Towards clinical-grade hESC culture and cryopreservation for tissue engineering purposes

Tissue engineering is a rapidly growing area which is envisioned to revolutionize current medical principles and practice. Tissue engineered constructs aim at restoring, maintaining, improving or replacing certain tissue functions that are compromised or lost either by congenitally linked abnormalities, injury, disease or aging. Several stem cell types are being considered for tissue engineering approaches, such as embryonic stem cells (ESC), mesenchymal stem cells and adult stem cells. In this thesis, the applicability of hESC for future tissue engineering purposes was further investigated. hESC are unspecialized, pluripotent stem cells that are capable of long-term self-renewal in vitro. The pluripotent nature of these cells combined with their unlimited proliferation capacity, turns hESC into one of the most interesting cell types not only for tissue engineering applications, but also for cell therapy, developmental research and drug discovery. Regardless of the countless possibilities for hESC in tissue engineering, there are some factors that currently limit the amount of experiments that can be performed and thus slow down the progression to clinical applications. Some of these limitations are inherently linked to the standard passaging technique, the animal-derived supportive feeder cells and animal-containing undefined culture media. Next to these limitations, there is also need for optimization of the cryopreservation process of hESC to ensure safe, largescale hESC banking opportunities. Initially, attempts were made to optimize the hESC passaging method. As hESC are routinely cultured on mouse embryonic fibroblast (MEF) feeder layers and split by manual mechanical passaging, progression to large-scale culture is limited due to the labor-intensive and time-consuming passaging method. Several single cell solutions have been suggested as alternatives for the manual mechanical passaging in literature, though there was no consensus on which solution or strategy would have the best outcome for optimization of the passaging method. Therefore, we explored the effect of 4 different dissociative solutions (TrypLE Express, Trypsin-EDTA, Cell Dissociation solution and Accutase) applied in two different approaches, on the quality and expansion of several hESC cell lines. Our comparative study had remarkable results: Cell Dissociation Solution, a mild non-enzymatic and animal-free solution, which was previously untested with hESC, had the best results regarding cell quality and expansion capacity. This new protocol allowed us to generate high amounts of hESC in a shorter time frame with sustained pluripotent capacity and in a much less labor-intensive and timeconsuming fashion. Secondly, we focused on optimization of the feeder layer and the elimination of all animal containing products from the culture. The MEFs can be substituted by human feeder cells, such as human foreskin fibroblasts (HFF). These HFFs should also be cultured in an animal substance-free medium, thus without fetal bovine serum (FBS), in order to limit the contamination risk of the hESC. The feasibility of plant hydrolysates as serum substitute in the HFF culture medium was evaluated as an initial step to total animal substance-free hESC culture. The application of Hypep Wheat hydrolysate combined with a small amount of ITS-X (Insulinetransferrine- selenium) solution and L-Glutamine led to an animal substancefree HFF culture, which sustained hESC culture. Lastly, we focused on the hESC cryopreservation method. hESC are routinely cryopreserved by vitrification in open pulled straws. This method does not allow safe large-scale hESC storage, due to the inherent requirements of the vitrification procedure and the LN2 contamination hazards. Slow freezingrapid thawing is an alternative technique for hESC cryopreservation which requires optimization to ensure high recovery ratios of the cells. A large comparative study with different combinations of DMSO (dimethylsulfoxide), EG (ethylene glycol) and HES (hydroxyethylstarch) was performed. HES is an extracellular cryoprotector which previously hadn’t been examined with hESC. By application of just 5% DMSO and 5% HES, we could obtain a cryopreservation protocol with high recovery rates and sustained quality, pluripotency and expansive behavior of the hESC. In a second cryopreservation article, we attempted to further optimize our protocol by making it completely animal substance-free. By application of a synthetic hydrolysate compound, the animal derived components in the cryopreservation holding medium were eliminated, resulting in a defined and animal substance-free hESC cryopreservation protocol which would be ideal for hESC tissue banking. These optimizations are a step forward to possible clinical progression of hESC in tissue engineering applications