. RESULTS: New immunosuppressive therapies were compared to CTLA4-Ig plus anti-CD154 mAb, used here as a control. The most effective was triple treatment with CTLA4-Ig, anti-CD154 mAb, and intracapsular CXCL12, and the next most effective was a non-depleting anti-CD4 mAb (YTS177.9) plus intracapsular CXCL12. Three additional regimens (CTLA4-Ig plus YTS177.9, YTS177.9 alone, and anti-OX40-Ligand mAb alone) significantly prolonged encapsulated API function. Dual treatment with CTLA4-Ig plus anti-CD40 mAb was as effective as CTLA4-Ig plus anti-CD154 mAb. Five other monotherapies and three combination therapies did not augment encapsulated API survival. Most peritoneal cytokines and chemokines were either absent or minimal. At necropsy, the capsules were intact, not fibrosed, and clean when function was maintained, but were coated with host cells if rejection had occurred. CONCLUSIONS: Multiple different immunotherapies which specifically inhibit CD4+ T cells, modulate T-cell trafficking, or interfere with antigen presentation can substitute for anti-CD154 mAb to prolong encapsulated islet xenograft function in diabetic NOD mice.

Microencapsulated adult porcine islets transplanted intraperitoneally in streptozotocin-diabetic non-human primates.

BACKGROUND: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression. METHODS: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG. RESULTS: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1ß, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure. CONCLUSIONS: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space.

mRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.

BACKGROUND: Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2 h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia. METHODS: We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions. RESULTS: Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo. CONCLUSIONS: The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT.

The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study.

BACKGROUND: Adult porcine islets (APIs) constitute a promising alternative to human islets in treating type 1 diabetes. The intrahepatic site has been used in preclinical primate studies of API xenografts; however, an estimated two-thirds of donor islets are destroyed after intraportal infusion due to a number of factors, including the instant blood-mediated inflammatory reaction (IBMIR), immunosuppressant toxicity, and poor reestablishment of extracellular matrix connections. Intraperitoneal (ip) transplantation of non-vascularized encapsulated islets offers several advantages over intrahepatic transplantation of free islets, including avoidance of IBMIR, immunoprotection, accommodation of a larger graft volume, and reduced risk of hemorrhage. However, there exists evidence that the peritoneal site is hypoxic, which likely impedes islet function. METHODS: We tested the effect of hypoxia (2%-5% oxygen or pO2 : 15.2-38.0 mm Hg) on free and encapsulated APIs over a period of 6 days in culture. Free and encapsulated APIs under normoxia served as controls. Islet viability was evaluated with a viability/cytotoxicity assay using calcein AM and ethidium bromide on days 1, 3, and 6 of culture. Alamar blue assay was used to measure the metabolic activity on days 1 and 6. Insulin in spent medium was assayed by ELISA on days 1 and 6. RESULTS: Viability staining indicated that free islet clusters lost their integrity and underwent severe necrosis under hypoxia; encapsulated islets remained intact, even when they began to undergo necrosis. Under hypoxia, the metabolic activity and insulin secretion (normalized to metabolic activity) of both free and encapsulated islets decreased relative to islets cultured under normoxic conditions. CONCLUSIONS: Hypoxia (2%-5% oxygen or pO2 : 15.2-38.0 mm Hg) affects the viability, metabolic activity, and insulin secretion of both free and encapsulated APIs over a six-day culture period. Encapsulation augments islet integrity under hypoxia, but it does not prevent loss of viability, metabolic activity, or insulin secretion.

Trichostatin A affects the secretion pathways of beta and intestinal endocrine cells.

Histone deacetylase inhibitors (HDACi) were recently identified as having significant clinical potential in reversing ß-cell functional inhibition caused by inflammation, a shared precursor of Type 1 and Type 2 diabetes. However, HDACi are highly complex and little is known of their direct effect on important cell secretion pathways for blood glucose regulation. The aims of the present study were to investigate the effect of HDACi on insulin secretion from ß-cells, GLP-1 secretion from L-cells, and recombinant insulin secretion from engineered L-cells. The ß-cell line ßTC-tet, L-cell line GLUTag, or recombinant insulin-secreting L-cell lines were exposed to Trichostatin A for 24h. Effects on insulin or GLP-1 mRNA, intracellular protein content, processing efficiency, and secretion were measured by real-time PCR, ELISA, and radioimmunoassay. HDACi increased secretion per viable cell in a dose-dependent manner for all cell types. Effects on mRNA levels were variable, but enhanced intracellular polypeptide content and secretion were comparable among cell types. Enhanced recombinant insulin secretion was sustained for seven days in alginate microencapsulated L-cells. HDACi enhances ß- and L-cell secretion fluxes in a way that could significantly improve blood glucose regulation in diabetes patients and holds potential as a novel method for enhancing insulin-secreting non-ß or ß-cell grafts.

