Human ex vivo models for organ fibrosis

Fibrosis is characterized by excessive deposition of extracellular matrix proteins and is considered a serious complication associated with aging and/or chronic injury. Fibrosis ultimately results in structural and functional organ deterioration. As anti-fibrotic drugs are currently not available, patients can only be treated by invasive surgical interventions, e.g. organ transplantation or resection of the affected tissue. Fibrotic diseases account for up to 45% of worldwide mortality. Animal models of chronic injury are widely utilized to study fibrosis. The initiation and progression of fibrosis in animals is accompanied by an extended period of considerable discomfort caused by specific operations or the administration of toxic substances or immune mediating compounds. About 100.000 animals per year are used in fibrosis research and these are only the one’s that were published. Although a multitude of studies have been undertaken to elucidate the mechanisms of fibrosis, the absence of anti-fibrotic drugs on the market still indicates lack of knowledge on the mechanisms of fibrosis and therewithal the existence of clear targets for treatment. The most relevant shortcoming of animal in vivo studies is however that they are not comparable to the human situation. It is known from our work and others that there are large species differences, therefore, extrapolation from animal to man is often not appropriate. Another drawback is the absence of non-invasive biomarkers to monitor the disease. Tissue biopsies are the gold standard, however, they are invasive and not practical in monitoring anti-fibrotic treatment. Therefore, a non-invasive and accurate diagnostic tool would better support diagnosis of fibrosis, follow-up and evaluation of future anti-fibrotic therapies. Although the exact mechanism is still poorly understood, emerging evidence suggests that fibrosis is a result of a multicellular process. Therefore, an ex vivo system is needed that almost perfectly resembles the in vivo environment. Precision-cut tissue slices represent an ex vivo tissue model that mimics the multicellular characteristics of organs in vivo. The results in human precision-cut liver slices are very promising and revealed that an ex vivo method in humans can be applied successfully to elucidate the mechanisms of liver fibrosis and to test pharmacological interventions. The use of precision-cut intestinal and kidney slices to unravel the pathogenesis of fibrosis ex vivo has not been explored so far. Because this tissue slice model is close to the in vivo situation, it will also give us the opportunity to identify specific biomarkers of fibrosis. Minor tissue injury normally lead to scarless wound healing, however, if the initial trigger persists and becomes chronic, fibrosis will develop and non-functional connective tissue is deposited. Fibrosis will eventually progress from early onset to end-stage fibrosis, a life threatening disease. Thus to understand the mechanism of fibrosis and to find novel therapeutic modalities it is important to study both early and late stages of fibrosis. In this proposal we will use diseased or healthy human leftover surgical material after resections. Precision-cut intestinal and kidney slices will be prepared from normal human bowel and kidney. The viability during culture will be evaluated by determining the ATP content and the morphology of the precision-cut tissue slices. Organ specific toxic compounds or (growth) factors (e.g. PDGF and TGF-beta) will be used to provoke a fibrotic response in intestinal and kidney slices. Precision-cut fibrotic tissue slices will be prepared of fibrotic strictures of Crohn’s disease patients, fibrotic kidneys and cirrhotic livers from patients receiving a transplant. To assess fibrosis in both stages, gene and protein expression of fibrosis markers will be determined, using techniques like ELISA’s for collagen breakdown products, fibrosis gene arrays and laser capture microscopy. These techniques will also be used to identify new non-invasive fibrosis markers. The impact we had and will have on animal testing is already visible in our own consortium, since we have brought together a group of people that studied fibrosis in other organs than liver. Previous studies on liver fibrosis in human liver slices have convinced them and they are eager to expand the use of human tissue slices to other organs and diseased tissues. In our laboratory, human liver slices are used on a regular basis. Fifty precent of the research in PhD theses from our laboratory is now solely human tissue based. In the past only papers from our laboratory were published with animal studies. From the moment we started with human tissue slicing, this has led to a 50 % reduction in animal use! Therefore, we firmly believe that introducing this human tissue technique for fibrosis research to national and international researchers will lead to a similar reduction in the near future.