Cerebrospinal fluid used as culture medium prior to autologous olfactory ensheathing cell transplantation*☆

Weijiang Wu1, 2, Qing Lan2, Hua Lu1, Aihua Zhu1, Yunzhao Jiang1, Ge Chen1, Guozhen Hui3

1Department of Neurosurgery, Wuxi Third People’s Hospital, Wuxi  214041, Jiangsu Province, China
2Second Affiliated Hospital, Soochow University, Suzhou  215004, Jiangsu Province, China
3First Affiliated Hospital, Soochow University, Suzhou  215006, Jiangsu Province, China

Weijiang Wu☆, Studying for doctorate, Associate chief physician, Master’s supervisor, Department of Neurosurgery, Wuxi Third People’s Hospital, Wuxi  214041, Jiangsu Province, China; Second Affiliated Hospital, Soochow University, Suzhou   215004, Jiangsu Province, China

Corresponding author: Qing Lan, Doctor, Chief physician, Doctoral supervisor, Second  Affiliated Hospital, Soochow University, Suzhou   215004, Jiangsu Province, China
szlq006@yahoo.com.cn

Supported by: the National Trauma Program (973 Program), No. 2005CB522600*

Abstract
BACKGROUND: Cerebrospinal fluid can be an inducer for neural stem cells in vitro, but few studies employ cerebrospinal fluid to culture olfactory ensheathing cells.
OBJECTIVE: To investigate the growth of nasal mucosa olfactory ensheathing cells in normal cerebrospinal fluid, and to analyze the feasibility of cerebrospinal fluid for culturing olfactory ensheathing cells used for transplantation.
DESIGN, TIME AND SETTING: A completely randomized, block design study was performed at the Cell Laboratory, Wuxi Third People’s Hospital, and Jiangsu Institute of Parasitic Diseases, China, in August 2008.
MATERIALS: Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) and fetal bovine serum (Gibco BRL, USA), mouse anti-rat P75 monoclonal antibody and rabbit anti-glial fibrillary acidic protein polyclonal anti body (Santa Cruz Biotechnology, USA), mouse anti-rat myelin basic protein monoclonal antibody (Cymbus, UK), mouse anti-rat microtubule-associated protein-2 monoclonal antibody (Transduction Laboratories, USA), FITC conjugated rabbit anti-mouse monoclonal antibody (Boster, China), TRITC conjugated goat anti-rabbit monoclonal antibody (Sigma, USA) were used.
METHODS: Nasal mucosa olfactory ensheathing cells were separately incubated in DMEM/F12, cerebrospinal fluid, and changing DMEM/F12 into cerebrospinal fluid. Adult female Sprague Dawley rat models of spinal hemisection were established. Nerve injury was repaired by transplantation of nasal mucosa olfactory ensheathing cells cultured in cerebrospinal fluid or DMEM/F12.
MAIN OUTCOME MEASURES: The proliferative ability of olfactory ensheathing cells cultured in cerebrospinal fluid was determined by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The morphology and purity of olfactory ensheathing cells were detected using immunohistochemistry. Animal behavior was evaluated by the Basso, Beattie and Bresnahan locomotor rating scale. Morphological repair was assessed by a horseradish peroxidase-tetramethylbenzidine retrograde tracer technique and immunohistochemistry.
RESULTS: Changing from DMEM/F12 to cerebrospinal fluid did not change overall culture morphology and purity on day 14. These cells also contributed to myelinization and the conduction velocity of regenerated axons, and improved motor abilities of denervated muscle fibers in rats with spinal cord injury. The recovery of behavioral function and neuronal regeneration was similar in the two groups.
CONCLUSION: Cerebrospinal fluid culture prior to autologous olfactory ensheathing cell transplantation is feasible for clinical use.
Key Words: cerebrospinal fluid; olfactory ensheathing cells; in vitro culture; cell transplantation

