Schwann Cell Viability Research Articles

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.

Toxic effects of bortezomib on primary sensory neurons and Schwann cells of adult mice.

The proteasome inhibitor bortezomib is nowadays first line treatment for multiple myeloma. One of the most significant adverse events is peripheral neuropathy, mainly involving sensory nerve fibers that can lead to withdrawal of treatment. Here we develop an in vitro model to compare the effects of bortezomib on primary sensory neurons and Schwann cells of adult mice. We observed that sensory neurons were more susceptible to bortezomib, and their viability was reduced at a concentration of 6 nM, that only affected Schwann cell proliferation but not survival. At concentration higher than 8 nM Schwann cell viability was also compromised. Already at low concentrations, surviving neurons presented alterations in neurite outgrowth. Neurites were shorter and had dystrophic appearance, with alterations in neurofilament staining. However, neurites were able to regrow after removing bortezomib from the medium, thus indicating reversibility of the neurotoxicity. We confirmed in vivo that bortezomib produced alterations in neurofilaments at early stages of the treatment. After an accumulated dose of 2 mg/kg bortezomib, dorsal root ganglia neurons of treated animals showed accumulation of neurofilament in the soma. To evaluate if this accumulation was related with alterations in axonal transport, we tested the ability of sensory neurons to retrogradely transport a retrotracer applied at the distal nerve. Treated animals showed a lower amount of retrotracer in the soma 24 h after its application to the tibial nerve, therefore suggesting that axonal transport was affected by bortezomib.

Cellulose/soy protein isolate composite membranes: evaluations of in vitro cytocompatibility with Schwann cells and in vivo toxicity to animals.

To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals. A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard. The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals. The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

Activation of Schwann cells in vitro by magnetic nanocomposites via applied magnetic field.

Schwann cells (SCs) are attractive seed cells in neural tissue engineering, but their application is limited by attenuated biological activities and impaired functions with aging. Therefore, it is important to explore an approach to enhance the viability and biological properties of SCs. In the present study, a magnetic composite made of magnetically responsive magnetic nanoparticles (MNPs) and a biodegradable chitosan–glycerophosphate polymer were prepared and characterized. It was further explored whether such magnetic nanocomposites via applied magnetic fields would regulate SC biological activities. The magnetization of the magnetic nanocomposite was measured by a vibrating sample magnetometer. The compositional characterization of the magnetic nanocomposite was examined by Fourier-transform infrared and X-ray diffraction. The tolerance of SCs to the magnetic fields was tested by flow-cytometry assay. The proliferation of cells was examined by a 5-ethynyl-2-deoxyuridine-labeling assay, a PrestoBlue assay, and a Live/Dead assay. Messenger ribonucleic acid of BDNF, GDNF, NT-3, and VEGF in SCs was assayed by quantitative real-time polymerase chain reaction. The amount of BDNF, GDNF, NT-3, and VEGF secreted from SCs was determined by enzyme-linked immunosorbent assay. It was found that magnetic nanocomposites containing 10% MNPs showed a cross-section diameter of 32.33±1.81 µm, porosity of 80.41%±0.72%, and magnetization of 5.691 emu/g at 8 kOe. The 10% MNP magnetic nanocomposites were able to support cell adhesion and spreading and further promote proliferation of SCs under magnetic field exposure. Interestingly, a magnetic field applied through the 10% MNP magnetic scaffold significantly increased the gene expression and protein secretion of BDNF, GDNF, NT-3, and VEGF. This work is the first stage in our understanding of how to precisely regulate the viability and biological properties of SCs in tissue-engineering grafts, which combined with additional molecular factors may lead to the development of new nerve grafts.

