Cell sorting supports numerous applications in biology and medicine, most of which fall into three categories: enumeration (how many cells of a particular type are present), expansion (e.g., for cell culture, bioproduction, or cell therapy), and analysis of cells or their molecular contents.
Today's cell sorters process millions of cells per hour, quantifying up to 10 or more fluorescent markers simultaneously at high sensitivity. Conventional cell sorters use an electrostatic droplet cell sorting technique called jet-in-air or cuvette-hybrid with fluorescence flow cytometry, in a configuration known as fluorescence-activated cell sorting (FACS). During this process, errors inevitably occur, for example in cell identification, collection, or through carryover contamination from previous experiments. These events lead to heterogeneous cell cultures or miscounted cell populations.
The implications of these errors, particularly from those resulting in cross-contamination, can be quite severe for clinical applications but they are troubling for basic research as well. Cell-line cross-contamination is a significant source of error in biology and medicine. A recent article identified more than 30,000 studies that used misidentified cells, and more than half a million citations of this work. The cost of irreproducibility in the life sciences has been estimated at $28 billion —a staggering number when you consider that the entire budget for the U.S. National Institutes of Health is just under $42 billion per year.
The most serious consequences of cross-contamination are evident in cell-based therapies, liquid biopsy, and high-throughput phenotypic screening.
For example, autologous chimeric antigen receptor T cell (CAR-T) based therapies require isolating rare T-cell phenotypes at 90% homogeneity from patients. Desired phenotypes are rare and may carry just a few identifying surface markers. Throughout the process of harvest, purification, T-cell re-programming, expansion, and re-infusion, therapeutic cell sorting must follow Good Manufacturing Practices with special emphasis on quality and purity. Clinicians must avoid at all costs the co-culturing of undesirable cells, whether they arise from instrument error or cross-contamination.
Today's cell sorters do many things very well but their suitability to critical medical applications is limited by uncertainties regarding throughput, reproducibility, and product homogeneity. A typical instrument based on the standard sorting method will collect cells at high purity, recovery, and viability at a rate of between 1,000 and 2,000 cells per second. This is well below what is required for therapy, which is around 140,000 cells/second. Throughput in these systems is limited by such factors as shear forces and pressure drops across the nozzle, which reduce cell yield, purity, and viability. These limitations apply to most single-stream cell sorters on the market today, including those employing microfluidics.
Cross-contamination during cell sorting can occur several ways, such as misidentification at the detection stage, errors when funneling cells to their destinations after sorting, or leftover material from previous experiments. Introduction of bacteria is also possible due to the open-air nature of flow methods.
Carryover is an issue with any flow-through instrument. Experiments that rely on precise measurements at high sensitivity are especially vulnerable. Manufacturers of FACS instrumentation are aware of this and have instituted rinsing or cleaning protocols to mitigate the most significant aspects of this problem. Some vendors boast very low carryover after using their instrument's standard rinse cycle, but even 1% carryover may be too much when protocols call for subsequent cell expansion. If the "leftover" cells grow faster than the desired cell populations, 1% can soon become 5%, 10%, or more. It therefore behooves operators to quantify (and limit) carryover from one sample to the next. Many regulated workflows require formal validation to assure that carryover does not exceed specifications.
Many companies involved in biomanufacturing have solved cross-contamination concerns by adopting single-use bioreactors. Could cell sorting follow a similar strategy? Since cytometry systems are too expensive to use only once, this seems unlikely without radical redesign of FACS.
Which is precisely what Tokyo-based On-chip Biotechnologies has accomplished with its On-chip Sort, microfluidic chip-based cell sorter. On-chip Sort isolates cells through microfluidic channels within its proprietary disposable microfluidic chips using their unique "flow shift" method, which avoids shear stresses, high pressure, collisions, or other events that might damage or alter cells. On-chip provides a sterilized environment for samples. Because it is used for only one experiment, then discarded, the potential for cross-contamination is zero. On-chip Sort fits inside a biosafety cabinet so samples can be handled under sterile condition. Aerosols—a source of environmental contamination—are also avoided due to the nature of the flow shift method.
Made from inexpensive materials through standard injection molding technology, On-chip Sort has been validated in numerous studies using up to six fluorescence channels. Most recently, Japanese researchers used this method to select sperm with a high likelihood of success in in vitro fertilization.
Cell sorting has become indispensable to researchers and clinicians. Whether the end-use is enumeration of cell populations, cell collection for future expansion, or fundamental proteomic or genomic analyses of populations or individual cells, investigators demand accuracy and, in many instances high viability and function. With critical applications the avoidance of cross-contamination becomes paramount. Single-use flow cassette offers a robust, highly accurate method for selecting rare cell populations with reduced risk of cross-contamination.
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