Autologous Schwann Cells Research Articles

Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects

Peripheral nerve injuries are a source of chronic disability. Incomplete recovery from such injuries results in motor and sensory dysfunction and the potential for the development of chronic pain. The repair of human peripheral nerve injuries with traditional surgical techniques has limited success, particularly when a damaged nerve segment needs to be replaced. An injury to a long segment of peripheral nerve is often repaired using autologous grafting of "noncritical" sensory nerve. Although extensive axonal regeneration can be observed extending into these grafts, recovery of function may be absent or incomplete if the axons fail to reach their intended target. The goal of this review was to summarize the progress that has occurred in developing an artificial neural prosthesis consisting of autologous Schwann cells (SCs), and to detail future directions required in translating this promising therapy to the clinic. In the authors' laboratory, methods are being explored to combine autologous SCs isolated using cell culture techniques with axon guidance channel (AGC) technology to develop the potential to repair critical gap length lesions within the peripheral nervous system. To test the clinical efficacy of such constructs, it is critically important to characterize the fate of the transplanted SCs with regard to cell survival, migration, differentiation, and myelin production. The authors sought to determine whether the use of SC-filled channels is superior or equivalent to strategies that are currently used clinically (for example, autologous nerve grafts). Finally, although many nerve repair paradigms demonstrate evidence of regeneration within the AGC, the authors further sought to determine if the regeneration observed was physiologically relevant by including electrophysiological, behavioral, and pain assessments. If successful, the development of this reparative approach will bring together techniques that are readily available for clinical use and should rapidly accelerate the process of bringing an effective nerve repair strategy to patients with peripheral nerve injury prior to the development of pain and chronic disability.

A Guidance Channel Seeded With Autologous Schwann Cells for Repair of Cauda Equina Injury in a Primate Model

Background/Objective: To evaluate an implantable guidance channel (GC) seeded with autologous Schwann cells to promote regeneration of transected spinal nerve root axons in a primate model.Methods: Schwann cells were obtained from sural nerve segments of monkeys (Macaca fascicularis; cynomolgus). Cells were cultured, purified, and seeded into a PAN/PVC GC. Approximately 3 weeks later, monkeys underwent laminectomy and dural opening. Nerve roots of the L4 through L7 segments were identified visually. The threshold voltage needed to elicit hindlimb muscle electromyography (EMG) after stimulation of intact nerve roots was determined. Segments of 2 or 3 nerve roots (each ∼8-15 mm in length) were excised. The GC containing Schwann cells was implanted between the proximal and distal stumps of these nerve roots and attached to the stumps with suture. Follow-up evaluation was conducted on 3 animals, with survival times of 9 to 14 months.Results: Upon reexposure of the implant site, subdural nerve root adhesions were noted in all 3 animals. Several of the implanted GC had collapsed and were characterized by thin strands of connective tissue attached to either end. In contrast, 3 of the 8 implanted GC were intact and had white, glossy cables entering and exiting the conduits. Electrical stimulation of the tissue cable in each of these 3 cases led to low-threshold evoked EMG responses, suggesting that muscles had been reinnervated by axons regenerating through the repair site and into the distal nerve stump. During harvesting of the GC implant, sharp transection led to spontaneous EMG in the same 3 roots showing a low threshold to electrical stimulation, whereas no EMG was seen when harvesting nerve roots with high thresholds to elicit EMG. Histology confirmed large numbers of myelinated axons at the midpoint of 2 GC judged to have reinnervated target muscles.Conclusions: We found a modest rate of successful regeneration and muscle reinnervation after treatment of nerve root transection with a Schwann cell-seeded, implanted synthetic GC. Newer treatments, which include the use of absorbable polymers, neurotrophins, and antiscar agents, may further improve spinal nerve regeneration for repair of cauda equina injury.