Bioplotting Alginate/Hyaluronic Acid Hydrogel Scaffolds with Structural Integrity and Preserved Schwann Cell Viability

Bioplotting is an emerging freeform scaffold fabrication technique useful for creating artificial tissue scaffolds containing living cells. Simultaneous maintenance of scaffold structural integrity and cell viability is a challenging task. In this article, we present strategies developed to bioplot alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells for potential use in peripheral nerve tissue engineering. The fabrication platform, upon which the scaffold is created, was coated with the polycation polyethylenimine to immobilize the first layer of the scaffold on the platform. Each layer was then dispensed into a bath containing calcium chloride to cross-link the alginate, polyvinyl alcohol, and low concentrations of polyethylenimine to improve the structural integrity of the resulting scaffolds while retaining biocompatibility. The present study illustrated that with these strategies, porous alginate/hyaluronic acid scaffolds can be fabricated with good structural integrity and long-term cell viability. Plotting method and speed were also important factors determining the printability of single-layered scaffolds. The methods developed and the results obtained from this study form the basis for a general bioplotting method by which more sophisticated alginate/hyaluronic acid scaffolds can be fabricated with the inclusion of living cells into desired structures.

Enhancement of primary neuronal cell proliferation using printing-transferred carbon nanotube sheets.

Artificial nerve guidance conduits (aNGCs) prepared from polymer scaffolds and carbon nanotubes (CNTs) possess unique chemical and physical properties, and have been widely used in preclinical trials to promote neuronal differentiation and growth. However, there have been only a few reports on the clinical applicability of CNT sheets for proliferation of primary neuronal cells due to safety concerns. The present study assesses the ability and potential applicability of multiwalled CNTs (MWNTs) composited with polydimethylsiloxane (PDMS) sheets to promote and enhance the proliferation of primary neuronal cells. In this study, the aqueous MWNT dispersion was filtered, and the PDMS/MWNT sheets were prepared using a simple printing transfer method. Characterization of PDMS/MWNT sheets demonstrated their unique physical properties such as superior mechanical strength and electroconductivity when compared with PDMS sheets. The effect of the PDMS/MWNT sheets on the neural cell proliferation and cytotoxicity was evaluated using MTT and alamar blue assays. Our results indicate the viability and proliferation of primary neuronal cells and Schwann cells in PDMS/MWNT sheets increased over twice when compared with a noncoated dish that is not usual in the primary neuronal cell growth control (p < 0.05). In addition, PDMS/MWNT sheets enhanced the adhesion and viability of the cells compared with poly-l-lysine coated dishes, which are most commonly used for improving cell adherence. Additionally, the PDMS/MWNT sheets exhibited excellent biocompatibility for culturing neuronal and Schwann cells. Overall, all assessments indicate that PDMS/MWNT sheets are ideal candidates for the development of artificial nerve conduits for clinical use following peripheral nerve injury.

The aligned core-sheath nanofibers with electrical conductivity for neural tissue engineering.

Currently, electroactive biomaterials have often been fabricated as tissue engineering scaffolds to provide electrical stimulation for neural tissue engineering. The goal of this work was to study the synergistic effect of electrical stimulation and nerve growth factor (NGF) on neuron growth. The composite meshes of polyaniline (PANi) and well-blended poly(l-lactic acid-co-ε-caprolactone)/silk fibroin (PS) incorporated with nerve growth factor (NGF) were prepared by coaxial electrospinning. The results showed that the increased concentration of PANi had a large effect on the fiber diameter, which was significantly reduced from 683 ± 138 nm to 411 ± 98 nm and then increased to 498 ± 100 nm. The contact angles and Young's modulus decreased to 28.3°± 5.4° and 7.2 ± 1.2 MPa, respectively, and the conductance increased to 30.5 ± 3.1 mS cm-1. The results of the viability and morphology of mouse Schwann cells on the nanofibrous meshes showed that PS-PANi-1 loaded with NGF exhibited the highest cell number after 5 days culture and the aligned nanofibers could guide cell orientation. The synergistic effects of electrical stimulation and NGF were also investigated via the growth and differentiation of rat pheochromocytoma 12 (PC12) cells. The scaffolds loaded with NGF under electrical stimulation could effectively support PC12 neurite outgrowth and increase the percentage of neurite-bearing cells as well as the median neurite length. More importantly, the NGF release from the conductive core-shell structure nanofiber could be increased by electrical stimulation. These promising results demonstrated that there was a potential use of this functional scaffold for nerve tissue regeneration.