INTRODUCTION
  
Olfactory mucosa-derived olfactory ensheathing cells (OECs) exhibited identical in vitro morphology and phenotypic characteristics as olfactory bulb-derived OECs. Olfactory mucosa-derived OECs can contribute to axonal regeneration and myelinization in the central nervous system of animal models[1-6], as well as repair of the central nervous system[7-10], indicating therapeutic potential for treating demyelinating disease or nerve injury. For example, transplantation of autologous OECs into the injured spinal cord of patients with complete, thoracic paraplegia is feasible and is safe up to 3 years post-implantation[11]. However, whether autologous OECs transplantation can avoid ethical problems? Numerous ethical problems involve various aspects, such as transplant type, posttransplantation rejection, oncogenicity of transplants and use of harmful reagents during culture. We tested the feasibility of culturing cells in autologous cerebrospinal fluid prior to transplantation to reduce the number of potential contaminants and determine if cell growth occurs normally in cerebrospinal fluid, addressing a clinical barrier to their use.
 

MATERIALS AND METHODS

Design
A completely randomized block design study.
Time and setting
Experiments were performed at the Cell Laboratory, Wuxi Third People’s Hospital, and Jiangsu Institute of Parasitic Diseases, China, in August 2008.
Materials
Olfactory mucosa were obtained from the adult nasal cavity during surgery on the sphenoid sinus: following conventional sterilization with povidone iodine, olfactory mucosa was peeled off from the back 1/3 of the middle turbinate, and then placed in a tube containing medium, which was stored in a clean ice bag. Protocols were performed in accordance with the Administrative Regulations on Medical Institution, formulated by the State Council of the People’s Republic of China[12]. Experiments were approved by the Ethics Committee of Wuxi Third People’s Hospital of China. Patients all signed informed consent.
Main reagents and equipment are as follows:

A total of 26 adult female Sprague Dawley rats aged 40 days and weighing (237.5 ± 18.3) g were supplied by Jiangsu Institute of Parasitic Diseases, China (license No. SCXK2007-0006). The rats were housed at 21 °C, 50% humidity, 15–20 lux illumination. Protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[13].
Methods
Cell culture methods
Olfactory mucosa were cultured as outlined in the flow chart. Following purification with a modified Nash differential adhesion method[14], cells were seeded at 1×106/mL in 9 wells of a poly-L-lysine-coated 24-well plate (1 mL/well). Whole medium (DMEM/F12 containing 10% fetal bovine serum) was added to 6 wells, 3 wells each for groups 1 and 2. Medium supplemented with forskolin and bovine pituitary extract was added to the remaining 3 wells as group 3. The remaining cell suspension was centrifuged to remove supernatant. Half of the cell colony was absorbed using a sterile capillary pipette. Following the addition of serum-free DMEM/F12, cells were incubated at 1 × 106/mL in 3 wells as group 4. The other cells were incubated in normal cerebrospinal fluid following filtration with 0.22 μm microporous membrane, adjusted to 1 × 106/mL, and seeded in 3 wells as group 5. Medium was replaced every 2–3 days. Cells were incubated in cerebrospinal fluid, rather than DMEM/F12, in group 2 on day 8.
Precise flowchart of cell culture is shown in Figure 1.
Effects of various culture methods on OECs
Cell growth was measured as the time needed to reach 80% confluence, and proliferation was determined by MTT assay[15-16] on day 14. OECs were treated with           5 mg/mL MTT for 4 hours, and then with 150 μL dimethyl sulfoxide in each well. Absorbance values were measured at 490 nm using a microplate reader. Cell morphology was observed daily and photographed under the inverted microscope. Immunofluorescence staining for expression of P75 and GFAP[17] was performed on day 14 following removal of the medium, cells were washed three times in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 30 minutes, treated with 0.25% TritonX-100 for 15 minutes, rinsed in PBS, and blocked in goat serum for 30 minutes. Cells were treated with mouse anti-rat P75 monoclonal antibody (1: 1 500) and rabbit anti-GFAP polyclonal antibody (1: 1 500) separately for 1.5 hours, washed in PBS, and then treated with FITC conjugated rabbit anti-mouse monoclonal antibody      (1: 1 000) and TRITC conjugated goat anti-rabbit monoclonal antibody (1: 1 000) for 1 hour at room temperature. Following rinsing, cells were observed under the fluorescence microscope (× 100). Ten fields from each section were randomly selected to count P75- and GFAP-positive cells to determine culture purity, and morphology was compared.
Animal model establishment and cell transplantation
OECs from groups 1 and 2 were suspended at 1×107/mL on day 14. Cells were incubated in Hoechst[18-19] at 37 °C for 30 minutes prior to transplantation, followed by two washes in DMEM/F12. Cells were digested and harvested at 1×107/mL for transplantation. Sprague-Dawley rats were randomly assigned to control group (n = 6), transplantation group 1 (n = 10) and transplantation group 2 (n = 10). All rats received a laminectomy at T10-12. A 3-mm diameter hole was made left of the spinal cord bordered by a central vein using a mini-aspirator to induce hemisection[20].
 

