Infoscience

Thesis

A novel cell culture approach for tissue engineering based on surface expansion

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 cultured three consecutive passages on the culture surface that has been expanded in steps of 5% every 6 h for 20 days, respectively. After nine weeks of expansion culture, MSC exhibited consistently high proliferation rates, fully retained their pluripotent stem cell character and were inducible to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. The cell surface expansion culture surface produced ∼10-times higher cell numbers compared with nine weeks of standard culture, which required nine events of passaging to attain the same culture surface. We propose our method to produce MSC for clinical cell therapy. In the third part of my thesis (Chapter 4) we implement our novel surface expansion method to culture fibroblasts with the aim to: 1) rapidly expand primary fibroblasts, 2) achieve higher cell yields than obtained with conventional culture, and 3) obtain homogeneous cell populations with non-fibrogenic character. The expansion of primary autologous dermal fibroblasts in culture is a pivotal step to obtain sufficiently high cell numbers for application in skin esthetics and for the treatment of large burn wounds. Dynamically enlarging the culture surface by 800% over 20 d, yielded ∼1.5 higher fibroblasts numbers than the equivalent three standard passages. By further optimizing the surface expansion speed according to the cell growth rates, the same high cell numbers were produced after only 9 d of culture. The highly compliant nature of the HESR surface had the beneficial effect of completely abolishing the formation of 'fibrotic' myofibroblasts. Hence, our novel method for fibroblast culture is faster and yields higher numbers of more homogeneous cells than standard culture passaging. It delivers a fibroblast quality and quantity that is appealing for cell therapeutic purposes. The aim of the last part of my thesis (Chapter 5) was to provide highly compliant silicone elastomers, such as the HESR, with a micropattern of cell adhesive proteins. In particular we aim to produce arrays of extracellular matrix proteins with a size and morphology to promote formation of single focal adhesions on highly elastic and expandable surfaces. This will allow future studies on cell mechanosensing through focal adhesions by discriminating between effects of focal adhesion size changes and stretch. An existing method to pattern rigid surfaces with proteins, so-called microcontact printing, is not suitable to transfer proteins onto surfaces that are softer than the stamp. We here establish a new transfer technique that is based on a stencil with micron-sized openings. The new stencil is used to expose the surface of compliant silicone substrates with Young's modulus of 10-100 kPa to a protein solution of fibronectin, collagen I and protein-repellent polymers, leaving behind the desired protein pattern. Fibroblasts were successfully attached onto the resulting focal islet arrays on compliant substrates.

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