Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture.

A strategy that seeks to combine the biophysical properties of inert encapsulation materials like alginate with the biochemical niche provided by pancreatic extracellular matrix (ECM)-derived biomaterials, could provide a physiomimetic pancreatic microenvironment for maintaining long-term islet viability and function in culture. Herein, we have demonstrated that incorporating human pancreatic decellularized ECM within alginate microcapsules results in a significant increase in Glucose Stimulation Index (GSI) and total insulin secreted by encapsulated human islets, compared to free islets and islets encapsulated in only alginate. ECM supplementation also resulted in long-term (58 days) maintenance of GSI levels, similar to that observed in free islets at the first time point (day 5). At early time points in culture, ECM promoted gene expression changes through ECM- and cell adhesion-mediated pathways, while it demonstrated a mitochondria-protective effect in the long-term. STATEMENT OF SIGNIFICANCE: The islet isolation process can damage the islet extracellular matrix, resulting in loss of viability and function. We have recently developed a detergent-free, DI-water based method for decellularization of human pancreas to produce a potent solubilized ECM. This ECM was added to alginate for microencapsulation of human islets, which resulted in significantly higher stimulation index and total insulin production, compared to only alginate capsules and free islets, over long-term culture. Using ECM to preserve islet health and function can improve transplantation outcomes, as well as provide novel materials and platforms for studying islet biology in microfluidic, organ-on-a-chip, bioreactor and 3D bioprinted systems.

Pancreas transplant versus islet transplant versus insulin pump therapy: in which patients and when?

PURPOSE OF REVIEW: The aim of the present review is to gather recent reports on the use of pancreas and islet transplantation and conventional insulin therapy for treating patients experiencing diabetes and its related complications. The present review directs attention to the current status, challenges and perspectives of these therapies and sheds light on potential future cellular therapies. RECENT FINDINGS: The risks and benefits of diabetes treatment modalities continue to evolve, altering the risk versus benefit calculation for patients. As continuous subcutaneous insulin infusion and monitoring technologies demonstrate increasing effectiveness in achieving better diabetes control and reducing hypoglycemia frequency, so are pancreas and islet transplantation improving and becoming more effective and safer. Both beta-cell replacement therapies, however, are limited by a dependence on immunosuppression and a shortage of cadaver donors, restricting more widespread and safer deployment. Based on the effectiveness of clinical beta-cell replacement for lengthening lifespan and improving quality of life, scientists are aggressively investigating alternative cell sources, transplant platforms, and means of preventing immunological damage of transplanted cells to overcome these principle limitations. SUMMARY: Essential goals of diabetes therapy are euglycemia, avoidance of hypoglycemia, and prevention or stabilization of end-organ damage. With these goals in mind, all therapeutic options should be considered.

Comprehensive characterization of the human pancreatic proteome for bioengineering applications.

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds.

Effect of alginate matrix engineered to mimic the pancreatic microenvironment on encapsulated islet function.

Islet transplantation is emerging as a therapeutic option for type 1 diabetes, albeit, only a small number of patients meeting very stringent criteria are eligible for the treatment because of the side effects of the necessary immunosuppressive therapy and the relatively short time frame of normoglycemia that most patients achieve. The challenge of the immune-suppressive regimen can be overcome through microencapsulation of the islets in a perm-selective coating of alginate microbeads with poly-l-lysine or poly- l-ornithine. In addition to other issues including the nutrient supply challenge of encapsulated islets a critical requirement for these cells has emerged as the need to engineer the microenvironment of the encapsulation matrix to mimic that of the native pancreatic scaffold that houses islet cells. That microenvironment includes biological and mechanical cues that support the viability and function of the cells. In this study, the alginate hydrogel was modified to mimic the pancreatic microenvironment by incorporation of extracellular matrix (ECM). Mechanical and biological changes in the encapsulating alginate matrix were made through stiffness modulation and incorporation of decellularized ECM, respectively. Islets were then encapsulated in this new biomimetic hydrogel and their insulin production was measured after 7 days in vitro. We found that manipulation of the alginate hydrogel matrix to simulate both physical and biological cues for the encapsulated islets enhances the mechanical strength of the encapsulated islet constructs as well as their function. Our data suggest that these modifications have the potential to improve the success rate of encapsulated islet transplantation.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Molecular Pathways Underlying Adaptive Repair of the Injured Kidney: Novel Donation After Cardiac Death and Acute Kidney Injury Platforms.

