ABSTRACT
BACKGROUND: The diversity of purification procedures resulting in various purities of olfactory ensheathing cells (OECs) used for grafting is considered to be relevant in the effectiveness of OECs transplant. It is important to develop a well-defined method which produces OECs of great purity and is easy to unify for the future standardization of research involving OECs.OBJECTIVE: To establish a method being easy to unify for purifying OECs to acquire highly and uniformly enriched population of OECs for standardized studies on cell transplantation.DESIGN: Randomized and controlled experiment.SETTING: Department of Orthopaedics, Affiliated Zhongda Hospital of Southeast University School of Clinical Medicine;Central Laboratory of Southeast University School of Clinical Medicine; Experimental Animal Center of Southeast University School of Clinical Medicine.MATERIALS: This experiment was carried out in the Central Laboratory of Southeast University School of Clinical Medicine from February to August 2006. Twenty-eight adult female SD rats weighing 200-250 g were selected in this study. The main reagents were detailed as follows: DMEM/F-12 (GIBCO); 2.5 g/L trypsin (GIBCO); poly-L-lysine (SIGMA); bovine pituitary extract (BPE, SIGMA); fetal bovine serum (FBS, Sijiqing Biological Agent Co., Ltd., Hangzhou);rabbit anti-low-affinity nerve growth factor receptor (anti-P75, SIGMA); biotinylated goat anti-rabbit IgG (Boster Bioengineering Co., Ltd., Wuhan); methyl thiazolyl tetrazolium (MTT) kit (SIGMA).METHODS: Primary cultures of OECs were separated from adult SD rats olfactory bulbs. At day 8 in vitro, the primary cultures were divided randomly into 4 groups, namely differential adhesion method group, immunoadsorption method group,the modified method group,and control group.①The cell suspension in the modified method group was seeded into uncoated flasks and incubated at 37 ℃ in 0.05 volume fraction of CO2 for 1 hours. The supematants were seeded into flasks that had been prepared as follows. The bottoms of these flasks were moistened with anti-P7s (1 mg/L) and were made to dry at 37 ℃, and then they were washed one time with DMEM/F-12. The supernatants were incubated on the anti-p75-treated flasks for 45 minutes at 37 ℃, 0.05 volume fraction of CO2. For removing unbound cells, the flasks were washed five times with DMEM/F-12. The bound cells were detached from the flasks with a cell scraper, centrifuged,and resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE. The cell suspension in differential anchoring method group or immunoadsorption method group was purified as previously described by Nash or Ramo'n-Cueto respectively. Three groups of cell suspensions resulted from the above three methods were seeded respectively onto poly-L-lysine-coated 24-well cell culture chambers and incubated for 14 days at 37 ℃ in 0.05 volume fraction of CO2. Without purification, the cell suspension in control group was also resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE and seeded onto poly-L-lysine-coated 24-well cell culture chambers and incubated under the same culture condition as the other groups.②Purity comparisons for the four groups were made at 2, 5, 8, 10, 12 and 14 days after the end of their respective manipulation to evaluate the effectiveness of the modified method. At per time point in each of the four groups, fifteen visual fields of cultures were selected randomly to count the At the day of 14, viabilities of OECs in the four groups were assessed by MTT assays.MAIN OUTCOME MEASURES: OECs purities at per time point and viabilities of OECs at the day of 14 in each of the four groups after the end of their respective manipulation.RESULTS:① The purities of OECs in the modified method group at each time point were greater (P＜0.05-0.01) than counterparts in the other three groups. OECs purities decreased with culture prolongation in all groups, but the changes of purities over the whole period of observation in the modified method group were the least. The last purities of OECs yielded from the modified method were still extremely great (92.1±1.2)%, whereas the parallels in the others were no more than (85.2±2.2)%.② There was no significant difference in viabilities of OECs between the modified method group and any of the others at the day of 14 (P=0.895).CONCLUSION: The modified method for purifying OECs from the adult rat olfactory bulb is highly effective without extra impairment on the viability of OECs and will be beneficial to the future standardization of research involving OECs.
ABSTRACT
[Objective]To investigate the effects of lysophosphatidic acid(LPA) on the morphology,proliferation and brain-derived neurotropic factor(BDNF) expression of olfactory ensheathing cells(OECs) in vitro.[Method]Primary cultures of OECs separated from adult rat olfactory bulbs were purified and cultured.Five experimental cultures were grown for a period of time in media with LPA at different concentrations,namely 1,5,10,20 and 50 ?mol/L,and the control culture was grown in the medium without LPA.Immunofluorescent staining was used to identify OECs and to observe their morphological changes.The proliferation of OECs was measured by MTT assay.Western blotting was used to detect the protein expression of BDNF.[Result]Exposure to LPA in medium induced the switch in the dominant morphology of OECs from process-bearing to flat morphology.This shift in morphology was reversed when LPA was removed from media.LPA at concentrations from 1 ?mol/L to 50 ?mol/L enhanced OECs proliferation,especially at the concentration of 10 ?m/L.Proliferation of OECs in all experimental cultures reached their respective significant peaks after 60 h of LPA treatment.There were significant upregulations in BDNF expression of OECs treated with LPA(1~50 ?m/L) compared with those in the control culture.[Conclusion]A reversible change from process-bearing to flat in morphology of OECs can be induced by LPA.LPA stimulates OECs proliferation in a time-and concentration-dependent manner.LPA upregulates BDNF expression of OECs.