RESUMEN
SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.
RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.
Asunto(s)
Humanos , Células del Tejido Conectivo , Técnicas de Cultivo de Célula/métodos , Bioingeniería/métodos , Encía/citología , Biología Celular , FibroblastosRESUMEN
Background Excessive elongation of axis and expansion of sclera is one of the hot topics in the study of the pathogenesis of high myopia.To establish a human scleral fibroblasts (HSFs)-collagen matrix culture model is helpful for understanding the reciprocal and adaptive interactions between HSFs and the collagen matrix in tissue.Objective The aim of this study was to establish a HSFs-seeded collagen three-dimension culture system that may mimic the sclera remolding in myopia.Methods HSFs were isolated and cultured from donor eyes by explant culture and purified by passages culture in vitro.The expressions of vimentin and keratin in the cells were detected by immunofluorescence technique to identify the cells.Rat tail tendon was obtained from 8-week-old SPF SD rats to prepare the collagen matrix.The mixed solution of 400 μl collagen matrix and 1 100 μl PBS,200 μ1 nutrient medium,50 μ1 NaOH and HSF suspension were mixed to prepare the collagen gel three-demension culture system.The growth and morphology of the cells in the culture system were observed under the inverted phase contrast microscope,and IPP-5 software was used to measure the contraction area of collagen gel,and the mechanical creep properties of the HSFs-seeded collagen matrix were measured by a biomechanics test instrument.Results HSFs emigrated from tissue 7 days after culture and passage could be performed 14 days after culture.The expression of keratin was absent in HSFs,while vimentin was positively expressed.The free-cell collagen gel was clear and unchanged in the experimental duration.However,the cells were obviously increased on the three-demension culture system and showed a tissue-like structure of net-like arrangement on dozens of layers.In 7-14 days after culture,the collagen gel area in a three-demension collagen matrix revealed a decrease of 90％.Duotriode-like and fusiform cells were seen 24 hours after culture.The biomechanical creep curve of HSFs-seeded collagen matrix consisted of the nonlinear section (0-100 seconds) and linear section (100-600 seconds),and the former appeared to be an elastic change of the gel under the temporal stress,and the latter was the creeping of the gel with the time.Conclusions Rat tail collagen appears to have a good biocompatibility to HSFs.HSFs-seeded collagen matrix can retain the mechanical creep properties,and it may be a good tool for the study on the relationship between HSFs and extracellular matrix or intercellular biological behaviour for scleral remodeling.
RESUMEN
Objective:To evaluate the cytotoxicity of four dentin filling materials and two dentin adhe-sives with a dentin barrier test and to compare the results with those in a conventional filter diffusion test in order to investigate the advantages of the dentin barrier test.Methods: Eugenol cement, zinc phos-phate cement, adhesive glass ionomer cement, composite resin and two self-etching adhesives ( REMI BOND and Adper Easy One) were tested.In the dentin barrier test, L929 mouse fibroblasts were three-dimensional cultured in polystyrene meshes.The dentin disks were cut from the human third molars, near the pulp and in parallel with the occlusal surface, and their permeability within the measurement area was evaluated by a hydraulic permeability device.A mesh with the cells was placed in the “pulp cavity” of the chamber and one dentin disk was put on the cell mesh and its “pulp side” was in contact with the mesh.The test materials and controls were in contact with the“occlusal side” of the dentine disks for 24 h.The cell viability was obtained with MTT assay and the results were expressed as a percentage of con-trol tissues.The Mann-Whitney U test was used to make the statistical analyses.In the filter diffusion test, after a 24 h contact between the test materials and the filters with monolayer cells, the filters were dyed and the grades of cytotoxicity were decided.Results:A mean permeability of the dentin disks near the pulp was 0.293 μL/(min· cm2· cmH2O)(1 cmH2O=0.098 kPa);In the dentin barrier test, Eu-genol cement, REMI BOND and Adper Easy One respectively reduced the cell survival rates to 82%, 63%and 54%.Other materials showed no or very low toxic reactions; In the filter diffusion test, the light-curing composite resin was moderately cytotoxic, the dental adhesive glass ionomer cement was mild-ly cytotoxic and the others were severely cytotoxic;All the six materials in the dentin barrier test had low-er cytotoxicity than in the filter diffusion test.Conclusion:The cytotoxicity of the test materials using the dentin barrier test with three-dimensional cell cultures is lower than that in the filter diffusion test, which has good correlation with the clinical situation.
