Background

Schwann cells (SCs) are a heterogeneous group of axon-ensheathing cells in the peripheral nervous system of all vertebrate species (Jessen et al., 2015). These nerve-resident neuroglial cells can be isolated and expanded in vitro using standard cell culture techniques. Human SC (hSC) cultures can be prepared using any type of nerve or ganglion from developing and adult organ donors [reviewed recently in Monje (2020)]. Culturing hSCs is a lengthier and more labor-intensive process than culturing SCs from rodents and other experimental animals. However, the hSCs obtained from an initial harvest can be amplified substantially in vitro to generate large numbers of cells for a wide range of experimental approaches (Figure 1). Once established, the hSC cultures can be managed in a manner similar to adherent cell lines, though there are limitations regarding the expandability of individual stocks.

Figure 1. Scalable workflow for the preparation of nerve-derived human Schwann cell (hSC) cultures. The protocols described in this article and associated manuscripts (Monje, 2023a and 2023b) address the following basic procedures: (1) tissue procurement, dissection, and culture of fascicles (pre-degeneration); (2) enzymatic dissociation, isolation, and culture of primary hSCs; (3) derivation of established hSC cultures and amplification via serial passaging; (4) routine manipulations in vitro (e.g., purification, cryopreservation, labeling, and gene delivery); and (5) quality control of identity and bioactivity of the cells to be used in experimentation. Our protocols are adaptable; for instance, the number of hSCs can be scaled up by increasing the size of the tissue specimens used for cell isolation (primary cultures) and expanding the populations in medium containing mitogenic factors and serum (established cultures).

SC cultures from human tissues have facilitated numerous discoveries over nearly four decades [reviewed in Guest et al. (2013); Monje et al. (2021); Vallejo et al. (2022)]. These cultures are accurate in vitro models for studying neural development, differentiation, regeneration, electrophysiology, and toxicology in normal and disease states. They are also valuable for cell therapy development to repair damage caused by trauma and neurodegenerative disease. Indeed, the transplantation of cultured hSCs from a patient’s sural nerve has been implemented as a strategy to treat spinal cord and peripheral nerve injuries in USA-FDA-regulated clinical trials (Levi et al., 2016; Anderson et al., 2017; Khan et al., 2021).

Empirical data have shown that highly viable, expandable hSC cultures can be established from tissues provided by donors > 60 years of age [reviewed in Bunge et al. (2017)]. Biospecimens from live donors are not required. In fact, hSC cultures from postmortem tissues are phenotypically indistinguishable from those obtained from live donors (Boyer et al., 1994; Casella et al., 1996; Bastidas et al., 2017). Importantly, in vitro cultured SCs from adult nerves retain their ability to proliferate in response to axon contact, promote axonal growth, and form a myelin sheath (Morrissey et al., 1991), which are key functions of SCs during nerve development, maturation, and repair (Jessen et al., 2015).

Two main obstacles must be overcome to establish SC cultures from humans: (1) harvesting sufficient proliferative hSCs from the source tissue; and (2) expanding these primary hSCs sufficiently while maintaining low levels of fibroblast contamination (Morrissey et al., 1995; Peng et al., 2020). Processing adult biospecimens for cell isolation is more time demanding and challenging than doing so from developing (embryonic, neonatal) nerves. This is at least in part due to the presence of multiple layers of connective tissue (CT) and extracellular matrix (ECM), both within and around the SC-enriched fascicles. In addition, the highly elaborate cellular architecture of mature myelinating and ensheathing (non-myelinating or Remak) SCs, which are among the largest cells in the body, impose a hard-to-overcome technical barrier for disintegrating the tissue without disturbing the integrity of the cells. Although isolating hSCs immediately after nerve harvesting is feasible under certain conditions (Weiss et al., 2016), we strongly recommend pre-culturing the tissues without dissociation to achieve more consistent cell yields (Bunge et al., 2017; Chu et al., 2022). We also recommend selective immunological purification protocols to tackle the problem of fibroblast overgrowth, although other methods are available to enrich hSCs over non-glial cell populations.

In the following sections, we present generic protocols for preparing primary hSC cultures by including procedures for: (1) the dissection and harvesting of fascicles, roots, and ganglia from adult human donors; (2) the pre-degeneration of nerve segments, an intermediate step that involves the culturing of intact tissues to enrich the number of hSCs; (3) the disintegration of cultured tissues by proteolytic enzymes and the plating of initial cell suspensions on an adherent substrate; and (4) the growth and management of primary hSCs at the desired quality and quantity (Figures 1 and 2). Relevant notes on the procurement, storage, and handling of patient-derived biospecimens are included along with two different protocols for processing large and small tissue biospecimens. Various recommendations are made for managing the hSC cultures during the initial stages of growth. Supportive data on the properties of hSC cultures prepared according to these methods can be found in our published studies (Monje et al., 2006, 2008 and 2018). Our methodologies were developed using assorted tissue sources from deidentified donors. Importantly, the methods presented here are intended for non-clinical research only and differ substantially from those used in preclinical research (Bastidas et al., 2017) and clinical trials (Khan et al., 2021). However, it should be mentioned that in vitro cultured hSCs from nerves, skin, and ganglia are expected to exhibit fairly similar phenotypic and functional characteristics regardless of the tissue of origin and the mode of preparation (Stratton et al., 2017; Monje et al., 2018 and 2020; Chu et al., 2022).

Figure 2. Isolation and culture of primary human Schwann cell (hSCs). The diagram depicts the overall cell culture strategy, highlighting the timeline for each procedure and the main steps involved, as illustrated by the representative images. This workflow starts with the bioprocessing of peripheral nerve tissues (steps 1–3) and ends with the establishment of confluent cultures of primary hSCs ready to use (step 11). The entire process can take at least three weeks, depending mostly on the length of the pre-degeneration phase (step 4, tissue culture).