3D Bioprinting of biomimetic pancreas with tunicate nanocellulose and human pancreatic islets

Diabetes is a disease that affects millions of people worldwide. The World Health Organization (WHO) is predicting that 400 million people will develop diabetes before 2030. Patients suffering from type 1 diabetes lack the hormone insulin, responsible for proper glucose regulation, which is produced by beta cells in the Islets of Langerhans localized in the pancreas. Insulin injection is the most common treatment to control blood sugar levels but does not cure the disease. One milestone for diabetes therapy is clinical islet transplantation. Today, this is a safe and effective treatment option for severe type 1 diabetes patients, but the method is hampered by loss of islets. Developing scaffolds for islet transplantation by 3D bioprinting technology could allow for transplantation outside the liver. Developing clinical grade biomaterials that can be used for this approach is urgently needed. In this innovation project we evaluated nanocellulose isolated from marine organisms, tunicates, in combination with alginate for delivery of human islets in 3D bioprinted scaffolds. The nanocellulose biosynthesized by tunicates is the most crystalline nanocellulose in nature. The processes for farming, purification and production of nanocellulose have been developed by Ocean Tunicell AS. The project leader, CELLHEAL AS, has been responsible for design and 3D Bioprinting of the device. NTNU has been responsible for evaluation of immune response of tunicate nanocellulose. Oslo University Hospital (OUS) has been responsible for testing devices with islets loaded into the biomaterial. Both the viability and functionality of islets were tested. The teams have made significant progress towards development of cell delivery devices using a novel biomaterial manufactured based on a starting material harvested from marine sources from the Norwegian coast. In work package H1, CELLHEAL designed implantable devices for delivery of human islets. The focus was on promoting vascularization for the implantable device. To test this, we used a grid-based design for the scaffolds and extensive characterization of these cell-free scaffolds showed good stability in vitro and good cell viability when printed with cells. 3D Bioprinting has been used to biofabricate vascular structures using a biomimetic LEAF design and computer simulation of flow has been conducted for evaluation of different prototypes. We have shown that we can promote binding of endothelial cells into the channels of the LEAF, a critical step in vascularization. In work package H2, Ocean TuniCell has developed isolation processes of nanocellulose from tunicates. The product, TuniCell, is manufactured as medical grade quality with endotoxin content under 0,5EU per ml and the aim was to show biocompatibility with insulin producing islets when combined with 20% of alginate. Ocean TuniCell has built a full-scale cleanroom production facility with GMP standard (good manufacturing practice) and has developed the whole process for isolation of cellulose and purification of nanocellulose from tunicates. Enzymatically prepared nanocellulose (ETC) was rheologically characterized and sent to project partners for biocompatibility (NTNU) and islet physiological testing (OUS). In work package H3, NTNU produced beads in the size range 500 - 800 micrometers with varying composition blends of tunicate nanocellulose and alginate. The beads, which formed gels with a mixture of Calcium and Barium, are stable in physiological solutions. NTNU further studied pericapsular fibrosis in a mouse model and inflammatory responses in human blood. The beads were found to have moderate cell coverage on the capsule surface after implantation, and this seems to correlate with activation of complement and coagulation in human blood, where the cytokine response was low. In work package H4, OUS studied the survival and function of insulin producing islets in 3D Bioprinted scaffolds using the TuniCell-based bioink. The distribution of islets in 3D bioprinted scaffolds was investigated to obtain equal distribution of islets. We found good viability of islets in 3D bioprinted scaffolds up to 14 days in culture. This was accompanied by good functionality (response to glucose stimulation). OUS tested different implantation sites of the cell-free 3D bioprinted scaffolds in vivo using an immunodeficient mouse model. OUS show that the Tunicell in alginate-based bioink could be crosslinked equally well with calcium-, barium- and strontium- chloride. The diffusion properties were investigated for molecules up to 70kDa, and good diffusion rates for molecules up to 5kDa were shown. This supported physiological transport processes of islets specific molecules in and out of the scaffolds (glucose (0.2 kda) and insulin (5kDa). In work package H5, we have initiated biocompatibility evaluation of 3D Bioprinted devices based on TuniCell in Wistar rats following the ISO 109936 standard.

The most important outcomes of this innovation project: -design of novel cell delivery device -process and material preparation of biocompatible nanocellulose from tunicates suitable for implantation and cell delivery -3D Bioprinting process to deliver pancreatic islets with tunicate nanocellulose biomaterial -advancement in development of technology for improved treatment of type 1 diabetes The most important impacts of this innovation project: -industrial development of a cGMP process for tunicate nanocellulose biomaterial -assessment of biocompatibility of tunicate nanocellulose for intended use -material and technology platform for cell therapy and tissue engineering -advancement in new treatment options for type 1 diabetes patients

There were 415 million people in the world with diabetes, type 1 and type 2 in 2015 and the number is expected to increase to 642 million in 2040. Annual cost of diabetes worldwide is more than 827 billion US$. Beta islets transplantation is used in several hospitals worldwide to help patients with diabetes type 1. It has however shortages such is limited cell supply and efficacy and needs of immunosuppression. Oslo University Hospital has developed islet isolation facility and islet manufacturing and is frequently transplanting human islets. The aim of the project is to design implantable biomimetic pancreas device and to biofabricate it using 3D Bioprinting with bioinks based on nanocellulose isolated from tunicates. The devices will be used to deliver pancreatic islets to treat type 1 diabetes patients. The bioink will be based on nanocellulose fibrils isolated from tunicates. Nanocellulose fibrils from wood and bacteria have been successfully used together with alginate as bioinks for 3D Bioprinting of living human tissue. Wood and bacteria derived nanocelluloses have however disadvantages due to high endotoxin levels. Dr Paul Gatenholm, founder of CELLHEAL AS is entrepreneur and world leader in using nanocellulose in biomedical applications and 3D Bioprinting expert. Nanocellulose produced by bacteria has unique biocompatibility due to low protein adsorption and hydrated surface. Nanocellulose isolated from tunicates by Ocean Bergen exhibits higher crystallinity and purity than bacterial cellulose. Biocompatibility of this potentially new biomaterial has not yet been determined. Dr Berit Strand at NTNU is expert in evaluation of immuno response of implanted biopolymers. She will be together with her colleagues leading task with aim to determine biocompatibility. Dr Hanne Scholz, Manager of islet transplantation unit and director of Oslo Regenerative Medicine Cluster will be responsible for evaluation of the 3D Bioprinted device in vitro and in vivo.