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

Background: Over 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 methods: Peripheral 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. Results: The 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 method: While 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. Conclusions: We describe a method for isolating SCs with high purity from both fresh and non-fresh peripheral nerves of adult horses.


Introduction
Axons in the peripheral nervous system are ensheathed by myelin formed by wrappings of the plasma membrane of Schwann cells, a plastic cell type which has important functions in both healthy and injured nerves (Bosch-Queralt, Fledrich, and Stassart, 2023;Cattin and Lloyd, 2016).In healthy nerves, differentiated myelinating and non-myelinating Schwann cells serve trophic support to the axons and regulate nerve impulse conduction.After an axonal injury, Schwann cells dedifferentiate in order to support axon outgrowth and nerve structure repair.When axonal regeneration is completed, the repair Schwann cell transforms back to its differentiated myelinating or non-myelinating phenotype.
Schwannopathy and demyelinating polyneuropathy is a feature in Acquired equine polyneuropathy (AEP) in Nordic horses (Hanche-Olsen et al., 2017).AEP is a neuromuscular disease with unknown etiology, characterized by bilateral knuckling in the metatarsophalangeal joints, primarily of the pelvic limbs (Gröndahl et al., 2012;Hanche-Olsen et al., 2008).The degree of severity in affected horses varies from intermittent tendency for knuckling to recumbency, and the reported mortality rate is 29-32 % (Gröndahl et al., 2012;Hanche-Olsen, 2017).The etiological agent causing AEP in Nordic horses is unknown, although histopathological findings of demyelinating polyneuropathy with Schwann cell inclusions and epidemiological data, suggests a triggering factor causing the Schwannopathy (Hanche-Olsen et al., 2017).
The plasticity of Schwann cells makes it possible to generate cultures of Schwann cells in monoculture or in coculture with neurons.Among various methods, Schwann cell cultures from different species can be established by the isolation of Schwann cells directly from nerve tissue or by induction from mesenchymal stromal cells sourced from bone marrow or adipose tissue (Andersen et al., 2016;Brooks et al., 2018;Cruz Villagrán et al., 2014;Ferreira et al., 2023;Haastert-Talini, 2012).Equine mesenchymal stromal cells derived from bone marrow and adipose tissue are able to differentiate into Schwann-like cells in vitro (Cruz Villagrán et al., 2014;Ferreira et al., 2023).When derived from nerve tissue, Schwann cells can be harvested from embryonic, postnatal, and adult peripheral nerves.Schwann cells have also successfully been cultivated from non-fresh human and rat nerves (Bastidas et al., 2017;Levi et al., 1994).
Primary cell cultures obtained from peripheral nerves usually consist of cocultures of Schwann cells from the nerve fascicles and endoneurial, perineurial and epineurial fibroblasts.The fibroblasts' high abundance and proliferation ratio pose a challenge when isolating Schwann cells, especially from adult peripheral nerves.Various protocols outline procedures to enhance the purity of Schwann cells through the utilization of diverse vessel coatings and selective medium formulations (Kaewkhaw, Scutt, and Haycock, 2012;Pietrucha-Dutczakv et al., 2014;Shojapour et al., 2018).Other techniques aim to remove fibroblasts from the culture by using complement lysis (Brockes, Fields, and Raff, 1979), magnetic beads (Andersen et al., 2016) or fluorescence activated cell sorting (FACS) (Shen et al., 2017).
When studying demyelinating diseases in large animals such as horses, animal welfare considerations often prevent the transport of the live animal to an animal hospital with suitable necropsy and laboratory facilities.Instead, the horse is humanely euthanized in the field.In these cases, it is sometimes possible to remove material immediately after euthanasia and subsequently transport the sample to a laboratory for further examination.
To our knowledge, there are no reports of the cultivation of neuronfree Schwann cells harvested from adult equine peripheral nerves.With regard to our long-time goal to understand the pathogenesis of AEP by studying Schwann cell behavior in vitro, the primary aim of the present study was to cultivate Schwann cells from adult equine peripheral nerves from non-neurological horses.Our second goal was to study the survival of Schwann cells in relation to fibroblasts in cultures from nerves mechanically and enzymatically treated immediately after euthanasia, compared to cultures from nerves kept at 4 • C for 14.5-19 h before further treatment, to simulate cases where material needs to be transported to a laboratory.

