Study of cellular processes by controlling single-cell microenvironment
Various cellular processes, such as endocytosis, calcium signaling, and morphology changes are affected by the microenvironment surrounding the cells, including the interactions between cells.
Despite advances in single-cell methods, which have provided excellent means to interrogate living cells, the technology for the control of the chemical environment around intact cells in tissue or cell cultures is scarce.
Fluicell has developed an easy-to-use microfluidic system called BioPen which enables a localized delivery of molecules in single cells experiments. Requiring a very small volume of solution, BioPen is particularly suitable for the delivery of expensive compounds such as antibodies or drugs.
Using an in-house microfluidic technology, the BioPen has been designed for targeting single-cells without contaminating the surrounding environment, enabling multiple protocols within the same culture dish. Combining a high precision pressure controller and a sophisticated software, BioPen allows a spatio-temporal control of the chemical environment of individual cells and offers the possibility to collect unique cell data responses.
CASE STUDY
Inducing the retraction of protrusion in single-cell
In 2018, Prof. Rizzo’s team published a significant article entitled “A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms” in Cell Reports journal. The published findings describe a new approach to quantify cellular processes in living cells more particularly to track enzyme activation through quantitative fluorescence microscopy.
In this study, they investigate Regulatory Light Chain (RLC) phosphorylation during retraction of cell protrusion using a Förster Resonance Energy Transfer (FRET) approach. To induce the retraction, they stimulate the cell protrusion with local delivery of a Platelet-Derived Growth Factor (PDGF) using the BioPen PRIME system.
PDGF stimulation resulted in the retraction of a cell protrusion initiating ∼5 min into the treatment (Figures 1A and 1B). The morphology of the cell outside of the treatment area did not undergo substantial alterations. Before retraction, the tip of the protrusion contained stable high-anisotropy phosphorylated regions (Figure 1B). Retraction proceeded through a multi-phasic process similar to those during random migration. Initially, cytoskeletal movements were observed 10–20 μm deep into the protrusion (Figure 1C, white arrows).
Figure 1: RLC Phosphorylation during Retraction of a Cell Protrusion
(A–C) Localized changes in RLC phosphorylation were stimulated using the BioPen to deliver 10 ng/mL PDGF to one portion of a REF52 cell expressing mCer3-RLC.
(A) Rhodamine (in red) was used to mark the area of stimulation for several minutes before addition of PDGF. The P image of mCer3-RLC presented (cyan).
(B) Fluorescence anisotropies are represented by the indicated color scale. Pixel intensities from the yellow bar (top right) were used to create a kymograph. Scale bar, 10 μm.
(C) Application of rhodamine (red) and PDGF (cyan) are indicated by the bars. The white arrows mark a PDGF-dependent shift in phosphorylated RLC that precedes an increase in the rate of retraction (pink arrow). Rapid myosin dephosphorylation during this phase is indicated by the blue arrow.
In summary, this case study proves that the BioPen system has been perfectly designed to stimulate only some part of the cell with the compound of interest without affecting the rest of the sample.
“The main uses of the Biopen is for stimulating migrating cells. The benefits when using the system is that it can stimulate a portion of a cell. The system provide us with unique data with it you are able to see how cell process are trapped” says Prof. Rizzo.”
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