Publication
Most tissue engineering approaches include the delivery of reparative cells to a damaged host tissue. Generally these cells are sought to be of a stem cell character since they retain a high potential for proliferation and differentiation into diverse phenotypes. To restrict cell function to the desired phenotype and to avoid anti-immune responses, autologous stem cells are frequently isolated from intact regions of the same (damaged) organ, such as keratinocytes from the basal layer of the skin and hair follicles or from the same germ layer origin such as mesenchymal stem cells (MSC) that are obtained from the bone marrow. This approach faces two major problems: 1) The number of source cells obtained from biopsies is generally too low for direct implantation, which requires cell multiplication in culture. 2) The standard culture conditions, including growth in plastic dishes, passaging, and the composition of the growth medium, never correspond to the natural microenvironment and influence cell differentiation, often resulting in non-desired phenotypes. The objective of my thesis was to provide a solution for both problems that is applicable for the average cell culture laboratory. Our technique reduces traumatic enzymatic passaging, provides constant cell densities to keep cells in the exponential growth phase and at the same time avoids contact inhibition of cell growth. This novel approach comprises the development of a new elastic culture surface, produced from a high extension silicone rubber (HESR) that we here for the first time applied for cell culture. The HESR is enlarged by a novel mechanical approach permitting to visualize and control cells under expansion. Untreated silicone, the polymer component of the HESR, does not promote cell attachment and must therefore be functionalized. In the first part of my thesis (Chapter 2) we establish a surface treatment for silicone membranes that will resist large surface expansions. We compare established coating techniques that have originally been developed to provide static silicone surfaces with cell adhesive molecules by 1) hydrophobic, 2) electrostatic and 3) covalent interactions. Cell attachment, spreading and proliferation was evaluated between these three treatments as a function of the coating in static and stretched conditions. We show that covalent immobilization of collagen type I is most suitable to stretch cells cultured on elastic silicone substrates. We applied this technique to functionalize the HESR culture surface in all later applications. The second central part of my thesis (Chapter 3) develops a new culture method consisting of a motorized device that isotropically expands the coated HESR surface to yet unmatched 1000% of its initial surface. Proof-of-principle of this method is given by culturing human MSC, which are sensitive to passaging during expansion in vitro and that are therapeutically relevant cells to repair bone, cartilage, and cardiovascular tissue. MSC were