Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations

Abstract

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75, O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach

“…Independent human SC cultures were analyzed for their relative content of p75 NFGR positive and negative cells prior to being enzymatically dissociated and separated via MACS. For this, we essentially followed the step-by-step protocol described in our recent paper 11 but introduced proper adaptations as per the content of fibroblasts to obtain the highest possible yields and purity in the eluted and retained fractions.…”

“…Achieving high purity was possible by performing two sequential rounds of MACS with the introduction of an intermediate step of cell culture (one to two days) to provide prompt adhesion to the retained fraction before attempting a second round of MACS (not shown). The incorporation of 10–20% FBS to the MACS buffer was efficacious to improve cell survival 11 .…”

“…The culture supernatant was collected by low speed centrifugation after each round of expansion. The time of collection was determined when it was visually appreciated that the medium turned yellow in color 11 . Every batch of hybridoma culture supernatant (herein referred to as ‘conditioned medium’) was tested for its labeling activity and specificity using positive control cells.…”

“…> 2 × 10 7 cells) was rarely needed but was possible by adjusting the volumes of primary and secondary antibodies, and using additional columns as dictated by the number of cells. Technical adjustments to this protocol for an increased speed of separation, enhanced purity and/or yields can be found in our recent publication 11 .…”

“…Magnetic-activated cell sorting (MACS) provides an advantage to rapidly (i.e., within 2 h) purify rat SCs at high efficiency without killing the fibroblasts 9 . However, we found that direct translation of purification methods from rats to humans was mostly ineffective 10 , 11 due to species-specific variations in the antigenic properties of the cells and other aspects of their growth control 12 . Consequently, our primary goal was to perform a phenotypic and functional analysis of peripheral nerve-derived human SC cultures to reveal the prevalent cell types in the populations and ascertain the most effective strategy for magnetic cell labeling, separation by MACS and characterization of the post-MACS cell products.…”