Treatment of chronic thoracic spinal cord injury patients with autologous Schwann cell transplantation: An interim report on safety considerations and possible outcomes

Several experimental studies have introduced Schwann cell transplantation as a means of recovery in animal models of spinal cord injury (SCI). The reported promising results together with the availability of autologous sources for Schwann cells indicate Schwann cell transplantation as a possible treatment for SCI. To address the safety and feasibility concerns we report 1-year follow-up of four patients aged between 22 and 43 years who had stable chronic (28-80 months) spinal cord injury at mid-thoracic level and treated with autologous Schwann cell transplantation. Purified Schwann cells used for transplantation were acquired from autologous sural nerve and cultured without the use of any specific mitogenic or growth factors. The patients were evaluated by means of American Spinal Injury Association (ASIA) criteria, sphincter, sexual function and Magnetic Resonance Imaging assessments for 1 year after transplantation. None of the patients were found to have any adverse effects indicating transfer of infection, neurological deterioration or other related clinical problems. Of the four patients, only one patient with incomplete SCI showed motor and sensory improvement 1 year after transplantation with extensive and continuous rehabilitation. All the four patients experienced transient paresthesia or increased muscle spasm after transplantation. Magnetic Resonance (MR) images of the patients did not show any visible changes or pathological findings after 1 year. This preliminary report shows that autologous Schwann cell transplantation is generally safe for the selected number of SCI patients but it does not prove beneficial effects. Further safety and outcome studies are recommended.

Bridging Critical Nerve Defects through an Acellular Homograft Seeded with Autologous Schwann Cells Obtained from a Regeneration Neuroma of the Proximal Stump

Over the last decade, several models have investigated the usefulness of different biologic and/or synthetic matrices as alternatives to conventional nerve grafts. Still, axonal regeneration did not occur over longer (> 3 cm) distances. One problem may be that a growth-promoting environment not only includes physical cues but also a rich spectrum of different growth factors only provided by reactive Schwann cells. In the current study, we investigated whether a hybrid graft consisting of first-generation autologous Schwann cells seeded onto an acellular auto- or homograft can aid regeneration across a critical nerve defect in a rat model. In this paradigm, Schwann cells were not expanded in vitro but harvested from the proximal stump neuroma at the time of reconstruction and seeded into either an acellular homo- or autograft. Regeneration was then quantitated with functional muscle testing, regular histology, histomorphometry, and retrograde tracing techniques 12 weeks after reconstruction. Results showed successful regeneration over the entire distance regardless of whether Schwann cells were transplanted onto auto- or homologous acellular matrix. Schwann cells did populate both grafts; however, only sensory axons persisted through the entire distance. The functional outcome was dismal with no motor and poor sensory recovery. Control group C with homologous matrix only without Schwann cells showed no signs of directed axonal regeneration. Control group D with autologous reverse graft showed excellent recovery, as was expected. The present experiment sought to create a hybrid graft where the proximal stump neuroma is used as a biological resource for autologous Schwann cells that are seeded unto an acellular matrix, thus providing both physical and chemical support to regenerating axons. The results are encouraging in that successful regeneration was observed over the entire distance; however, only sensory axons had enough regenerative potential to also make end-organ contact. For motor axons, further refinements in conduit preparation have to be done.

Use of Cold-Preserved Allografts Seeded with Autologous Schwann Cells in the Treatment of a Long-Gap Peripheral Nerve Injury

Limitations in autogenous tissue have inspired the study of alternative materials for repair of complex peripheral nerve injuries. Cadaveric allografts are one potential reconstructive material, but their use requires systemic immunosuppression. Cold preservation (> or =7 weeks) renders allografts devoid of antigens, but these acellular substrates generally fail in supporting regeneration beyond 3 cm. In this study, the authors evaluated the reconstruction of extensive nonhuman primate peripheral nerve defects using 7-week cold-preserved allografts repopulated with cultured autologous Schwann cells. Ten outbred Macaca fascicularis primates were paired based on maximal genetic disparity as measured by similarity index assay. A total of 14 ulnar nerve defects measuring 6 cm were successfully reconstructed using autografts (n = 5), fresh allografts (n = 2), cold-preserved allografts (n = 3), or cold-preserved allografts seeded with autogenous Schwann cells (n = 4). Recipient immunoreactivity was evaluated by means of enzyme-linked immunosorbent spot assay, and nerves were harvested at 6 months for histologic and histomorphometric analysis. Cytokine production in response to cold-preserved allografts and cold-preserved allografts seeded with autologous Schwann cells was similar to that observed for autografts. Schwann cell-repopulated cold-preserved grafts demonstrated significantly enhanced fiber counts, nerve density, and percentage nerve (p < 0.05) compared with unseeded cold-preserved grafts at 6 months after reconstruction. Cold-preserved allografts seeded with autologous Schwann cells were well-tolerated in unrelated recipients and supported significant regeneration across 6-cm peripheral nerve defects. Use of cold-preserved allogeneic nerve tissue supplemented with autogenous Schwann cells poses a potentially safe and effective alternative to the use of autologous tissue in the reconstruction of extensive nerve injuries.

