Kyushu University develops CyTOR method to extract cytoplasm in 5 seconds by bursting cells

Mid-size drugs, which are attracting attention as next-generation pharmaceuticals, hold great potential for treating cancer and rare diseases. Like small-molecule drugs, these medications enter cells in the body and exert their effects by binding to specific targets. However, only complex and time-consuming methods were able to measure how much of the drug enters cells and how long it remains. Moreover, testing with diseased cells was not possible. A research group led by Associate Professor Takayuki Kawai of the Department of Chemistry, Graduate School of Science at Kyushu University developed a new method called CyTOR that uses the osmotic pressure of water to instantly burst cells and quickly extract their contents. With this method, drugs inside cells can be extracted in just 5 seconds through simple operations and accurately measured using liquid chromatography-mass spectrometry (LC-MS). When four types of drugs were administered to cells and examined, differences in permeability and efflux were found, consistent with previous research results. This will become a new cell membrane permeability assay method for efficiently advancing drug discovery research. The study was published in Analytical Chemistry.

The CyTOR procedure is extremely simple, requiring only sprinkling hypotonic solution (ultrapure water) on cultured cells. Because an osmotic pressure difference is created between the sprinkled hypotonic solution and the cell interior, cells burst within a short time and cytoplasm can be rapidly extracted. While methods using osmotic pressure differences to extract proteins from cytoplasm have been developed previously, they require the use of surfactants or long operation times, causing drugs present outside the cytoplasm (such as in cell membranes) to also be eluted. CyTOR selectively extracts only drugs in the cytoplasm through rapid extraction in 30 seconds, and when combined with LC-MS, enables high-precision measurement of intracellular drug concentrations.

To first confirm whether cells could be burst with ultrapure water to collect only cytoplasm, cells were treated with two reagents, calcein-AM and CellMask, to stain the cytoplasm and cell membrane separately. Subsequently, when cells were burst using ultrapure water, the cytoplasm marker disappeared in just 5 seconds after ultrapure water addition, while the cell membrane marker remained almost unchanged even after 1 minute. This demonstrated that CyTOR enables rapid and selective collection of cytoplasm.

Next, when theoretically predicting how intracellular drug concentration would change over time after adding the drug to the medium, it was found that this could be expressed as a function of drug permeability rate and time. By fitting this function to actual experimental data and determining the constants, the ease of drug entry (membrane permeability from outside to inside cells) and ease of exit (permeability from inside to outside cells) could ultimately be calculated.

Ease of drug entry and exit can be classified into three patterns: easy entry and easy exit, difficult entry and easy exit, and difficult entry and difficult exit. HeLa cells were cultured in medium containing the small-molecule drugs corresponding to each of the three patterns - i.e., propranolol, cimetidine, and atenolol - along with cyclosporine A as a mid-size molecule. At several culture times, drugs extracted by CyTOR were analyzed by LC-MS. When the proportion of penetration into cells relative to the drug added (drug permeability rate) was calculated and plotted against culture time, the ease of entry and exit for each drug showed trends highly consistent with previous reports, demonstrating that drug permeability can be conveniently measured using cancer cells.

CyTOR is an innovative technology that enables rapid and high-precision quantification of intracellular drugs, which was previously difficult. The research group plans to pursue improvements toward high-throughput and automated analysis by introducing automatic cell volume measurement technology and algorithms for simultaneous analysis of multiple drugs. Furthermore, because CyTOR can be applied not only to drugs but also to analysis of intracellular metabolites and chemical probes, it has the potential to contribute to understanding molecular dynamics in life science research in general.

Kawai commented: "The cell membrane is a wall that separates the inside and outside of cells, but it has the property of allowing the passage of substances beneficial to the cell while blocking foreign substances like drugs. Confirming whether drugs truly penetrate diseased cells is extremely important. In this research, we developed a method to instantly burst cells and easily extract internal drugs by using osmotic pressure, which had always been a blind spot. Because it is simple, it is a useful technique that anyone can use, and we hope that many people will utilize it."

Journal Information
Publication: Analytical Chemistry
Title: Quantifying Cell Membrane Permeability of Small to Mid-Size Drugs Using Quick Osmotic Cytoplasm Extraction
DOI: 10.1021/acs.analchem.5c01064