A highly versatile biopolymer-based platform for the maturation of human pluripotent stem cell-derived cardiomyocytes enables functional analysis in vitro and 3D printing of heart patches.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent a valuable tool for in vitro modeling of the cardiac niche and possess great potential in tissue engineering applications. However, conventional polystyrene-based cell culture substrates have adverse effects on cardiomyocytes in vitro due to the stress applied by a stiff substrate on contractile cells. Ultra-high viscosity alginates offer a unique versatility as tunable substrates for cardiac cell cultures due to their biocompatibility, flexible biofunctionalization, and stability. In this work, we analyzed the effect of alginate substrates on hPSC-CM maturity and functionality. Alginate substrates in high-throughput compatible culture formats fostered a more mature gene expression and enabled the simultaneous assessment of chronotropic and inotropic effects upon beta-adrenergic stimulation. Furthermore, we produced 3D-printed alginate scaffolds with differing mechanical properties and plated hPSC-CMs on the surface of these to create Heart Patches for tissue engineering applications. These exhibited synchronous macro-contractions in concert with more mature gene expression patterns and extensive intracellular alignment of sarcomeric structures. In conclusion, the combination of biofunctionalized alginates and human cardiomyocytes represents a valuable tool for both in vitro modeling and regenerative medicine, due to its beneficial effects on cardiomyocyte physiology, the possibility to analyze cardiac contractility, and its applicability as Heart Patches.

The development of alginate-based amperometric nanoreactors for biochemical profiling of living yeast cells.

This study describes the development of a one-pot electrochemical miniaturized system for simultaneous cultivation and monitoring of the oxidative status of living cells. This system consisted of screen-printed electrodes modified by electroplated Pd-NPs as an electrocatalyst (i) and living yeast cells (Saccharomyces cerevisiae) (ii) immobilized on the cytocompatible alginate layer (iii). Briefly, during the course of electrochemical investigations a novel electroactive compound methylhydrazine derivative as a secondary metabolite and result of microbial activity was found in yeast cells and used as a signaling molecule for their biochemical profiling. Under the optimized experimental conditions the signal corresponding to the found electroactive secondary metabolite formed in medium of living cells was measured without sample collecting, transport, storage or pre-treatment steps (i.e. extraction, pre-concentration, chemical derivatization or labeling). The electrochemical dependencies, which were derived by a miniaturized electroanalytical system, were fully validated in a conventional three-electrode system under inert atmosphere (Ar) and in the presence of oxygen (air, O2). It is believed that the proposed one-pot nanoreactors serving simultaneously as nanofermenters and amperometric detectors for the quantification of secondary metabolites formed in medium of living cells can significantly enhance the understanding of ongoing fermentation processes in the future and our knowledge on the biochemistry of yeasts.

Effective surface-based cryopreservation of human embryonic stem cells by vitrification.

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing. Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.

A comparative study of freezing single cells and spheroids: towards a new model system for optimizing freezing protocols for cryobanking of human tumours.

Cryopreservation of human tumour cells and tissue is a valuable tool for retrospective analysis and for the transport and handling of biopsy material. Tumour tissue consists of different cell types, which have different optimal freezing conditions, and extracellular matrix. A well-defined and authentic model system is required for developing new freezing protocols and media. This work describes the use of L929 and PC-3 spheroids as new model systems for freezing human tumours. Cell suspension and spheroids were frozen in different vessels (1 ml cryovials and a special, cryo-compatible 30 x 25 microl multi well plate) at slow rate (1 degrees C/min). Freezing media were combinations of culture or tumour transport medium (Liforlab) with the cryoprotective agents, Me(2)SO, trehalose and modified starch. We also present a new method of evaluating the viability of three dimensional multicellular systems to compare thawed spheroids objectively. Best viability (70%) of L929 spheroids occurred with a combination of Liforlab and starch hydrolysis product. The best cryopreservation results for spheroids were found with extracellular cryoprotectants, while optimum viability of single cells was achieved with Me(2)SO.