Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells

Abstract

Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable for the generation of human 3D neural in vitro models, which can be used to feed high-throughput screening platforms, contributing to expand the available in vitro tools for drug screening and toxicological studies.

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells

“…Cells were cultured as spheroids in stirred-tank bioreactors, displaying high cell viability ( Fig 1A). This culture system presents several advantages considering our experimental design, as it allows for a fast and multiple sampling while providing controlled conditions (temperature, pH and pO 2 ) that maximize the biological reproducibility between replicates [12]. Cell spheroids of hiPSC and hNSC maintained their phenotype features for the 3 days of the experiment.…”

“…hiPSC 1, hiPSC 2, hNSC 1 and hNSC 2 were used for these experiments at cell passage number P40, P36, P12 and P34, respectively (four bioreactor runs in total). Bioreactor temperature was set to 37˚C, dissolved oxygen to 15%, pH to 7.4, aeration rate to 0.1 vvm and the agitation rate to a range from 70 to 100 rpm [12,42]. Perfusion was initiated after inoculation and interrupted just before the perturbation experiment.…”

Bringing Neural Cell Therapies to the Clinic: Past and Future Strategies

“…High impeller speeds increase oxygen supply and create homogeneity of the culture environment but at the cost of raising shear force that can damage cells. Software control of the bioreactor can monitor and dynamically adjust important parameters of the culture, potentially allowing higher cell densities with better viability and quality while saving resources 45 . While most work for NSCs has been in suspension, alternative adherent strategies have also been pursued.…”

The A818–6 system as an in-vitro model for studying the role of the transportome in pancreatic cancer

“…Hitherto, 3-dimensional (3D) culturing is not intensively investigated [24] partly because the assessment and scalability of the biological behaviour of the cells under 3D culturing condition is still problematic. However, it is generally accepted that the in vitro organotypic 3D cell culture system better resembles the physiological condition of the tissues in vivo, in regards of i) architectural organization and the processes of glandular lumen formation, ii) cell-to-cell interaction, and iii) the role of cancer genes in cell polarity, therefore, allowing the investigation of the different aspects of tumour biology and pathophysiology [25].…”