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

Ji-Hey Lim Department of Clinical Sciences, College of Veterinary Medicine, and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27606.

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 DVM, PhD
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Natasha J. Olby Department of Clinical Sciences, College of Veterinary Medicine, and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27606.

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 VET MB, PhD

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Abstract

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.

Abstract

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.

The ability of mammals to recover from severe injury to the CNS is notoriously poor as a consequence of a physiologic environment that inhibits axonal regeneration.1,2 By contrast, axonal regeneration in the PNS is robust, in large part owing to the behavior of Schwann cells,3 which are the myelin-forming glial cells of the PNS. Following injury, Schwann cells synthesize surface cell adhesion molecules,4 produce basement membrane composed of extracellular matrix proteins such as collagen,5 and increase the synthesis of neurotrophic factors and their receptors.6,7

In research dating back to the 1980s,8 it has been shown repeatedly that CNS axons can regenerate into peripheral nerve grafts and Schwann cell–coated implants. There has been extensive investigation of transplantation of Schwann cells as a treatment for spinal cord injury,9–12 and now clinical trials involving humans and dogs are underway.

Numerous studies have revealed benefit from Schwann cell grafts in rodents with experimentally induced spinal cord injury10,13,14 or spinal cord demyelination.15 More recently, an autologous Schwann cell transplantation trial11 involving humans revealed that such transplantation is safe and can improve autonomic, motor, and sensory function. In addition, tissue engineering strategies involving nerve guidance conduits combined with Schwann cell transplants have provided a viable therapeutic option for peripheral nerve injuries in which the nerve defect is too large to repair.16–19

The strong experimental evidence of benefit from Schwann cell transplants for injuries of both the PNS and CNS is supportive of moving forward to clinical trials. To allow translation of this work to clinical applications for dogs, culture techniques that allow timely generation of large numbers of pure, autologous Schwann cells are needed. Moreover, it is important that harvesting of cells from patients has no detrimental effects. Although many reports20,21 exist of Schwann cell culture for various species, culture conditions and growth rate differ depending on species and developmental stage.22 In adult nerves, the epineurium and perineurium are fully developed and contain more connective tissue than immature nerves.23,24 Consequently, cultures of Schwann cells from adult nerves have more contamination from fibroblasts than do those from embryonic or neonatal tissues25 and purification is necessary. In addition, the effects of mitogens used to amplify Schwann cells differ among species.26, 27

A few studies27,28 have been conducted to evaluate the isolation of Schwann cells from the sciatic nerve of dogs. However, biopsy of the sciatic nerve can result in neurologic deficits and pain.29 The purpose of the study reported here was to investigate methods of isolation, purification, and amplification of Schwann cells from biopsy specimens of the dorsal cutaneous branches of the cervical nerves of dogs. The goal was to develop an optimal method for producing autologous Schwann cells for clinical use in dogs with injuries of the CNS and PNS.

Materials and Methods

Animals and nerve biopsy specimens

One Beagle and 2 mixed-breed dogs (2 females and 1 male) between 12 and 24 months of age were used as a source of tissues for the study. All dogs had been euthanized with pentobarbital sodium for reasons unrelated to the study, and tissue specimens were obtained within 3 hours after euthanasia.

All specimens were collected in aseptic conditions. A midline skin incision was made in the dorsal cervical region, and the subcutaneous tissue was dissected to reveal the paired segmental dorsal cutaneous cervical nerves lying on the surface of the muscles. The nerves were completely transected on the midline and dissected peripherally as far as possible. Approximately 3- to 4-cm-long pieces of nerve tissue were excised and transferred to ice-cold DMEMa containing 1% penicillin-streptomycin.a The connective tissues and vessels were removed, and single nerve fascicles were pulled out under a tissue dissection microscope.b Each dorsal cutaneous cervical nerve specimen was cut into 1-cm-long sections weighing between 15 and 20 mg of wet weight prior to predegeneration. Some nerve specimens were reserved for immunostaining.