Synthesis, characterization and biological evaluation of poly [LA-co-(Glc-alt-Lys)] for nerve regeneration scaffold

A novel nerve repairing material poly [LA-co-(Glc-alt-Lys)] (PLGL) was synthesized. The viability and growth of Schwann cells (SCs) co-cultured with poly (D, Llactic acid) (PDLLA) films (control group) and PLGL films were evaluated by MTTassay and SEM observation. Then, contact angle measurement, histological assessment and enzyme-linked immunosorbent assay (ELISA) testing on inflammatory-related cytokines such as IL-10 and TGF-β1 were performed. The results showed that, compared with PDLLA, PLGL films possesses better hydrophilicity, biocompatibility, degradation property and less inflammatory reaction. The present study indicated that PLGL scaffolds would meet the requirements of artificial nerve scaffold and have a potential application in the fields of nerve regeneration.

Interleukin-17 impedes Schwann cell-mediated myelination

BackgroundPro-inflammatory cytokines are known to have deleterious effects on Schwann cells (SCs). Interleukin 17 (IL-17) is a potent pro-inflammatory cytokine that exhibits relevant effects during inflammation in the peripheral nervous system (PNS), and IL-17-secreting cells have been reported within the endoneurium in proximity to the SCs.MethodsHere, we analyzed the effects of IL-17 on myelination and the immunological properties of SCs. Dorsal root ganglia (DRG) co-cultures containing neurons and SCs from BL6 mice were used to define the impact of IL-17 on myelination and on SC differentiation; primary SCs were analyzed for RNA and protein expression to define the putative immunological alignment of the SCs.ResultsSCs were found to functionally express the IL-17 receptors A and B. In DRG cultures, stimulation with IL-17 resulted in reduced myelin synthesis, while pro-myelin gene expression was suppressed at the mRNA level. Neuronal outgrowth and SC viability, as well as structural myelin formation, remained unaffected. Co-cultures exhibited SC-relevant pro-inflammatory markers, such as matrix metalloproteinase 9 and SCs significantly increased the expression of the major histocompatibility complex (MHC) I and exhibited a slight, nonsignificant increase in expression of MHCII, and a transporter associated with antigen presentation (TAP) II molecules relevant for antigen processing and presentation.ConclusionsIL-17 may act as a myelin-suppressive mediator in the peripheral nerve, directly propagating SC-mediated demyelination, paralleled by an inflammatory alignment of the SCs. Further analyses are warranted to elucidate the role of IL-17 during inflammation in the PNS in vivo, which could be useful in the development of target therapies.

Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis

Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

When Schwann Cells Conspire with Mitochondria, Neighboring Axons Are under Attack by Glia-Derived Neurotoxic Lipids

In this issue of Neuron, Viader etal. (2013) report on the production of toxic lipids by Schwann cells deficient in mitochondrial respiration, which are capable of destroying neighboring axons. This may have implications for understanding the neurobiology and treatment of mitochondrial neuropathies.

In Vitro Antiseptic Effects on Viability of Neuronal and Schwann Cells

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Ferulic Acid Enhances Peripheral Nerve Regeneration across Long Gaps

This study investigated the effect of ferulic acid (FA) on peripheral nerve injury. In the in vitro test, the effect of FA on viability of Schwann cells was studied. In the in vivo test, right sciatic nerves of the rats were transected, and a 15 mm nerve defect was created. A nerve conduit made of silicone rubber tube filled with FA (5 and 25 μg/mL), or saline (control), was implanted into the nerve defect. Results show that the number of proliferating Schwann cells increased significantly in the FA-treated group at 25 μg/mL compared to that in the control group. After 8 weeks, the FA-treated group at 25 μg/mL had a higher rate of successful regeneration across the wide gap, a significantly calcitonin gene-related peptide (CGRP) staining of the lamina I-II regions in the dorsal horn ipsilateral to the injury, a significantly diminished number of macrophages recruited, and a significantly shortening of the latency and an acceleration of the nerve conductive velocity (NCV) of the evoked muscle action potentials (MAPs) compared with the controls. In summary, the FA may be useful in the development of future strategies for the treatment of peripheral nerve injury.

