Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To culture astrocytes of human optic nerve and establish the cell lines for further study of healing process after optic nerve trauma. Methods Astrocytes of infantile optic nerve were cultured by tissue inoculation or tissue digestion with 0.25 % trypsin and 0.06% EDTA. The second and fourth passage cells were stained with HE and anti-GFAP, S-100 protein, vimentin, and CD34 antibodies. Results The trypsinized astrocytes of infantile optic nerver eached confluence in 7 days. The cultured cells were in polygonal shape with processes and the cytoplasm was abundant. These cells were positive in GFAP, S-100 protein and vimentin staining, and negative in CD34 staining. Conclusions Astrocytes of human optic nerve can be successfully cultured by trypsinization rather than tissue inoculation. (Chin J Ocul Fundus Dis, 2001,17:144-146)
OBJECTIVE: To construct a tissue engineering skin containing melanocytes by employing tissue engineering method. METHODS: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected by Dopa staining, transmission electron microscope (TEM) and S-100 immunohistochemical staining. RESULTS: Melanocytes can be detected in the basal layer of the constructed tissue engineering skin. The results of TEM showed that the melanocytes were in good conditions. CONCLUSION: The artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects.
Objective To locate sinoatrial node (SAN) in suckl ing pigs, to develop a rel iable method for isolation, purification and cultivation of SAN cells and to observe the compatibil ity of SAN cells and Col I fiber scaffold. Methods Five newborn purebred ChangBaiShan suckl ing pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, wereused. Multi-channels electrophysiological recorder was appl ied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 × 105 cells/mL) were co-cultured with prewetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM). Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% (P lt; 0.01), and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% (P lt; 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively (P gt; 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a rel iable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibil ity.
To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for cl inical islet transplantation. Methods Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection (CSCI), and subsequently, digested with low concentration (0.5 mg/mL)of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation thatwas prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon (DTZ)stain, and propidium iodide (PI)/fluorescein diacetate (FDA) double stain was used to check viabil ity of islets. The endocrine secretory function was assessed by insul in secretion in either low (2.8 mmol/L) or high (25.0 mmol/L) glucose incubation after 3 days of culture in RPMI1640 media. Results Average islet digestion time of 5 experiments was (13.8 ± 1.6) min. Before purification, average isolated number was (5 626 ± 422) islets, and the number was significantly reduced to (2 914 ± 485) islets after purification (P lt; 0.01). The average recovery rate was 51.6% ± 6.0%, and the average yield was (583 ± 97) islets/pancreas. The average purity and viabil ity of islets were 90.2% ± 3.4% and 81.6% ± 7.0%, respectively. After 3 days of culture, insul in secretion of the islets was (116.1 ± 17.4) EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [(39.7 ± 7.5) EU/L, P lt; 0.01)]. The average insul in stimulation index was 3.0 ± 0.4. Conclusion The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
Objective To discuss the applycation possibility of themicroscopic stripping technique used in the primary culture of human embryonicesophagus squamous epithelial cells, and of the methodds for the isolation, depuration and subculture of the esophagus epithelial cells in vitro. Methods The squamous epithelial cells wereobtained from the esophagus mucous membrane of the 20-week abortion fetus through the microscopic stripping technique, and were digested with trypsin. Then, the morphological, immunohistochemical observation and the growth curve of the isolated cells were studied. Results The isolated cells were spherical in the cell suspension and spherical-like or polygon-like after attachment to the culture flask.The squamous epithelial specialized cytokeratin staining was bly positive. And the morphological studies by the transmission electron microscopy indicated that the cultured cells were squamous epithelial cells. The squamous epithelial cells reached the peak level 3-4 days after the transfer of the culture. The absorbanceat 3 and 4 days was significantly higher than that at 1,2,5 and 6 days (P＜0.05). Conclusion A large mumber of squamous epithelial cells can be available with the microscopic stripping technique and the digestion method. Thecultured squamous epithelial cells can be proliferated quickly, and fit for the tissue engineering study.
Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth state of primary culture of adult rat retinal Muuml;ller cells. Methods Peritoneal exudative cells were gained from adult rats, which were identified with specifically biological marker of macrophage (CD68). The phagocytosis was evaluated by the ink particles experiment. Retinal Muuml;ller cells of adult rats were cultured by enzyme digestion method, and identified by GFAP and vimentin immunocytochemically. As the feeder cells, peritoneal exudative cells were cocultured with Muuml;ller cells. The proliferation cycle of Muuml;ller cells was assayed by flow cytometry. One-step TUNEL staining was employed to detect the apoptotic Muuml;ller cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a favourable phagocytic ability for the ink particles. The primary cultured Muuml;ller cells adhered to the wall of flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocultured with feeder cells, the Muuml;ller cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4.172, Plt;0.001; G2/M phase, t=3.562, Plt;0.01） and less apoptotic rate (t=3.804, Plt;0.01). The growing cycle was cut down from 25-30 days to 1822 days for the firstgeneration cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocultured with primary culture of retinal Muuml;ller cells, which can shorten the culture period of Muuml;ller cells in adult rats.
Objective To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to valuate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell prol iferation and phenotype. Methods The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) andco-cultured with notochord cells (group B) (1 ∶ 1), and cell prol iferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. Results The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 μm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 μm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P gt; 0.05). The co-cultured cells (group B) increased in cell prol iferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell prol iferation in group B was more than that in group A after 4-day culture (P lt; 0.05). The cocultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. Conclusion The notochord cells are conducive for the prol iferation and phenotypekeeping of the chondroid cells and may play a key role in preventing degeneration of the disc.