Immunostaining of dorsal cervical cutaneous nerves

Dorsal cervical cutaneous nerve specimens were processed for immunohistochemical analysis to identify cells. Tissues were fixed for 10 minutes in 4% paraformaldehyde, then washed and made permeable by soaking in 0.25% Triton X-100 in PBST for 10 minutes. Tissues were blocked for 1 hour at room temperature (approx 23°C) in 10% bovine serum albumin in PBST and then incubated overnight at 4°C with primary antibodies (polyclonal rabbit anti-GFAPc and monoclonal mouse anti-human S100 βd). The next day, tissues were incubated with secondary antibodies (goat anti-rabbit Alexa 488e and goat anti-mouse conjugated Cy3e) in PBST for 1 hour at room temperature. Isotype-matched control specimens were used. Nerve specimens were placed on glass slides and then mounted with mounting medium with DAPIf and examined with a fluorescence microscope.g

In vitro predegeneration, cell isolation, and primary culture

Predegeneration of nerve specimens was performed by use of a method modified from that in a previous report.30 Nerve specimens were placed in the wells of precoatedh 6-well plates and incubated dry for 15 minutes at 37°C, and then SCM with 10% fetal bovine serumi was added. The SCM consisted of melanocyte growth mediumj supplemented with bovine pituitary extracts (5 µg/mL) and basic fibroblast growth factore (10 ng/mL). The cultures were maintained at 37°C and 5% CO2 in a humidified tissue culture incubator, with half of the medium changed every other day. After 8 to 10 days of predegeneration, nerve specimens were collected and enzymatically dissociated for 30 minutes at 37°C, with dissociation medium composed of collagenase type IVa (125 U/mL) and dispasek (1.25 U/mL) in DMEM with 1% penicillin-streptomycin. Dissociation was inactivated by use of 10% fetal bovine serum in DMEM.

Pellets of dissociated cells were suspended in SCM, and the total number of viable cells was determined by staining with 0.4% trypan blue and manual counting with a hemacytometer. Suspended single cells were cultured in SCM on PLOl-coated, 6-well plates at a concentration of 1 × 105 viable cells/well. The cultures were maintained at 37°C and 5% CO2 in a humidified tissue culture incubator until cells reached confluence. Cells were subsequently harvested for SC purification. Some cells were reserved for quantification of purity.

Purification of Schwann cells

Primary cell cultures were purified by use of magnetic beads coupled with rat anti-CD90m (fibroblast marker) in accordance with manufacturer's protocol. Briefly, magnetic beads conjugated to sheep anti–rat IgGn were washed in 0.1% bovine serum albumin in 1X PBS solution 3 times (10 µL of beads in 500 µL of 0.1% bovine serum albumin). Ten microliters of washed beads was then added to 300 µL of a solution of rat anti–CD90 antibody (diluted 1:300 in melanocyte growth medium) and rotated for 45 minutes at 4°C. Ten microliters of beads/1 × 105 cells were used. Cells were enzymatically detached, inactivated, and resuspended in SCM. The cell suspension was incubated for 45 minutes at 4°C with the anti-CD90–coupled magnetic beads to allow the beads to bind to CD90 expressed on fibroblasts. Immunolabeled cells were then removed by use of a magneto and discarded.

Purified cells were seeded on a PLO-coated, 6-well plate with SCM and 10% fetal bovine serum to allow cell adherence for 1 day. The next day, the culture medium was replaced with SCM and 10μM Ara-Cp for 48 hours, after which the culture medium was replaced with SCM only. Half of the SCM was replaced every other day until cells reached confluence.

Quantification of Schwann cell purity

Schwann cell purity was determined before immunopurification, immediately after immunopurification, and following 2 passages of subculture after purification. Cells were seeded on a PLO-coated, 8-chamber slideq at a density of 5 × 103 cells/chamber and cultured for 24 hours. Cells were fixed for 10 minutes in 4% paraformaldehyde, then washed 3 times with ice-cold PBS solution with 0.1% PBST. Cells were then blocked for 1 hour at room temperature in 10% goat serum in PBST with 0.3% Triton X-100 and incubated overnight at 4°C with primary antibodies (rat anti-canine CD90 to identify fibroblasts and rabbit anti-human p75r to identify Schwann cells). The p75 was used in cell cultures because it is expressed on the cell surface, allowing optimal staining. The intracellular marker S100 β was used for dorsal nerve root evaluation because it provided optimal staining of the dissected nerve fascicles.