Sample collection
Samples were collected from horses (n = 5) that were referred for necropsy due to non-neurological reasons, with the consent of the owners to use the material for research purposes.The horses were two Standardbreds and three Coldblooded trotters, with an age ranging from 3-15 years.No approval of an animal ethics committee was required as the samples were taken postmortem.The left deep fibular nerve (n.peroneus profundus sinister) (hereafter called N1) was obtained under aseptic conditions shortly after euthanasia using pentobarbital, or stunning with a captive bolt gun followed by bleeding.The nerve measured 8 cm (+/-0.5 cm) and was placed in a 50 ml Falcon tube with ice-cold Leibovitz's L-15 medium (L-15, catalog number L5520, Sigma-Aldrich, Germany) containing gentamicin (25 µg/ml, catalog number 15750037, ThermoFisher, USA).To simulate collection and transport in field conditions, the same nerve on the opposite limb (hereafter called N2) was collected in situ within its muscle tissue (m.extensor digitalis longus and m. extensor digitalis lateralis).The muscle tissue containing the nerve was wrapped in a surgical drape and placed at 4 • C for 14.5-19 h.Thereafter N2 was collected and placed in ice-cold L-15 medium containing gentamicin (25 µg/ml).

Establishing primary Schwann cell cultures
Primary cell cultures were prepared following the method described by Andersen et al. (2016) with minor modifications.Inside a horizontal laminar flow hood, the nerve was transferred to a 100 mm culture dish and handled on ice under a stereo microscope.The epineurium was removed with Dumont No 5 forceps.The nerve was subsequently divided in two, and each segment was placed in cooled L-15 medium in a 60 mm culture dish and teased for 35-40 min into individual nerve fibers using No 5 forceps.The nerve fibers were transferred to a new mm culture dish containing fresh medium every 10 min.When all fascicles were separated into finely separated, cotton like fibers, the medium was replaced with 5 ml 0.25 % Dispase II (catalog number D4693, Sigma-Aldrich) and 0.05 % type I collagenase (catalog number 177018029, ThermoFisher) prepared in high-glucose Dulbecco's Modified Eagle Medium (catalog number D6429, DMEM, Sigma-Aldrich) to digest fibers harvested from a nerve segment measuring approximately 4 cm.The mechanically teased nerve fibers were incubated at 37 • C in a 5 % CO 2 incubator for 14-18 h.
The enzymatic process was stopped by adding 8.5 ml 40 % heatinactivated fetal bovine serum (FBS, catalog number 16140071, Ther-moFisher) in Hanks' Balanced Salt Solution (HBSS, catalog number H6648, Sigma-Aldrich).The dissociated fibers were transferred to a Falcon tube together with the medium, and the same medium was used to rinse the dish to collect the remaining cells.The suspension was centrifuged at 200 xg for 10 min at 4 • C. The supernatant was removed, and the cells were resuspended in DMEM containing 10 % FBS, 1 % mM L-alanyl-L-glutamine dipeptide in 0.85 % NaCl (catalog number 35050061, ThermoFisher), 1 % penicillin-streptomycin (catalog number 15140122, ThermoFisher), and 25 µg/ml gentamicin (low proliferation medium).The suspension was centrifuged at 200 xg for 5 min at 4 • C. The supernatant was removed, and the cells were resuspended in 4 ml low proliferation medium containing 10 nM neuregulin (catalog number 78071.1,Stemcell Technologies, UK) and 2 µM forskolin (catalog number F3917, Sigma-Aldrich) (growth medium).The cells were plated in growth medium onto two 100 mm culture plates in droplets, using a glass pipette.The plates were precoated with Poly-L-Lysine (PLL, catalog number P4832, Sigma-Aldrich) and laminin (catalog number 11243217001, Sigma-Aldrich), or laminin or PLL alone.The culture dishes were transferred to a 5 % CO 2 incubator and the cells incubated at 37 • C for 24 h without disturbing the droplets.After 27-75 h the culture dishes were refilled with 8 ml growth medium.Thereafter, media changes of 10 ml fresh growth medium were performed every 2-3 days.