The control group received a gelatin sponge and 8 μL medium (DMEM/F12+10% fetal bovine serum) in the hole. 2 μL medium was injected 1 mm superior and inferior to the wound surface via multiple points using a glass pinhead (75 μm tip diameter). A small fascia was cut to repair the spinal dura mater. The muscular layer and skin were sutured. In the transplantation groups, 8 μL OEC suspension was infused into the gelatin filled in the hole, and 2 μL cell suspension was injected 1 mm superior and inferior to the wound surface via multiple points. The wound was sutured. All rats received daily gentamicin 10 000 U via intramuscular injection for 2 weeks, and cyclosporine A 10 mg/kg via intraperitoneal injection for 1 month. Padding in the cage was replaced twice a day. The rats received bladder massage regularly until spontaneous voiding.
Behavioral measurements
Motor function of the hindlimb hip, knee, and ankle joint were determined using the Basso, Beattie and Bresnahan locomotor rating scale[21] prior to surgery, and at 12 hours, 3, 7, 14, 21 and 28 days following surgery. A score of 21 is normal activity.
Horseradish peroxidase tracing
The wounds of all rats was incised again on day 28 to expose spinal meninges, and 30% horseradish peroxidase (0.5 μL) was infused 5 mm inferior using a microsyringe[22]. Two days later, all rats were sacrificed by overdose anesthesia and fixed in 4% paraformaldehyde. The brain and spinal cord were placed in 30% sucrose at 4 °C overnight. Tissues from the cortical hindlimb area, the magnocellular division of the red nucleus, and the injury area of the spinal cord were permeabilized with xylene, embedded in paraffin, and sliced into serial sections along the longitudinal axis and horizontal axis. The presence of labeled cells in the defect region of the spinal cord was observed under a fluorescence microscope.
Immunohistochemical analysis
Sections from the injured region of the spinal cord underwent immunocytochemical staining for expression of P75, microtubule-associated protein-2, and myelin basic protein[23]. Sectiones were dried, deparaffinized in graded concentrations of xylene and ethanol, treated with 3% H2O2 for 5–10 minutes to inactivate endogenous enzyme, washed in distilled water, and subjected to antigen retrieval at high temperature. After adding blocking serum at 37 °C for 30 minutes, sections were separately treated with mouse anti-rat P75 monoclonal antibody (1: 1 500), mouse anti-rat microtubule- associated protein-2 monoclonal antibody (1: 1 000), and mouse anti-rat myelin basic protein monoclonal antibody (1: 1 000) at 37 °C for 1.5 hours. Sections were washed in PBS and incubated in biotinylated goat anti-rat mouse monoclonal antibody (1: 1 000) at 20 °C for 20 minutes, rinsed in PBS, treated with streptavidin biotin peroxidase complex for 20 minutes, developed by 3, 3’-diaminobenzidine, counterstained with hematoxylin, and observed under an inverted phase contrast microscope.
Quantitative analysis of horseradish peroxidase- tetramethylbenzidine retrograde tracer in motor neurons
Tissues from the cortical hindlimb area and the magnocellular division of the red nucleus were sectioned into 6-μm serial paraffin slices colored with tetramethylbenzidine. Cortical motor neurons and magnocellular red nucleus neurons were counted using a phase contrast microscope (× 100). The average number of rat cortical motor neurons and magnocellular red nucleus neurons was calculated in each group[24].
The experimental procedure of olfactory ensheathing cell transplantation for spinal and repair is shown in Figure 2.
 

