Cell sorting

Fluorescence-activated

Fluorescence-Activated Cell Sorting is also known as flow cytometry cell sorting, or by the acronym FACS, which is a trademark of Becton Dickinson and Company. Fluorescence-activated cell sorting utilizes flow cytometry to separate cells based on morphological parameters and the expression of multiple extracellular and intracellular proteins. This method allows multiparameter cell sorting and involves encapsulating cells into small liquid droplets which are selectively given electric charges and sorted by an external electric field. Fluorescence-activated cell sorting has several systems that work together to achieve successful sorting of events of interest. These include fluidic, optical, and electrostatic systems. The fluidic system has to establish a precisely timed break-off from the liquid stream in small uniform droplets, so that droplets containing individual cells can then be deflected electrostatically^[2] Based on the invention of Richard Sweet,^[3] droplet formation of the liquid jet of a cell sorter is stabilized by vibrations of an ultrasonic transducer at the exit of the nozzle orifice. The disturbances grow exponentially and lead to breakup of the jet in droplets with precise timing. A cell of interest that should be sorted is measured at the sensing zone and moves down the stream to the breakoff point. During the separation of the droplet with the cell in it from the intact liquid jet, a voltage pulse is given to the liquid jet so that droplets containing the cells of interest can be deflected in an electric field between two deflection plates for sorting. The droplets are then caught by collection tubes or vessels placed below the deflection plates.^[2] Flow cytometry cell-sorting yields very high specificity according to one or several surface markers, but one limitation is constituted by the number of cells that can be processed during a work day. For this reason pre-enrichment of the population of interest by immunomagnetic cell sorting is often considered, especially when the target cells are comparatively rare and a large batch of cells must be processed. Moreover, flow cytometry cell-sorters are complex instruments that are generally used only by well-trained staff in flow cytometry facilities or well-equipped laboratories and, since they are normally large in size, it is not always possible to place them inside a biological safety cabinet. Therefore, it is not always possible to ensure sample sterility and, since the fluidic systems can be cleaned but are not single-use, there is the possibility of cross-contamination among samples. Another aspect to be considered is that droplet-generation inside the instrument could lead to aerosol formations that are hazardous to the operator when using infectious samples. These last considerations are of particular importance when cell sorting is used for clinical applications -- for example cell therapy -- and should be performed under Good Manufacturing Practice (GMP) conditions. Researchers can use a variety of fluorescent dyes to design multi-color panels to achieve successful, simultaneous sorting of multiple, precisely defined cell types. Diagram A shows fluorescence-activated cell sorting of negative cell selection (undesired group) and diagram B shows FACS of positive cell selection (desired group).

Immunomagnetic cell sorting

MACS

Immunomagnetic cell sorting is also known as immunomagnetic cell separation, immunomagnetic cell enrichment, or magnetic-activated cell sorting, and commonly known by the acronym MACS which is a trademark of Miltenyi GmbH. Immunomagnetic cell sorting is based on separation of beads passing a magnetic field. A variety of companies offer different solutions for enrichment or depletion of cell populations. Immunomagnetic cell sorting provides a method of enriching a heterogeneous mixture of cells based on cell-surface protein expression (antigens). This technology is based on the attachment of small, inert, supra-magnetic particles to mAbs specific for antigens on the target cell population. Cells labelled to these antibody-bead conjugates are then separated via a column containing a ferromagnetic matrix. By applying a magnetic field to the matrix, the beads stick to the matrix inside the column and the bead-carrying cells are held back from passing through. Unlabelled cells can pass through the matrix and are collected in the flow-through. To elute the trapped cells from the column, the magnetic field is simply removed. Immunomagnetic cell sorting therefore enables different strategies for positive enrichment or depletion of cells.^[2] Immunomagnetic beads are small and usually do not interfere with downstream assays, however for some applications it may be necessary to remove them. Using this separation method up-scaling the cell numbers does not significantly increase processing times, and the sterility of the sample is guaranteed if the cell sorting is performed inside a biosafety cabinet. On the other hand, this technique allows cells to separate based only on a single marker, and cannot discriminate between different levels of protein expression (quantitative analysis). Immunomagnetic cell sorting has shown to be beneficial when used with NPC (neural progenitor cell) cultures in particular, as it is easier to manage and causes minimal damage to live cells.^[5]