Dual factor delivery of CXCL12 and Exendin-4 for improved survival and function of encapsulated beta cells under hypoxic conditions.

A bioartifical pancreas (BAP) remains a promising approach for treating insulin-dependent diabetes. Several obstacles to the clinical implementation of a BAP remain, including hypoxia following implantation. Within native pancreatic islets, CXCL12 and glucagon-like peptide-1 (GLP-1) act in a paracrine fashion to promote the survival, function, and proliferation of ß-cells. This work sought to investigate if the presentation of CXCL12 and delivery of a GLP-1 receptor analog, Exendin-4 (Ex-4), alone and in combination, conferred pro-survival and insulinotropic effects on an encapsulated ß-cell line, ßTC-tet, cultured under hypoxic conditions of 7.6 mmHg O2 . Our findings indicate that presentation of CXCL12 in the encapsulation matrix completely abrogated apoptosis under hypoxic conditions. Delivery of Ex-4 increased insulin secretion rate under both normoxic and hypoxic conditions, and additionally reduced apoptosis under hypoxic conditions. Furthermore, presentation of CXCL12 combined with Ex-4 delivery significantly increased insulin secretion rate under hypoxic conditions compared to delivery of Ex-4 alone. These findings demonstrate that the presentation of CXCL12 combined with the delivery of Ex-4 may constitute a promising strategy for supporting ß-cell function and survival following transplantation.

Encapsulated Cells for the Treatment of Diabetes: Danger of Acute Hypoglycemia Following Injury?

Transplants comprised of encapsulated islets have shown promise in treating insulin-dependent diabetes. A question raised in the scientific and clinical communities is whether the insulin released from an implanted encapsulation device damaged in an accident could cause a serious hypoglycemic event. In this commentary, we consider the different types of damage that a device can sustain, including the encapsulation membrane and the islets within, and the amount of insulin released in each case. We conclude that the probability that device damage would cause an adverse hypoglycemic event is indeed very low.

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells.

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-ß cells that secrete insulin in response to physiologic stimuli. A normal pancreatic ß cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal ß cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.

Mathematical modeling of cryoprotectant addition and removal for the cryopreservation of engineered or natural tissues.

Long-term storage of natural tissues or tissue-engineered constructs is critical to allow off-the-shelf availability. Vitrification is a method of cryopreservation that eliminates ice formation, as ice may be detrimental to the function of natural or bioartificial tissues. In order to achieve the vitreous state, high concentrations of CPAs must be added and later removed. The high concentrations may be deleterious to cells as the CPAs are cytotoxic and single-step addition or removal will result in excessive osmotic excursions and cell death. A previously described mathematical model accounting for the mass transfer of CPAs through the sample matrix and cell membrane was expanded to incorporate heat transfer and CPA cytotoxicity. Simulations were performed for two systems, an encapsulated system of insulin-secreting cells and articular cartilage, each with different transport properties, geometry and size. Cytotoxicity and mass transfer are dependent on temperature, with a higher temperature allowing more rapid mass transfer but also causing increased cytotoxicity. The effects of temperature are exacerbated for articular cartilage, which has larger dimensions and slower mass transport through the matrix. Simulations indicate that addition and removal at 4°C is preferable to 25°C, as cell death is higher at 25°C due to increased cytotoxicity in spite of the faster mass transport. Additionally, the model indicates that less cytotoxic CPAs, especially at high temperature, would significantly improve the cryopreservation outcome. Overall, the mathematical model allows the design of addition and removal protocols that insure CPA equilibration throughout the sample while still minimizing CPA exposure and maximizing cell survival.

Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions.

