The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay

Abstract

Cytometric studies utilizing flow cytometry or multi-well culture plate fluorometry are often limited by a deficit in temporal resolution and a lack of single cell consideration. Unfortunately, many cellular processes, including signaling, motility, and molecular transport, occur transiently over relatively short periods of time and at different magnitudes between cells. Here we demonstrate the multitrap nanophysiometer (MTNP), a low-volume microfluidic platform housing an array of cell traps, as an effective tool that can be used to study individual unattached cells over time with precise control over the intercellular microenvironment. We show how the MTNP platform can be used for hematologic cancer cell characterization by measuring single T cell levels of CRAC channel modulation, non-translational motility, and ABC-transporter inhibition via a calcein-AM efflux assay. The transporter data indicate that Jurkat T cells exposed to indomethacin continue to accumulate fluorescent calcein for over 60 minutes after calcein-AM is removed from the extracellular space.

Nanoparticles

“…One good example of this is the encapsulation of Calcein Acetomethoxy (CAM) into nanoparticles to produce a cell viability sensor. CAM is a dye that requires cleavage of the acetomethoxy group by intracellular esterases to fluoresce 107 Similar work with Nano sensor platforms has been done in the detection of nitric oxide, a secondary messenger that has critical interactions with several neurological, immune and other physiological processes 106 Protein and peptide detection is another common use of nanoparticle sensors with quantum dot based systems showing promise. Mattoussi was able to leverage q-dots to create a system that could detect the proteolytic activity of a number of enzymes 112 .…”

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

“…Perfusion derived gradients are generally generated by an array of hierarchically ramified microchannels, upstream of the culture chamber [65,66]. A hydrodynamic junction can be used for diffusive mix, creating a progressive gradient over the length of the resulting channel [60,66]. Diffusion-based gradients are a result of a source-sink arrangement siding the chamber, whereby a directional movement by diffusion is observed from the compartment containing higher concentration to the one with lower concentration [61,67].…”

“…In such systems, species in solution are delivered through perfusive or diffusive fluidic regimes. Perfusion derived gradients are generally generated by an array of hierarchically ramified microchannels, upstream of the culture chamber [65,66]. A hydrodynamic junction can be used for diffusive mix, creating a progressive gradient over the length of the resulting channel [60,66].…”

Metronomic doses and drug schematic combination response tested within microfluidic models for the treatment of breast cancer cells (JIMT-1)

“…In fact, staining cells inside microfluidic cultivation devices is a powerful tool to detect intracellular changes for live cell imaging. It allows the determination of changes in cell states such as distinguishing live or dead cells, lysed or senescent cells, and other dynamic changes in cell state 25,26,27,28 . For that reason, being able to develop new approaches for cell analyzes will allow this heterogeneity to be evaluated.…”