OBJECTIVE: To test the hypothesis that gene expression profiling in peripheral blood from patients who have undergone kidney transplantation (KT) will provide mechanistic insights regarding graft repair and regeneration. BACKGROUND: Renal grafts obtained from living donors (LD) typically function immediately, whereas organs from donation after cardiac death (DCD) or acute kidney injury (AKI) donors may experience delayed function with eventual recovery. Thus, recipients of LD, DCD, and AKI kidneys were studied to provide a more complete understanding of the molecular basis for renal recovery. METHODS: Peripheral blood was collected from LD and DCD/AKI recipients before transplant and throughout the first 30 days thereafter. Total RNA was isolated and assayed on whole genome microarrays. RESULTS: Comparison of longitudinal gene expression between LD and AKI/DCD revealed 2 clusters, representing 141 differentially expressed transcripts. A subset of 11 transcripts was found to be differentially expressed in AKI/DCD versus LD. In all recipients, the most robust gene expression changes were observed in the first day after transplantation. After day 1, gene expression profiles differed depending upon the source of the graft. In patients receiving LD grafts, the expression of most genes did not remain markedly elevated beyond the first day post-KT. In the AKI/DCD groups, elevations in gene expression were maintained for at least 5 days post-KT. In all recipients, the pattern of coordinate gene overexpression subsided by 28 to 30 days. CONCLUSIONS: Gene expression in peripheral blood of AKI/DCD recipients offers a novel platform to understand the potential mechanisms and timing of kidney repair and regeneration after transplantation.

BioSphincters to treat Fecal Incontinence in Nonhuman Primates.

Loss of anorectal resting pressure due to internal anal sphincter (IAS) dysfunctionality causes uncontrolled fecal soiling and leads to passive fecal incontinence (FI). The study is focused on immediate and long-term safety and potential efficacy of bioengineered IAS BioSphincters to treat passive FI in a clinically relevant large animal model of passive FI. Passive FI was successfully developed in Non-Human Primates (NHPs) model. The implantation of autologous intrinsically innervated functional constructs resolved the fecal soiling, restored the resting pressure and Recto Anal Inhibitory Reflex (RAIR) within 1-month. These results were sustained with time, and efficacy was preserved up to 12-months. The histological studies validated manometric results with the regeneration of a well-organized neuro-muscular population in IAS. The control groups (non-treated and sham) remained affected by poor anal hygiene, lower resting pressure, and reduced RAIR throughout the study. The pathological assessment of implants, blood, and the vital organs confirmed biocompatibility without any adverse effect after implantation. This regenerative approach of implanting intrinsically innervated IAS BioSphincters has the potential to offer a better quality of life to the patients suffering from FI.

Extracellular matrix-based hydrogels obtained from human tissues: a work still in progress.

PURPOSE OF REVIEW: The current review summarizes contemporary decellularization and hydrogel manufacturing strategies in the field of tissue engineering and regenerative medicine. RECENT FINDINGS: Decellularized extracellular matrix (ECM) bioscaffolds are a valuable biomaterial that can be purposed into various forms of synthetic tissues such as hydrogels. ECM-based hydrogels can be of animal or human origin. The use of human tissues as a source for ECM hydrogels in the clinical setting is still in its infancy and current literature is scant and anecdotal, resulting in inconclusive results. SUMMARY: Thus far the methods used to obtain hydrogels from human tissues remains a work in progress. Gelation, the most complex technique in obtaining hydrogels, is challenging due to remarkable heterogeneity of the tissues secondary to interindividual variability. Age, sex, ethnicity, and preexisting conditions are factors that dramatically undermine the technical feasibility of the gelation process. This is contrasted with animals whose well defined anatomical and histological characteristics have been selectively bred for the goal of manufacturing hydrogels.

