Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system.

BACKGROUND AIMS: Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). METHODS: The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-µm polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. RESULTS: Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. CONCLUSIONS: This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.

Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.

Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies.

A method for clonal analysis of epidermal growth factor-responsive neural progenitors.

Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10-20 ng/ml EGF for 12-14 days. This gave a cloning efficiency of 2.12%+/-0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10-14 and 21-28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells.

Titration of fluorochrome-conjugated antibodies for labeling cell surface markers on live cells.

Nonspecific antibody binding is best eliminated by optimizing the amount and concentration of the antibody. An antibody titration assay should be applied to determine the antibody amount and concentration resulting in the highest signal of the positive population and the lowest signal of the negative population. While conventional antibody titration protocols focus on the concentration of the antibody, this protocol for antibody titration considers the antibody concentration, as well as the antibody amount. Thus, it is designed to find the optimal antibody concentration for labeling antigens expressed on the surface membrane of live cells, while nonspecific antibody binding is kept to a minimum.

Considerations for the control of background fluorescence in clinical flow cytometry.

Accurate measurement of antigen-positive cells by flow cytometry can be hampered by background fluorescence of antigen-negative cells and other particles (e.g., debris). This article focuses on three major causes of background (autofluorescence, spectral overlap, and undesirable antibody binding) by reviewing individual aspects of flow cytometric measurements that contribute to these causes. The appropriate use of controls facilitates a thorough understanding of these contributing factors as well as the development of robust cell labeling protocols intended for routine flow cytometric analysis. We present a set of recommendations that enables the user to develop an optimized cell labeling protocol that minimizes background and maximizes the ability to reliably distinguish between a positive and a negative population of cells. These recommendations are also intended to augment existing guidelines designed to aid in the formulation of a consensus regarding the utility of flow cytometry for the analysis of clinical samples.