The following day, cells were incubated for 1 hour with secondary antibodies (goat anti–rat Alexa 488 and goat anti–rabbit conjugated Cy3) and counterstained with DAPI. Isotype-matched control samples were used for each antibody and had negative results of staining in every instance. Images were obtained with 20X magnification by use of a fluoromicroscope,s and immunoreactive cells were counted in 5 randomly selected representative microscope fields. Data were expressed as percentages of the total number of cells within the evaluated fields.31

Expansion of Schwann cells with rhGGF2

After 2 passages of subculture, the effect of various concentrations of rhGGF2t on Schwann cell expansion was examined. To test for proliferation of Schwann cells, 2,000 cells were seeded into each well on a PLO-coated, 96-well microplate and cultured in SCM for 24 hours to allow cell attachment. The next day, rhGGF2 was added in various concentrations (0, 20, 40, 60, 80, 100, 150, and 200 ng/mL), and then cells in the microplate were cultured for another 3 days. Cell proliferation was evaluated by use of an MTS assayu in accordance with the manufacturer's instructions. Twenty microliters of MTS assay solution was added to each well, and microplates were incubated for 1 to 2 hours at 37°C. After colorimetric changes appeared, absorbance was measured with a microplate readerv at a wavelength of 490 nm.

To analyze cell doubling time, concentrations of rhGGF2 were selected on the basis of the results of the MTS assay (40, 60, 80, and 100 ng/mL). Then, 1 × 105 cells were seeded on PLO-coated, 6-well plates with each concentration of rhGGF2 in SCM. Half the medium was changed every other day until cells were confluent. Cells were then harvested, counted, and replated up to the third passage. To identify cell doubling time, the cell culture duration and final cell number for each passage were noted. Cell doubling number and time were calculated for each passage in accordance with a previously reported method.31 Doubling times were expressed as mean ± SD.

Statistical analysis

Assays for establishment of MTS and doubling time values were performed in triplicate for each dog for each rhGGF2 concentration. Summary data are reported as mean ± SD. The effect of rhGGF2 concentration on both MTS and doubling time values was examined by means of mixed-model factorial ANOVA, with dog included as a random effect and rhGGF2 concentration included as a fixed effect.w Values of P < 0.05 were considered significant.

Results

Dorsal cutaneous cervical nerves in dogs

The dorsal cervical nerves were easily made visible, and biopsy specimens were easily obtained from the 3 canine cadavers. A dissection microscope was used to aid dissection of the surrounding vessels, connective tissues, and fat without problem, yielding nerve fascicles (Figure 1). Mean ± SD wet weight of nerve fascicles obtained from the biopsy specimens was 16.8 ± 2.8 mg (Table 1). Immunohistochemical staining revealed that single nerve fascicles expressed S100 β (myelinating Schwann cells) and GFAP (nonmyelinating Schwann cells).

Figure 1—
Figure 1—

Representative photographs (A and B), dissection microscopic image (C), and bright-field microscopic images (D–G) of various preparations of dorsal cervical cutaneous nerve specimens harvested from the cadavers of 3 young adult dogs. A—Dorsal cervical cutaneous peripheral nerves are easily visible on dissection. B—Isolated nerve fascicles are surrounded with vessels, fat, and connective tissues. C—Nerve fascicles are shown dissected from surrounding tissues. D and F—The perineurium has been removed from the nerve fascicles. The GFAP (green) and S100 β (red) stains run mostly longitudinally along each fiber. Bar = 100 µm. E and G—At higher magnification, small nonmyelinating Schwann cells appear green (GFAP) and Schwann cells appear red (S100 β). Bar = 20 μm.

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1166

Table 1—

Cell yields after predegeneration and primary culture of dorsal cervical cutaneous nerve specimens harvested from the cadavers of 3 young adult dogs.

