The Research of Individual Cells
One of our main goals at the Jerome Schottenstein Center, since the early nineties, has been the development of technologies and methods suitable for the measurement and analysis of single cells within a population. The significance of single cell analysis lies in the understanding that analysis of a population of cells at single cell resolution enables defining the heterogeneity within a population and identifying phenomena, subpopulations and rare cells overlooked by bulk sample measurements. The single cell analysis approach is manifested at various levels of the cell component, including DNA, mRNA, proteins, metabolites, and cell phenotype, and is implemented using microfluidic devices, microwell arrays, microdroplets, single cell mass spectrometry and other techniques. These methods are currently applied on cell lines, primary cells and tissue samples.
During the last few years, single cell analysis has become a major research issue. Due to recognition of the importance of this particular issue, there is a persistent increase in the number of researchers and grant proposals in this field. For instance, the National Institutes of Health (NIH) Common Fund announces awards for Single Cell Analysis: "The (NIH) plans to invest more than $90 million over five years, contingent upon the availability of funds, to accelerate the development and application of single cell analysis across a variety of fields. The goal is to understand what makes individual cells unique and to pave the way for medical treatments that are based on disease mechanisms at the cellular level. Supported by the NIH Common Fund, NIH plans to support 26 awards as part of three initiatives of the Single Cell Analysis Program (SCAP)."
The heterogeneity within a population of cells could be inherent in the nature of the cells; for instance, cells from different tissues or several differentiation stages which have a different phenotype due to the expression of different sets of genes. It could also result from the stochastic quality of gene expression which causes stochastic natural events in apparently homogeneous populations of cells. This can be observed in cell division, proliferation, drug efficacy, and in the onset time of drug response, apoptosis, cell signaling, transcription, receptor expression, concentration of critical metabolites and ions, etc. In addition, cells in the population are not synchronized, making it difficult to notice phenomena such as cell cycle stage, circadian clock translocation, secreted protein and ion oscillation, transcriptional cycles and cell signaling dynamics – unless cells are measured and analyzed at single cell resolution. Hence, from a biological and clinical point of view, there are great advantages in recognizing, characterizing and sorting rare cells or subpopulations of cells.
One of the earliest and most commonly used methods for single cell analysis is flow cytometry (FC), in which one is able to analyze about 100,000 cells in a few seconds and make comparisons according to their size, granularity and fluorescence parameters at an end time point. Using this method, one can also sort cells according to predefined parameters (FACS). The main drawback of FC is its inability to handle dynamic, kinetic, long lasting measurements, including tracking spatiotemporal protein movements within a single cell. The most adequate instrument for kinetic intracellular localization is the fluorescent microscope. It enables monitoring intracellular protein localization kinetics of several different labeled proteins in parallel, in a few single cells at the same time. But long lasting time-dependent and localization measurements of 1,000-10,000 cells at the same time are very complicated to accomplish for adherent and, of course, for non-adherent cells. Adherent cells have a dynamic movement on the surface, and cell boundaries are difficult to determine for analysis, while non-adherent cells, like blood cells which grow in suspension, lack a fixed location. Thus, cells must maintain their location for long periods of time in order to facilitate monitoring the kinetic parameters of few thousands of cells in parallel, under the microscope.
In the course of years, several techniques have been developed for retaining/trapping cells at defined locations over time, including patch clamp, hydrodynamics, optical tweezers, dielectrophoretics, magnetics, acoustics, droplets, and other techniques as well, each with its own advantages and drawbacks. The simplest and most straightforward among these techniques, is the micro-well array structure/miniaturized multiwell array chip, an approach wherein walls are constructed around each cell in order to create a physical separation between the cells and constrain their location without tethering. In this system, each cell maintains its location and has accurate coordinates/spatial landmark, so it could be monitored through time following various manipulations such as medium exchange, multiple staining, washing, drug treatment, and the like.
In the Jerome Schottenstein Center we devote our time to developing, characterizing and validating novel means, methodologies and assays to allow controlled, long-term culturing, monitoring and high content analysis of numerous individual cells within populations. These means and readouts are currently being applied in various biological and medical research projects.