Trypsinization of equine primary Schwann cells for passaging or cryopreservation
When the cell culture of equine primary Schwann cells (P0) reached 70-90 % confluence, the cells were trypsinized prior to subsequent passaging or cryopreservation of passage 1 (P1) cells.The growth medium was removed, and the cells were washed with 5 ml Dulbecco's phosphate-buffered saline (DPBS, catalog number 14190136, Thermo-Fisher).Trypsinization was performed by adding 2.5 ml 0.05 % trypsin EDTA (catalog number 59428 C, Sigma-Aldrich) in HBSS and the culture dish was placed in a 5 % CO 2 incubator at 37 • C for 1 min.The cells were then monitored under a phase contrast microscope during trypsinization, occasionally stirring the vessel carefully with circular movements.When the cells started to detach, trypsinization was stopped by adding E.F.Kvigstad et al. low proliferation medium.The number of viable cells was determined by counting Erythrosin B (catalog number L13002, BioNordika, Norway) negative cells using a Countess II FL Automated cell counter (ThermoFisher).The cells were collected by centrifugation at 200 xg for 10 min at 4 • C.
For passaging of cells, the supernatant was removed and cells resuspended in growth medium before replating onto new pre-coated vessels.The cells were incubated at 37 • C and 5 % CO 2 until desired confluency was reached.
For cryopreservation, the collected cells were resuspended in ice cold low proliferation medium containing 10 % dimethyl sulfoxide (DMSO, catalog number 02196055, MP Biomedicals, USA) at a density of 1.5 × 10 6 cells per ml.One ml cell suspension was transferred to cryogenic vials and immediately placed in a freezing container holding isopropanol, and placed at -80 • C.After 24 h, the vials were transferred to liquid nitrogen.

Culture of passaged equine Schwann cells from cryopreserved stocks
Frozen stocks of cells were thawed in a 37 • C bead bath until approximately 75 % of the volume was melted.The cells were diluted 1:10 in low proliferation medium prior to centrifugation at 200 xg for 10 min at 4 • C. The supernatant was removed, and the collected cells were resuspended in growth medium before seeded as P1 cells.For assessment of Schwann cell purity in cultures from N1 and N2, cells were replated onto 100 mm or 60 mm laminin-coated culture plates.To evaluate the effect of vessel coatings, N1 derived cells from the five horses were plated onto 6-well plates coated with PLL and laminin, or laminin or PLL alone.For immunocytochemistry, P1 cells were passaged and seeded onto PLL and laminin-coated eight well-glass chambered slides (catalog number PEZGS0816, Sigma-Aldrich) as P2 cells.The cells were incubated at 37 • C and 5 % CO 2 .

Coating of cell culture dishes
Culture dishes were precoated with 0.01 % PLL and 0.7 µg laminin per cm 2 .Culture vessels were incubated with PLL for 1 h in room temperature and washed three times with distilled water before drying under a laminar flow hood for 2 h.Laminin coated vessels were incubated with laminin for 1 h in room temperature, washed with distilled water and dried for 30 min under a laminar flow hood.Poly-L-Lysine and laminin-coated vessels were initially coated with PLL, followed by laminin.

Flow cytometry
After trypsinization and centrifugation, cells were resuspended in DPBS and distributed in samples containing 10 6 cells.According to the manufacturer's protocol, Live-Dead staining (Zombie Violet™ Fixable Viability Kit, catalog number 423113, BioLegend®, San Diego, CA, US) was used to exclude dead cells.The samples were washed two times: 0.5 % Bovine Serum Albumin (BSA, catalog number 805030, Bio-Rad, USA) and 0.005 % azide (catalog number 1.06688, Sigma-Aldrich) in DPBS (flow buffer) was added, samples were centrifugated at 800 xg in 1 min, and the supernatant was removed.The cells were incubated for 60 min on ice with anti-human CD90 (5E10) phycoerythrin-conjugated monoclonal antibody (1:20, catalog number A15794, ThermoFisher) and antihuman CD271 (NGFR5) allophycocyanin-conjugated monoclonal antibody (1:15, catalog number AFC-6XPNF0, Nordic BioSite) diluted in flow buffer.Cells were washed, resuspended in flow buffer, and analyzed using a Cytoflex XL flow cytometer (Beckman Coulter) and Kaluza software (Beckman Coulter).Singlets were gated based on side scatter area versus height, and forward scatter area versus side scatter area was used to exclude debris.For evaluation of Schwann cell purity in cultures derived from N1 and N2 cryopreserved stocks, dead cells were excluded by gating on Zombie Violet negative events.Live cells were then subdivided into CD271 -/CD90 -cells, CD271 + /CD90 -cells, CD271 -/ CD90 + cells and CD271 + /CD90 + cells by using unstained cells, single stained and fluorescence minus one controls.To assess the impact of coating treatments on Schwann cell purity and viability, CD271 + cells were identified by setting a gate based on the above mentioned strategy.Subsequently, live CD271 + cells were distinguished by gating on Zombie violet negative CD271 + cells.

