Purpose of Review Beta cell replacement therapy as a form of islet transplantation is a promising alternative therapy with the possibility to make selected patients with type 1 diabetes (T1D) insulin independent. However, this technique faces challenges such as extensive activation of the host immune system post-transplantation, lifelong need for immunosuppression, and the scarcity of islet donor pancreas. Advancement in tissue engineering strategies can improve these challenges and allow for a more widespread application of this therapy. This review will discuss the recent development and clinical translation of tissue engineering strategies in beta cell replacement therapy. Recent Findings Tissue engineering offers innovative solutions for producing unlimited glucose responsive cells and fabrication of appropriate devices/scaffolds for transplantation applications. Generation of pancreatic organoids with supporting cells in biocompatible biomaterials is a powerful technique to improve the function of insulin-producing cell clusters. Fabrication of physical barriers such as encapsulation strategies can protect the cells from the host immune system and allow for graft retrieval, although this strategy still faces major challenges to fully restore physiological glucose regulation. Summary The three main components of tissue engineering strategies including the generation of stem cell-derived insulin-producing cells and organoids and the possibilities for therapeutic delivery of cell-seeded devices to extra-hepatic sites need to come together in order to provide safe and functional insulin-producing devices for clinical beta cell replacement therapy.

Objective: Storage at temperatures as low as −80 °C and below (cryopreservation) is considered a method for long-term preservation of cells and tissues, and especially blood vessel segments, which are to be used for clinical operations such as transplantation. However, the freezing and thawing processes themselves can induce injuries to the cells and tissue by damaging the structure and consequently functionality of the cryopreserved tissue. In addition, the level of damage is dependent on the rate of cooling and warming used during the freezing-thawing process. Current methods for monitoring the viability and integrity of cells and tissues after going through the freezing-thawing cycle are usually invasive and destructive to the cells and tissues. Therefore, employing monitoring methods which are not destructive to the cryopreserved tissues, such as bioimpedance measurement techniques, is necessary. In this study we aimed to design a bioimpedance measurement setup to detect changes in venous segments after freezing-thawing cycles in a noninvasive manner. Approach: A bioimpedance spectroscopy measurement technique with a two-electrode setup was employed to monitor ovine jugular vein segments after each cycle during a process of seven freezing-thawing cycles. Main results: The results demonstrated changes in the impedance spectra of the measured venous segments after each freezing-thawing cycle. Significance: This indicates that bioimpedance spectroscopy has the potential to be developed into a novel method for non-invasive and non-destructive monitoring of the viability of complex tissue after cryopreservation.

No abstract

Intra-portal islet transplantation is the method of choice for treatment of insulin dependent type 1 diabetes, but its outcome is hindered by limited islet survival due to the immunological and metabolic stress post transplantation. Adipose-derived stromal cells (ASCs) promise to improve significantly the islet micro-environment but an efficient long-term delivery method has not been achieved. We therefore explore the potential of generating ASC enriched islet transplant structure by 3D bioprinting. Here, we fabricate a double-layered 3D bioprinted scaffold for islets and ASCs by using alginate-nanofibrillated cellulose bioink. We demonstrate the diffusion properties of the scaffold and report that human ASCs increase the islet viability, preserve the endocrine function, and reduce pro-inflammatory cytokines secretion in vitro. Intraperitoneal implantation of the ASCs and islets in 3D bioprinted scaffold improve the long-term function of islets in diabetic mice. Our data reveals an important role for ASCs on the islet micro-environment. We suggest a novel cell therapy approach of ASCs combined with islets in a 3D structure with a potential for clinical beta cell replacement therapies at extrahepatic sites.

