Growth factors regional patterned and photoimmobilized scaffold applied to bone tissue regeneration.

Bone diseases such as osteomalacia, osteoporosis, and osteomyelitis are major illnesses that threaten the health of human. This study aimed to provide an idea at the molecular level of material properties determined with UV specific surface approaches. The tert-butyl hydroperoxide (t-BHP) exposure aging model bone mesenchymal stem cells (BMSCs) were reverted by using a poly-hybrid scaffold (PS), which is a carbon nanotube (CNT) coated polycaprolactone (PCL) and polylactic acid (PLA) scaffold, combined with insulin-like growth factor-1 (IGF). Then, the region-specific PS photo-immobilized with different growth factors (GFs) was obtained by interference and diffraction of ultraviolet (UV) light. Additionally, the reverted BMSCs were regionally pattern differentiated into three kinds of cells on the GF immobilized PS (GFs/PS). In vivo, the GFs/PS accelerate bone healing in injured Sprague-Dawley (SD) rats. The data showed that GFs/PS effectively promoted the differentiation of reverted BMSCs in the designated area on 21st day. These results suggest region-specific interface immobilization of GFs concurrently differentiating reverted BMSCs into three different cells in the same scaffold. This method might be considered as a short-time, low cost, and simple operational approach to scaffold modification for tissue regeneration in the future.

Application of double network of gellan gum and pullulan for bone marrow stem cells differentiation towards chondrogenesis by controlling viscous substrates.

Hydrogels have a large amount of water that provides a cartilage-like environment and is used in tissue engineering with biocompatibility and adequate degradation rates. In order to differentiate stem cells, it is necessary to adjust the characteristics of the matrix such as stiffness, stress-relaxing time, and microenvironment. Double network (DN) hydrogels provide differences in cellular biological behavior and have interpenetrating networks that combine the advantages of the components. In this study, by varying the viscous substrate of pullulan (PL), the DN hydrogels of gellan gum (GG) and PL were prepared to determine the cartilage differentiation of bone marrow stem cell (BMSC). The characteristics of GG/PL hydrogel were investigated by examining the swelling ratio, weight loss, sol fraction, compressive modulus, and gelation temperature. The viability, proliferation, and toxicity of BMSCs encapsulated in hydrogels were evaluated. Cartilage phenotype and cartilage differentiation were confirmed by morphology, GAG content, and cartilage-specific gene expression. Overall results demonstrate that GG/PL hydrogels can form cartilage differentiation of BMSCs and can be applied for tissue engineering purposes.

Enhanced osteogenesis of human mesenchymal stem cells by self-assembled peptide hydrogel functionalized with glutamic acid templated peptides.

Self-assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three-dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self-assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL-1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real-time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.

Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

Cell subtype-dependent formation of breast tumor spheroids and their variable responses to chemotherapeutics within microfluidics-generated 3D microgels with tunable mechanics.

Tumor spheroids have been considered valuable miniaturized three dimensional (3D) tissue models for fundamental biological investigation as well as drug screening applications. Most tumor spheroids are generated utilizing the inherent aggregate behavior of tumor cells, and the effect of microenvironmental factors such as extracellular matrix (ECM) on tumor spheroid formation has not been extensively elucidated to date. Herein, uniform-sized spherical microgels encapsulated with different subtypes of breast tumor cells, based on tumor aggressiveness, are developed by flow-focusing microfluidics technology. Mechanical properties of microgels are controlled in a wide range via polymer concentration, and their influence on tumor physiology and spheroid formation is shown to be highly dependent on cell subtype. Specifically, the formation of polyploid/multinucleated giant cancer cells is a key early step in determining initial proliferation and eventual tumor spheroid generation within microgels with varying mechanics. In addition, chemotherapeutic screening performed on these tumor spheroids in microgels also display significantly variable cytotoxic effects based on microgel mechanics for each cell subtype, further highlighting the importance of microenvironmental factors on tumor spheroid physiology.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

YestroSens, a field-portable S. cerevisiae biosensor device for the detection of endocrine-disrupting chemicals: Reliability and stability.

