PUB - Publikationen an der Universität Bielefeld

The production of biopharmaceuticals represents one of the most important aspects of today’s biotechnological functions. In industrial-scale manufacturing facilities mostly mammalian cell lines are applied in stirred-tank bioreactor-based bioprocesses to yield diverse products ranging from monoclonal antibodies to growth factors or pharmaceutical enzymes. Predominantly, Chinese hamster ovary (CHO) cells are utilized in these bioproduction processes, as they can easily be genetically modified and thereby are able to express biopharmaceuticals that are human-compatible, are well-established in industrial-scale cultivation, and achieve high product titers. Yet especially CHO cells’ genetical instability represents a serious challenge. Due to random mutations, cellular traits like growth behavior, morphology, and productivity can be affected. Besides genetical variations also environmental gradients during cultivation or stochastic biological processes like cell cycle distribution or biochemical stoichiometry can lead to cell-to-cell heterogeneity inside an isogenic population and thereby result in severe disadvantages for process robustness or even failure of bioproduction processes. Despite already available analytical methods for the investigation of single cells like e.g., flow cytometry, no convenient tool for the systematic analysis and cultivation of mammalian suspension cells under bioprocess relevant environmental conditions is existent. Thus, the aim of this thesis was to develop a suitable platform to perform long-term cultivation of CHO suspension cells on single-cell level and generate first insights into cell-to-cell heterogeneity of industrially relevant mammalian suspension cells to investigate its impact on bioproduction processes. Based on a thorough survey of state-of-the-art single-cell tools and their respective qualities, a polydimethylsiloxane (PDMS)-glass-based microfluidic single-cell cultivation (MSCC) device was designed. In consecutive steps device fabrication and preparation, biocompatibility tests of applied materials, preculture and cultivation medium handling, device inoculation techniques as well as the adjustment of the applied periphery concerning microscopy, pumping and consumables were performed and optimized for the cultivation of CHO suspension cells. Eventually, this microfluidic cultivation device allows the long-term cultivation of single cells under precise environmental control. With the application of live cell imaging, cellular behavior can be investigated with particularly high spatio-temporal resolution, as individual cells are trapped in specific cultivation chambers, and thus single-cell dynamics become accessible. Applying the microfluidic cultivation device, first proof-of-concept studies were performed to confirm its technical applicability. Here, for the first time CHO-K1 suspension cells were cultivated for 150 h in a MSCC device and growth characteristics were analyzed on single-cell level. The resulting growth rates between 0.85 and 1.16 d-1 resembled the cell line’s growth in customary cultivation approaches like shake flasks, already indicating the devices feasibility to properly resemble bigger cultivation scales. Additionally, first cellular heterogeneity in division time and morphology was detectable based on the high degree of spatio-temporal resolution of cellular behavior provided by the microfluidic cultivation device, which strongly advice continuative systematic studies of cell-to-cell heterogeneity.  In a next step, growth, morphology, and production behavior from cells cultivated in the MSCC device were systematically compared to larger cultivation approaches like shake flasks and laboratory-scale stirred-tank bioreactors. This systematic validation is mandatory to prove the transferability of gained knowledge between scales since MSCC ultimately should function as a miniaturization tool for these approaches. Analyzing growth characteristics showed matching specific growth rates across all cultivation scales. Furthermore, cellular diameters throughout the analyzed populations showed highly comparable distributions in MSCC, shake flasks and bioreactors. Determining eGFP expression as an exemplary readout for the cells’ productivity underlines the high comparability across the respective scales as well. However, MSCC provided valuable additional insights into single-cell fluorescence dynamics; time intervals of single-cell fluorescence increase and decrease could be observed, that were masked behind flow cytometric analysis of shake flasks and bioreactors. Since CHO suspension cells are randomly motile inside the microfluidic device, constant loss of cells during long-term cultivation makes single-cell studies challenging. Escaping cells also cause a discrepancy between specific growth rates determined on population level by counting the cell number in each chamber at a specific time, and growth rate determination on single-cell level by investigating single-cell doubling times. Therefore, concluding the technical development of the microfluidic cultivation device, the existing setup was optimized regarding its potential to retain trapped cells inside its cultivation chambers. The newly developed trapping concept, which is based on a physical barrier introduced into the entrance of each cultivation chamber, led to reliable cell retention and thus enables quantitative single-cell studies. Designing, validating, and optimizing the presented microfluidic cultivation device resulted in a fully applicable MSCC tool. In a next step the advanced platform was tested for first applications to address biological questions. In this context, MSCC was implemented to identify cellular heterogeneity in hyperosmotic stress response of CHO cells, to quantify the migratory behavior of human cardiac stem cells under different cultivation conditions, and to analyze cell cycle progression and cell cycle heterogeneity in CHO cells. Here, investigating these processes on single-cell level resulted in new insights. Yet the current state of the microfluidic cultivation device still holds some limitations e.g., the inevitable application of complex medium compounds to guarantee single-cell growth and nearly no automated image analysis. Solving these limitations would enable additional application fields of the microfluidic cultivation device introduced in this thesis. Some potential bioprocess related future applications are presented to propose how MSCC of mammalian suspension cells can be integrated into already established bioprocess research and development workflows. The results presented in this thesis clearly show that the developed microfluid cultivation device is suited for the miniaturization of CHO suspension cell cultivation. Besides reproducible long-term cultivation, its high potential for the investigation of cellular heterogeneity was not only illustrated by detecting varying division times, but also by investigating fluctuating fluorophore expression, heterogeneous stress response, and varying cell cycle progression. Furthermore, its applicability in investigating other cellular characteristics like single-cell migration has been shown evidently. With these findings, this work represents the fundament for future investigations of bioprocess relevant research questions in the field of cell-to-cell heterogeneity.