Injury of the cornea is a complex biological process. Regeneration of the corneal stroma can be facilitated by the presence of mesenchymal stromal cells (MSCs) and application of tissue equivalents. A new tissue‐engineering strategy for corneal stroma regeneration is presented using cellularized 3D bioprinted hydrogel constructs implanted into organ cultured porcine corneas using femtosecond laser‐assisted intrastromal keratoplasty. The ex vivo cultured, MSC‐loaded 3D bioprinted structures remain intact, support cell survival, and contain de novo synthesized extracellular matrix components and migrating cells throughout the observation period. At day 14 postimplantation, the cellularized tissue equivalents contain few or no cells, as demonstrated by optical coherence tomography imaging and immunofluorescent staining. This study successfully combines a laboratory‐based method with modern, patient‐care practice to produce a cell‐laden tissue equivalent for corneal implantation. Optimal bioink composition and cellularization of tissue equivalents are essential in fine‐tuning a method to promote the current technique as a future treatment modality.

Introduction: After the first-in-human pilot study which showed safety of the pre-vascularized Sernova Cell Pouch (SCP) in the subcutaneous space, we modified islet transplantation (ITx) conditions for improved engraftment in the SCP. Methods: Two sets of the SCP were implanted in the abdominal anterior rectus sheath in seven patients with longstanding type 1 diabetes mellitus, problematic hypoglycemia and no stimulated C-peptide. Only highly purified islets were used for ITx and islets were suspended in the patient's own serum. Immunosuppression was initiated 1 month later followed by a marginal dose ITx after another month. Small sentinel SCPs were explanted for histopathological evaluation 3 months after each ITx. Results: Seven patients were submitted to 21 study related surgeries with a wound infection in 2 patients after SCP implantation with only one patient requiring device excision. The first subject presented with persistent stimulated serum C-peptide at 6 months after 1st and 2nd ITx into SCP. After 2nd ITx, glucose control improved substantially including reaching optimal target values for CGM with only 5% of Time Below Range (TBR). Subsequent intraportal ITx allowed for insulin independence currently maintained for over 15 months. The second patient at 3 months after 2nd ITx had positive stimulated serum C-peptide (0.48 ng/mL) with reduction of HbA1c from 10.6% to 7.6%, decreased insulin requirement from 49 to 28 u/day, improved CGM with TBR <4%, and reduction in Time Above Range (TAR) from 76% to 48%. To date, stimulated C-peptide has been detected for over 9 months. Three additional patients recently received ITx and await evaluation. Conclusion: Persistent islet graft function with sustained blood levels of C-peptide, reduction of HbA1c, improved CGM parameters, reduction of SHEs, and decreased total daily insulin requirement was achieved in the first 2 patients after ITx into SCPs implanted into abdominal wall. Significantly improved islet engraftment and clinical outcomes occurred using a modified approach for ITx into SCP.

Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31+CD45−) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugation in vitro using both adipose-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs.

Purpose: Regeneration of the cornea due to trauma or disease is a complex biological process, involving a delicate balance between cell differentiation, migration and a multitude of secreted factors and signalling molecules. We hereby present a tissue‐engineering technique as a possible first step towards getting a bench‐to‐bedside approach to assist corneal regeneration and ultimately, corneal transplantation. Methods: 3D extrusion‐based bioprinting and femtosecond laser‐assisted intrastromal keratoplasty (FLISK) were used in the study. Ex vivo cultured mesenchymal stem cells (MSCs) derived from adipose tissue, bone marrow and corneal stroma were bioprinted in a complex, collagen I‐containing hydrogel. The transparent, cell‐loaded nanocellulose and alginate‐based 3D constructs were then implanted into a pig cornea organ culture model. The 3D printed intrastromal constructs were first cultivated ex vivo, while the de novo extracellular matrix (ECM) synthesis and migration of cells was followed by optical coherence tomography and immunohistochemistry. Results: The ex vivo cultured, MSC‐loaded 3D bioprinted constructs supported cell survival and propagation for all three types of MSCs used. The transparency of the corneal stroma substitute was not compromised by the presence of MSCs in the bioprinted matrix. At day 14, the 3D‐bioprinted scaffolds appeared intact, as analysed by optical coherence tomography, while immunofluorescent staining using ECM and MSCs markers demonstrated the cellular presence in the constructs. Conclusions: Optimization of the bioink composition, cellular content and properties of the bioprinted material to be used in corneal transplantation and regeneration studies are essential in fine‐tuning the technique towards its clinical use.