Farming, industry and urbanization lead to increases in the concentrations of potentially harmful compounds in waste, surface and drinking waters. One example of such pollution are estrogens, the steroidal female reproductive hormones. Already at a few nanograms per litre, these hormones can trigger endocrine disruption and cause acute and chronic health problems in humans and wildlife. Here, we present a Saccharomyces cerevisiae estrogen biosensor capable of detecting estradiol, as well as ethinylestradiol, at concentrations of 1 nM. After an initial characterization of the sensor strain performance in an optimal laboratory setting, we focused on developing a biosensor device. We addressed current limitations of biosensors, such as the requirement of the cells for a liquid growth matrix, controlled storage conditions required to preserve cell viability, and the usually required bulky, as well as expensive, laboratory equipment. Our study provides significant new insights into the field of applied biosensors. The system presented in this work takes microorganism-based analytics one step closer to field application in decentralized locations.

Dual delivery of encapsulated BM-MSCs and BMP-2 improves osteogenic differentiation and new bone formation.

Stem cell-based therapies provide a promising approach for bone repair. In the present work, we developed a novel 3D vehicle system for dual-delivery of encapsulated bone marrow mesenchymal stem cells (BM-MSCs) and bone morphogenetic protein-2 (BMP-2) for treatment of large bone defects. The vehicle system consists of sodium alginate microcapsules and polylactic acid (PLLA) microspheres. BM-MSCs are encapsulated in the microcapsules, and BMP-2 proteins are encapsulated in the PLLA microspheres. This vehicle system acted as a multicore structure for sustained release of BMP-2, which enabled pulsed dosing induction of osteogenic differentiation of the co-embedded BM-MSCs. in vitro experiments showed that the loaded BMP-2 was constitutively released up to 30 days. Bioactivity of the incorporated BMP-2 in the microspheres was preserved and osteogenic differentiation of the BM-MSCs in the microcapsules was improved. In vivo, osteogenesis studies demonstrated that satisfactory degree of repair of a rat calvarial defect was achieved with the delivery of either encapsulated BM-MSCs alone or encapsulated BMP-2 alone. Transplantation of encapsulated both BM-MSCs and BMP-2 exhibited the greatest repair potential following 4- or 8-weeks treatment. In conclusion, microencapsulation of BM-MSCs and BMP-2 promoted the maturity of newly generated bone and improved new bone formation. Transplantation of BM-MSCs and BMP-2 in our novel 3-D vehicle system is a promising strategy for regenerative therapies of large bone defects.

Injectable stem cell-laden supramolecular hydrogels enhance in situ osteochondral regeneration via the sustained co-delivery of hydrophilic and hydrophobic chondrogenic molecules.

Hydrogels have been widely used as the carrier material of therapeutic cell and drugs for articular cartilage repair. We previously demonstrated a unique host-guest macromer (HGM) approach to prepare mechanically resilient, self-healing and injectable supramolecular gelatin hydrogels free of chemical crosslinking. In this work, we show that compared with conventional hydrogels our supramolecular gelatin hydrogels mediate more sustained release of small molecular (kartogenin) and proteinaceous (TGF-ß1) chondrogenic agents, leading to enhanced chondrogenesis of the encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro and in vivo. More importantly, the supramolecular nature of our hydrogels allows injection of the pre-fabricated hydrogels containing the encapsulated hBMSCs and chondrogenic agents, and our data show that the injection process has little negative impact on the viability and chondrogenesis of the encapsulated cells and subsequent neocartilage development. Furthermore, the stem cell-laden supramolecular hydrogels administered via injection through a needle effectively promote the regeneration of both hyaline cartilage and subchondral bone in the rat osteochondral defect model. These results demonstrate that our supramolecular HGM hydrogels are promising delivery biomaterials of therapeutic agents and cells for cartilage repair via minimally invasive procedures. This unique capability of injecting cell-laden hydrogels to target sites will greatly facilitate stem cell therapies.

3D-3 Tumor Models in Drug Discovery for Analysis of Immune Cell Infiltration.

The cross talk between tumor cells and other cells present in the tumor microenvironment such as stromal and immune cells highly influences the behavior and progression of disease. Understanding the underlying mechanisms of interaction is a prerequisite to develop new treatment strategies and to prevent or at least reduce therapy failure in the future. Specific reactivation of the patient's immune system is one of the major goals today. However, standard two-dimensional (2D) cell culture techniques lack the necessary complexity to address related questions. Novel three-dimensional (3D) in vitro models-embedded in a matrix or encapsulated in alginate-recapitulate the in vivo situation much better. Cross talk between different cell types can be studied starting from co-cultures. As cancer immune modulation is becoming a major research topic, 3D in vitro models represent an important tool to address immune regulatory/modulatory questions for T, NK, and other cells of the immune system. The 3D systems consisting of tumor cells, fibroblasts, and immune cells (3D-3) already proved as a reliable tool for us. For instance, we made use of those models to study the molecular mechanisms of the cross talk of non-small cell lung cancer (NSCLC) and fibroblasts, to unveil macrophage plasticity in the tumor microenvironment and to mirror drug responses in vivo. Generation of those 3D models and how to use them to study immune cell infiltration and activation will be described in the present book chapter.