Main outcome measures
The following parameters were measured: proliferative ability, morphology, purity, and recovery of nerve function.
Statistical analysis
Using a completely randomized design, analysis of variance and Chi-square test, the data were analyzed using SAS 9.1.1 statistical software (SAS, USA, No. M3P020206). A value of P < 0.05 was considered statistically significant.

RESULTS

Morphological observation of olfactory mucosa- derived OECs
Olfactory mucosa-derived OECs had distinct morphology. For example, at 1–3 days following adhesion, dispersed spindle cells with strong refraction were visible, with a few tripolar and quadripolar cells, in contrast to images

on day 14 (Figures 3A, B). From 7–10 days, cells proliferated rapidly, mainly bipolar spindle cells that showed OEC characteristics and could be easily separated from fibroblasts (Figures 3C, D). No significant difference was detected in cell morphology during growth and at 2 weeks following staining in the 5 groups.
Effects of culture methods on OECs growth and adhesion
Cells were 80% confluent in groups 1, 2, and 3 following 5-day culture, and in groups 4 and 5 following 7-day culture. Cells distributed densely and cell division occurred in groups 1, 2, and 3. However, cells in groups 4 and 5 were dispersed. At 7–10 days, abundant large, flat fibroblasts were visible in groups 1, 2, and 3, but few fibroblasts were observed in groups 4 and 5 (Figures 3C, D). The time to 80% confluence was shortest in group 3 (Figure 4), with significant differences compared with group 1 (P < 0.05). Groups 4 and 5 were longer than group 1 (P < 0.05), but groups 4 and 5 were similar, as were groups 1 and 2.
Effects of media on cell proliferation
On day 14, cell proliferation was largest in group 3 (P < 0.05), and weakest in groups 4 and 5. Groups 4 and 5 proliferated faster than group 1 (P < 0.05), but groups 4 and 5 were the same, as were groups 1 and 2 (Figure 5).

Changes in cell purity
On day 14, P75 and GFAP immunohistochemistry was used to determine culture purity, measured as the average percentage staining of both markers (Figure 6). Cell purities were 76.55%, 79.12%, 75.43%, 81.59%, and 83.06% in groups 1, 2, 3, 4, and 5, respectively. No difference was determined among the 5 groups (P > 0.05, Chi-square test).

Basso, Beattie and Bresnahan (BBB) locomotor rating scale in rats
The BBB score was 21 points in normal rats. Spinal cord hemisection disrupted hindlimb function and caused limb contraction in the control group, but cell transplantation ameliorated this contraction. Transplantation increased behavioral recovery faster than controls (P < 0.05), but all transplantation groups were similar (P > 0.05) (Table 1).

Immunohistochemistry detection in transplant and spinal cord repair
Fluorescence microscopy of spinal cord sections demonstrated that transplanted cells with green fluorescence were visible in cell transplantation groups (Figure 7A). Immunohistochemistry showed that P75-positive bipolar or multipolar cells were found in cell transplantation groups. Fibers positive for myelin basic protein were visible in broken ends of fractured bone, and myelin basic protein-positive particles were also seen in multipolar cells in cell transplantation groups. Many neurons positive for microtubule-associated protein-2 were seen in injured spinal cords in the cell transplantation groups (Figure 7B). Many glial scars were found, but no staining for P75, microtubule-associated protein-2, or myelin basic protein was seen in the control group.

Retrograde tracing of cortical motor neurons and red nucleus motor neurons
On the normal side, horseradish peroxidase-labeled neurons were primarily located on the magnocellular division of the red nucleus and cortical motor area. No horseradish peroxidase-labeled neurons were found on the damaged side in the control group. No significant difference was detected in horseradish peroxidase-labeled neurons between cell transplantation groups (P > 0.05) (Table 2, Figure 8).

