Schwann Cell Purity Research Articles

Cultivation of Schwann cells from fresh and non-fresh adult equine peripheral nerves

BackgroundOver the past 25 years, acquired equine polyneuropathy (AEP) has emerged as a neurological disease in Scandinavian horses. This condition is characterized by histopathological features including the presence of Schwann cell (SC) inclusions. Cultivated equine SCs would serve as a valuable resource for investigations of factors triggering this Schwannopathy. Ideally, cells should be sampled for cultivation from fresh nerves immediately after death of the animal, however the availability of fresh material is limited, due to the inconsistent case load and the inherent technical and practical challenges to collection of samples in the field. This study aimed to cultivate SCs from adult equine peripheral nerves and assess their ability to survive in sampled nerve material over time to simulate harvesting of SCs in field situations. New methodsPeripheral nerves from five non-neurological horses were used. After euthanasia, both fresh and non-fresh nerve samples were harvested from each horse. Flow cytometry was employed to confirm the cellular identity and to determine the SC purity. ResultsThe results revealed successful establishment of SC cultures from adult equine peripheral nerves, with the potential to achieve high SC purity from both fresh and non-fresh nerve samples. Comparison with existing methodWhile most SC isolation methods focus on harvest of cells from fresh nerve materials from laboratory animals, our approach highlights the possibility of utilizing SC cultures from field-harvested and transported nerve samples from horses. ConclusionsWe describe a method for isolating SCs with high purity from both fresh and non-fresh peripheral nerves of adult horses.

Differentiation of Human Induced Pluripotent Stem Cells into Mature and Myelinating Schwann Cells.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days. The SC identity was characterized by expression of SRY-Box Transcription factor 10 (SOX10), S100b, glial fibrillary acidic protein (GFAP), P75, growth-associated protein 43 (GAP43), and early growth response 2 (EGR2) markers. The SC purity reached 87% as assessed by flow cytometry using the specific SOX10 marker, and 69% based on S100b expression. When SCs were cocultured with iPSC-derived motor neurons two-dimensionally or three-dimensionally (3D), they also expressed the markers of myelin MBP, MPZ, and gliomedin. Likewise, when they were seeded on the opposite side of a porous collagen sponge from motor neurons in the 3D model, they were able to migrate through it and colocalize with motor axons after 8 weeks of maturation. Moreover, they were shown by transmission electron microscopy to form myelin sheaths around motor axons. These results suggest that the use of autologous iPSC-derived SCs for clinical applications such as the repair of peripheral nerve damage, the treatment of spinal cord injuries, or for demyelinating diseases could be a valuable option. Impact Statement Peripheral nerve injuries can cause the complete paralysis of the upper or lower limbs, which considerably reduces the quality of life of patients. To repair this injury, many approaches have been developed by tissue engineering. Combining biomaterials with Schwann cells (SCs) has been shown to be an effective solution for stimulating nerve regeneration. However, the challenge faced concerns the strategy for obtaining autologous SCs to treat patients. A promising approach is to differentiate them from the patient's own cells, previously induced into pluripotent stem cells. We propose a fast culture method to generate functional SCs differentiated from induced pluripotent stem cells.

An RNA-sequencing transcriptome of the rodent Schwann cell response to peripheral nerve injury

BackgroundThe important contribution of glia to mechanisms of injury and repair of the nervous system is increasingly recognized. In stark contrast to the central nervous system (CNS), the peripheral nervous system (PNS) has a remarkable capacity for regeneration after injury. Schwann cells are recognized as key contributors to PNS regeneration, but the molecular underpinnings of the Schwann cell response to injury and how they interact with the inflammatory response remain incompletely understood.MethodsWe completed bulk RNA-sequencing of Schwann cells purified acutely using immunopanning from the naïve and injured rodent sciatic nerve at 3, 5, and 7 days post-injury. We used qRT-PCR and in situ hybridization to assess cell purity and probe dataset integrity. Finally, we used bioinformatic analysis to probe Schwann cell-specific injury-induced modulation of cellular pathways.ResultsOur data confirm Schwann cell purity and validate RNAseq dataset integrity. Bioinformatic analysis identifies discrete modules of genes that follow distinct patterns of regulation in the 1st days after injury and their corresponding molecular pathways. These findings enable improved differentiation of myeloid and glial components of neuroinflammation after peripheral nerve injury and highlight novel molecular aspects of the Schwann cell injury response such as acute downregulation of the AGE/RAGE pathway and of secreted molecules Sparcl1 and Sema5a.ConclusionsWe provide a helpful resource for further deciphering the Schwann cell injury response and a depth of transcriptional data that can complement the findings of recent single cell sequencing approaches. As more data become available on the response of CNS glia to injury, we anticipate that this dataset will provide a valuable platform for understanding key differences in the PNS and CNS glial responses to injury and for designing approaches to ameliorate CNS regeneration.

Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeutic intervention. A high purity of isolated SC cultures used for experimentation must be demonstrated to exclude that novel findings are derived from a contaminating fibroblasts population. In addition, information about the SC proliferation status is an important parameter to be determined in response to different treatments. The evaluation of SC purity and proliferation, however, usually depends on the time consuming, manual assessment of immunofluorescence stainings or comes with the sacrifice of a large amount of SCs for flow cytometry analysis. We here show that rat SC culture derived cytospins stained for SC marker SOX10, proliferation marker EdU, intermediate filament vimentin and DAPI allowed the determination of SC identity and proliferation by requiring only a small number of cells. Furthermore, the CellProfiler software was used to develop an automated image analysis pipeline that quantified SCs and proliferating SCs from the obtained immunofluorescence images. By comparing the results of total cell count, SC purity and SC proliferation rate between manual counting and the CellProfiler output, we demonstrated applicability and reliability of the established pipeline. In conclusion, we here combined the cytospin technique, a multi-colour immunofluorescence staining panel, and an automated image analysis pipeline to enable the quantification of SC purity and SC proliferation from small cell aliquots. This procedure represents a solid read-out to simplify and standardize the quantification of primary SC culture purity and proliferation.

Detailed Protocols for the Isolation, Culture, Enrichment and Immunostaining of Primary Human Schwann Cells.

This chapter emphasizes detailed protocols for the effective establishment of highly enriched human Schwann cell cultures and their characterization via immunostaining. The Schwann cells are isolated from immediately dissociated fascicle tissue and expanded prior to purification. Two purification methods are described that use either fluorescence-activated cell sorting for the Schwann cell marker TNR16 (p75NTR) or a less-manipulative two-step enrichment exploiting the differential adhesion properties of Schwann cells and fibroblasts, which is especially useful for low Schwann cell numbers. In addition, a method to determine Schwann cell purity via stained cytospin slides is introduced. Together with an immunofluorescence staining procedure for the combined analysis of extra- and intracellular markers, this chapter provides a solid basis to study human primary Schwann cells.

A novel method for obtaining highly enriched Schwann cell populations from mature monkey nerves based on in vitro pre‑degeneration.

Schwann cells (SCs) are indispensable for cell therapy and tissue engineering of the peripheral nervous system. Easy access to activated, highly proliferative SCs is necessary for clinical applications. The present study developed a fast, efficient method for obtaining highly purified SCs from the peripheral nerve of a mature Rhesus monkey. The common peroneal nerves of 4-year-old Rhesus monkeys were harvested and subjected to in vitro pre-degeneration in a modified SC culture medium (SCCM). The nerve pieces were subsequently treated enzymatically to dissociate the cells and then cultured for 2 days in SCCM. Cultured cells were treated with purification medium containing Ara-C to assist in restricting the overgrowth of fibroblast-like cells, for 24 h. After another 24-h cultivation period, the cells were subsequently treated with a multiplex collagenase, which enabled SC detachment over fibroblast detachment, and thereby facilitated SC isolation. Finally, SCs were cultured in SCCM. The cell yield was determined by cell counting following enzyme digestion and SC purity was determined from the percentage of SCs with respect to the total number of cells. Following purification, 96.3±3.9% of cells were identified as SCs. In vitro pre-degeneration in the presence of basic-fibroblast growth factor, heregulin β1 and forskolin maximized the purity and yield of SCs that could be obtained from monkey peroneal nerves. The present study identified a novel technique that can efficiently isolate and purify SCs from mature monkey nerves based on in vitro pre-degeneration.