Regenerative immunology: the immunological reaction to biomaterials.

Regenerative medicine promises to meet two of the most urgent needs of modern organ transplantation, namely immunosuppression-free transplantation and an inexhaustible source of organs. Ideally, bioengineered organs would be manufactured from a patient's own biomaterials-both cells and the supporting scaffolding materials in which cells would be embedded and allowed to mature to eventually regenerate the organ in question. While some groups are focusing on the feasibility of this approach, few are focusing on the immunogenicity of the scaffolds that are being developed for organ bioengineering purposes. This review will succinctly discuss progress in the understanding of immunological characteristics and behavior of different scaffolds currently under development, with emphasis on the extracellular matrix scaffolds obtained decellularized animal or human organs which seem to provide the ideal template for bioengineering purposes.

Transplantation of a Human Tissue-Engineered Bowel in an Athymic Rat Model.

Intestinal failure is a serious clinical condition characterized by loss of motility, absorptive function, and malnutrition. Current treatments do not provide the optimal solution for patients due to the numerous resulting complications. A bioengineered bowel that contains the necessary cellular components provides a viable option for patients. In this study, human tissue-engineered bowel (hTEB) was developed using a technique, whereby human-sourced smooth muscle cells were aligned and neoinnervated using human-sourced neural progenitor cells, resulting in the formation of intrinsically innervated smooth muscle sheets. The sheets were then rolled around hollow tubular chitosan scaffolds and implanted in the omentum of athymic rats for neovascularization. Four weeks later, biopsies of hTEB showed vascularization, normal cell alignment, phenotype, and function. During the biopsy procedure, hTEB was transplanted into the same rat's native intestine. The rats gained weight and 6 weeks later, hTEB was harvested for studies. hTEB was healthy in color with normal diameter and with digested food in the lumen, indicating propulsion of luminal content through the hTEB. Histological studies indicated neomucosa with evidence of crypts and villi structures. This study provides proof of concept that hTEB could provide a viable treatment to lengthen the gut for patients with gastrointestinal disorders.

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation.

Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

A step towards clinical application of acellular matrix: A clue from macrophage polarization.

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.

Autologous Cells for Kidney Bioengineering.

Worldwide, increasing numbers of patients are developing end-stage renal disease, and at present, the only treatment options are dialysis or kidney transplantation. Dialysis is associated with increased morbidity and mortality, poor life quality and high economic costs. Transplantation is by far the better option, but there are insufficient numbers of donor kidneys available. Therefore, there is an urgent need to explore alternative approaches. In this review, we discuss how this problem could potentially be addressed by using autologous cells and appropriate scaffolds to develop 'bioengineered' kidneys for transplantation. In particular, we will highlight recent breakthroughs in pluripotent stem cell biology that have led to the development of autologous renal progenitor cells capable of differentiating to all renal cell types and will discuss how these cells could be combined with appropriate scaffolds to develop a bioengineered kidney.

Extracellular matrix scaffolds as a platform for kidney regeneration.

Chronic and end stage renal disease (ESRD) have reached pandemic levels and pose a substantial public health burden. Unfortunately, available therapies lack efficacy in preventing progression to its end stage phase. Regenerative medicine promises to restore function of diseased organs among which the kidney, through two possible approaches: firstly, the maximization of the innate ability of tissues to repair or regenerate following injury; secondly, the ex vivo bio-fabrication of the organ in question. When regenerative medicine is applied to the setting of chronic or ESRD, it is intuitive that endeavors to improve renal repair, promote nephrogenesis in damaged kidneys, or the de novo engineering of transplantable kidneys, could have a major impact on the current management of this pandemic. Among the different regenerative medicine technologies currently under development, cell-on-scaffold seeding technology (CSST) - involving cells seeded throughout supporting scaffold structures made from biomaterials - is the most favorable candidate in the context of realistic clinical application. In this review, we outline and describe current investigations taking place in the field of CSST as it pertains to the restoration of kidney function.

