Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting

www.nature.com/scientificreports OPEN Received: 17 August 2018 Accepted: 22 November 2018 Published: xx xx xxxx Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting Bryan A. Sutermaster 1,2 & Eric M. Darling1,2,3,4 Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer’s recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL− cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7–9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4–6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications. Cell sorting, enrichment, and purification methods are powerful tools enabling the isolation of cellular subpopulations for basic science and clinical applications. The stromal vascular fraction (SVF), or vascular-associated cellular component, of lipoaspirate has been identified as an attractive cell source for both basic science and translational study as it contains subpopulations of adipose-derived stem cells (ASCs) and other progenitors1,2. Compared to other stem cell niches like bone marrow and muscle, adipose tissue contains higher percentages of differentiable cells, and can be isolated with ease and little donor site morbidity3. As the SVF is comprised of a heterogeneous cell population, in vitro plating/expansion or cell separation techniques are required to isolate ASCs from non-stem cell types4–6. Plating and expansion is a time consuming process not compatible with single-surgery procedures. More rapid cell separation techniques are needed for time-sensitive applications. Subpopulations of ASCs and other progenitors can be fluorescently tagged based on biochemical markers and subsequently isolated from other cell types in the SVF by cell sorting techniques7–12. The gold standard for cell separation is fluorescence-activated cell sorting (FACS). While FACS is capable of processing millions of cells and isolating multiple, high purity subpopulations, it is also relatively time consuming for very large cell numbers and requires expensive machinery. A related technology, magnetic-activated cell sorting (MACS), relies on direct (primary antibody-conjugated microbead) or indirect (primary antibody plus secondary antibody-conjugated microbead) magnetic labeling of cells prior to separation in a magnetic field13. MACS is also used to select for cell populations using surface markers but is less time consuming and requires less expensive equipment than FACS. However, it lacks the sensitivity and cell-specific data provided by a fluorescence-based system and is not easily compatible with multiple-marker profiles. Surprisingly, measures of cell throughput and yield, viabilities, and 1 Center for Biomedical Engineering, Brown University, Providence, USA. 2Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, USA. 3Department of Orthopaedics, Brown University, Providence, USA. 4School of Engineering, Brown University, Providence, USA. Correspondence and requests for materials should be addressed to E.M.D. (email: ) SCiEntifiC REportS | (2019) 9:227 | DOI:10.1038/s41598-018-36698-1 1 www.nature.com/scientificreports/ processing time between FACS and MACS are largely unreported, making it difficult to compare the practicality of the two techniques for a given application. Cell separation techniques for ASCs often employ multiple surface markers to specifically define the cell type, as a single, definitive marker has yet to be identified14,15. A general ASC definition proposed by the International Federation of Adipose Therapeutics and Science (IFATS) includes positive/negative expression for four surface markers (CD34+/CD31−/CD45−/CD235a−), with an additional four markers for increased specificity (CD13, CD73, CD90, and CD105)15. These restrictive definitions result in very small numbers of enriched, yet still heterogeneous, cells such that the population input to FACS must be extremely large to acquire therapeutically relevant numbers (~106–108) as output16–22. Less restrictive surface marker profiles may enable isolation of larger cell populations and prove advantageous for regenerative medicine applications. One such marker, alkaline phosphatase liver/bone/kidney (ALPL), is a membrane bound protein involved in early matrix mineralization during osteogenesis and may be a useful target for identifying stem cell subpopulations, particularly for end applications of bone regeneration23–28. Previously, groups have isolated subpopulations of induced pluripotent stem cells and jaw periosteal cells based on ALPL expression that were capable of increased osteogenesis, though this has not yet been demonstrated with primary SVF cells29,30. The objective of this study was to quantify the processing times, cell yields and viabilities of MACS and FACS separations using defined mixtures of osteogenically primed SVF cells and A375 human melanoma cells based on their expression of ALPL. To accomplish this, primary SVF cells were first expanded and osteogenically stimulated to upregulate expression of the ALPL marker in responsive cell types. After priming, SVF cells were mixed in defined ratios with A375 cells (0:1, 1:3, 1:1, 3:1, 1:0) and separated based on ALPL expression using FACS or MACS. Processing time and cell throughput, yield, and viability for ALPL+ and ALPL− groups were quantified and compared between the two sorting methods. Effort was made to identify and reconcile discrepancies between the two approaches to better inform researchers using these techniques for cell enrichment/purification studies. Methods SVF Isolation and Culture. Primary, human lipoaspirate was procured from the breast and abdomen of one, informed and consenting, (...truncated)