Glycolaldehyde Induces Cytotoxicity and Increases Glutathione and Multidrug-Resistance-Associated Protein Levels in Schwann Cells

Schwann cell injury is observed in diabetic neuropathy. It is speculated that glycolaldehyde (GA), a precursor of advanced glycation end products (AGEs), contributes to the pathogenesis and development of diabetic neuropathy. Here, we demonstrated for the first time that GA at near-physiological concentration decreased the viability of rat Schwann cells. In contrast, methylglyoxal, glyoxal, and 3-deoxyglucosone, all of which are AGE precursors, had no effects on cell viability. It is well known that methylglyoxal causes oxidative damage. In the present study, however, GA failed to induce reactive oxygen species production in Schwann cells. The addition of glutathione (GSH) or N-acetyl-L-cysteine protected Schwann cells from the loss of viability induced by GA. Moreover, GA increased intracellular GSH level and γ-glutamylcysteine synthetase mRNA level. Flow cytometric analysis revealed that GA increased multidrug-resistance-associated protein 1 (MRP1) level as well. Moreover, we demonstrated that the knockdown of MRP1 with small interfering RNA (siRNA) enhanced the loss of cell viability induced by GA. Taken together, these findings suggest that MRP1, together with GSH, plays an important role in the GA-induced toxicity in Schwann cells.

The Beneficial Effect of Ginsenoside Rg1 on Schwann Cells Subjected to Hydrogen Peroxide Induced Oxidative Injury

Ginsenoside Rg1 (GRg1) has been considered to have therapeutic potential in promoting peripheral nerve regeneration and functional recovery after sciatic nerve injuries. However, the mechanism underlying the beneficial effect of GRg1 on peripheral nerve regeneration is currently unclear. The possible effect of GRg1 on Schwann cells (SCs), which were subjected to oxidative injury after nerve injury, might contribute to the beneficial effect of GRg1 on nerve regeneration. The present study was designed to investigate the potential beneficial effect of GRg1 on SCs exposed to oxidative injury. The oxidative injury to SCs was induced by hydrogen peroxide. The effect of GRg1 (50 μM) on SCs exposed to oxidative injury was measured by the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) in SCs. The cell number and cell viability of SCs were evaluated through fluorescence observation and MTT assay. The apoptosis of SCs induced by oxidative injury was evaluated by an apoptosis assay. The expression and secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were evaluated using RT-PCR, Western blotting, and an ELISA method. We found that GRg1 significantly up-regulated the level of SOD, GSH and CAT, and decreased the level of MDA in SCs treated with hydrogen peroxide. In addition, GRg1 has been shown to be able to inhibit the proapoptotic effect of hydrogen peroxide, as well as inhibit the detrimental effect of hydrogen peroxide on cell number and cell viability. Furthermore, GRg1 also increased the mRNA levels, protein levels and secretion of NGF and BDNF in SCs after incubation of hydrogen peroxide. Further study showed that preincubation with H89 (a PKA inhibitor) significantly inhibited the effects induced by hydrogen peroxide, indicating that the PKA pathway might be involved in the antioxidant effect and neurotrophic factors (NTFs) promoting effect of GRg1. In addition, a short-term in vivo study was performed to confirm and validate the antioxidant effect and nerve regeneration-promoting effect of GRg1 in a sciatic crush injury model in rats. We found that GRg1 significantly increased SOD, CAT and GSH, decreased MDA, as well as promoted nerve regeneration after crush injury. In conclusion, the present study showed that GRg1 is capable of helping SCs recover from the oxidative insult induced by hydrogen peroxide, which might account, at least in part, for the beneficial effect of GRg1 on nerve regeneration.