Nerve Regeneration across a 2-cm Gap in the Rat Median Nerve Using a Resorbable Nerve Conduit Filled with Schwann Cells

In a rat model, nerve regeneration was evaluated across a 2-cm defect in the median nerve using a resorbable artificial nerve conduit. The aim of this study was to develop an artificial, biocompatible nerve guide to induce regeneration in the peripheral nervous system. The authors compared a nerve conduit of trimethylenecarbonate-co-epsilon-caprolactone (TMC/CL) filled with autologous Schwann cells with an empty hollow conduit and with an autologous nerve graft. Non-operated animals served as controls. Regeneration was evaluated with the grasping test, histological analysis of the nerve, muscle weight analysis (flexor digitorum sublimis muscle), and electrophysiologic examination.

Peripheral Nerve Tissue Engineering: Autologous Schwann Cells vs. Transdifferentiated Mesenchymal Stem Cells

Peripheral Nerve Tissue Engineering: Autologous Schwann Cells vs. Transdifferentiated Mesenchymal Stem Cells

Peripheral Nerve Tissue Engineering: Autologous Schwann Cells vs. Transdifferentiated Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) were evaluated as an alternative source for tissue engineering of peripheral nerves. MSCs, transdifferentiated MSCs, or Schwann cells cultured from male rats were grafted into devitalized autologous muscle conduits bridging a 2-cm sciatic nerve gap in female rats. The differentiation potential of MSCs and transformed cultivated MSCs into Schwann cell-like cells was exploited using a cocktail of cytokines. Polymerase chain reaction of the SRY gene confirmed the presence of the implanted cells in the grafts. After 6 weeks, regeneration was monitored clinically, histologically, and morphometrically. Autologous nerves and cell-free muscle grafts were used as control. Revascularization studies suggested that transdifferentiated MSCs, in contrast to undifferentiated MSCs, facilitated neo-angiogenesis and did not influence macrophage recruitment. Autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate regeneration was noted in the Schwann cell-groups and, albeit with restrictions, in the transdifferentiated MSC groups, whereas regeneration in the MSC group and in the cell-free group was impaired. The results indicate that transdifferentiated MSCs implanted into devitalized muscle grafts are able to support peripheral nerve regeneration to some extent, and offer a potential for new therapeutic strategies.

Autologous adult human Schwann cells genetically modified to provide alternative cellular transplants in peripheral nerve regeneration

The purpose of this study was to provide genetically modified adult human Schwann cells as tools for cell transplantation in peripheral nerve repair. The application of transfected autologous Schwann cells overexpressing regeneration-promoting proteins, for example, neurotrophic or growth factors, is a promising approach in the aforementioned context. To achieve an optimal clinical outcome, it is highly important to perform enrichment, genetic modification, and retransplantation of cells in a short time. To enable the development of these autologous cellular prostheses, the authors tested the properties of adult human Schwann cells obtained from differently treated human peripheral nerve biopsy samples. The use of "cold jet," a fast and effective enrichment procedure, as well as selective, serum-free culture conditions, resulted in very pure adult human Schwann cell cultures. Using an optimized electroporation protocol, as many as 48.4% of adult human Schwann cells were successfully transfected. The authors present a very fast protocol to establish adult human Schwann cell cultures that demonstrably express plasmid proteins after plasmid DNA insertion by electroporation. These autologous human Schwann cells transfected to enhance the endogenous production of regeneration-supporting proteins will likely constitute a major component of tissue-engineered peripheral nerve grafts.

