Abstract
Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.
Highlights
The global trend towards improved in vitro three-dimensional tissue models in biomedical research has led to an increased application of advanced stem cell technologies using iPSC- and embryonic stem cells
The Vitamin A derivatives retinoic acid (RA) and EC23 are commonly used for triggering stem cell differentiation to a neuronal phenotype [39], elevated levels of both molecules are known to induce neurotoxic effects [40,41]
Untreated embryoid body sizes increased by 30% over four days resulting in maximum diameter of 734.1 ± 33.6 μm
Summary
The global trend towards improved in vitro three-dimensional tissue models in biomedical research has led to an increased application of advanced stem cell technologies using iPSC- and embryonic stem cells Due to their inherent pluripotency and self-renewal capacity, embryonic stem cells (ESCs)–the inner cell mass of a blastocyst–provide an indispensable and powerful tool in basic and applied research. By cultivating embryonic stem cells in three-dimensional (3D) cellular aggregates, early stages of embryonic development can be recapitulated in vitro in the form of so-called embryoid bodies (EBs). These in vitro derived structures give rise to all three primary germ layers, including the ectoderm, the mesoderm, and the endoderm [1–3]. The study of early embryonic events is of particular importance to gain deeper insights into processes that influence cell fate decisions ranging from cell-to-cell interactions, cell communication such as neuralizing signals, biochemical cues, and systemic circulatory factors such as growth factors, etc. [5–8]
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