DISCUSSION

Olfactory mucosa-derived OECs are difficult to culture and purify compared with olfactory bulb-derived OECs. Olfactory mucosa-derived OECs rely on serum in early cultures, and cell culture tests have confirmed that increased serum concentration contributes to cell adherence and proliferation. Serum is not critical when cells enter the logarithmic phase. Therefore, before entering logarithmic phase, the culture condition was replaced: autologous olfactory mucosa-derived OECs were incubated in cerebrospinal fluid, which could avoid harmful factors entering human bodies, such as fetal bovine serum and bovine pituitary extract. Thus, clinical tests will be more in accordance with ethical requirements.

Cerebrospinal fluid contains electrolytes, proteins, sugars, and growth factors, such as basic fibroblast growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor[25-26]. Cerebrospinal fluid is an ideal medium for OECs in the reproductive cycle, as it ensures normal proliferation prior to autologous transplantation. Cerebrospinal fluid alone produced normal adhesion and proliferation. Forskolin and bovine pituitary extract in conventional medium contributes to in vitro growth and amplification of exogenous OECs, but serum-free or cerebrospinal fluid cultures had higher cell purity. OECs purity in this study was slightly lower than other olfactory bulb-derived OECs (approximately 90%)[27]. Komiyama et al[28] used the serum-free culture method, but OECs rely on serum at an early stage, thus reducing the final cell number.
OECs cultured in media with cerebrospinal fluid promote axonal regeneration, demonstrated behaviorally and immunohistochemically, in animal tests to a similar extent as other culturing methods. Myelin basic protein and microtubule-associated protein-2 staining of neurons were similar in the damaged regions after transplantation, indicating that cerebrospinal fluid did not impact posttransplant results, and is feasible in the clinic. Myelin basic protein indicates axonal regeneration is occurring, and horseradish peroxidase-tetramethylbenzidine retrograde tracer technique verifies axonal regeneration. Behavioral improvements indicated nerve regeneration and improvements in nerve conduction. Thus, OECs transplantation improved neural regeneration. As ensheathing cells, OECs can produce myelinization of regenerating axons, resulting in the recovery of action potential conduction, and assist the recovery of denervated muscle fibers.
Cell transplantation for central nervous system diseases involves decisions of cell culture methods prior to autologous cell transplantation. The molecular mechanisms underlying axonal regeneration downstream of OECs require further study. In vitro tests and animal tests of cell therapy are satisfactory, but outcomes of clinical tests are disappointing. The interaction between transplanted cells and their environment is a key focus for this and future studies.