Isolation and Purification of Schwann Cells from Human Peripheral Nerves via the Cold Jet Technique

Schwann cells (SCs) play an important role in peripheral nerve regeneration. However, established methods for SC preparation have limitations in that they are non-specific and time-consuming. The purpose of this study was to assess the amount and purity of SCs isolated from human peripheral nerve tissue using the cold jet technique. The cold jet technique is an SC isolation and purification method that exploits the different responses of the SCs and fibroblasts to temperature shock. Specifically, the temperature shock causes specific detachment of SCs from fibroblasts. A total of 32 human peripheral nerve tissues (sensory and motor) were obtained from reconstruction procedures for oral cancer. After the length and weight of nerve segments were measured, the nerve fascicles were digested and SCs were isolated and purified by the cold jet technique. The purity of SCs was confirmed by morphology, RT-PCR, immunohistochemistry and flow cytometry. Phasecontrast photomicrographs, S100 mRNA expression rates and immunohistochemical staining show that the SC purification rate was greater with the cold jet technique. Flow cytometry showed the percentage (%) gated value increased from 56.10 to 95.95%. On average, 2.48 ± 2.57 × 106 SCs/g or 0.16 ± 0.18 × 106 SCs/cm were harvested efficiently from human peripheral nerve segments. The cold jet technique was an efficient method for SC isolation and purification from the human peripheral nerves. The determined amount and the level of purity of the SC harvested by this method may be helpful for tissue engineering or cell therapy

Generation of pure cultures of autologous Schwann cells by use of biopsy specimens of the dorsal cutaneous branches of the cervical nerves of young adult dogs

OBJECTIVE To identify an optimal technique for isolation, purification, and amplification of Schwann cells (SCs) from biopsy specimens of the dorsal cutaneous branches of the cervical nerves of dogs. SAMPLE Biopsy specimens of dorsal cervical cutaneous nerves from the cadavers of three 1- to 2-year-old dogs. PROCEDURES Nerve specimens were dissected, predegenerated, and dissociated to isolate single cells. After culture to enhance SC growth, cells were immunopurified by use of magnetic beads. Cell purity was evaluated by assessing expression of cell surface antigens p75 (to detect SCs) and CD90 (to detect fibroblasts). Effects of various concentrations of recombinant human glial growth factor 2 (rhGGF2) on SC proliferation were tested. Cell doubling time was assessed in SC cultures with selected concentrations of rhGGF2. RESULTS Mean ± SD wet weight of nerve fascicles obtained from the biopsy specimens was 16.8 ± 2.8 mg. A mean predegeneration period of 8.6 days yielded approximately 6,000 cells/mg of nerve tissue, and primary culture yielded 43,000 cells/mg of nerve tissue in a mean of 11 days, of which 39.9 ± 9.1% expressed p75. Immunopurification with magnetic beads yielded a mean of 85.4 ± 1.9% p75-positive cells. Two passages of subculture with 10μM cytosine arabinoside further enhanced SC purity to a mean of 97.8 ± 1.2% p75-positive cells. Finally, rhGGF2 supplementation at a range of 40 to 100 ng/mL increased the SC proliferation rate up to 3-fold. CONCLUSIONS AND CLINICAL RELEVANCE SCs could be cultured from biopsy specimens of dorsal cervical cutaneous nerves and purified and expanded to generate adequate numbers for autologous transplants to treat dogs with spinal cord and peripheral nerve injuries.

Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(P<0.01). Immunofluorescence staining showed that the SCs purity was (95.73±1.51)% in the improved method group,(84.66±2.68)% in the hemi-explants-culture method group,and (74.50±4.23)% in the explants-culture method group(P<0.01). Conclusion The improved enzyme digestion combined with explants-culture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

Efficacy of optimized in vitro predegeneration period on the cell count and purity of canine Schwann cell cultures.

Predegeneration is a standard technique to obtain mitotically activated and enriched cultures of Schwann cells (SCs). This study, for the first time, evaluated the impact of various duration of predegeneration on cell yield and enrichment of SCs from dog peripheral nerve. Dog sural nerves were subjected to 5, 10, 15 day-long in vitro predegeneration. The total cell yield and the purity of SCs were evaluated in each group on the first and seventh day after plating. The maximum and minimum numbers of cells were counted in 15 day-long predegene-ration and control groups which underwent no predegeneration. The 10 day-long in vitro predegeneration group with 80±0.5% SCs enrichment had the best purity after plating day and could maintain its purity with elapsing on cultures. 10 day-long predegeneration results in the higher cell number and the better and prolonged purity of SCs in culture.