Kidney transplantation, bioengineering and regeneration: an originally immunology-based discipline destined to transition towards ad hoc organ manufacturing and repair.

Kidney transplantation (KT), as a modality of renal replacement therapy (RRT), has been shown to be both economically and functionally superior to dialysis for the treatment of end-stage renal disease (ESRD). Progress in KT is limited by two major barriers: a) a chronic and burgeoning shortage of transplantable organs and b) the need for chronic immunosuppression following transplantation. Although ground-breaking advances in transplant immunology have improved patient survival and graft durability, a new pathway of innovation is needed in order to overcome current obstacles. Regenerative medicine (RM) holds the potential to shift the paradigm in RRT, through organ bioengineering. Manufactured organs represent a potentially inexhaustible source of transplantable grafts that would bypass the need for immunosuppressive drugs by using autologous cells to repopulate extracellular matrix (ECM) scaffolds. This overview discusses the current status of renal transplantation while reviewing the most promising innovations in RM therapy as applied to RRT.

Sudden death of a patient with polycystic kidneys due to acute inferior vena cava thrombosis.

A management algorithm for large renal cyst in autosomal dominant polycystic kidney disease (ADPKD) is lacking despite the potential to cause widespread medical and surgical complications. We report the case of a 37-year-old gentleman with ADPKD and large (>5 cm diameter) cysts who suffered sudden death due to autopsy-proven inferior vena cava and pulmonary arterial thrombosis. In this article, we discuss the possible pathophysiological factors at play in this catostrophic complication of ADPKD. We also review available literature to establish the prevalence of such a complication and also establish current thoughts and opinions as to the optimal management strategy for giant cysts in the context of ADPKD.

Heterogeneity of Scaffold Biomaterials in Tissue Engineering.

Tissue engineering (TE) offers a potential solution for the shortage of transplantable organs and the need for novel methods of tissue repair. Methods of TE have advanced significantly in recent years, but there are challenges to using engineered tissues and organs including but not limited to: biocompatibility, immunogenicity, biodegradation, and toxicity. Analysis of biomaterials used as scaffolds may, however, elucidate how TE can be enhanced. Ideally, biomaterials should closely mimic the characteristics of desired organ, their function and their in vivo environments. A review of biomaterials used in TE highlighted natural polymers, synthetic polymers, and decellularized organs as sources of scaffolding. Studies of discarded organs supported that decellularization offers a remedy to reducing waste of donor organs, but does not yet provide an effective solution to organ demand because it has shown varied success in vivo depending on organ complexity and physiological requirements. Review of polymer-based scaffolds revealed that a composite scaffold formed by copolymerization is more effective than single polymer scaffolds because it allows copolymers to offset disadvantages a single polymer may possess. Selection of biomaterials for use in TE is essential for transplant success. There is not, however, a singular biomaterial that is universally optimal.

Tissue-Engineering Approaches to Restore Kidney Function.

Kidney transplantation for the treatment of chronic kidney disease has established outcome and quality of life. However, its implementation is severely limited by a chronic shortage of donor organs; consequently, most candidates remain on dialysis and on the waiting list while accruing further morbidity and mortality. Furthermore, those patients that do receive kidney transplants are committed to a life-long regimen of immunosuppressive drugs that also carry significant adverse risk profiles. The disciplines of tissue engineering and regenerative medicine have the potential to produce alternative therapies which circumvent the obstacles posed by organ shortage and immunorejection. This review paper describes some of the most promising tissue-engineering solutions currently under investigation for the treatment of acute and chronic kidney diseases. The various stem cell therapies, whole embryo transplantation, and bioengineering with ECM scaffolds are outlined and summarized.