Repair of peripheral nerve defect by a scroll of amnion derivative compound with cultured autogenous Schwann cell in a rat model

To test a nerve bridge substitute for peripheral nerve repair by tissue-engineering approach. An artificial nerve fabricated with a scroll of amnion derivative (ZQ membrane) and cultured autogenous Schwann cell was sutured to bridge sciatic nerve defect of 2.5 cm in length in rats. The specimens were assessed with tracking study, histology, electrophysiological technique, NF200, and synaptophysin-38 (SYP) immuno histochemical staining 3 months postoperatively. The regenerated nerve sprouted 3 months after the operation. The regenerated nerve fibers were plentiful and could grow into the recipient nerve and target muscle's motor end plate (MEP) areas to reinnervate target muscle, and reconstruct function of nerve-muscle junction. Functional recovery could reach to 40%-60% of normal control. Nerve-muscle conduction velocity (N-MCV) arrived at 21.77 +/- 1.15 m/s. A tissue engineering material fabricated with a scroll of ZQ membrane and cultured autologous Schwann cell may be a useful substitute for nerve repair.

Nerve regeneration across a 2-cm gap in the rat median nerve using a resorbable nerve conduit filled with Schwann cells

In a rat model, nerve regeneration was evaluated across a 2-cm defect in the median nerve by using a resorbable artificial nerve conduit. The aim of this study was to develop an artificial, biocompatible nerve guide to induce regeneration in the peripheral nervous system. The authors compared a nerve conduit of trimethylenecarbonate-co-epsilon-caprolactone (TMC/CL) filled with autologous Schwann cells with both an empty hollow conduit and an autologous nerve graft. Animals that did not undergo surgery served as the control group. Nerve regeneration was evaluated with the grasping test, histological analysis of the nerve, muscle weight analysis (flexor digitorum superficialis muscle), and electrophysiological examination. After an observation period of 9 months, regeneration occurred only in animals that had received an autologous graft or a Schwann cell containing nerve conduit. No signs of regeneration were found in animals supplied with the empty conduit. Results of this study reveal the important role of Schwann cells in the regeneration process across a 2-cm defect in the rat median nerve. Furthermore, Schwann cell-filled nerve conduits induced functional recovery, as demonstrated in the grasping test, that was comparable with that of the autologous graft 9 months after implantation.

Enhancing Central Nervous System Remyelination in Multiple Sclerosis

Recent studies on adult neural stem cells and the developmental biology of myelination have generated the expectation that neural precursors can repair the damaged central nervous system of multiple sclerosis patients where the endogenous remyelination process has failed. As a result, many laboratories are engaged in translational studies in which the goal is to design ways to promote remyelination and repair. Here we raise issues highlighted by prior experimental and human work that should be considered lest these studies become "lost in translation."

Study on the reconstruction of rhesus tissue-engineered peripheral nerve using acellular allogenic nerve with implanted autologous mesenchymal stem cells or Schwann cells

目的 评价用自体源骨髓间充质干细胞(MSCs)或许旺细胞(SCs)作种子细胞,与去细胞同种异体神经构建的组织工程化外周神经修复猕猴尺神经4 cm缺损的实验效果,为临床研究提供资料.方法分别用4种移植物桥接猕猴4 cm尺神经缺损.A组:种植自体MSCs的去细胞同种异体神经;B组:种植自体SCs的去细胞同种异体神经;C组:去细胞同种异体神经;D组:自体神经移植.通过功能学、神经电生理学及组织学指标评价各组修复神经缺损的效果.结果 A、B组术后6个月能引起小鱼际肌群产生复合动作电位的潜伏期、复合动作电位的最大振幅、神经传导速度和再生的神经纤维数目与自体神经移植组相比无统计学意义(P＞0.05);但A、B、C、D组分别与C组相比,差异有统计学意义(P＞0.05).结论用自体源SCs或MSCs作种子细胞,与去细胞同种异体神经构建的组织工程化外周神经修复猕猴4 cm尺神经缺损,均能取得与自体神经移植相近的效果。

Graft repair of a peripheral nerve without the sacrifice of a healthy donor nerve by the use of acutely dissociated autologous Schwann cells

We hypothesised that pieces of nerve that are normally trimmed away during graft repair could be a source of Schwann cells and used to create an autologous cell-containing nerve graft. To test the idea a transsection injury was made on the rat sciatic nerve. Seven days later, pieces from the proximal and distal stumps were used for immediate isolation of its non-neuronal cells. These cells, of which 80% were Schwann cells, were injected into a silicone tube bridging a 10 mm gap on the same nerve. Tubes containing cells showed superior regeneration of nerve fibres across the gap compared with tubes with no cells. This new repair technique is rapid, does not require the sacrifice of a healthy donor nerve, or the time-consuming culturing and expansion of Schwann cells. The method has the potential to replace current methods for repair of extended nerve injuries.

