BACKGROUND: Both density gradient centrifugation and adherence method arc frequently used to isolate bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the approaches to isolate, culture and identify the rabbit BMSCs in vitro by the combination of den,ity gradient centrifugation and adherence method. DESIGN, TIME AND SETTING: Contrast cytological study, which was performed in Central Laboratory of Shanghai 6th People's Hospital between October 2007 and March 2008.MATERIALS: Six 2-week-old rabbits were selected for BMSCs preparation and primary culture; Percoll separating medium (1.073 kg/L) was also used for this study.METHODS: BMSCs were separated and purified with Percoll separating medium by density gradient centrifugation and adherence method. The three-, five-, seven-, and nine-passage BMSCs were counted for growth curve. MAIN OUTCOME MEASURES: Morphological features and growth states of primary and passage cells were observed under inverted microscope. Indirect immunofluorescence of CD44 and CD34 antibodies were used to examine the stem cells. CD44 staining was positive, and CD34 staining was negative, suggesting the extracting and purifying cells were BMSCs. RESULTS: The passage BMSCs were uniformly distributed like fusiform shape, which were more uniform than primary cultured cells. The BMSCs grew productively and proliferated rapidly; meanwhile, the nucleolus was clear, caryopla.sm was in a large proportion, morphological features were uniform, ceils like bostrychoid or whirlpool were arranged parallelly, and the five-pa.ssage cells were not changed remarkably. Proliferation was decreased gradually with the passage increasing; especially, the proliferation of three-five-passage cells was the strongest. The separated cells expressed CD44 but not CD34. CONCLUSION: High-purified rabbit BMSCs are obtained by both density gradient centrifugation and adherence method.

Objective This study investigates feasibility of replacing urinary epidermal cells with foreskin epidermal cells to reconstruct engineered anterior urethra with acellular collagen matrix. Methods A cellular collagen matrices were generated from allogeneic rabbit bladder submucosa.In 6 rabbits,autologous foreskin epidermal cells isolated,in vitro expanded and labeled with Brdu before seeding onto a tubular collagen matrix of 2 cm long.In 12 male rabbits, urethral mucous defect was created and urethroplasty was performed with tubular collagen matrix seeded with epidermal cells (n=6) in experimental group or matrix without cells seeding (n=6) in control group.Urethrography was performed at 1,2,and 6 months postoperatively.Urethras grafts were harvested and analyzed grossly and histologically at the same time points. Results In control group, gross and urethrography demonstrated urethral stricture of repaired defects at all time points.Histology showed a single layer of epidermal cell with unorganized and crassitude muscle fiber bundles in submucosa layer.In contrast, urethrography showed a patency of repaired urethral defect with the maintenance of a wide urethral caliber in experimental group without sign of strictures at any time points. Histologically,the graft seeded with epidermal cell had a multi-layers epidermal cells by 1 month,which demonstrated the presence of a epidermal cells layer with a normal submucosa structure.The immunoflorescent staining of Brdu confirmed the presence of implanted epidermal cells.In addition, the implanted cells also express keratin when stained with anti-pancytokeratins AE1/AE3 antibody.At 2 month,histology showed relatively normal urethral architecture with vessel abundance in submucosa structure.The staining of Brdu showed decreased at implanted epidermal cells and epithelial phenotypes were confirmed immunocytochemically using pancytokeratins AE1/AE3.At 6 month,the graft structure show multi-layers epidermal cells without staining of Brdu. Conclusions Urethra reconstruction was better achieved with cell seeded acellular collagen matrix when compare with acellular matrices alone.Foreskin epidermal cells seems succeeded in replacing urethral epidermal cells for urethra reconstruction.