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Cell sorting for everyone: From the core lab to your lab
When cell-sorting technology first arrived in the late 1970s, it was used by academics and the medical research community to detect, count and characterize cells from the body and environment. As cell-sorting technology evolved and improved, it also became more complex, unfortunately making it less accessible to many researchers. Fortunately, recent advances in instrumentation have led to the development of much smaller, significantly less expensive and much easier-to-use cell-sorting devices, meaning that even basic researchers can now access this useful technology.
Early cell sorters were based on fundamental principles such as sheath flow and impedance, which are still used in modern instrumentation. Flow cytometry entered the age of fluorescence-based detection in the 1960s, about the same time the utility of collecting cells was demonstrated. Although it would be more than a decade until the development of devices resembling modern cell sorters, advancements and discoveries in engineering and biology throughout the 1970s and 1980s were instrumental in the development of today’s cell-sorting technologies.
The introduction of fluorescence measurement to flow cytometers is arguably the most important advancement in the field to this day. Fluorescence detection of target particles greatly improved the ability to characterize cells and enhanced quantitative measurements. The first flow cytometers to use fluorescence measurement principles—built in the late 1960s and early 1970s—employed either argon ion lasers or arc lamps for the detection of DNA content in cells. While still rudimentary by today’s standards, these early systems successfully demonstrated the ability to detect tumor abnormalities and study cell cycles in the lab.
In 1965, Mack Fulwyler, a physicist at Lawrence Livermore National Laboratories, built and demonstrated the first cell sorter: a device with the ability to sort cells using Coulter principles and inkjet printing technology, resulting in a patent and publication in Science. Later, the combination of now-proven fluorescent detection with new technologies being developed at Stanford University in the 1970s ultimately led to the development of the first cell-sorting device that resembles the instruments in use today.
Leonard Herzenberg’s team at Stanford understood that fluorescence-based characterization followed by the isolation of cells for further study would be very beneficial to the research community. The basic principles of sorting cells based on their fluorescent signatures were developed and improved in the early 1970s, resulting in the release of the first commercially available cell sorter in 1974.
Over the next 10 years, the cancer research and diagnostic communities showed increased interest in the analysis of cellular DNA content. Additionally, immunology researchers started using antibodies labeled with fluorescent dyes, which opened up new possibilities for the application of flow cytometers and cell sorters. The addition of a second laser to early instrumentation, in combination with dye-labeled antibodies, then allowed researchers to both analyze and sort very closely related cells, which proved to be an incredibly powerful technique in both the research and medical communities. During this time, the use of flow cytometry and cell sorting exploded as the world struggled to understand how to cope with a new, devastating condition that would come to be known as AIDS.
Research and competition drives innovation
Flow cytometry and cell sorting quickly matured into useful technologies with demonstrated value to researchers and clinicians. Early cell sorters and cytometers had sensitivities that were adequate for existing uses when both cells and detectable surface markers were abundant. These new tools shined when compared with traditional microscopy; their ability to analyze or isolate thousands of cells every second provided a far more efficient way to analyze a large sample of cells. Researchers wanted more.
In the 1980s, advances in medical research and new discoveries in immunology drove the demand for faster, more sensitive instruments. Cell biologists needed the ability to distinguish and isolate different classes and subclasses of cells. They also wanted to isolate rare cell events within samples of tens of millions of cells. By the late 1990s, researcher demand, new technology and market competition had driven critical improvements in cell-sorting instrumentation. New companies with novel approaches to fluorescent detection and sorting had introduced their own products. Advances in electronics and computing power enabled samples to be analyzed and sorted 10 to 100 times faster than a decade earlier. In addition, advances in both laser and detection technologies led to sorting devices equipped with more than eight different lasers and 15–20 detection channels for multicolor sorting. While these advancements were necessary to meet an essential scientific need, the added capability and flexibility came with a cost and complexity that ultimately limited the use of cell sorting.
Access to cell sorting for research
The availability of a centralized research (core) facility dictates access to cell sorting in the majority of academic, private and for-profit research institutes. Typically, any instrument or system that has a high cost of ownership, requires special training to operate and is not routinely used within a basic research laboratory, is operated by a core facility. In the case of cell sorting, the upfront cost to own and operate a cell-sorting instrument capable of handling a wide range of immunology work (greater than four lasers and six-color detection) starts at $500,000 and can run as high as $1 million. This price does not include the ongoing cost of annual service contracts and salary for a skilled technician for daily set-up and operation. However, having high-end cell-sorting devices in the core facility provides many researchers with access to powerful instrumentation and the expertise needed to run them on an as-needed basis.
The ability of sophisticated cell sorters in core facilities to isolate pure populations of multiple target cell types from a heterogeneous sample is becoming increasingly valuable in many research areas. Conversely, not all users need the multicolor assay capabilities that are typically utilized in immunology research. The increasing popularity of cell sorters in broader research areas has resulted in wait lists of at least two to three weeks at many core facilities.
Anywhere from 30 to 70 percent of cell sorting in many core facilities is low-complexity work, requiring at most four-color and one- or two-population sorting. Although this kind of work can be completed using the same type of cell-sorting device that handles work requiring more than six colors and more than two populations, acquiring another sophisticated cell sorter is an impractical way of expanding capacity. What is the core facility to do? Apply for additional grants and funding and/or hire another operator to increase sorting hours?
New advances enable greater adoption
Fortunately for the core facility as well as any lab that regularly relies on flow cytometry and cell sorting, simpler, more compact cell-sorting systems have recently been introduced that are also more cost-effective and easier to use. These new cell sorters additionally feature a high level of automation, allowing for capacity increases without requiring more technician time. These devices offer up to four-color detection and sorting of one to two populations, which is adequate to handle most low-complexity work. While these new systems have reduced color capability, they can match the speed, sensitivity, purity and efficiency of high-end systems.
The development of flow cytometry and cell-sorting technologies began over 50 years ago, and significant advancements since then have greatly improved the capabilities of these powerful analytical instruments in the lab and clinic. More recent developments appear poised to enable greater adoption and provide more researchers with access to this powerful technique now and in the future.
Steve Kulisch manages the cell biology unit at Bio-Rad Laboratories, which this year launched the S3 cell sorter, a high-performance, user-friendly cell sorter designed for individual and core labs. He started at Bio-Rad in 2000 as a product manager and became head of marketing for Bio-Rad’s Gene Expression Division in 2005.