ÉLŐ SEJTEK ADHÉZIÓJÁNAK MONITOROZÁSA OPTIKAI BIOSZENZOROKKAL

Egy újszerű, nagy áteresztőképességű optikai bioszenzor, az Epic BenchTop (BT) segítségével azt vizsgáltuk, hogyan függ a rákos sejtek kiterülésének kinetikája a integrin-ligandum RGD-motívumok átlagos felületi sűrűségétől (νRGD) [1]. A biologiallag inaktív PLL-g-PEG kopolimer, és az RGD-funkcionalizált PLL-g-PEG-RGD vegyített oldatait használtuk a felületi bevonatok elkészítéséhez, νRGD-t négy nagységrenden keresztül hangoltuk. Modell sejtvonalként az erősen adherens HeLa rákos sejtvonalat használtuk, a kiterülés kinetikáját az Epic BT bioszenzorral egyedülálló minőségben rögzítettük. A kapott görbéket kinetikai elemzésnek vetettük alá: az adatokat a logisztikus egyenlettel illesztettük meg, hogy meghatározhassuk, hogyan függ a felületi ligandsúrúségtől a sejtkiterülés sebességi állandója (r), illetve a maximális bioszenzor jel (∆λmax). Eredményeink szerint r nem függ νRGD-től, átlagos értéke 0.062±0.004 min-1. Ezzel szemben ∆λmax, ami egyenesen arányos a kiterült sejt átlagos kontaktterületével, növekedett, ahogy νRGD-t növeltük. Ezt a viselkedést egy egyszerű monovalens kötési reakcióval leírva meghatároztuk a PLL-PEG-RGD molekula RGD-motívuma és a sejt integrinjei közötti kölcsönhatás két dimenziós disszociációs állandóját, mely 1753 μm-2-nek adódott. Ebből egyszerűen származtatható a kapcsolat 3D-s disszociációs állandója, melynek értéke ~30 μM. Mindezen eredményehez egyedülló módon teljesen noninvazív és jelölésmentes kísérleteken keresztül jutottunk

Automated single cell sorting and deposition in submicroliter drops

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4–0.7 ll with a sorting speed of 15–20 s per cell. After picking NE- 4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 9463% and 5467% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.564% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology

Automated single cell sorting and deposition in submicroliter drops

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology. © 2014 AIP Publishing LLC

Nanonewton scale adhesion force measurements on biotinylated microbeads with a robotic micropipette

Binding force between biomolecules has a crucial role in most biological processes. Receptor-ligand interactions transmit physical forces and signals simultaneously. Previously, we employed a robotic micropipette both in live cell and microbead adhesion studies to explore the adhesion force of biomolecules such as cell surface receptors including specific integrins on immune cells. Here we apply standard computational fluid dynamics simulations to reveal the detailed physical background of the flow generated by the micropipette when probing microbead adhesion on functionalized surfaces. Measuring the aspiration pressure needed to pick up the biotinylated 10 μm beads on avidin coated surfaces and converting it to a hydrodynamic lifting force on the basis of simulations, we found an unbinding force of 12 ± 2 nN, when targeting the beads manually; robotic targeting resulted in 9 ± 4 nN (mean ± SD). We measured and simulated the effect of the targeting offset, when the microbead was out of the axis (off-axis)of the micropipette. According to the simulations, the higher offset resulted in a higher lifting force acting on the bead. Considering this effect, we could readily correct the impact of the targeting offset to renormalize the experimental data. Horizontal force and torque also appeared in simulations in case of a targeting offset. Surprisingly, simulations show that the lifting force acting on the bead reaches a maximum at a flow rate of ~ 5 μl/s if the targeting offset is not very high (<5 μm). Further increasing the flow rate decreases the lifting force. We attribute this effect to the spherical geometry of the bead. We predict that higher flow rates cannot increase the hydrodynamic lifting force acting on the precisely targeted microbead, setting a fundamental force limit (16 nN in our setup) for manipulating microbeads with a micropipette perpendicular to the supporting surface. In order to extend the force range, we propose the offset targeting of microbeads

Label-Free Optical Monitoring Of The Adhesion And Spreading Of Human Cells: High Throughput Analysis With Superior Sensitivity And Time Resolution

Here, we briefly discuss the past, present, and possible future of label-free optical biosensors in cell adhesion research. Currently available optical biosensors possess outstanding potentials still not rightfully recognized and still waiting to be fully exploited in the field. Thus, during the description we give special emphasis to the advantages the state-of-the-art optical cell-based biosensors possess as compared to microscope- or force- measurement based techniques that are currently much more generally used to characterize cell adhesion. To name here only a few, they enable label-free detection close to a planar sensor surface, have high sensitivity, and generate superior quality kinetic data. Such information-rich kinetic data, in turn, can be subjected to in-depth comparative and kinetic analysis. To exemplify the importance of in-depth kinetic analysis, we review a recent study, in which the Epic BenchTop high-throughput optical biosensor was used to measure the dependence of adhesion kinetics on the surface density of integrin ligands. Based on the kinetically analyzed data, a model enabling the label-free determination of the dissociation constant for the interaction between adhesion ligands and their native cell membrane receptors has been constructed

Single-cell adhesivity distribution of glycocalyx digested cancer cells from high spatial resolution label-free biosensor measurements

The glycocalyx is a cell surface sugar layer of most cell types that greatly influences the interaction of cells with their environment. Its components are glycolipids, glycoproteins, and oligosaccharides. Interestingly, cancer cells have a thicker glycocalyx layer compared to healthy cells, but to date, there has been no con- sensus in the literature on the exact role of cell surface polysaccharides and their derivatives in cellular adhesion and signaling. In our previous work we discovered that specific glycocalyx components of cancer cells regulate the kinetics and strength of adhesion on RGD (arginine-glycine-aspartic acid) peptide- coated surfaces [1]. Depending on the employed enzyme concentration digesting specific components both adhesion strengthening and weakening could be observed by monitoring the averaged behavior of thousands of cells. The enzyme chondroitinase ABC (ChrABC) was used to digest the chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate components in the glycocalyx of cancer cells. In the present work, a high spatial resolution label-free optical biosensor was employed to monitor the adhesivity of cancer cells both at the single-cell and population level. Population-level distributions of single-cell adhesivity were first recorded and analyzed when ChrABC was added to the adhering cells. At relatively low and high ChrABC concentrations subpopulations with remarkably large and weak adhesivity were identified. The changes in the adhesivity distribution due to the enzyme treatment were analyzed and the subpopulations most affected by the enzyme treatment were highlighted. The presented results open up new directions in glycocalyx related cell adhesion research and in the development of more meaningful targeted cancer treatments affecting adhesion