No abstract

Repeated passaging in conventional cell culture reduces pluripotency and proliferation capacity of human mesenchymal stem cells (MSC). We introduce an innovative cell culture method whereby the culture surface is dynamically enlarged during cell proliferation. This approach maintains constantly high cell density while preventing contact inhibition of growth. A highly elastic culture surface was enlarged in steps of 5% over the course of a 20-day culture period to 800% of the initial surface area. Nine weeks of dynamic expansion culture produced 10-fold more MSC compared with conventional culture, with one-third the number of trypsin passages. After 9 weeks, MSC continued to proliferate under dynamic expansion but ceased to grow in conventional culture. Dynamic expansion culture fully retained the multipotent character of MSC, which could be induced to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. Development of an undesired fibrogenic myofibroblast phenotype was suppressed. Hence, our novel method can rapidly provide the high number of autologous, multipotent, and nonfibrogenic MSC needed for successful regenerative medicine.

a b s t r a c tOver the past decade, various implantable devices have been developed to treat diseases that were previously difficult to manage such diabetes, chronic pain, and neurodegenerative disorders. However, translation of these novel technologies into clinical practice is often difficult because fibrotic encapsulation and/or rejection impairs device function after body implantation. Ideally, cells of the host tissue should perceive the surface of the implant being similar to the normal extracellular matrix. Here, we developed an innovative approach to provide implant surfaces with adhesive protein micropatterns. The patterns were designed to promote adhesion of fibroblasts and macrophages by simultaneously suppressing fibrogenic activation of both cell types. In a rat model, subcutaneously implanted silicone pads provided with the novel micropatterns caused 6-fold lower formation of inflammatory giant cells compared with clinical grade, uncoated, or collagen-coated silicone implants. We further show that micropatterning of implants resulted in 2e3-fold reduced numbers of pro-fibrotic myofibroblast by inhibiting their mechanical activation. Our novel approach allows controlled cell attachment to implant surfaces, representing a critical advance for enhanced biointegration of implantable medical devices.

To be applied in sufficient numbers for regenerative medicine, primary mesenchymal stem cells (MSCs) need to be amplified in culture. Standard cell culture involves regular passing because MSC proliferation in size-limited culture vessels stagnates due to contact inhibition of growth. The use of harmful enzymes for passaging and the mechanical properties of standard culture vessels change the MSC phenotype. Initially, fast growing multipotent and regenerative MSCs will turn into slowly growing cells with reduced multipotency and fibrotic character. We here describe an innovative culture system that maintains overall constant cell densities which are near-optimal for proliferation, while preventing contact-inhibition of cell growth. This is achieved by dynamically enlarging a novel highly elastic culture dish using a motorized mechanical device and adapting the culture surface to the increasing cell numbers. Dynamic MSC culture expansion reduces the number of enzymatic passages by a factor of 3 and delivers higher MSC yields than conventional culture. On the expanded culture surface, MSCs maintain stem cell characteristics and high growth rates over months and are still inducible to follow different lineages thereafter.