Scale-down of CHO cell cultivation from shake flasks based on oxygen mass transfer allows application of parallelized, non-invasive, and time-resolved monitoring of the oxygen transfer rate in 48-well microtiter plates.

Cultivating Chinese hamster ovary (CHO) cells in microtiter plates (MTPs) with time-resolved monitoring of the oxygen transfer rate (OTR) is highly desirable to provide process insights at increased throughput. However, monitoring of the OTR in MTPs has not been demonstrated for CHO cells, yet. Hence, a CHO cultivation process was transferred from shake flasks to MTPs to enable monitoring of the OTR in each individual well of a 48-well MTP. For this, the cultivation of an industrially relevant, antibody-producing cell line was transferred from shake flask to MTP based on the volumetric oxygen mass transfer coefficient (kL a). Culture behavior was well comparable (deviation of the final IgG titer less than 10%). Monitoring of the OTR in 48-well MTPs was then used to derive the cytotoxicity of dimethyl sulfoxide (DMSO) based on a dose-response curve in a single experiment using a second CHO cell line. Logistic fitting of the dose-response curve determined after 100 h was used to determine the DMSO concentration that resulted in a cytotoxicity of 50% (IC50). A DMSO concentration of 2.70% ± 0.25% was determined, which agrees with the IC50 previously determined in shake flasks (2.39% ± 0.1%). Non-invasive, parallelized, and time-resolved monitoring of the OTR of CHO cells in MTPs was demonstrated and offers excellent potential to speed up process development and assess cytotoxicity.

Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation.

The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production.

The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing.

Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures.

Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications.

Joint analysis of extracellular spike waveforms and neuronal network bursts.

BACKGROUND: Neuronal networks are routinely assessed based on extracellular electrophysiological microelectrode array (MEA) measurements by spike sorting, and spike and burst statistics. We propose to jointly analyze sorted spikes and detected bursts, and hypothesize that the obtained spike type compositions of the bursts can provide new information on the functional networks. NEW METHOD: Spikes are detected and sorted to obtain spike types and bursts are detected. In the proposed joint analysis, each burst spike is associated with a spike type, and the spike type compositions of the bursts are assessed. RESULTS: The proposed method was tested with simulations and MEA measurements of in vitro human stem cell derived neuronal networks under different pharmacological treatments. The results show that the treatments altered the spike type compositions of the bursts. For example, 6-cyano-7-nitroquinoxaline-2,3-dione almost completely abolished two types of spikes which had composed the bursts in the baseline, while bursts of spikes of two other types appeared more frequently. This phenomenon was not observable by spike sorting or burst analysis alone, but was revealed by the proposed joint analysis. COMPARISON WITH EXISTING METHODS: The existing methods do not provide the information obtainable with the proposed method: for the first time, the spike type compositions of bursts are analyzed. CONCLUSIONS: We showed that the proposed method provides useful and novel information, including the possible changes in the spike type compositions of the bursts due to external factors. Our method can be employed on any data exhibiting sortable action potential waveforms and detectable bursts.