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves.

Schwann cells (SCs), the supporting cells of the peripheral nerves, are indispensable for regenerating the peripheral and central nervous system. Copious preparation of these cells in a well-defined manner is to be a privileged position. SCs cultivation is overwhelmed by contaminating fibroblasts which are often outgrowing as the predominant cell type in an in vitro culture. This study introduces a technically simple and efficient procedure for SCs isolation and enrichment based on implementing recombinant and defined supplements. Collected adult rat sciatic nerves were cultured for 10days as in vitro predegeneration. After dissociation and plating, the medium changed to knockout serum replacement supplemented DMDM/F12 medium containing various growth factors. The whole procedure took 3weeks and SCs purity was then evaluated through implementing specific cytoplasmic and membranous markers. The viability of enriched SCs were evaluated by MTT assay. Within 10days, over 99% homogenous SCs were achieved and confirmed through immunofluorescence staining and flow-cytometry for P75(NTR) and S100 markers, respectively. MTT data revealed that the viability and metabolic activities of purified SCs were increased in expansion medium. This study provides a technically easy and efficient method with the benefits of not utilizing bovine serum or other animal products for SCs isolation and enrichment.

Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.

Dorsal root ganglion-derived Schwann cells combined with poly(lactic-co-glycolic acid)/chitosan conduits for the repair of sciatic nerve defects in rats.

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and purification of Schwann cells are complicated by contamination with fibroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly purified Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells (> 95%) using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly(lactic-co-glycolic acid)/chitosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were significantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental findings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly(lactic-co-glycolic acid)/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

A new protocol for cultivation of predegenerated adult rat Schwann cells

The purpose of this study was to optimize the methodology of cultivation of predegenerated Schwann cells (SCs). SCs were isolated from 7-day-predegenerated sciatic nerves of adult rats. We applied commercially available culture medium for cultivation of endothelial cells endothelial cell culture medium (EBM-2) instead of Dulbecco's Modified Eagle's Medium commonly used to culture adult Schwann cells. Additionally, cell culture medium was supplemented with factors specifically supporting SCs growth as: bovine pituitary extract (5 μg/ml), heregulin (40 ng/ml) and insulin (2.5 ng/ml). Similarly to the reports of others authors, we did not observe any beneficial effects of Forskolin application, so we didn't supplement our medium with it. Cell culture purity was determined by counting the ratio of GFAP, N-Cadherin and NGFR p75-positive cells to total number of cells. About 94-97 % of cells were confirmed as Schwann cells. As a result, we obtained sufficient number and purity of Schwann cells to be applied in different experimental models in rats. EBM-2 medium coated with fibronectin was the best for cultivation of adult rat Schwann cells.

Two-photon polymerization-generated and micromolding-replicated 3D scaffolds for peripheral neural tissue engineering applications

In this study, we explore the production of well-defined macroscopic scaffolds with two-photon polymerization (2PP) and their use as neural tissue engineering scaffolds. We also demonstrate that these 3D scaffolds can be replicated via soft lithography, which increases production efficiency. Photopolymerizable polylactic acid (PLA) was used to produce scaffolds by 2PP and soft lithography. We assessed the biocompatibility of these scaffolds using an SH-SY5Y human neuronal cell line and primary cultured rat Schwann cells (of direct relevance to the repair of nerve injuries). A Comet assay with SH-SY5Y human neuronal cells revealed minimal DNA damage after washing the photocured material for 7 days in ethanol. Additionally, thin films and 3D scaffolds of the photocured PLA sustained a high degree of Schwann cell purity (99%), enabled proliferation over 7 days and provided a suitable substrate for supporting Schwann cell adhesion such that bi-polar and tri-polar morphologies were observed. Evidence of orthogonally aligned and organized actin thin filaments and the formation of focal contacts were observed for the majority of Schwann cells. In summary, this work supports the use of PLA as a suitable material for supporting Schwann cell growth and in turn use of 3D soft lithography for the synthesis of neural scaffolds in nerve repair.