Immunocytochemistry
For evaluation of CD271 and CD90 antigen expression, cells were seeded on to eight well-glass chambered slides (catalog number PEZGS0816, Sigma-Aldrich) precoated with PLL and laminin in growth medium with a density of 50.000 cells per well.One day after plating, growth medium was removed, and the cells were washed three times in DPBS and fixed in 4 % paraformaldehyde (catalog number 19210, Electron Microscopy Science, USA) in DPBS for 15 min at room temperature.The cells were rinsed in 0.5 % BSA and 0.005 % azide in DPBS three times, each time for a 5-minute incubation.Thereafter, the cells were incubated for 1 h at room temperature with anti-human CD90 (5E10) phycoerythrin-conjugated monoclonal antibody (1:100, catalog number A15794, ThermoFisher) and anti-human CD271 (NGFR5) allophycocyanin-conjugated monoclonal antibody (1:15, catalog number AFC-6XPNF0, Nordic BioSite) diluted in 0.5 % BSA and 0.005 % azide in DPBS.The cells were washed three times with DPBS before incubation with Hoechst 33342 (1:5000, catalog number B2261, Sigma-Aldrich) for 10 min at room temperature.The cells were washed with DPBS three times and mounted with Fluoromount G (catalog number 00-4958-02, ThermoFisher).Wells where antibodies were omitted were used as negative controls.The samples were analyzed using a Stellaris 5 confocal microscope (Leica, Wetzlar, Germany).

Statistical analysis
Statistical analysis was performed using STATA/SE 16.0 (StataCorp LLC, College Station, Texas, USA).Due to a low number of observations and a non-normal distribution of the data, the descriptive data are presented as the median with range.

Cultivation of primary Schwann cell cultures from horses
The cell cultures were monitored by phase contrast microscopy (Fig. 1).Cells attached to the vessel 1-3 days after plating.After drop plating, cells harvested from N1 reached confluence locally in the original droplets in 5.5 days (median; 4.5-9 days) and 5.5 days (median; 4-15.5 days) in cultures of cells harvested from N2.
Cryopreserved N1 and N2 Schwann cells from horse No. 1 were thawed and replated onto laminin coated vessels.Schwann cells from N1 appeared with longer and more prominent needle shaped processes (Fig. 2a) compared to Schwann cells harvested from N2 (Fig. 2b).Fibroblasts were more evident in cultures from N2 (Fig. 2b), growing among and under Schwann cells.
In passaged cultures with a high Schwann cell purity, the cells aligned to one another in bundles, making a fingerprint-like pattern, when reaching confluence (Fig. 3).The Schwann cell purity in these cultures was above 90 %, as measured by flow cytometry analysis.

Analysis of Schwann cell purity in cultures from N1 and N2 from cryopreserved stocks
Cryopreserved stocks of Schwann cells and fibroblasts were thawed and replated as P1 cells onto laminin-coated 60 mm or 100 mm culture plates.Prior to assessment of Schwann cell purity, the cells were trypsinized and immunostained.CD271 was used as a Schwann cell marker and CD90 as a fibroblast marker.The percentage of Schwann cells (CD271 + /CD90 -cells) and fibroblasts (CD271 -/CD90 + cells) in cultures from each of the five horses included in the study is presented in Table 1.The purity of Schwann cells (CD271 + /CD90 -cells) was 89.5 % (median; 45.5-98.5 %) in cultures from N1 compared to 70.3 % (median; 54.9-97.3) in cultures from N2.The percentages of CD271 + /CD90 -cells and CD271 -/CD90 + cells in a fluorescence intensity dot plot, exemplified by cells of horse No. 3, is illustrated in Fig. 4.