Acutely-dissociated Schwann cells used in tendon autografts for bridging nerve defects in rats: A new principle for tissue engineering in nerve reconstruction

A new method of acute dissociation of Schwann cells was used to study the effect of addition of such cells to a tendon autograft--a recently-described graft material--on peripheral nerve regeneration in rats. Autologous Schwann cells were obtained from enzymatic dissociation of predegenerated nerves. The tendon autografts were supplied with Schwann cells through brief in vitro coincubation. Schwann cell-free tendon autografts were used as controls. Axonal outgrowth was measured immunohistochemically after four, seven, and 10 days. At seven days, outgrowth was significantly longer in the pretreated autografts. The use of acutely-dissociated Schwann cells is a new approach to tissue engineering in nerve reconstruction, and may abolish the need for time-consuming culture of Schwann cells.

Intravitreal transplants of Schwann cells and fibroblasts promote the survival of axotomized retinal ganglion cells in rats

Schwann cells (SCs) are considered one of the major cellular components to maintain the integrity of the peripheral nervous system (PNS) neurons after injury. Intravitreal transplant of peripheral nerves or Schwann cells has been shown to enhance the regenerative ability of retinal ganglion cells (RGCs). In the present study, we compared the effects of intravitreal transplants of Schwann cells and fibroblasts, two major components of peripheral nerves, on the survival of retinal ganglion cells in adult rats after optic nerve (ON) transection. Purified Schwann cells and fibroblasts from neonatal sciatic nerves were injected into the vitreous body of adult rats. Three days after the injection, the optic nerves were transected intraorbitally. After 1 week or 1 month, surviving retinal ganglion cells were retrogradely labelled with Fluoro-Gold (FG) and the number of surviving retinal ganglion cells was counted. The retinas were further processed for 200-kDa neurofilament RT-97 immunohistochemistry. It was found that intravitreally injected- Schwann cells and -fibroblasts delayed the death of axotomized retinal ganglion cells for 1 week. In addition, in the animal group with 1 month survival time after optic nerve transection, those received a larger number of Schwann cells had more surviving retinal ganglion cells and more profusely ramified axonal processes near the optic disc. These findings reveal that both Schwann cells and fibroblasts isolated from the peripheral nerve can promote retinal ganglion cell survival after optic nerve transection, presumably by secreting neurotrophic factors. In addition, the data also demonstrate that Schwann cells could promote intraretinal axonal sprouting. Our findings demonstrate a remarkable glial source of neurotrophic factors with potential clinical applications, as autologous Schwann cells and fibroblasts can be feasibly obtained from peripheral nerves.

Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve

The intrinsic capacity of Schwann cells to promote regeneration after limited peripheral nerve lesions has been successfully transferred to extensive peripheral nerve injuries and central nervous system lesions by autologous transplantation strategies. However, both the intrinsic ability of axotomized neurons to regenerate and the permissiveness of the parenchyma surrounding the acute injury site diminish over time. Therefore, the autologous transplantation mode requires a fast and effective method to isolate Schwann cells from peripheral nerve biopsies. Here, we report a method to purify p75 low affinity nerve growth factor receptor (p75LNGFr) expressing Schwann cells from peripheral nerve biopsies in adult rats using magnetic-activated cell separation (MACS). After 1 week of nerve degeneration in culture, nerve fragments were dissociated resulting in mixed cultures containing Schwann cells and fibroblasts. After incubation with specific anti-p75LNGFr antibodies and secondary magnetic bead conjugated antibodies followed by one cycle of MACS, 95% pure Schwann cell cultures were generated as confirmed by flow-cytometry and immunocytochemistry. In contrast to established methods, MACS separation of p75LNGFr expressing cells allows the reliable purification of Schwann cells within 9 days after biopsy employing direct selection of Schwann cells rather than fibroblast depletion assays. Therefore, this method represents an effective and fast means to generate autologous Schwann cells for clinical transplantation strategies aiming for axon repair and remyelination.

A new approach to CNS repair using chimeric peripheral nerve grafts.