   Nerve yield Postculture yieldPostpurification yield
SubjectSpecimen wet weight (mg)Duration of predegeneration (d)Duration of culture (d)Total cells (X 105)Cells/mg (X 104)Total cells (X 105)Cells/mg (X 104)  
Dog 115107.5576.44.22.61.7
Dog 215.5812.58.11095.83.32.1
Dog 320810.55.216633.21.6
Mean ± SD16.8 ± 2.88.7 ± 1.210.2 ± 2.56.1 ± 1.711 ± 4.57.1 ± 1.64.3 ± 1.43.0 ± 0.31.8 ± 1.3

Isolation and purification of Schwann cells

After 8 to 10 days of in vitro predegeneration, dorsal cutaneous cervical nerve specimens provided a mean ± SD yield of 6.1 ± 1.7 × 103 cells/mg of nerve tissue. Mean cell yields after primary culture for 7 to 16 days were 4.3 ± 1.4 × 104 cells/mg of nerve tissue. After purification with magnetic beads, the mean total number of cells decreased from 7.1 ± 1.6 × 105 to 3.0 ± 0.3 × 105, with a mean yield of CD90-negative cells of 1.8 ± 1.3 × 104 cells/mg of nerve tissue (Table 1).

Primary cultures were a mixed population of fibroblasts with flat, polymorphic cells and Schwann cells with bipolar or tripolar shapes and smaller nuclei (Figure 2). After purification, cells with typical Schwann cell morphology became more dominant. In primary culture, a mean 39.8 ± 9.1% of cells expressed p75 and a mean 50.8 ± 9.1% of cells expressed CD90. After purification, the mean proportion of p75-positive cells increased to 85.4 ± 1.9% and that of CD90-positive cells decreased to 14.1 ± 12.9%. After 2 passages of subculture with antimitotic treatment, p75-positive cells accounted for a mean 97.8 ± 1.2% of cells and the mean proportion of CD90-positive cells decreased to 1.5 ± 0.7%. Overall, populations of Schwann cells were isolated with a mean purity of 85.4 ± 1.9% at mean yield of 1.8 ± 1.3 × 104 cells/mg of nerve tissue in 3 weeks. With 2 additional passages with antimitotic treatment, mean Schwann cell purity increased up to 97.8 ± 1.2%.

Figure 2—
Figure 2—

Representative immunofluoresence microscopic images of nerve cells isolated from specimens described in Figure 1 after predegeneration (A), purification through magnetic bead cell separation (B), and 2 passages after purification (C) as well as percentage of cells expressing p75 (for Schwann cells) and CD90 (for fibroblasts) at various points of processing (D). A—In primary culture, a mixed population of fibroblasts (green) with flat, polymorphic cytoplasm and Schwann cells (red) with bipolar morphology and smaller nuclei are evident. B—After purification, Schwann cells are more dominant than fibroblasts. C—After 2 passages, most cells express p75. Bar = 50 μm in all microscopic images. See Figure 1 for stain information.

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1166

Expansion of Schwann cells with rhGGF2

After 2 passages, the effect of a range of concentrations of rhGGF2 on Schwann cell proliferation was tested. In general, proliferation rates increased in a concentration-dependent manner (Figure 3). The results were normalized to control (0 mg of rhGGF2/mL) values. No significant (random effect predictions, P > 0.05) difference was identified among the 3 dogs from which specimens originated. The effect of rhGGF2 was significant (P = 0.006). All cells cultured with rhGGF2 had a significantly higher proliferation rate than did control cells. The proliferation rate as a result of exposure to rhGGF2 at 80 and 100 ng/mL was approximately triple that of the control value, and the proliferation rate at a concentration of 80 ng/mL was significantly (P = 0.01) higher than that at 20 ng/mL. The proliferation rate decreased and was more variable at rrGGF2 concentrations > 100 ng/mL.

Figure 3—
Figure 3—

Mean proliferation rate ratios at various concentrations of rhGGF2 in an MTS assay for Schwann cells isolated from dorsal cervical cutaneous nerve specimens harvested from the cadavers of 3 young adult dogs. Each value represents the mean of 3 observations for each dog. Rate ratios were calculated as the Schwann cell proliferation ratio normalized to that of the control (0 ng of rhGGF2/mL) ratio. Error bars represent SD. *Value differs significantly (P < 0.05) from the control value. The proliferation rate of Schwann cells cultured in rhGGF2 at a concentration of 80 ng/mL was significantly higher than that for cells cultured in rhGGF2 at a concentration of 20 ng/mL (bracket).