RESUMEN
Objective: To isolate and identify tumor-like stem cells from human osteosarcoma cell line U2-OS. Methods: Tumor sphere cells were isolated from osteosarcoma cell line with serum-free culturing techniques. Flow cytometry was used for detecting the positive rates of Stro-1 protein in U2-OS cell line. The proliferation ability of sarcospheres was estimated by MTT assay. Stro-1+ cells were isolated using magnetic activated cell sorting system. RT-PCR was used to detect the transcription of Stro-1 mRNA in Stro-1+ cells. The Stro-1+ cells and Stro-1- cells were inoculated into NOD/SCID mice. Results: The results of flow cytometry showed that the percentage of Stro-1+ cells was 5.67% in U2-OS cells. Stro-1+ cells had capability of proliferation and differentiation. The over-expression of Stro-1 mRNA was observed in Stro-1+ cells. Inoculation of Stro-1+ cells induced osteosarcoma formation in NOD-SCID mice. Conclusion: Osteosarcoma-like stem cells exist in human osteosarcoma cell line U2-OS. They have self-renewal and multi-directional differentiation abilities.
RESUMEN
Objective:To establish a method for isolation,purification,and culture of human multipotent adult progenitor cells(MAPCs) in vitro,so as to lay a foundation for the application of hMAPCs in tissue-engineering research and clinical medicine.Methods: The mononuclear cells were obtained from bone marrow of healthy volunteers by gradient centrifugation and were subjected to adherence culture.The adherent cells were then subjected to magnetic activated cell sorting(MACS)(depletion selection with CD45 and GlyA microbeads).Then the purity of selected cells was identified by flow cytometer.The growth of the purified cells was observed and the expression of CD29,CD44,CD34,and HLA-DR was analyzed by flow cytometers.Results: (1)Averagely(5-10)?10~(4)/ml hMAPCs could be separated from 1?10~(6)/ml bone marrow mononuclear cells by MACS.The cell viability was similar before(96.7?1.7%) and after(96.0?2.4%) isolation.(2)The isolated MAPCs grew well in the self-designed culture medium and could be passaged for 20 generation.(3)The purity of the CD45~-and GlyA~-cells separated from bone marrow adherent cells was more than 98% as determined by flow cytometer.(4) In hMAPCs,the positive rate was 99.2% for CD29,98.3% for CD44,1.2% for CD34,and 5.3% for HLA-DR.Conclusion:(1)The bone marrow-derived hMAPCs can be purified by MACS through depletion selection of CD45 and GlyA microbeads.(2)The hMAPCs can be expanded in vitro in self-designed medium,maintaining a non-differentiation state for a long time.
RESUMEN
Objective:To investigate a method of isolating and culturing the epithelial cells from human fistula tube.Methods:We separated epithelial cells of human fistula tube by tissue block method and cultured the epithelial cells.The morphology of the cells was studied with a phase contrast microscope and the living characteristics after subculture of the cells were observed.We identified the cells using immunocytochemical assessment.Results:The culturing cells grew on the wall appeared after 24-36 hours.The living characteristics of the cultured cells were quickly into the logarithms growth period between 7 and 9 days.The identified cells were keratin positive and vimentin negative by using immunocytochemistry.Conclusion:The epithelial cells of human fistula tube could be cultured stably in vitro by the method.It could be possible to study the healing mechanism of human fistula tube based on the results of this study.