Collection and culture of human connective tissue cells from gingival explant technique for oral tissue bioengineering / Colección de células de tejido conectivo humano a partir de la técnica de explante gingival para bioingeniería de tejidos orales

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.

Advances in Bioculture Technology of Bupleuri Radix and Biosynthesis Pathways of Saikosaponins / 中国实验方剂学杂志

Synthetic biology is an emerging discipline that analyzes the biosynthesis pathways of active constituents in traditional Chinese medicine and explores genes involved in biosynthesis. Bupleuri Radix is one of the most commonly used Chinese medicinal materials with remarkable medicinal value, its index component is saikosaponins, which has significant anti-inflammatory, anti-viral and anti-tumor activities. However, the current wild resources of Bupleuri Radix have been destroyed, and there were some problems in the process of artificial cultivation. The application of biological culture technology and synthetic biology can expand the sources of saikosaponins and protect resources of Bupleuri Radix. The culture conditions of different plants can be followed without a fixed pattern, and the biosynthetic pathways of different medicinal active ingredients are also inconsistent. At present, there is no review report on the culture technology of Bupleuri Radix and the research on the biosynthesis pathway of saikosaponins. This paper introduces the research progress of biological culture techniques, such as callus culture, adventitious root culture, hairy root culture and suspension cell culture used in synthetic biology, and the biosynthesis pathway of saikosaponins and its key enzyme functional genes. It is suggested to optimize the biological culture technology of Bupleuri Radix by referring to the tissue culture technology of other traditional root medicinal materials, so as to provide a reference for the in-depth study on the biosynthesis pathway and metabolic regulation of saikosaponins.

Establishment of human scleral fibroblasts-seeded collagen matrix and its mechanical properties / 中华实验眼科杂志

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.

The effect of spirulina (Arthrospira platensis) (Oscillatoriales: Cyanobacteria) on the experimental breeding of Pseudosuccinea columella (Basommatophora: Lymnaeidae) / Efecto de la espirulina (Arthrospira platensis) (Oscillatoriales: Cyanobacteria) en la cría experimental de Pseudosuccinea columella (Basommatophora: Lymnaeidae)

Snails of the family Lymnaeidae, as Pseudosuccinea columella, are the intermediate hosts of Fasciola hepatica, the causative agent of fasciolosis in human and livestock all over the world. A thorough knowledge of snail biology is essential for describing the transmission dynamics and for controlling this disease. Since food quality has had a significant effect on snail growth, fecundity and fertility, in this study we evaluated the use of spirulina (Arthrospira platensis) as a food resource for the artificial breeding of P. columella, an invasive snail and the main intermediate host of F. hepatica in Northeastern Argentina. The main purpose was to measure the effect of spirulina on fitness parameters such as survival rate, growth rate, size at first reproduction, lifetime fecundity and viable offspring. A total of 20 676 newly-laid F2 eggs were used; half of them were fed with lettuce (treatment L) and the other half with lettuce plus spirulina (treatment L+S). In comparison with P. columella snails fed only with lettuce, we found that P. columella fed with lettuce plus spirulina: 1) showed higher survival rates, 2) grew faster and showed higher growth increments, 3) attained sexual maturity earlier in time (L+S:60 days vs. L:120 days) and at a smaller size (L+S:4.8mm vs. L:8.2mm), 4) had a longer reproductive period (L+S:150 days vs. L:90 days), 5) produced a higher number of eggs/snail (L+S:29.6 vs. L:13.3), and 6) showed a higher offspring hatching rate (L+S:70% vs. L:40%). The supplementation of P. columella diet with commercial spirulina enhances it fitness and improved the artificial breeding of this species. Spirulina may have a direct positive effect on P. columella development by consuming it, along with an indirect positive effect by improving the water quality. This rearing technique provided large number of reproducing adults and a continuous production of offspring, which are essential for developing future experimental studies in order to improve our knowledge on P. columella biology.

Los caracoles de la familia Lymneidae, como Pseudosuccinea columella, actúan como hospedadores intermediarios de Fasciola hepatica, el agente etiológico de la fasciolosis, zoonosis que afecta al ganado y al hombre en todo el mundo. Conocer profundamente las características biológicas de estos caracoles resulta esencial para describir la dinámica de transmisión y controlar esta parasitosis. La calidad del alimento afecta significativamente el crecimiento, la fecundidad y la fertilidad de estos caracoles. En este estudio, evaluamos la utilización de la espirulina (Arthrospira platensis) como fuente de alimento para la cría artificial de P. columella, una especie invasora que actúa como el principal hospedero intermediario de F. hepatica en el Noreste Argentino. El objetivo principal de este trabajo fue medir el efecto de la espirulina en parámetros del fitness tales como: tasa de supervivencia, tasa de crecimiento, tamaño que alcanzan a la madurez sexual, duración del período fértil, fecundidad y viabilidad de la descendencia. Se utilizaron 20 767 huevos F2 recién puestos; la mitad de ellos fue alimentado con lechuga (tratamiento L) y la otra mitad con lechuga mas espirulina (tratamiento L+S). En comparación con las P. columella alimentadas solamente con lechuga, las P. columella alimentadas con lechuga mas espirulina: 1) presentaron mayores tasas de supervivencia, 2) alcanzaron mayores tamaños y en menor tiempo, 3) alcanzaron la madurez sexual antes de tiempo (L+S:60 días vs. L:120 días) y a menor tamaño (L+S:4.8mm vs. L:8.2mm), 4) tuvieron un período reproductivo más largo (L+S:150 días vs. L:90 días), 5) produjeron mayor cantidad de huevos/caracol (L+S:29.6 vs. L:13.3) y 6) su descendencia tuvo una mayor tasa de eclosión (L+S:70% vs. L:40%). La incorporación de espirulina como suplemento alimenticio mejoró significativamente los parámetros biológicos de P. columella y maximizó la cría artificial de esta especie en laboratorio. La espirulina podría tener un efecto positivo directo sobre el desarrollo de P. columella mediante su consumo, pero también podría tener un efecto positivo indirecto al mejorar la calidad del agua. Con la técnica de cría desarrollada en este trabajo se obtiene una gran cantidad de caracoles adultos reproductores y una continua producción de huevos que son esenciales para desarrollar futuros estudios que permitan aumentar nuestro conocimiento sobre la biología de P. columella.