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1166

On the basis of the MTS assay results, the doubling time of Schwann cells in culture was evaluated at rhGGF2 concentrations of 40, 60, 80, and 100 ng/mL, revealing no significant (random effect predictions, P > 0.05) difference among the 3 dogs. The effect of rhGGF2 was significant (P < 0.001). Mean doubling times at 0, 40, 60, 80, and 100 ng/mL were 2.8 ± 0.3 days, 1.6 ± 0.1 days, 1.6 ± 0.1 days, 1.6 ± 0.1 days, and 1.8 ± 0.1 days at first passage, respectively; 6.2 ± 0.8 days, 2.0 ± 0.1 days, 1.9 ± 0.1 days, 1.8 ± 0.1 days, and 1.9 ± 0.0 days, respectively, at second passage; and 14.0 ± 2.8 days, 2.8 ± 0.4 days, 1.9 ± 0.1 days, 2.0 ± 0.0 days, and 2.1 ± 0.1 days, respectively, at third passage. Doubling time of control cells was significantly (P < 0.001) prolonged, compared with doubling times for rhGGF2-exposed groups at each passage, but did not differ significantly among groups of cells treated with rhGGF2 (Figure 4).

Figure 4—
Figure 4—

Mean doubling times of the Schwann cells described in Figure 3 at various passages and concentrations of rhGGF2. Error bars represent SD. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1166

Discussion

In the present study, Schwann cells were successfully cultured from dorsal cervical cutaneous nerve specimens from cadavers of young adult dogs. A mean of 0.7 million cells were generated within 3 weeks from single nerve fascicles (originating from two 3- to 4-cm-long strips of nerve) by means of predegeneration and primary culture. Approximately 40% of the cells expressed the Schwann cell marker p75. Use of magnetic bead–based cell separation and subculture with antimitotic treatment allowed us to increase purity to approximately 97% Schwann cells. The addition of rhGGF2 to the culture medium resulted in a 3-fold increase in cell proliferation rate, whereas cell doubling time remained stable through 3 passages. The combination of rhGGF2 and magnetic bead sorting could be used to rapidly expand and purify cultures of Schwann cells from adult dogs.

Many studies17,20,32 in the early 1990s involved the isolation of Schwann cells from rodents, primates, and humans. In rodent studies,32 Schwann cells were obtained from sciatic nerve biopsy specimens and frequently from fetal or neonatal rather than adult sources.32 The ability to generate autologous Schwann cells is important for patients to avoid immune rejection or the need for immunosuppression, and concerns exist about the functional consequences of sciatic nerve biopsy. In humans, sural nerves and phrenic nerves have been used as safe nerve biopsy sites for isolation of Schwann cells.25,33 Also, samples obtained from the brachial plexus during reconstructive surgery or biopsy for diagnostic purposes have been used to generate adequate numbers of Schwann cells.34

In the study reported here, the dorsal cervical cutaneous sensory nerves in dogs were used for biopsy. Dorsal cutaneous nerves are part of the medial branch of the dorsal ramus of spinal nerves and usually consist of single or a couple of nerve fascicles.35 These nerves are easily accessible and are frequently transected during the dorsal approach to the cervical vertebrae with no consequences to the patient.36 We also limited biopsy sites to 2 nerves, which could be obtained through a small (2 to 3 cm) skin incision on the dorsal aspect of the neck. Two nerve strips approximately 3 to 4 cm in length weighed approximately 16 mg and yielded clinically relevant numbers of Schwann cells. One downside to this approach was that the nerves are sensory, and data exist to suggest that Schwann cells from sensory nerves are more likely to support sensory axons.37 Although the importance of matching the Schwann cell source with the intended therapeutic application needs to be investigated, it has now been demonstrated that addition of a brain-derived growth factor can remove the phenotypic specificity of Schwann cells.37,38

Obtaining a large, pure population of Schwann cells from fairly small biopsy specimens of peripheral nerves is challenging. Dissociation of neonatal sciatic nerves yields high numbers of pure Schwann cells in a serum-containing medium.39 However, direct dissociation of adult peripheral nerves results in fairly poor yields of Schwann cells. One study40 revealed that only 600 cells/mg of nerve tissue were obtained from adult rat sciatic nerve specimens after fibroblasts were eliminated. On the other hand, in vitro culture of nerve specimens before dissociation enhances the Schwann cell yield.25,34 In a study25 in which direct dissociation was compared with multiple explantation by placing human adult phrenic nerve specimens in culture, immediate and direct dissociation yielded mostly fibroblast-like cells. However, the multiple explantation technique yielded 2 × 104 cells/mg of nerve tissue with 98% purity after 6 to 7 weeks in vitro. In the same study,25 immediate dissociation of adult rat sciatic nerve specimens yielded approximately 1,000 cells/mg of nerve tissue, with only 10% to 40% purity. In addition, other investigators34 report that in vitro predegeneration of human nerve biopsy specimens by culturing for 7 to 14 days resulted in a mean of 2.1 × 104 cells/mg of nerve tissue (range, 1 to 7 × 104 cells/mg), with > 85% purity. They also report that approximately 7,000 cells/mg of nerve tissue can be obtained from canine sciatic nerve specimens with the same protocol.27

