Measuring the density of a cell can reveal an excellent deal concerning the cell’s state. As cells proliferate, differentiate, or bear cell demise, they could acquire or lose water and different molecules, which is revealed by adjustments in density.
Monitoring these tiny adjustments in cells’ bodily state is troublesome to do at a big scale, particularly with single-cell decision, however a workforce of Massachusetts Institute of Know-how (MIT) researchers has now discovered a solution to measure cell density rapidly and precisely, measuring as much as 30,000 cells in a single hour.
The researchers additionally confirmed that density adjustments might be used to make worthwhile predictions, together with whether or not immune cells resembling T cells have grow to be activated to kill tumors, or whether or not tumor cells are prone to a selected drug.
“These predictions are all based mostly on taking a look at very small adjustments within the bodily properties of cells, which may let you know how they’re going to reply,” mentioned Scott Manalis, PhD, the David H. Koch Professor of Engineering within the departments of Organic Engineering and Mechanical Engineering, and a member of the Koch Institute for Integrative Most cancers Analysis. Senior creator Manalis, along with lead creator, MIT researcher Weida (Richard) Wu, PhD, and colleagues, described their research in Nature Biomedical Engineering, in a paper titled, “Excessive-throughput single-cell density measurements allow dynamic profiling of immune cell and drug response from affected person samples.” Of their paper, they concluded, “Our technique reveals sudden habits in molecular crowding throughout cell state transitions and suggests density as a biomarker for useful precision drugs.”
As cells enter new states, their molecular contents, together with lipids, proteins, and nucleic acids, can grow to be roughly crowded. Measuring the density of a cell presents an oblique view of this crowding. “Cell density, the ratio of cell mass to quantity, is an indicator of molecular crowding and a basic determinant of cell state and performance,” the authors wrote. “The coupling between crowding stage and cell physiology makes cell density a key proxy for characterizing basic mobile processes resembling proliferation, apoptosis, metabolic shifts, and differentiation, indicating its potential as a biomarker for mobile health and drug response. Nonetheless, they identified, “… current density measurements lack the precision or throughput to quantify refined variations in cell states, significantly in main samples.”
The brand new density measurement method reported by Manalis et al. builds on work within the Manalis lab over the previous 20 years on applied sciences for making measurements of cells and tiny particles. In 2007, the lab developed a microfluidic gadget often called a suspended microchannel resonator (SMR), which consists of a microchannel throughout a tiny silicon cantilever that vibrates at a selected frequency. As a cell passes by the channel, the frequency of the vibration adjustments barely, and the magnitude of that change can be utilized to calculate the cell’s mass.
In 2011, the researchers tailored the method to measure the density of cells. To realize that, cells are despatched by the gadget twice, suspended in two liquids of various densities. A cell’s buoyant mass (its mass because it floats in fluid) is dependent upon its absolute mass and quantity, so by measuring two totally different buoyant plenty for a cell, its mass, quantity, and density might be calculated.
That method works nicely, however swapping fluids and flowing cells by each is time-consuming, so it may well solely be used to measure a couple of hundred cells at a time. “… the throughput of this strategy can also be restricted to some hundred cells per experiment as a result of it requires cells to be sequentially measured in two varieties of fluids,” the workforce famous.
To create a sooner, extra streamlined system, the researchers mixed their SMR gadget with a fluorescent microscope, which allows measurements of cell quantity. The microscope is positioned on the entrance to the resonator, and cells circulate by the gadget whereas floating in a fluorescent dye that may’t be absorbed by cells. When cells cross by the microscope, the dip within the fluorescent sign can be utilized to find out the amount of the cell.
After that quantity measurement is taken, the cells circulate into the resonator, which measures their mass. This course of, which permits for fast calculation of density, can be utilized to measure as much as 30,000 cells in an hour. “As an alternative of making an attempt to circulate the cells forwards and backwards at the very least twice by the cantilever to get cell density, we wished to attempt to create a technique to do a streamlined measurement, so the cells solely have to cross by the cantilever as soon as,” Wu defined. “From a cell’s mass and quantity, we are able to then derive its density, with out compromising the throughput or the precision.” The authors additional defined, “Right here, we current a fluorescence exclusion-coupled SMR (fxSMR) platform that concurrently measures single-cell buoyant mass and quantity, which permits us to profile cell density with a throughput of over 30,000 cells per hour and a precision of 0.03% (0.0003 g ml−1) for cells bigger than 12 μm in diameter.”
The researchers used their new method to trace what occurs to the density of T cells after they’re activated by signaling molecules. “We carried out each day measurements of CD8+ T cells from two human donors after anti-CD3 and anti-CD28 activation,” they famous. As T cells transition from a quiescent state to an lively state, they acquire new molecules, in addition to water, the researchers discovered. From their pre-activation state to the primary day of activation, the densities of the cells dropped from a mean of 1.08 g/mL to 1.06 g/mL. Which means the cells have gotten much less crowded, as they acquire water sooner than they acquire different molecules.
“That is suggesting that cell density could be very possible reflecting a rise in mobile water content material because the cells transit from a quiescent, non-proliferative state to a high-growth state,” Wu commented. “These information are pointing to the notion that cell density is an fascinating biomarker that’s altering throughout T-cell activation and should have useful relevance to how nicely the T cells may proliferate.”
Travera, a clinical-stage firm co-founded by Manalis, is engaged on utilizing the SMR mass measurements to foretell whether or not particular person most cancers sufferers’ T cells will reply to medication which are anticipated to stimulate a robust antitumor immune response. The corporate has additionally begun utilizing the density measurement method, and preliminary research have discovered that utilizing mass and density measurements collectively provides a way more correct prediction than utilizing both one alone. “Each mass and density are revealing one thing concerning the general competency of the immune cells,” Manalis mentioned.
One other potential software for this strategy is predicting how tumor cells will reply to various kinds of most cancers medication. In earlier work, Manalis confirmed that monitoring adjustments in cell mass after therapy can predict whether or not a tumor cell is present process drug-induced apoptosis. Within the new research, the workforce discovered that density may additionally reveal these responses. “Since adjustments in cell density can reveal state transitions associated to cell proliferation, we sought to find out whether or not it might be used as a biomarker for assessing the ex vivo therapy response of most cancers cells,” they wrote.
In these experiments, the researchers handled pancreatic most cancers cells with one drug that the cells are prone to, and a distinct drug to which they’re resistant. The workforce discovered that density adjustments after therapy precisely mirrored the cells’ recognized responses to therapy. “We seize one thing concerning the cells that’s extremely predictive throughout the first couple of days after they get taken out from the tumor,” Wu famous. “Cell density is a fast biomarker to foretell in vivo drug response in a really well timed method.”
Manalis’ lab is now engaged on utilizing measurements of cell mass and density as a solution to consider the health of cells used to synthesize complicated proteins resembling therapeutic antibodies. “As cells are producing these proteins, we are able to study from these markers of cell health and metabolic state to attempt to make predictions about how nicely these cells can produce these proteins, and hopefully sooner or later additionally information design and management methods to even additional enhance the yield of those complicated proteins,” Wu famous.
Of their paper the workforce concluded, “Along with its potential for exploration of the biology of density homeostasis, our strategy might present a much-needed technique for useful precision drugs in sufferers … With our high-throughput strategy, we imagine that many medication can now be extra readily profiled on affected person samples inside a hyperacute time window, thereby enabling scientific research for assessing the effectiveness of density response in guiding affected person therapy.”