Strategies for controlling egress of therapeutic cells from hydrogel microcapsules.

Endothelial progenitor cells and human mesenchymal stem cells (hMSCs) have shown great regenerative potential to repair damaged tissue; however, their injection in vivo results in low retention and poor cell survival. Early clinical research has focussed on cell encapsulation to improve viability and integration of delivered cells. However, this strategy has been limited by the inability to reproduce large volumes of standardized microcapsules and the lack of information on cell-specific egress and timed release from hydrogel microcapsules. Here, we address both of these limitations. First, we use a droplet microfluidic platform to generate monodisperse agarose microcapsules, and second we encapsulate and characterize egress of therapeutically relevant cells (human umbilical vein endothelial cells, endothelial progenitor cells, and hMSCs). With increased temporal resolution, we demonstrate distinct differences in egress between cell types. Importantly, therapeutic cells (hMSCs) egress quickly, in <6 hr following encapsulation. Further, we examined potential escape mechanisms and showed that proliferation can be exploited by cells for microcapsule translocation. We also systematically characterized the egress of fibroblasts (as model cells) following alterations to the microcapsules. Specifically, we show that microcapsule size and hydrogel density impact cell egress efficiency. Overall, our results demonstrate the need for characterization of cell-specific egress and tuning of the cocoon microenvironment prior to delivery, for timely release and successful engraftment.

Layer-by-Layer Cerium Oxide Nanoparticle Coating for Antioxidant Protection of Encapsulated Beta Cells.

In type 1 diabetes, the replacement of the destroyed beta cells could restore physiological glucose regulation and eliminate the need for exogenous insulin. Immunoisolation of these foreign cellular transplants via biomaterial encapsulation is widely used to prevent graft rejection. While highly effective in blocking direct cell-to-cell contact, nonspecific inflammatory reactions to the implant lead to the overproduction of reactive oxygen species, which contribute to foreign body reaction and encapsulated cell loss. For antioxidant protection, cerium oxide nanoparticles (CONPs) are a self-renewable, ubiquitous, free radical scavenger currently explored in several biomedical applications. Herein, 2-12 alternating layers of CONP/alginate are assembled onto alginate microbeads containing beta cells using a layer-by-layer (LbL) technique. The resulting nanocomposite coatings demonstrate robust antioxidant activity. The degree of cytoprotection correlates with layer number, indicating tunable antioxidant protection. Coating of alginate beads with 12 layers of CONP/alginate provides complete protection to the entrapped beta cells from exposure to 100 × 10-6 m H2 O2 , with no significant changes in cellular metabolic activity, oxidant capacity, or insulin secretion dynamics, when compared to untreated controls. The flexibility of this LbL method, as well as its nanoscale profile, provides a versatile approach for imparting antioxidant protection to numerous biomedical implants, including beta cell transplantation.

Method for measuring extracellular flux from intact polarized epithelial monolayers.

Purpose: The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating. Methods: Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates. Results: Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls. Conclusions: We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.

TGF-ß1-Modified Hyaluronic Acid/Poly(glycidol) Hydrogels for Chondrogenic Differentiation of Human Mesenchymal Stromal Cells.

In cartilage regeneration, the biomimetic functionalization of hydrogels with growth factors is a promising approach to improve the in vivo performance and furthermore the clinical potential of these materials. In order to achieve this without compromising network properties, multifunctional linear poly(glycidol) acrylate (PG-Acr) is synthesized and utilized as crosslinker for hydrogel formation with thiol-functionalized hyaluronic acid via Michael-type addition. As proof-of-principle for a bioactivation, transforming growth factor-beta 1 (TGF-ß1) is covalently bound to PG-Acr via Traut's reagent which does not compromise the hydrogel gelation and swelling behavior. Human mesenchymal stromal cells (MSCs) embedded within these bioactive hydrogels show a distinct dose-dependent chondrogenesis. Covalent incorporation of TGF-ß1 significantly enhances the chondrogenic differentiation of MSCs compared to hydrogels with supplemented noncovalently bound TGF-ß1. The observed chondrogenic response is similar to standard cell culture with TGF-ß1 addition with each medium change. In general, multifunctional PG-Acr offers the opportunity to introduce a range of biomimetic modifications (peptides, growth factors) into hydrogels and, thus, appears as an attractive potential material for various applications in regenerative medicine.