Proliferation of Schwann cells is stimulated during Wallerian degeneration in vivo.41 Both multiple explantation of nerve pieces and in vitro predegeneration appear to induce a Wallerian degeneration–like process and are reportedly highly effective at increasing Schwann cell populations.20,41,42 In the present study, 8 to 10 days of in vitro predegeneration yielded approximately 6,000 cells/mg of nerve tissue, consistent with findings in another study27 involving dogs. Additional primary culture and purification yielded 1.8 × 104 cells/mg of nerve tissue with approximately 85% purity within 3 weeks.

Dulbecco modified Eagle medium with 10% fetal bovine serum has long been known to be suitable for proliferation of Schwann cells.43 However, cells isolated from adult peripheral nerve specimens are heterogeneous and include a high number of fibroblasts.23,24 Because these culture conditions are suitable for fibroblasts, which grow much faster than Schwann cells, fibroblast overgrowth results.

In several studies, attempts have been made to isolate purified adult Schwann cells. Highly purified Schwann cells can be obtained from sciatic nerve specimens from rats by culture in an oligodendrocyte growth medium containing < 2.5% fetal bovine serum.44 Isolation of Schwann cells by fluorescence-activated cell sorting based on specific binding properties of the extracellular domain of Necl1 has also been described.45 Although fluorescence-activated cell sorting has been widely used as an effective method to purify cells, concerns remain with regard to contamination and reduction in cell viability while sorting.46

More recently, a protocol has been reported21,30 for obtaining highly purified Schwann cells from rat, human, and canine sciatic nerve specimens by culture in serum-free melanocyte growth medium supplemented with mitogens and a rapid washing step (cold jet). However, the cold-jet technique was less effective in the purification of Schwann cells from adult dogs, compared with the purification of Schwann cells from other species. The same research group reported that fibroblasts could be removed from canine nerve specimens via magnetic bead–based cell separation.27,47

Treatment with an antimitotic agent such as Ara-C can also enhance the purity of Schwann cell populations by up to 95%.48 In the present study, we modified previously reported techniques by combining immunopurification and antimitotic treatment. An increase in Schwann cell purity can reportedly be achieved by immunopurification with 1mM Ara-C for 4 days during subculture.27 By this method, a purity of primary cells of approximately 30% can be increased to 60% after purification and up to 80% in sequential passages and purification.27 Compared with these reported findings,27 results of the present study indicated a similar purity (approx 43%) and initial cell numbers (approx 6,000 cells/mg of nerve tissue) after in vitro predegeneration. Immunopurification via magnetic bead–based cell separation followed by culture resulted in a high purity (approx 85%). The Ara-C reportedly eliminates a large number of fibroblasts without affecting Schwann cells in rat nerve specimens, whereas it has a toxic effect on Schwann cells in adult human peripheral nerve specimens.30,48 In the present study, Schwann cell cultures were exposed to 10μM Ara-C for 48 hours, which was 0.01 times the concentration used in the previous study.30

In the study reported here, concentrations of rhGGF2 were identified that would be useful for canine Schwann cell expansion. Glial growth factor 2 is a secreted isoform of neuregulin (neuroregulin-1), which is known for its stimulatory effects on oligodendrocytes and Schwann cells.49 Several growth factors such heregulin, forskolin, and rhGGF2 are potent mitogens for adult Schwann cells and have been used successfully to expand Schwann cell populations.21,50,51 Although the effects of rhGGF2 have been investigated in rodents and humans,50–52 to the authors’ knowledge, no reports exist of its use for culture of Schwann cells for dogs.

