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| DZF-Project: Michael Sittinger |
In vitro synthesis of vital human tissues using resorbable biocompatible polymer fleeces and perfusion culture systems to replace large animal experiments
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Michael Sittinger, Gerd-R Burmester, Jesus Bujia, Will W Minuth, Claus Hammer
Department of Rheumatology, Charité, Berlin, Tucholskystrasse 2, 10117 Berlin, Germany
Michael.Sittinger(at)charite.de
Keywords: Arthritis model, artificial tissues, cell culture
Begin and End of the Project: 1994 - 1998
Background and Aim
Background: The isolation of mammalian cells and their culture in monolayer system is a basic procedure for investigations in cell biology, intercellular molecular signals and the etiology of many diseases. Furthermore, the effects of drugs and all kinds of substances on the human body are mainly evaluated by their influence on cultured cells. However, isolated cells can only simulate a small part of the body’s tremendous complexity. The major problem of human cells in culture is the phenomenon of dedifferentiation. The cells are isolated from their tissue specific extracellular matrix and after suspension in growth medium they adhere at the bottom of polystyrene dishes. Cells can grow and proliferate in these culture dishes to a confluent cell layer. However, the cells frequently lose their morphology as well as their biochemical and functional properties. Such dedifferentiated cells behave completely different compared to the cells in their original tissue environment. A good example of this phenomenon are cartilage cells. After a few days in monolayer culture they begin to change their appearance to a fibroblast like morphology. The typical formation of chondrons and pericellular matrix is not seen in culture. Biochemical investigations reveal a switch of collagen synthesis. Thus, instead of the cartilage typical collagen type II, cultured chondrocytes mainly synthesize collagen type I which is absent from normal cartilage. The reason for the process of dedifferentiation may be caused by cell adherence to an unsuitable substrate, unstable nutrient supply, the two-dimensional cell growth and most importantly the lack of formation of an appropriate extracellular matrix. These limitations of cell culture still demand a great number of animal experiments. In the case of osteoarthritis most experiments are done in dogs. For rheumatoid arthritis, normally primates are used for model systems.
The deficiency of the tissue specific characters of conventional cultured cells demand the development of new improved culture systems considering mainly a three-dimensional growth also leading to an embedding of the cells in their own secreted and accumulated extracellular matrix.
Aim: The aim of the research project is the engineering of human tissues in vitro. In-vitro model systems for the investigation of autoimmune diseases against cartilage tissues will be applied. The engineered tissues should provide an advanced tool to analyze the effects of drugs on the pathogenetic situation in cartilage destruction. Animal experiments using primates and dogs for the investigation and treatment of human diseases such as osteoarthritis or rheumatoid arthritis will be replaced by complex in vitro tissue systems. Therefore, our previous research activity was primarily focused on the in vitro formation of human cartilage tissue. The properties of the engineered tissue will be approximated to the cartilage found in vivo. In a first step, cells from biopsies will be amplified using efficient monolayer techniques. In a second step, cells will be arranged in three-dimensional cultures to form artificial cartilage and synovial tissues.
Methods and Results
Three-dimensional cultures of chondrocytes and synovial fibroblasts were established using fibrinogen and thrombin for cell immobilization. Cartilage samples were dissected, washed and subsequently digested using collagenase and hyaluronidase. The resulting cell suspension was sieved through nylon filters and cell vitality was verified by trypan-blue staining. Chondrocytes were then suspended in culture medium and mixed with fibrinogen. The mixture was then transferred into 96-well plates and polymerized by adding dilutions of thrombin. These 3-dimensional chondrocyte cultures were incubated for 3 weeks to allow synthesis of extracellular matrix. Subsequently, artificial cartilage tissues were transferred from 96-well to 48-well plates. For artificial pannus formation, synovial cells from primary cultures (containing macrophages/monocytes and fibroblast) and subcultures of synovial fibroblasts were diluted in culture medium and fibrinogen and then given into the 48-well plates to enclose the centrally positioned cylinders of artificial cartilage tissues. The surrounding 3D synovial culture was then also polymerized with thrombin to form interacting artificial cartilage and synovial tissues for further analyses. Additionally, larger cultures were prepared 6-well plates and the cultures mechanically stabilized using polymer fleeces for handling. Extended culture times up to 85 days were performed in perfusion culture for efficient and convenient supply of nutrients.
The results generally have shown a homogenous distribution of cells in the artificial tissues. Staining with alcian blue, safranin O and azan confirmed the formation of extracellular matrix in the 3D chondrocyte cultures formed in well plates. Compared to native cartilage, the in vitro formed matrix appeared to be of significantly lower density. In microscopic evaluation of the interacting cultures, the vertical border zone of the two different tissues was clearly visible. After 4-6 days synovial cells derived from inflammatory tissues began to invade the artificial cartilage cylinders. This infiltrative behaviour continued for up to 3 week. Synovial cells from non-inflammatory synovial tissues did not show invasive migration towards the cartilage cylinders. Therefore, the interacting artificial tissues have shown similar behaviour to the destructive situation in vivo of patients with rheumatoid arthritis.
Conclusions and Relevance for 3R
Novel concepts for the artificial synthesis of vital human tissues and organs provide new possibilities for the synthesis of biochemical factors with great importance for the treatment of many diseases. The development of adequate in vitro models and assay systems is of great interest for the replacement of experiments with mammals. Thus far, in diseases like osteoarthritis or rheumatoid arthritis animals like primates (collagen type II arthritis) and dogs (osteoarthritis) are interesting models frequently used for investigation. In these and many other diseases the tissue forming extracellular matrix plays a major role in the etiology of the diseases. Therefore, cell cultures in monolayer lacking the tissue specific extracellular matrix offer only very limited possibilities to simulate the situation found in vivo. In contrast, in vitro synthesized tissues combined with co-cultures and an automated stabilized supply of nutrients or other biologically important substances provide far more complex model systems to investigate developmental pathways and the etiology of diseases. In artificial tissue constructs, histological alterations and developments can be included as measurable parameters. Interactions of different cell and tissue types in the presence of important test substances or under the influence of physical factors may be investigated or routinely analyzed.
Artificial tissues can simulate pathologic features in knees of arthritis patients. Interacting engineered cartilage and synovial tissues have shown that such systems could be applicable for valuable assays for basic research and drug testing and may ultimately reduce and partly replace experiments in dogs and primates. |
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