Immunocytochemistry
To evaluate the specificity of antibodies used in the flow cytometry analysis to determine the fraction of fibroblasts and Schwann cells in equine Schwann cell cultures, immunocytochemistry was used (Fig. 5).Fibroblasts expressing CD90 antigen had a flattened appearance with extended processes and dispersed, large nucleus.CD271 positive Schwann cells often exhibited a spindle-shaped, bipolar morphology with long needle-like processes and a condensed smaller oval nucleus.Nonetheless, some Schwann cells resembled fibroblasts, with an expanded phenotype and a bigger and rounded nucleus.

Evaluation of the influence on Schwann cell purity and viability by coating treatments
Viability was defined as maintenance of plasma membrane integrity by using Zombie Violet Viability kit.Gating strategy used to determine the percentage of CD271 + cells and viable CD271 + cells corresponding to different vessel coating is shown in Fig. 6a.The percentage of CD271 + cells was 82.3 % (median; 45.8-96.7),82.3 % (median; 34.4-86.7)and 73.4 % (median; 36.8-96.2) in cultures maintained in wells precoated with laminin, PLL and laminin, and PLL, respectively (Fig. 6b).Cell viability was high (>99 %), in all types of vessel coating (Fig. 6c).

Discussion
The objective of our study was to cultivate Schwann cells derived from adult equine peripheral nerves, both from fresh and non-fresh material, in order to generate cryopreserved material for further hostpathogen/toxin studies.Previous studies have demonstrated the ability of equine mesenchymal stem cells derived from bone marrow and adipose tissue to transdifferentiate into Schwann-like cells in vitro (Cruz Villagrán et al., 2014;Ferreira et al., 2023).To our knowledge, the present study is the first publication of cultivation of Schwann cells derived from adult equine peripheral nerves.
Published methods for cultivating primary Schwann cells from other species are transferable to horses.In this study, we have used a protocol published by Andersen et al. (2016), although we have deviated somewhat from the strict procedure of precoating of vessels.Using laminin and PLL for coating of vessels prior to plating facilitates adhesion of Schwann cells (Monje, 2020).Laminin, a component of nerve tissue extracellular matrix, is recognized by Schwann cell-expressed integrins (Einheber et al., 1993;Hall et al., 1990;Niessen et al., 1994), and PLL is a positively charged substrate which enhances binding of negatively charged cell membranes.For financial and practical reasons in this study, some of the vessels used for drop plating of initial cultures were precoated with laminin or PLL only.When compared in phase-contrast microscopy, there were few or no visual differences in Schwann cell purity between cultures precoated with both PLL and laminin compared to vessels precoated with PLL or laminin alone.Therefore, when the primary cell cultures reached confluency locally in the areas of the droplets, cells from vessels of different precoating were collected and cryopreserved or subcultivated to new laminin-coated vessels.The Schwann cell purity observed by visual inspection was confirmed by flow cytometry in cultures cultivated on PLL and laminin, and PLL or laminin alone.Our findings reveal a median CD271 + cell percentage of 82.3 % in cultures on PLL and laminin, and laminin alone, in contrast to 73.4 % on PLL alone.Equine Schwann cells, similar to Schwann cells of rodent origin, can be cultivated on different coating substrates, in contrast to human Schwann cells, which need laminin to attain higher proliferation rates (reviewed in (Monje, 2020)).Further investigation is required to assess whether coating treatments affect the proliferation rate of equine Schwann cells.
A challenge in veterinary medicine and research, is the limited availability of fresh sample material from diseased animals, as this needs   to be collected immediately after euthanasia.Unfortunately, a significant proportion of cases are lost from sampling as the animal cannot be transported while alive to a necropsy facility, due to the severity of the clinical signs.Our findings suggest that nerve samples obtained from horses euthanized in the field can be chilled and transported to a laboratory for further processing and Schwann cell cultivation, providing material that can be cryopreserved for later analysis.These results align with studies in primary human Schwann cell cultures which can be harvested from postmortem nerve samples (Bastidas et al., 2017).The delayed time interval chosen for harvesting from non-fresh nerve samples corresponds to common express delivery times to domestic and many international destinations, highlighting the implications of these results.Using flow cytometry, we demonstrated the ability to generate primary equine Schwann cell cultures with purity exceeding 90 % from both fresh and non-fresh material (Table 1) from non-neurological horses.We have also recently cultivated Schwann cells from horses diagnosed with acute and chronic peripheral neuropathies, with a delay in time between euthanasia and nerve sampling (unpublished observations).