Proliferation rates of Schwann cells in the present study were significantly increased relative to control values when rhGGF2 concentrations of 20 to 200 ng/mL were used. In addition, a concentration of 80 ng/mL yielded significantly higher proliferation rates than did a concentration of 20 ng/mL, whereas concentrations > 100 ng/mL appeared to result in a reduction in efficacy, although this difference was not significant. These findings are comparable to findings reported for rodent Schwann cells.53 However, before this particular protocol is used to expand Schwann cell populations for transplantation in dogs, the effect of exogenous rhGGF2 on chromosomal stability in long-term culture should be examined.

We were able to generate Schwann cells from small strips of nerve fascicles harvested from dorsal cervical cutaneous sensory nerves of canine cadavers. A mean wet weight of 16 mg of nerve fascicles were obtained from two 3- to 4-cm-long nerve strips. A mean 8.6 days of in vitro predegeneration yielded approximately 6,000 cells/mg of nerve tissue, and subsequent primary culture yielded 43,000 cells/mg of nerve tissue in a mean of 11 days. These primary cells comprised a mean of 39.9 ± 9.1% p75-expressing Schwann cells. Immunopurification by use of magnetic beads yielded a mean of 85.4 ± 1.9% p75-positive cells. Two passages of subculture with 10μM Ara-C for 48 hours further enhanced Schwann cell 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 Schwann cell proliferation rate by up to 3 times and maintained cell quality throughout the cell passages. Although additional studies are needed to investigate the functionality of cultured Schwann cells, these findings suggested that autologous canine Schwann cells can be obtained, purified, and expanded for potential use to treat dogs with axonal injury both within the spinal cord and peripheral nerves.

Acknowledgments

Supported by the American Kennel Club Canine Heath Foundation.

Presented in abstract form at the 32th Annual Forum of the American College of Veterinary Internal Medicine, Nashville, June 2014.

ABBREVIATIONS

Ara-C

Cytosine arabinoside

DAPI

4′,6-diamidino-2-phenylindole

DMEM

Dulbecco modified Eagle medium

GFAP

Glial fibrillary acidic protein

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

PBST

PBS solution with 0.1% Tween 20

PLO

Poly-L-ornithine

PNS

Peripheral nervous system

rhGGF2

Recombinant human glial growth factor 2

SCM

Schwann cell culture medium

Footnotes

a.

Gibco BRL, Grand Island, NY.

b.

Olympus dissecting microscope SZ30, Olympus Corp, Buffalo Grove, Ill.

c.

DAKO, Carpinteria, Calif.

d.

SantaCruz Biotechnology Inc, Dallas, Tex.

e.

Invitrogen, Carlsbad, Calif.

f.

VECTASHILD mounting medium with DAPI, Vector Laboratories, Burlingame, Calif.

g.

AZ100 fluorescence microscope, Nikon Instruments Inc, Melville, NY.

h.

Matrigel, BD Biosciences, San Jose, Calif.

i.

Corning Inc, Corning, NY.

j.

PromoCell GmbH, Heidelberg, Germany.

k.

STEMCELL Tech, Vancouver, Canada.

l.

Poly-l-ornithine hydrobromide, Sigma-Aldrich Corp, St Louis, Mo.

m.

Rat anti-canine Thy1-antibody (anti-Cdw 90) canine, Serotec, Raleigh, NC.

n.

Dynabeads sheep anti-rat IgG, Dynal Biotech, Invitrogen, Carlsbad, Calif.

o.

Dynal MPC-S, Dynal Biotech, Invitrogen, Carlsbad, Calif.

p.

Ara-C, Sigma-Aldrich Corp, St Louis, Mo.

q.

Lab-Tek chamber slides, Thermo Scientific, Rochester, NY.

r.

Rabbit-anti-human p75, SantaCruz Biotechnology Inc, Dallas, Tex.

s.

Fluoromicroscope AZ100 macro/microscope, Nikon Corp, Fukasawa, Japan.

t.

Acorda Therpeutics, Ardsley, NY.

u.

CellTiter 96 AQueous one solution cell proliferation assay, Promega Corp, Fitchburg, Wis.

v.

TECAN SUNRISE remote microplate reader, Tecan Group Ltd, Männedorf, Switzerland.

w.

SAS, version 9.2, SAS Institute Inc, Cary, NC.

References

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