CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones

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Recent advances in nanobiotechnology and high-throughput molecular techniques for systems biomedicine

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnologybased materials and living cells in both in vitro and in vivo settings.     

Cover Image,Volume 116, Number 5, May 2019

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm ². We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm ², which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm ², whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm ² was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.     

Devices and techniques used to obtain and analyze three-dimensional cell cultures

Cell cultures are indispensable for both basic and applied research. Advancements in cell culture and analysis increase their utility for basic research and translational applications. A marked development in this direction is advent of three-dimensional (3D) cultures. The extent of advancement in 3D cell culture methods over the past decade has warranted referring to a single cell type being cultured as an aggregate or spheroid using simple scaffolds as “traditional.” In recent years, the development of “next-generation” devices has enabled cultured cells to mimic their natural environments much better than the traditional 3D culture systems. Automated platforms like chip-based devices, magnetic- and acoustics-based assembly devices, di-electrophoresis (DEP), micro pocket cultures (MPoC), and 3D bio-printing provide a dynamic environment compared to the rather static conditions of the traditional simple scaffold-based 3D cultures. Chip-based technologies, which are centered on principles of microfluidics, are revolutionizing the ways in which cell culture and analysis can be compacted into table-top instruments. A parallel evolution in analytical devices enabled efficient assessment of various complex physiological and pathological endpoints. This is augmented by concurrent development of software enabling rapid large-scale automated data acquisition and analysis like image cytometry, elastography, optical coherence tomography, surface-enhanced Raman scattering (SERS), and biosensors. The techniques and devices utilized for the purpose of 3D cell culture and subsequent analysis depend primarily on the requirement of the study. We present here an in-depth account of the devices for obtaining and analyzing 3D cell cultures.