Cryoprotectant (CPA) cytotoxicity constitutes a challenge in developing cryopreservation protocols, specifically in vitrification where high CPA concentrations are necessary to achieve the ice-free, vitreous state. Few cytotoxicity studies have investigated vitrification-relevant concentrations of CPAs, and the benefits and disadvantages of cocktail solutions and of incorporating non-permeating solutes have not been fully evaluated. In this study, we address these issues by determining the cytotoxicity kinetics for dimethylsulfoxide (Me(2)SO) and 1,2-propanediol (PD) on alginate-encapsulated ßTC-tet mouse insulinomas for a range of concentrations and temperatures. Cytotoxicity kinetics were also determined for two cocktails, DPS (3M Me(2)SO+3M PD+0.5M sucrose) and PEG400 (1M Me(2)SO+5M PD+0.34M poly(ethylene)glycol with M.W. of 400). PD was found to be more cytotoxic than Me(2)SO at higher concentrations and temperatures. This was reflected in PEG400 being more cytotoxic at room temperature than PEG400 at 4°C or DPS at either temperature. Addition of non-permeating solutes increased the cytotoxicity of cocktails. Furthermore, results indicate that CPA cytotoxicity may not be additive and that combining CPAs may increase cytotoxicity synergistically. Finally, when comparing cytotoxic effects towards encapsulated HepG2 and ßTC-tet cells, and towards ßTC-tet cells in capsules and in monolayers, CPAs appear more cytotoxic towards cells with higher metabolic activity. The incorporation of these results in the rational design of CPA addition/removal processes in vitrification is discussed.

Noninvasive Fluorine-19 Magnetic Resonance Relaxometry Measurement of the Partial Pressure of Oxygen in Acellular Perfluorochemical-loaded Alginate Microcapsules Implanted in the Peritoneal Cavity of Nonhuman Primates.

BACKGROUND: We have utilized a noninvasive technique for measuring the partial pressure of oxygen (pO2) in alginate microcapsules implanted intraperitoneally in healthy nonhuman primates (NHPs). Average pO2 is important for determining if a transplant site and capsules with certain passive diffusion characteristics can support the islet viability, metabolic activity, and dose necessary to reverse diabetes. METHODS: Perfluoro-15-crown-5-ether alginate capsules were infused intraperitoneally into 3 healthy NHPs. Peritoneal pO2 levels were measured on days 0 and 7 using fluorine-19 magnetic resonance relaxometry and a fiber-optic probe. Fluorine-19 MRI was used to determine the locations of capsules within the peritoneal space on days 0 and 7. Gross and histologic evaluations of the capsules were used to assess their biocompatibility postmortem. RESULTS: At day 0 immediately after infusion of capsules equilibrated to room air, capsules were concentrated near the infusion site, and the pO2 measurement using magnetic resonance relaxometry was 147 ± 9 mm Hg. On day 7 after capsules were dispersed throughout the peritoneal cavity, the pO2 level was 61 ± 11 mm Hg. Measurements using the fiber-optic oxygen sensor were 132 ± 7.5 mm Hg (day 0) and 89 ± 6.1 mm Hg (day 7). Perfluoro-15-crown-5-ether capsules retrieved on day 7 were intact and free-floating without host cell attachment, although the numbers of peritoneal CD20 B cells, CD4 and CD8 T cells, and CD14 macrophages increased consistent with a mild foreign body reaction. CONCLUSIONS: The peritoneal pO2 of normal NHPs is relatively low and we predict would decrease further when encapsulated islets are transplanted intraperitoneally.

Use of magnetic nanoparticles to monitor alginate-encapsulated betaTC-tet cells.

Noninvasive monitoring of tissue-engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide-based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for betaTC-tet cells encapsulated within alginate/poly-L-lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION-labeled betaTC-tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed.

Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L-cells.

A tissue engineered pancreatic substitute (TEPS) consisting of insulin-producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose-based constructs which contained either a single cell suspension of GLUTag-INS cells, a suspension of pre-aggregated GLUTag-INS spheroids, or GLUTag-INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag-INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non-encapsulated GLUTag-INS cells. Though improvements in the expression level of GLUTag-INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L-cells upon incorporation into an implantable three-dimensional TEPS.

Insulin-secreting L-cells for the treatment of insulin-dependent diabetes.

