Mm Gepp

Dispensing of very low volumes of ultra high viscosity alginate gels: a new tool for encapsulation of adherent cells and rapid prototyping of scaffolds and implants

We present a tool for dispensing very low volumes (20 nL or more) of ultra high viscosity (UHV) medical-grade alginate hydrogels. It uses a modified piezo-driven micrometering valve, integrated into a versatile system that allows fast prototyping of encapsulation procedures and scaffold production. Valves show excellent dispensing properties for UHV alginate in concentrations of 0.4% and 0.7% and also for aqueous liquids. An optimized process flow provides excellent handling of biological samples under sterile conditions. This technique allows the encapsulation of adherent cells and structuring of substrates for biotechnology and regenerative medicine. A variety of cell lines showed at least 70% viability after encapsulation (including cell lines that are relevant in regenerative medicine like Hep G2), and time-lapse analysis revealed cells proliferating and showing limited motility under alginate spots. Cells show metabolic activity, gene product expression, and physiological function. Encapsulated cells have contact with the substrate and can exchange metabolites while being isolated from macromolecules in the environment. Contactless dispensing allows structuring of substrates with alginate, isolation and transfer of cell-alginate complexes, and the dispensing of biological active hydrogels like extracellular matrix-derived gels.

Nanoparticle-mediated gene transfer from electrophoretically coated metal surfaces.

The transfer of genetic information into living cells is a powerful tool to manipulate their protein expression by the regulation of protein synthesis. This can be used for the treatment of genetically caused diseases (gene therapy). However, the systemic application of genes is associated with a number of problems, such as a targeted gene delivery and potential side effects. Here we present a method for the spatial application of nanoparticle-based gene therapy. Titanium was electrophoretically coated with DNA-functionalized calcium phosphate nanoparticles. NIH3T3 cells and HeLa cells were transfected with pcDNA3-EGFP. We monitored the transfection in vitro by fluorescence microscopy, flow cytometry, and Western Blot analysis. By coating a transparent substrate, i.e., indium tin oxide (ITO), with nanoparticles, we followed the transfection by live cell imaging.

3D printing of hydrogels in a temperature controlled environment with high spatial resolution

Abstract There is great hope in 3D printing techniques to create patient specific scaffolds for therapeutic applications. The majority of these approaches rely on materials that both give support to cells and effectively mimic a tissue specific microenvironment. Hydrogels provide an exceptional support for cells but their physicochemical properties are not suited for conventional additive layer manufacturing. Their low viscosity and resulting fluidic nature inhibit voluminous 3D deposition and lead to crude printing accuracy. To enhance mechanical features, hydrogels are often chemically modified and/or mixed with additives; however it is not clear whether these changes induce effects on cellular behavior or if in vivo applications are at risk. Certainly it increases the complexity of scaffold systems. To circumvent these obstacles, we aimed for a 3D printing technique which is capable of creating scaffolds out of unmodified, pure hydrogels. Here we present a new method to produce alginate scaffolds in a viscosity- independent manner with high spatial resolution. This is achieved by printing in a sub-zero environment which leads to fast freezing of the hydrogels, thus preserving the printed shape and circumventing any viscosity dependent flows. This enables the user to create scaffolds which are able to reflect soft or stiff cell niches.

Labbag® - a versatile bag-based cultivation system for expansion, differentiation and cryopreservation of human stem cells

Abstract Novel approaches in regenerative medicine and tissue engineering are highly promoted by human induced pluripotent stem cells. Typical workflows include the expansion, differentiation and/or cryopreservation of these cells. Often, the differentiation, cytotoxicity tests or disease modelling rely on multicellular aggregates. Here, we present the novel bag-based system “Labbag®” to address workflows required for these applications. As a unique feature, the Labbag® provides a chemical spot pattern to allow the formation of several hundreds of hanging droplets within a few seconds with standard laboratory equipment. In this study, we describe the general concept of the Labbag® and show aggregate formation of stem cells as first stem cell workflow successfully transferred.