Experimental demonstration of bindingless signal delivery in human cells via microfluidics

Abstract

The cellular signal transduction is commonly believed to rely on the direct “contact” or “binding” of the participating molecule reaction that depends positively on the corresponding molecule concentrations. In living systems, however, it is somewhat difficult to precisely match the corresponding rapid “binding,” depending on the probability of molecular collision, existing in the cellular receptor-ligand interactions. Thus, a question arises that if there is another mechanism (i.e., bindingless) that could promote this signal communication. According to this hypothesis, we report a cellular model based on the examination of intracellular calcium concentration to explore whether the unidentified signal delivery in cells exists, via a microfluidic device. This device was designed to isolate the cells from directly contacting with the corresponding ligands/molecules by the particular polydimethylsiloxane (PDMS) membranes with different thicknesses. Results show a significant increment of calcium mobilization in human prostate cancer PC-3 cells by the stimulation of endothelin-1, even up to a separated distance of 95 μm. In addition, these stimulated signals exhibited a bump-shaped characteristics depending on the membrane thickness. When the PDMS membrane is capped by SiO2, a particular trait that resembles the ballistic signal conduction was observed. A theoretical model was developed to describe the signal transport process across the PDMS membrane. Taken together, these results indicate that the unidentified signal (ligand structural information) delivery could occur in cells and be examined by the proposed approach, exhibiting a bindingless communication manner. Moreover, this approach and our finding may offer new opportunities to establish a robust and cost-effective platform for the study of cellular biology and new drug development.

Observation of “wired” cell communication over 10-μm and 20-μm poly(dimethylsiloxane) barriers in tetracycline inducible expression systems

“…5 In this work, however, we did not observe the similar property by using the Tet inducible expression system, in which the estimated mean free path is less than 10 lm (Figure 4). The minimum and optimized membrane thickness we can fabricate was 10 lm that is uniform and compatible for cell culture in this system.…”

“…To explore whether a "wired" communication exists in the Tet and TetR interaction in cells afforded by bindingless signal delivery, 5 we followed the procedure illustrated in Figure 1(a) to examine this phenomenon (for details, refer Section II). The bindingless means that the interaction between Tet and TetR does not result from the molecular collision and binding process.…”

“…5 Briefly, two fabricated PDMS microchannels and a customized membrane with various thicknesses (10 lm, 20 lm, and 35 lm) were aligned and then permanently bounded together (Figure 1(b)). The size was 600-lm width  85-lm height for the top channel and 8000-lm width  40-lm height for the bottom channel.…”

“…In living systems, however, a perfect match between ligands/agonists and receptors may be questioned owing to the relatively low binding probability dominated by molecule collision. 5 Therefore, we hypothesize that another mechanism exists, cooperatively contributes to this interaction, and behaves as bindingless and "wired" signal communication.…”

“…5 To further examine our hypothesis described above, we utilize a microfluidic approach combined with a green fluorescent protein (GFP) inducible expression HEK 293T cell line by using a T-REx TM system. It is a Tet-regulated mammalian expression system that uses regulatory elements from E. coli Tn10-encoded Tet resistance operon, which contains two expression plasmids-pcDNA5/TO and pcDNA6/TR.…”