NPC cultures are especially difficult to work with because live brain cells are sensitive and tend to contaminate each other.^[5] In order to get clearer results, labs need cleaner materials, meaning more pure NPC lines.^[5] A study done in 2019 (with the funding support of New York Stem Cell Foundation and the Association for Frontotemporal Degeneration) found immunomagnetic cell sorting to be a cheap, simple way to yield such purity with minimal damage to the cell lines, therefore maintaining better quality cells, collecting more homogeneous NPCs, and increasing their chances of finding effective treatments for neurological disorders.^[5] They used both the immunomagnetic and fluorescence-activated methods to filter out CD271- (useful markers for mesenchymal stem cells) and CD133+ (markers for cancer stem cells) to compare viability of each method.^[5]

Immunomagnetic cell sorting has also been used in assistance with reproduction (artificial insemination) and retinal transplant treatment.^[6][7] In the case of assisting reproduction, apoptotic sperm cells (dead or damaged cells) are separated out so more non-apoptotic sperm (non-fragmented) cells can be collected and used to increase the subject's chances of fertility.^[6] This type of treatment has shown to be more effective when done repeatedly, increasing the amount of non-apoptotic cells present during insemination.^[6]

A 2018 study done in France (with the support of multiple individuals and agencies including: the Institut de la Vision in Paris and the Retina France Association) used rats and the immunomagnetic cell-sorting method to show that photoreceptors (cells in the retina which respond to light) may be transplanted to cure blindness.^[7] In this process, the microbeads were attached to the CD73 enzyme to assist in the separation of PRs (photoreceptors) from retinal organoids.^[7] When a CD73+ antigen expressed itself with RCVRN+ cells (calcium-binding proteins in the eye), it showed researchers that this combination of CD73+ and RCVRN+ could be used with post-mitotic PR precursors for repair.^[7] Although the study could not verify success in humans, they have the foundation for further research based on the success of pairing non-damaged photoreceptors with a CD73 antigen and the transplantation in rats.^[7] This success in cell separation and pairing through transplantation shows promise for a potential cure for retinal diseases including total blindness. So far, only partial vision repair has been reported.^[7]

Microfluidic devices

Due to various limitations of fluorescence-activated and immunomagnetic cell sorting devices, a wide range of microfluidic cell-sorting devices have emerged. A few of these are now commercially available or in commercial development. Research into microfluidic cell-sorter designs often employ soft lithography techniques utilizing materials such as polydimethylsiloxane (PDMS).

A key benefit of microfluidic sorters is the potential to perform fluorescence-activated cell sorting in a closed single-use sterile cartridge. Such a closed cartridge would prevent the exposure of an operator to biohazards through the droplets that are emitted by FACS systems. Other benefits include reduced impact on cell viability due to reduced hydrodynamic stress on the cells. Some published devices show the potential for multi-way sorting, decreased cost of a cartridge due to low-cost manufacturing methods, lower power consumption, and smaller-sized footprints, with some devices being the size of a credit card. Some have achieved high-purity outputs and rates of up to around 50,000 cells/s.^{[8][9][10][11]}

Microfluidic cell-sorters can be divided into two categories: active and passive. Active devices deflect individual cells by the cytometric measurements of the cells, made in real time. Passive devices exploit physical differences between cells in how they interact with the fluid flow or surfaces.

Other antibody-based methods of cell separation

Several methods of cell separation and enrichment using antibodies were employed before fluorescence-based and immunomagnetic cell sorting gained popularity.^[43] These included antibody- and complement-mediated cell separation, polystyrene immunoaffinity devices, and the CellPro CEPRATE® SC System which employed immobilised antibodies in a porous column. The latter was the first FDA-approved medical device for the separation of hematopoietic stem cells.

A newer cell separation technique employing antibodies is buoyancy-activated cell sorting (BACS) is a separation technique in which microbubbles bind to cells through antibodies binding to the surface of cells. The targeted cells are then removed from a biological sample through flotation.^[44]