Dentin barrier cytotoxicity test with three-dimensional cell cultures / 北京大学学报(医学版)

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.

Trypanosoma cruzi, hemocultura: uma abordagem prática / Trypanosoma cruzi, hemoculture: a practical approach

JUSTIFICATIVA E OBJETIVOS: A hemocultura permanece útil para diagnosticar a doença de Chagas crônica. O objetivo deste estudo foi comparar a técnica "clássica" e a "modificada" mais usada para essa finalidade no Brasil. MÉTODO: A inoculação de "creme leucocitário" com sedimento de sangue centrifugado caracteriza ambas as técnicas. Amostras provenientes de dois grupos de 137 pacientes chagásicos crônicos foram examinadas. A "clássica" utiliza 10 mL de sangue; a "modificada", 30 mL. RESULTADOS: A técnica "modificada" mostrou-se mais sensível do que a "clássica". CONCLUSÃO: Sugere-se o uso rotineiro da técnica "modificada".

BACKGROUND AND OBJECTIVES: Hemoculture remains a valuable tool for the diagnosis of chronic Chagas disease. We tested the two techniques routinely used in Brazil. METHOD: The "modified" technique includes the inoculation of "buffy coat" and centrifuged blood sediment from a 30 mL sample, the serum being washed out after centrifugation and replaced by equal volume of culture medium; the "classic" one requires the inoculation of centrifuged blood sediment plus "buffy coat" from a 10 mL sample. RESULTS: A higher sensitivity of the "modified" technique was observed when the results of two samples of 137 chronic Chagas disease patients were compared. CONCLUSION: The routine use of the "modified" technique should be preferred.

Isolation and identification of tumor-like stem cells from human osteosarcoma cell line / 肿瘤

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.

Preliminary study of the cultivation and growth characteristics of human salivary gland epithelial cells / 实用口腔医学杂志

Objective:To establish a culture method of human salivary gland epithelial cells and to study their growth characteristics in vitro.Methods:Tissue explant technic was employed to culture human salivary gland epithelial cells in serum free medium (SFM),1∶1 DMEM/F12 and 1∶1 DMEM/F12 containing 25 ml/L fetal boven serum (FBS) respectively.The morphology of the cultured cells was observed by phase contract microscope.The cell growth was studied by cell counting.The cells were identified by HE staining,PAS staining and SP staining.Results:Growth of human salivary gland epithelial cells was observed in primary culture in the three kinds of medium in all 10 cases. The cultured cells were epidermoid, positive for cytokeratin, negative for Vimentin and positive for PAS staining. The cells in SFM could be subcultured for five passages,while only for one passage in the other two kinds of medium.Conclusion:SFM is superior to serum free 1∶1 DMEM/F12 or 1∶1 DMEM/F12 containing 25 ml/L FBS for the culture of human salivary gland epithelial cells.

In vitro purification and culture of multipotent adult progenitor cells from human bone marrow by magnetic activated cell sorting / 第二军医大学学报

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.

Cultivation and in vitro identification of epithelial cells from human fistula tube / 中国现代普通外科进展

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.

Observation of In Vitro Interaction of Furosemide On Stria Vascularis Cultureds From Guinea Pig / 听力学及言语疾病杂志

Objective To observe the effect of furosemide on stria vascularis cultivated in vitro from guinea pig, to explore the probable mechanism of furosemide ototoxicity.Methods 20 healthy pigmented guinea pigs was randomly divided into two groups: Furosemide group (n=16), control group (n=4). The stria vascularis explant were cultured for 24 hours using the explant-culture technique. Experimental groups were added into imediately different final concentration of Furosemide (60ug/ml?300ug/ml?600ug/ml?1250ug/ml?2500ug/ml) differently, and continuously cultivated 30 minutes or 90 minutes , then observed structure feature of the cultivated stria vascularis .Results Furosemide group did not show pathological changes such as edema of stria vascularis,shrinkage of the intermediate cells and enlargement of intercellular spaces. No significant changes were found in the stria vascularis structure features between furosemide group and control group. Conclusion Furosemide did not induce remarkable edema on cultivated stria vascularis from guinea pig.These findings suggested that furosemide might induce ototoxicity by indirect mechanism.