Fabrication of Silk Fibroin/P(LLA‐CL) Aligned Nanofibrous Scaffolds for Nerve Tissue Engineering

AbstractAligned nanofibers are an attractive option in neural tissue engineering due to providing topographic cues for cell and axonal growth. Aligned and radom 25:75 wt% silk fibroin/poly[(L‐lactic acid)‐co‐(ε‐caprolactone)] nanofibrous scaffolds are fabricated via electrospinning. The mechanical properties of aligned scaffolds present a strong anisotropy, with much higher tensile strength in parallel than in perpendicular direction. Schwann cell viability studies show that aligned scaffolds significantly promote cell growth and the direction of SC elongation is parallel to the direction of fibers for aligned scaffolds. These results suggest that aligned nanofibrous scaffolds might be potential candidates for nerve tissue engineering.magnified image

Local Neurotoxicity and Myotoxicity Evaluation of Cyclodextrin Complexes of Bupivacaine and Ropivacaine

Bupivacaine (BVC) and ropivacaine (RVC) are local anesthetics widely used in surgical procedures. In previous studies, inclusion complexes of BVC or RVC in hydroxypropyl-β-cyclodextrin (HP-β-CD) increased differential nervous blockade, compared to the plain anesthetic solutions. In this study we evaluated the local neural and muscular toxicity of these new formulations containing 0.5% BVC or RVC complexed with HP-β-CD (BVC(HP-β-CD) and RVC(HP-β-CD)). Schwann cell viability was assessed by determination of mitochondrial dehydrogenase activity, and histopathological evaluation of the rat sciatic nerve was used to identify local neurotoxic effects (48 hours and 7 days after the treatments). Evaluations of serum creatine kinase levels and the histopathology of rat gastrocnemius muscle (48 hours after treatment) were also performed. Schwann cell toxicity evaluations revealed no significant differences between complexed and plain local anesthetic formulations. However, use of the complexed local anesthetics reduced serum creatine kinase levels 5.5-fold, relative to the plain formulations. The differences were significant at P < 0.05 (BVC) and P < 0.01 (RVC). The histopathological muscle evaluation showed that differences between groups treated with local anesthetics (BVC or RVC) and their respective complexed formulations (BVC(HP-β-CD) or RVC(HP-β-CD)) were significant (P < 0.05). We concluded that the new formulations presented a lower myotoxicity and a similar cytotoxic effect when compared to plain local anesthetic solutions.

High glucose levels increase the expression of neurotrophic factors associated with p-p42/p44 MAPK in Schwann cells in vitro

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus. One contributing factor to DPN is altered neurotrophism due to changes in the synthesis and expression of neurotrophins. Schwann cells (SCs) are the myelin-forming cells of the peripheral nervous system that promote nerve regeneration through the expression and secretion of neurotrophic factors (NTFs). Therefore, in this study, using SCs cultured in the presence of high levels of glucose for 24 h, with and without the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor, PD98059, we investigated the effect of high glucose levels on SCs over a short period of time. The cultured cells were evaluated using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst staining, immunocytochemistry, reverse transcriptase-polymerase chain reaction and western blot analysis. High glucose levels did not promote morphological abnormalities or decrease the viability of SCs. However, high glucose levels enhanced the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and induced the activation of p42/p44 MAPK in cultured SCs in a dose-dependent manner. Additionally, the phosphorylation of p42/p44 MAPK may be associated with the expression of NTFs by SCs exposed to high glucose conditions; the excessive activation of p42/p44 MAPK inhibited the expression of NTFs. These observations demonstrate that exposure to high glucose levels lead to acutely elevated levels of NGF and BDNF in SCs over a short period of time, which may be involved in the p42/p44 MAPK pathway.

Schwann cell metabolic activity in various short-term holding conditions: implications for improved nerve graft viability.

Strategies for improvement of nerve regeneration and optimal conditions to prevent Schwann cell (SC) loss within a nerve transplant procedure are critical. The purpose of this study was to examine SC viability, which plays an important role in peripheral nerve regeneration, under various incubation conditions up to three hours. To address this issue, Schwann cell metabolic activity was determined using different independent test methods. The following experimental conditions were compared: SCs prepared from nerves were incubated in (1) isotonic saline solution (2) Dulbecco's modified Eagles medium as used for cell culturing, (3) Hannover bioreactor medium, and (4) Leibovitz's medium. SC metabolic activity of excised rat sciatic nerve was determined at 4°C, 18°C, and 37°C over 3 hrs. The results indicate that SC activity was optimized by the usage of Leibovitz's medium or HBRM at 37°C. Greater SC viability at the time of surgical nerve grafting could contribute to improved axonal regeneration and remyelination after nerve transplantation, and thus more successful functional recovery.