Forsøksdyr: Fibrotic reactions towards alginate capsules; a study on the effect of novel capsule materials as a mean to reduce the fibrosis currently limiting the use of alginate capsules in cell therapy.

Godkjenningsdato 21.06.2018

Godkjenningsperiode 01.07.2018-01.10.2019

Alginates are polysaccharides that form hydrogels under physiological conditions. Alginates are generally biocompatible. In cell therapy, alginate gels restrict access to immune cells and has been proposed as a functional cure for Type 1 diabetes. This disease causes the autoimmune destruction of pancreatic beta-cells and insulin deficiency. Transplantation of insulin producing tissues restores endogenous insulin production, but it requires immunosuppressive treatment with adverse health effects. Encapsulating insulin producing cells in alginate will mitigate the need for immunosuppressant therapy.

A current limitation of the use of alginates in cell therapy is fibrotic overgrowth, which causes graft failure following transplantation of pancreatic tissue. Alginates can be tailored by enzymatic and chemical modifications. We aim to investigate the host-graft responses to gel beads of modified alginates with the implantation into a C57BL/6J mouse strain that provokes immune responses towards microcapsules and is relevant for reactions observed in monkeys and humans. Our hypothesis is that the modification will reduce the fibrotic reactions towards the biomaterial.

The study will consist of a pilot experiment using 30 mice to qualitatively assess the host-graft responses, and a subsequent main study of selected alginate capsules showing minimal/no fibrosis using up to 70 mice (8-12 animals per capsule design), giving a total of 100 mice. Empty capsules will be implanted into the peritoneal cavity of mice and explanted within two-four weeks (end of experiment). The implantation procedure to be performed is considered to be mild with few adverse effects (minor degree of pain quantified in minutes). The procedure involves a small incision (maximum 1 cm) through the abdominal wall and peritoneum, where the capsules will be injected into the intraperitoneal cavity. The incision site will be closed with one stitch. Isofluran (4% induction, 2.5 % inhalation) will be used as anesthesia (reconvalence period of animals is 10 minutes), and xylocain before implantation of capsules (0.02 ml subcutaneously at operation site) to hinder discomfort after the surgical treatment. The cage setup will initially be 5 mice/cage, but if the mice exhibit aggressive/stressful behavior they will be moved to separate cages to mitigate stress responses. If the animals display abnormal behavior or signs of distress/pain (mouse grimace scale), inadequate wound healing at the site of injection before the end of experiment, the animal will be euthanized. The animals will be euthanized at the end of the experiment, before opening the animal and evaluation of capsules. The method of euthanization will be cervical dislocation with anesthesia or CO2.

In vitro assays, particularly the human whole blood model, are supplemental to in vivo studies. There are no current methods that can replace in vivo models of fibrotic response to alginate beads, as such reactions are complex and involve more cell types than found in blood, as well as a longer time-span. The current animal study will be correlated to investigations on protein adhesion on alginate beads incubated in human plasma, also complement and cytokine responses in whole blood, to study the transferability of the in vivo results to the in vitro findings.