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complications associated with diabetes. An autologous cell source is especially attractive for regulatory and ethical reasons in addition to eliminating the need for immunosuppression. This study uses non-beta-cells, genetically modified for physiologic insulin secretion. Enteroendocrine L-cells, exhibit regulated secretion in response to physiologic stimuli and their endogenous products are fully compatible with prandial metabolism. Murine GLUTag L-cells were transfected with a plasmid co-expressing human insulin and neomycin resistance and the stable cell line, GLUTag-INS, was established. Secretion properties of GLUTag-INS cells were investigated in vitro through induced secretion tests using meat hydrolysate or 3-isobutyl-1-methylxanthine and forskolin as secretagogues. GLUTag-INS cells rapidly co-secreted recombinant insulin and endogenous glucagon-like peptide in response to metabolic cues from the surrounding medium and demonstrated efficient processing of proinsulin to insulin.

Advanced Cell and Tissue Biomanufacturing.

This position paper assesses state-of-the-art advanced biomanufacturing and identifies paths forward to advance this emerging field in biotechnology and biomedical engineering, including new research opportunities and translational and corporate activities. The vision for the field is to see advanced biomanufacturing emerge as a discipline in academic and industrial communities as well as a technological opportunity to spur research and industry growth. To navigate this vision, the paths to move forward and to identify major barriers were a focal point of discussions at a National Science Foundation-sponsored workshop focused on the topic. Some of the major needs include but are not limited to the integration of specific scientific and engineering disciplines and guidance from regulatory agencies, infrastructure requirements, and strategies for reliable systems integration. Some of the recommendations, major targets, and opportunities were also outlined, including some "grand challenges" to spur interest and progress in the field based on the participants at the workshop. Many of these recommendations have been expanded, materialized, and adopted by the field. For instance, the formation of an initial collaboration network in the community was established. This report provides suggestions for the opportunities and challenges to help move the field of advanced biomanufacturing forward. The field is in the early stages of effecting science and technology in biomanufacturing with a bright and important future impact evident based on the rapid scientific advances in recent years and industry progress.

Bioluminescence tracking of alginate micro-encapsulated cell transplants.

Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba2+ crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.

Alginate microencapsulation of human mesenchymal stem cells as a strategy to enhance paracrine-mediated vascular recovery after hindlimb ischaemia.

Stem cell-based therapies hold great promise as a clinically viable approach for vascular regeneration. Preclinical studies have been very encouraging and early clinical trials have suggested favourable outcomes. However, significant challenges remain in terms of optimizing cell retention and maintenance of the paracrine effects of implanted cells. To address these issues, we have proposed the use of a cellular encapsulation approach to enhance vascular regeneration. We contained human mesenchymal stem cells (hMSCs) in biocompatible alginate microcapsules for therapeutic treatment in the setting of murine hindlimb ischaemia. This approach supported the paracrine pro-angiogenic activity of hMSCs, prevented incorporation of hMSCs into the host tissue and markedly enhanced their therapeutic effect. While injection of non-encapsulated hMSCs resulted in a 22 ± 10% increase in vascular density and no increase in perfusion, treatment with encapsulated hMSCs resulted in a 70 ± 8% increase in vascular density and 21 ± 7% increase in perfusion. The described cellular encapsulation strategy may help to better define the mechanisms responsible for the beneficial effects of cell-based therapies and provide a therapeutic strategy for inducing vascular growth in the adult. As hMSCs are relatively easy to isolate from patients, and alginate is biocompatible and already used in clinical applications, therapeutic cell encapsulation for vascular repair represents a highly translatable platform for cell-based therapy in humans.

Effects of cryopreservation on cell viability and insulin secretion in a model tissue-engineered pancreatic substitute (TEPS).

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 betaTC3 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.

Preproinsulin mRNA engineering and its application to the regulation of insulin secretion from human hepatomas.

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include non-beta cells genetically engineered to secrete insulin in response to physiologic cues; responsiveness can be introduced at the transcriptional level to regulate preproinsulin (PPI) mRNA biosynthesis. However, these cells exhibit sluggish secretion dynamics, which is not appropriate for achieving euglycemia in higher animals and, eventually, humans. In this work, we have engineered the PPI mRNA so as to destabilize it through nonsense-mediated mRNA decay (NMD). When expressed under transcriptional regulation in HepG2 hepatomas, the engineered PPI mRNA level and of the insulin secretion rate declined faster upon switching off transcription, compared to the one-copy non-engineered control. Our work provides a simple and straightforward method to improve the dynamics of transcriptionally regulated insulin secretion, which can be a useful tool in developing cell-based therapies for IDD.