In general, when examined under a phase contrast microscope, Schwann cells exhibit a spindle-shaped, phase-bright morphology, while fibroblasts appear as large polygonal flat cells.In this study, we demonstrate that equine Schwann cells can form a fingerprint-like pattern in culture, which also has been described in established Schwann cell cultures from rodents and humans (Andersen et al., 2016;Weiss et al., 2016).The fibroblast-like appearance of some equine Schwann cells in our study is analogous to observations in human Schwann cell cultures (Peng et al., 2020).Equine Schwann cells in cultures with high purity (Fig. 2a) possessed more of long needle-like processes compared to Schwann cells in cultures with high fibroblast contamination (Fig. 2b).Schwann cell morphology is known to be influenced by environmental factors like cell density (Monje, 2020), coating treatments (Vleggeert-Lankamp et al., 2004) and medium formulation (Monje et al., 2009).Therefore, the degree of fibroblast contamination may be a contributing factor to the observed phenotypic differences.
In the present study, there is important variability in the degree of Schwann cell purity (45.8-95.8%) between independent cultures.Several factors may contribute to this phenomenon, including variations in the quantity of connective tissue in peripheral nerves among the horses included in the study, the technical skills of the operator when teasing nerve samples into individual fibers, and the enzymatic digestion time of the fascicles.Also, the ratio between the amount of teased nerve fibers and the volume of enzymatic solution may differ across nerve samples, thereby affecting seeding density at drop plating (Shan et al., 2022).
It may be an advantage to have as pure a cell culture as possible when studying Schwann cells in vitro to prevent fibroblasts from outgrowing the culture.Also, when Schwann cells are used as transplants in regenerative medicine, pure SC cultures with minimal fibroblast contamination may be necessary as the fibroblasts might increase scar tissue formation (Kanno et al., 2015).Nevertheless, other studies demonstrate that when fibroblasts are co-transplanted with Schwann cells, they collaborate to better support nerve regeneration (Wang et al., 2017).Fibroblasts and Schwann cells coexist in nerve tissue in vivo, leading to the speculation that the presence of fibroblasts in a culture with Schwann cells may resemble the in vivo state more closely than Schwann cell monocultures.Research indicates that Schwann cells that were cocultured with nerve fibroblasts exhibited increased cell proliferation (Li et al., 2022) and enhanced migration ability (Dreesmann et al., 2009;Li et al., 2022;Zhang et al., 2016).When primary Schwann cell cultures are intended as a research tool for studying Schwann cell biology or as a model for analyzing host-pathogen interactions, the presence of fibroblasts together with Schwann cells may cause bias for interpretation.A pure culture of Schwann cells is often an advantage   when using e.g.western blot analysis, proteomics or transcriptomics as analyzing tools.However, in morphological and single cell studies, immunolabeling can be used to identify the cells and a minor portion of fibroblasts should not have a significant impact.
In our study, the omission of a purification step prevented the possibility to obtain a Schwann cell-enriched culture from both N1 and N2 nerve samples.To increase Schwann cell purity, FACS can be employed as an immunoselection method, utilizing fluorescently tagged CD271 and CD90 antibodies.In the present study, these antibodies have been shown to be suitable for the detection of equine Schwann cells and fibroblasts respectively.A disadvantage using FACS is that it requires a high cell count which can be a challenge to achieve at a low passage number.It is desirable to study the cell cultures at the lowest possible cell passage, since Schwann cells undergo cellular changes as they reach higher passages (Funk, Fricke, and Schlosshauer, 2007).This includes changes in proliferation rate, interactions with the extracellular matrix and the ability to grow in absence of serum.Furthermore, dorsal root ganglion cells were observed to exhibit longer axonal extensions when cocultured with Schwann cells at higher passages, compared to those at lower passages.An alternative is to employ FACS for the separation of Schwann cells from fibroblasts following the completion of the experiment and prior to downstream analysis techniques, such as proteomics, exclusively focusing on this specific cell type.Other selective immunological purification techniques, such as complement lysis and magnetic beads, are possible alternatives to further enhance Schwann cell purity.
In conclusion, techniques for cultivation of Schwann cells from adult equine peripheral nerves were established.We also showed that research material can be transported from the field, by demonstrating that Schwann cells from adult equine peripheral nerves can be isolated and cultured in vitro after a delay of up to 19 h at 4 • C. Our long-term goal is to use equine Schwann cell cultures to study host-pathogen interactions in neuropathies in horses.