Application of Hydrogels in Cartilage Tissue Engineering.

BACKGROUND: Cartilage has limited ability for self-repairing, prompting the search for cartilage substitutes that can repair cartilage defects. Hydrogels have attracted attention as cartilage substitutes, since their mechanical properties, swelling ability and lubricating behavior are similar to extracellular matrix of articular cartilage. Hydrogels can be of natural, synthetic or hybrid origin, and hydrogels can encapsulate stem cells and/or be loaded with growth factors to promote cell differentiation into a chondrogenic phenotype. OBJECTIVE AND RESULTS: This review summarizes basic research advances in using hydrogels to repair cartilage defects. The raw materials, stem cells and growth factors used to prepare hydrogels are discussed. CONCLUSION: Substantial success has been achieved in small animal models of cartilage repair and regeneration, but further research is needed to improve hydrogels' mechanical properties and their integration with surrounding tissues.

PEG hydrogel containing calcium-releasing particles and mesenchymal stromal cells promote vessel maturation.

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. STATEMENT OF SIGNIFICANCE: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.

Calcium deposition in photocrosslinked poly(Pro-Hyp-Gly) hydrogels encapsulated rat bone marrow stromal cells.

Reproducing the features of the extracellular matrix is important for fabricating three-dimensional (3D) scaffolds for tissue regeneration. A collagen-like polypeptide, poly(Pro-Hyp-Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro-Hyp-Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible-light photocrosslinking at various concentrations of methacrylated poly(Pro-Hyp-Gly) (20-50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro-Hyp-Gly) with 5-min irradiation. These findings demonstrate that poly(Pro-Hyp-Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro-Hyp-Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.

Investigation of the role of alginate containing high guluronic acid on osteogenic differentiation capacity of human umbilical cord Wharton's jelly mesenchymal stem cells.

BACKGROUND AND AIM: Cell encapsulation using biodegradable material has promising results for tissue engineering. Since pressure is an effective factor on stem cell behaviour and various concentrations of alginate create different pressures on the cells, therefore our goal was to evaluate the mechanical effect of 1/2% (w/v) and 1/8% (w/v) alginate containing high guluronic acid on viability and osteogenic capacity of HUCWJ cells. METHODS: Cell viability was evaluated by MTT assay after 1, 7 and 14 days. Alkaline phosphatase activity was evaluated by alkaline phosphatase assay kit after 14 and 21 days. Alizarin red S staining was performed for calcium deposition among histological section. RESULTS: MTT assay showed significant difference in the mean of viability rates between groups in day 14 (p < 0.05). Alizarin red S staining was higher in the group 1.8%. In addition, there was statistically significant higher ALP activity in the group 1.8% compared to the group 1.2%.

Co-encapsulation and co-transplantation of mesenchymal stem cells reduces pericapsular fibrosis and improves encapsulated islet survival and function when allografted.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

Engaging Cold to Upregulate Cell Proliferation in Alginate-Encapsulated Liver Spheroids.

For many years, the impact of hyper- and hypothermia on mammalian cells has been examined. With the exception of short, low temperature storage, which has uses in areas such as preservation for transplantation or regenerative medicine, advantages for the use of low temperature treatment in hepatocytes have not been previously reported. We have observed that alginate-encapsulated HepG2 liver spheroids that are cryopreserved or experience a cold reduction in temperature (≤10°C) for periods between 1 and 90 min display an enhanced cell proliferation during culture 7-16 days post-treatment compared with untreated samples. Following 8-12 days post-treatment, alginate-encapsulated liver spheroids experienced a cell density of 1.71 ± 0.35 times that of control samples (p < 0.001). This effect occurred in samples with a variety of cold treatments. This low temperature treatment offers a simple method to rapidly increase cell proliferation rates for extended culture systems, such as bioartificial liver devices. This would allow the manufacture of required biomass more rapidly, and to a higher cell density, reducing final required biomass volume. This could enable bioartificial liver devices to be prepared more cheaply, making them a more cost effective treatment.