We have examined whether transplanted freeze-thawed peripheral nerve (PN) sheaths repopulated ex vivo with purified adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Cultured adult SCs were derived from donor rats or from the host animals themselves. We also transplanted PN sheaths filled with neonatal SCs or donor adult olfactory ensheathing glia (OEG). 100,000 cells were injected into 1.5-cm lengths of freeze-thawed PN. After 2 days in culture, repopulated PN segments were grafted onto the transected optic nerve of adult Fischer rats. Three weeks later, 6% fluorogold (FG) was applied to distal PN. Retrogradely labeled RGCs were counted in retinal wholemounts and PN grafts were processed for immunohistochemistry. As expected, there was no RGC axon regeneration in cell-free grafts. Regrowth was also absent in neonatal SC- and adult OEG-filled grafts, which contained only small numbers of surviving donor cells. Many cells were, however, seen in adult SC repopulated PN grafts, intermingled with pan-neurofilament(+) and GAP-43(+) fibers. SCs were aligned along the grafts and were S-100(+), p75(+). Ultrastructurally, SCs were associated with myelinated and unmyelinated axons. Hundreds of FG-labeled RGCs were seen in retinas of rats with congeneic or allogeneic PN sheaths repopulated with either donor or autologous (host-derived) adult SCs. Intraocular CNTF injections significantly increased the number of regenerating RGCs in donor and autologous adult SC groups. The use of chimeric grafts to bridge CNS tissue defects could provide a clinical alternative to using multiple PN autografts, the harvesting of which would exacerbate peripheral dysfunction in already injured patients.

Effect of Allogeneic Schwann Cell Transplantation on Peripheral Nerve Regeneration

Transplantation of allogeneic Schwann cells (SC) would make it feasible to reconstruct immediately peripheral nerve defects, compared to using autologous SC; however, this treatment modality has not been adequately evaluated. The aim of this study was to characterize and compare the effects of allogeneic versus syngeneic SC transplantation following peripheral nerve injury. Polyhydroxybutyrate conduits were used to bridge a 10-mm gap in the rat sciatic nerve. The conduits were filled with alginate hydrogel with or without cultured allogeneic or syngeneic genetically labeled SC, without the use of immunosuppressive therapy, and examined after 2, 3, and 6 weeks with 5-bromo-4-chloro-3-indoyl-β-d-galactosidase chemical staining and immunohistochemistry to quantify SC migration into the conduit, axonal regeneration, the state of SC differentiation, and the expression of major histocompatibility complexes (MHC) I and II, as well as to quantify macrophage and B- and T-lymphocyte infiltration. Allogeneic SC were rejected by 6 weeks, whereas syngeneic SC could still be identified. Allogeneic and syngeneic SC equally enhanced the axonal regeneration distance but the quantity of axons was greater using syngeneic SC. The ingrowth of SC into the conduits containing allogeneic SC was similar to that observed in the presence of syngeneic SC, indicating the absence of deleterious immune response. SC continued to express phenotypic markers of nonmyelination and these were highest in conduits with allogeneic SC. Expression of MHC I and II was higher in the conduits with allogeneic SC at 3 weeks and without significant difference in the number of macrophages and lymphocytes, except at 6 weeks, when there was a larger number of lymphocytes using syngeneic SC. In conclusion, allogeneic SC enhanced axonal regeneration distance and did not induce a deleterious immune response. In a clinical setting the immediate availability of allogeneic SC for transplantation may compensate for the better outcome achieved by the use of autologous SC that require a longer preparation time in culture.

Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C)

Attempts to design the nerve cellular prostheses have focused on the production of autologous Schwann cells expanded in vitro as the essential component in the regeneration process of injured peripheral nerves . To obtain human Schwann cells of high quality we tested a short enzymatic dissociation protocol that optimized cellular viability levels. We also assessed patterns of bromodeoxyuridine (BrdU) incorporation in both Schwann cells and fibroblasts in the presence or absence of the antimitotic Ara-C, an enrichment option for adult human Schwann cell cultures. The Ara-C treated cultures showed a significantly higher Schwann cell percentage (95%), compared with that obtained in the absence of Ara-C (70%), indicating that this antimitotic acts to eliminate fibroblasts in each one of the applied pulses (four pulses). However, we have observed that the use of this antimitotic during prolonged periods of time produced a cumulative effect causing Schwann cell cytotoxicity. Therefore, we consider that our enzymatic dissociation technique and the application of only two pulses of Ara-C to the cultures are enough to achieve enrichment of adult human Schwann cells in culture.