Funding
This study was funded by grant number H-18-47-409/NFR298735 from the Swedish-Norwegian Foundation for Equine Research/Research Council of Norway, with contributions from the Norwegian Equine

Fig. 1 .
Fig. 1.Phase-contrast images of primary cells isolated from N1 and N2 from horse No.1 at different time points.(a) Cells attaching to vessel one day (1 d) after drop plating.(b-c) Five days (5 d) after plating, cell growth varies within the same vessel.(d) Cells from N2 five days after drop plating.

Fig. 2 .
Fig. 2. Phase-contrast images of thawed cells (P1) from N1 (a) and N2 (b) from horse No.1.The bottom images are of a higher magnification than the top images.The cultures consist of two types of cells: Schwann cells (black arrows) and fibroblasts (white arrows).Schwann cells are spindle shaped, phase-bright, and bipolar or tripolar cells, while fibroblasts are flattened polygonal cells with a large amount cytoplasm.In the culture from N2 (b), fibroblasts are more evident.Schwann cells in cultures from N1 (a) show more prominent needle shaped processes (arrowheads) compared to Schwann cells in cultures from N2.

Fig. 3 .
Fig. 3. Phase-contrast images of Schwann cell culture at confluence.Cells align to one another (a) making a fingerprint-like pattern (b).

Fig. 4 .
Fig. 4. Flow cytometric analysis data of Schwann cell purity using CD271 as a Schwann cell marker and CD90 as a fibroblast marker, from horse No. 3 at passage 1.In N1, 89.45 % of the cells were CD271 positive and CD90 negative (lower right quadrant), while 9.25 % were CD90 positive and CD271 negative (upper left quadrant) (a).In N2, 70.31 % of the cells were CD271 positive and CD90 negative (lower right quadrant), while 27.82 % were CD90 positive and CD271 negative (upper left quadrant) (b).

Fig. 5 .
Fig. 5. Detection of equine Schwann cells and fibroblasts by immunocytochemistry.Passage culture (P2) of Schwann cells were positive for CD271 (red) and negative for CD90 (green) immunostaining.Fibroblasts were negative for CD271 and positive for CD90 immunostaining.The nuclei were located by Hoechst 33342 (blue).Schwann cells exhibited two phenotypes: spindle-shaped and elongated (arrow) or extended and flattened (arrowhead).

Fig. 6 .
Fig. 6.The effect of vessel coating on cultivated equine Schwann cell purity and viability measured by flow cytometry, using CD271 antibody for detection of Schwann cells and Zombie Violet Cell Viability Kit for exclusion of non-viable cells.Images showing gating strategy used to determine the percentage of viable CD271 + cells for each coating condition tested: laminin (left panels), PLL and laminin (middle panels) and PLL (right panels) (a).The median percentage of CD271 + cells was 82.3 %, 82.3 % and 73.4 % in cultures cultivated on laminin, PLL and laminin, and PLL, respectively (n = 5) (b).Median percentage of live CD271 + cells was 99.5 %, 99.8 % and 99.5 % in cultures cultivated on laminin, PLL and laminin, and PLL, respectively (n = 5) (c).Error bars represent the range.