REFERENCES

[1] Au E, Roskams AJ. Olfactory ensheathing cells of the lamina propria in vivo and in vitro. Glia. 2003;41(3):224-236.
[2] Lindsay SL, Riddell JS, Barnett SC. Olfactory mucosa for transplant-mediated repair: A complex tissue for a complex injury? Glia. 2009;58(2):125-134.
[3] Iwatsuki K, Yoshimine T, Kishima H, et al. Transplantation of olfactory mucosa following spinal cord injury promotes recovery in rats. Neuroreport. 2008;19(13):1249-1252.
[4] Féron F. Current cell therapy strategies for repairing the central nervous system. Rev Neurol (Paris). 2007;163 Spec No 1: 3S23-30.
[5] Lima C, Pratas-Vital J, Escada P, et al. Olfactory mucosa autografts in human spinal cord injury: a pilot clinical study. J Spinal Cord Med. 2006;29(3):191-206.
[6] Féron F, Perry C, Cochrane J, et al. Autologous olfactory ensheathing cell transplantation in human spinal cord injury. Brain. 2005;128(Pt 12):2951-2960.
[7] Richter MW, Fletcher PA, Liu J, et al. Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord. J Neurosci. 2005;25(46):10700-10711.
[8] Lu P, Yang H, Culbertson M, et al. Olfactory ensheathing cells do not exhibit unique migratory or axonal growth-promoting properties after spinal cord injury. J Neurosci. 2006;26(43): 11120-11130.
[9] Deng C, Gorrie C, Hayward I, et al. Survival and migration of human and rat olfactory ensheathing cells in intact and injured spinal cord. J Neurosci Res. 2006;83(7):1201-1212.
[10] Toft A, Scott DT, Barnett SC, et al. Electrophysiological evidence that olfactory cell transplants improve function after spinal cord injury. Brain. 2007;130(Pt 4):970-984.
[11] Mackay-Sim A, Féron F, Cochrane J, et al. Autologous olfactory ensheathing cell transplantation in human paraplegia: a 3-year clinical trial. Brain. 2008;131(Pt 9):2376-2386.
[12] State Council of the People's Republic of China. Administrative Regulations on Medical Institution.1994-09-01.
[13] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[14] Nash HH, Borke RC, Anders JJ, et al. New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb. Glia. 2001;34(2):81-87.
[15] Ji Z, Qian J, Han J, et al. Effect of olfactory ensheathing cells on growth of spinal cord neurons and its protective effect on neurons after injury in vitro. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(1):21-25.
[16] Chen Q, Zhang JH, Chen JT, et al. MTT assay for detecting osteosarcoma cell apoptosis. Nan Fang Yi Ke Da Xue Xue Bao. 2009;29(9):1899-1901.
[17] Collazos-Castro JE, Mu?etón-Gómez VC, Nieto-Sampedro M. Olfactory glia transplantation into cervical spinal cord contusion injuries. J Neurosurg Spine. 2005;3(4):308-317.
[18] Feng L, Meng H, Wu F, et al. Olfactory ensheathing cells conditioned medium prevented apoptosis induced by 6-OHDA in PC12 cells through modulation of intrinsic apoptotic pathways. Int J Dev Neurosci. 2008;26(3-4):323-329.
[19] Yu XD, Luo ZJ, Zhang L, et al. Effects of olfactory ensheathing cells on hydrogen peroxide-induced apoptosis in cultured dorsal root ganglion neurons. Chin Med J (Engl). 2007;120(16): 1438-1443.
[20] Boido M, Rupa R, Garbossa D, et al. Embryonic and adult stem cells promote raphespinal axon outgrowth and improve functional outcome following spinal hemisection in mice. Eur J Neurosci. 2009;30(5):833-846.
[21] Lebedev SV, Timofeyev SV, Zharkov AV, et al. Exercise tests and BBB method for evaluation of motor disorders in rats after contusion spinal injury. Bull Exp Biol Med. 2008;146(4):489-494.
[22] Cheng SY, Ruan HZ, Wu XG. Olfactory ensheathing cells enhance functional recovery of injured sciatic nerve. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2003;17(1):18-21.
[23] Shen HY, Yin DZ, Tang Y, et al. Influence of cryopreserved olfactory ensheathing cells transplantation on axonal regeneration in spinal cord of adult rats. Chin J Traumatol. 2004;7(3):179-183.
[24] Lu X, Geng X, Zhang L, et al.The methodology for labeling the distal cerebrospinal fluid-contacting neurons in rats. J Neurosci Methods. 2008;168(1):98-103.
[25] Grundstr?m E, Lindholm D, Johansson A, et al. GDNF but not BDNF is increased in cerebrospinal fluid in amyotrophic lateral sclerosis. Neuroreport. 2000;11(8):1781-1783.
[26] Straten G, Eschweiler GW, Maetzler W, et al. Glial cell-line derived neurotrophic factor (GDNF) concentrations in cerebrospinal fluid and serum of patients with early Alzheimer's disease and normal controls. J Alzheimers Dis. 2009;18(2): 331-337.
[27] Wu WJ, Hui GZ, Lu H, et al. Effect of different harvesting and culturing methods on the purity of human fetal olfactory ensheathing cells. Zhonghua Shi Yan Wai Ke Za Zhi. 2004;21(7): 841-844.
[28] Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods. 2003;122(2):195-200.
 (Edited by Wang ZH/Qiu Y/Song LP)