Purification of Schwann cells from adult rats by differential detachment

Differential detachment by collagenase treatment is a new and efficient method for Schwann cell (SC) purification. As its effect on adult animals remains unclear, we have investigated the possibility of SC purification from adult rats. To avoid any systematic bias, Schwann cell purity before and after purification were compared by morphology, immunostaining of P75(NTR) and S100 and flow cytometric analysis. The final SC purities reached 99% as confirmed by three independent analyses SC purity and the cell yields were above 10(6) cells after two rounds of purification. The method of differential detachment is also suitable for SC purification in adult rats and could be useful for research and clinical applications.

Application of melanocyte growh medium in the culture of Schwann cells from adult SD rats:an experimental study

Objective To develop a simple and effective method to obtain highly purified Schwann cells. Methods After enzymatic digestion of Predegenerated sciatic nerves from 20 adult SD rats were with 1 week ion, the suspension were cultured in group A (DMEM/F12 medium) and group B (melanocyte growth medium) . cold jet as the passage method. S-100 stain for detection of Schwann cell purity. Results The cell yields and purity of group B was beyond 90%. Conclusion The combination of melanocyte growth medium and cold jet provides a convenient mean to obtain considerable Schwann cells with purity. Key words: Schwann cells; Cell culture techniques; Melanocyte growth medium

Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment

Schwann cell purification is usually difficult due to the contamination of fibroblasts, which often become a predominant cell type in Schwann cell culture in vitro. We have developed a novel and efficient method to enrich Schwann cells by differential detachment of two types of cells. In the culture, cells were treated with a multiplex collagenase and the Schwann cells were found to detach faster than fibroblasts and thus Schwann cells could be easily isolated. Within 5 days, Schwann cell purity could reach above 99%, which was confirmed by immunostaining characterization and flow cytometric analysis. In addition, this efficient method can reach a high cell yield after two rounds of differential detachment procedures and does not require antimitotic treatment or special equipments as often needed by other reported methods.

A method for isolation and cultivation of adult Schwann cells for nerve conduit.

It has been found that one of the methods to repair peripheral nervous system or even central nervous system injury is to use Schwann cells as nerve regeneration promoters. Therefore, it seems necessary to look for a way to obtain activated Schwann cells, with a sufficient amount of numbers and purity, in a short time for clinical applications. However, the previous methods using mitogens are not much clinically acceptable, and other methods that do not require mitogens, fail to isolate adult Schwann cells effectively or require a long period of time. In this study, Schwann cells were isolated from predegenerated sciatic nerves of adult rat (one to three nerves per primary culture) and subcultivated two times in a week with the 10%fetal bovine serum supplementation. Thereafter, Dulbecco's Modified Eagle's Medium media supplemented with 10%, 5%, 2.5%, 1.25%, and 0.625% fetal bovine serum were employed to determine their influence on the density and purity of Schwann cells after a 10-day period of cultivation. The concentrations of fetal bovine serum less than 10% immediately stimulated some morphological changes to happen in Schwann cells but not fibroblasts. Finally, Schwann cells acquired their normal shape on day 6 when fibroblasts just began to alter and die. Our results demonstrated that total cell density was highly significant (P<0.05) in the medium supplemented with 10% fetal bovine serum (950 cells/mm(1)) while purity was significant (P<0.05) in the medium supplemented with 2.5% fetal bovine serum (97%) in comparison with other concentrations of fetal bovine serum.

Variable galactocerebroside expression by human Schwann cells in dissociated and peripheral nerve explant cultures

It has been well established that rat Schwann cells down regulate their cell-surface expression of galactocerebroside (GalC) in vitro under normal cell culture conditions. To determine whether human Schwann cells exhibit a similar down-regulation of GalC in vitro we examined GalC expression in dissociated human Schwann cell cultures derived from normal adult peripheral nerve. Twenty-four hours post-dissociation up to 63% of human Schwann cells were found to express detectable levels of GalC on their surface whereas less than 8% of the Schwann cells expressed detectable levels of GalC at 14 days post-dissociation. In contrast, after nearly 3 months of peripheral nerve explant culture, greater than 30% of human Schwann cells still retained their GalC expression. A similar pattern was also observed when analyzing Schwann cell purity with dissociated cultures exhibiting a rapid decrease in Schwann cell purity under normal culturing conditions although Schwann cell purity was found to be largely unaffected during the period of peripheral nerve explant culture. In summary, we found there was less variation in both GalC expression and Schwann cell purity with time in peripheral nerve explant cultures than dissociated cultures.