Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy

Abstract

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena.

Functional blood cell analysis by label-free biosensors and single-cell technologies

“…Studying population level heterogeneity at the single-cell level using these novel biophysical technologies, and identifying important subpopulations will potentially open up new research directions with important aspects in medical diagnostics, and further treatment. Singlecell optical biosensors [33,390] and high-throughput robotic FluidFM [333] were already successfully demonstrated in monitoring population distributions of single-cell biological parameters in a label-free and noninvasive manner. Importantly, concerning adhesion strength, a lognormal distribution profile was revealed [33,333,390].…”

“…Singlecell optical biosensors [33,390] and high-throughput robotic FluidFM [333] were already successfully demonstrated in monitoring population distributions of single-cell biological parameters in a label-free and noninvasive manner. Importantly, concerning adhesion strength, a lognormal distribution profile was revealed [33,333,390]. Therefore, treating the cell population as normally distributed or measuring only a few cells can easily result in misleading conclusions.…”

Single-cell temporal transcriptomics from tiny cytoplasmic biopsies

“…While the physical world is built up from elementary particles with well-defined properties, the building blocks of life are largely heterogeneous. Even the single-cell populations of cell lines are heterogeneous with a relatively wide distribution of properties depending on the actual cell cycle state of individual cells ( Nagy et al., 2022 ). Investigating and understanding cellular heterogeneity and identifying and characterizing possible sub-populations at the tissue and cellular level are becoming increasingly important nowadays.…”

“…At the end of the cantilever either a 2–8 μm large opening (microprobe) or a sharp hollow tip (nanosyringe) can be found. The microprobes are mostly used for cell adhesion measurements ( Nagy et al., 2022 ; Sztilkovics et al., 2020 ), and the nanosyringes find applications in single-cell injections ( Guillaume-Gentil et al., 2014 ) and extractions ( Guillaume-Gentil et al., 2016 ). In the later applications, the sharp hollow needle is thrust into the targeted cell and by employing over- or under-pressure liquid material can be injected into or extracted from the cytoplasm (or even the nucleus).…”

“…The most advanced robotic FluidFM setup can even handle several petri dishes at the same time with the possibility of automatic probe washing and exchange. While mainly demonstrated in cell-adhesion studies ( Nagy et al., 2022 ; Sztilkovics et al., 2020 ) and surface printing of functionalized polymers ( Saftics et al., 2019 ), these automatized and robust platforms are expected to upgrade Live-seq and other related single-cell injection/extraction studies to a next level and open up novel directions in the biological applications of FluidFM.…”