Comparison of primary keloid fibroblast cultivation methods and the characteristics of fibroblasts cultured from keloids, keloid-surrounding tissues, and normal skin tissues / Comparación de los métodos de cultivo de fibroblastos queloides primarios y las características de los fibroblastos cultivados a partir de queloides, tejidos circundantes queloides y tejidos cutáneos normales

SUMMARY: The establishment of primary keloid fibroblast culture has always been a fundamental measure for studying mechanisms of keloid disease. The quality of the primary cell culture can directly affect the results of further experiments. This study was performed to investigate the optimal growth conditions, including the optimal storage time and collagenase treatment time, for in vitro cell culture models and the suitable methods for epidermis-dermis separation in different tissues. Keloid tissues, keloid-surrounding tissues, and normal skin tissues were collected from patients, for primary fibroblast culture. Two methods, tissue explant and collagenase digestion, were deployed and compared. Expression levels of the keloid-related genes α -SMA, Col1, and Col3 were assessed in cells cultured using both methods, to verify the qualities of the primary cells. A comparative analysis was conducted between the two methods and among the three different tissues used. Bacterial and lipid contamination was immediately minimized after the samples were processed. Different methods of epidermis removal and different durations of collagenase digestion were required in different tissues to generate optimal results. Real-time PCR results showed that the mRNA expression levels of keloid-related genes in cultured fibroblasts correlated to their in vivo expression profile, as previously reported in other studies. The results of this study have revealed several key points in the culture of primary keloid fibroblasts and demonstrated the correlation in gene expression between in vivo keloid fibroblasts and in vitro primary keloid fibroblasts.

RESUMEN: La identificación de un cultivo de fibroblastos queloides primarios, siempre ha sido una medida fundamental para estudiar los mecanismos de la enfermedad queloide. La calidad del cultivo de células primarias puede afectar directamente los resultados de otros experimentos. Este estudio se realizó para investigar las condiciones óptimas de crecimiento, incluido el tiempo óptimo de almacenamiento y el tiempo de tratamiento con colagenasa, para modelos de cultivo celular in vitro y los métodos adecuados para la separación epidermis-dermis en diferentes tejidos. Se recogieron de los pacientes tejidos queloides, tejidos circundantes queloides y tejidos cutáneos normales, para cultivo primario de fibroblastos. Se implementaron y compararon dos métodos, explante de tejido y digestión con colagenasa. Los niveles de expresión de los genes relacionados con queloides α -SMA, Col1 y Col3 se evaluaron en células cultivadas usando ambos métodos, para verificar las cualidades de las células primarias. Se realizó un análisis comparativo entre los dos métodos y entre los tres tejidos diferentes utilizados. La contaminación de bacterias y lípidos se minimizó inmediatamente después de que se procesaron las muestras. Se requirieron varios métodos de eliminación de la epidermis y diferentes tiempos de digestión con colagenasa en los tejidos para generar resultados óptimos. Los resultados de la PCR en tiempo real mostraron que los niveles de expresión de ARNm de genes relacionados con queloides en fibroblastos cultivados se correlacionaban con su perfil de expresión in vivo, como se informó en estudios anteriores. Los resultados de este studio indicaron varios puntos clave en el cultivo de fibroblastos queloides primarios y han demostrado la correlación en la expresión génica entre fibroblastos queloides in vivo y fibroblastos queloides primarios in vitro.

Optimización de cultivos primarios de células de carcinoma renal de células claras como modelo "in vitro" para estudios metabólicos y determinantes de progresión neoplásica / Optimization of primary cultures of clear cell renal carcinoma cells as an "in vitro" model for metabolic studies and determinants of neoplastic progression

El objetivo del presente estudio fue optimizar la implementación de cultivos primarios a partir de muestras de carcinoma renal de células claras (CRCC) para comprobar la conservación del fenotipo lipogénico contra cortes fijados del mismo origen. Se utilizaron muestras de pacientes con CRCC, evaluándose diversas metodologías y condiciones experimentales de digestión de muestras, adherencia y despegue celular, fenotipo lipogénico, potencial de clonación, proliferación y capacidad de migración. El mayor rendimiento y viabilidad celular se verificó mediante digestión con colagenasa. La adherencia inicial se logró a las 24 hs de incubación, utilizando placas plásticas de cultivo, recubiertas con colágeno comercial y gelatina 0,2% en la mayoría de las muestras analizadas (60% de los casos). Se obtuvieron monocapas, con potencial de migración, en un 40% de los casos, tras 5 ± 1 días de incubación. El promedio de subcultivos fue de 3 ± 1. Este estudio permitió estandarizar cultivos primarios de CRCC comprobándose la conservación de la fenotipia lipogénica, logrando de dicha manera una herramienta importante y útil para el estudio de la biología tumoral y el ensayo de nuevas terapéuticas

The aim of this study was to optimize the implementation of primary cultures from samples of renal clear cell carcinoma (CRCC) to check the conservation of the lipogenic phenotype. CRCC Patient samples were used, in order to evaluate different methodologies and the experimental conditions of sample digestion, cell adhesion and lipogenic phenotype, proliferation and migration ability. The highest yield in cell number and viability was assessed using collagenase digestion. The initial adhesion was achieved after 24 hours of incubation in plastic plates recoverd with commercial collagen or 0.2% gelatin (60% of cases). Monolayers, with migration potential, were obtained in 40% of all cases, after 5 ± 1 days of incubation. The subcultures average was 3 ± 1. This study allowed us to standardize primary cultures of CRCC and check the conservation of the lipogenic phenotyping, achieving in this way an important and useful tool to study the tumor biology.

O objetivo deste trabalho foi otimizar a implementação de culturas primárias de amostras de carcinoma de células claras renal (CRCC) para verificar conservação fenótipo lipogenic contra os cortes previstos a mesma origem. As amostras dos pacientes foram utilizados CRCC, avaliando diferentes metodologias e as condições experimentais da digestão de amostras, adesão celular e fenótipo clonagem potencial take-lipogenic, proliferação e capacidade de migração. O maior rendimento e a viabilidade celular foi avaliada por digestão com colagenase. A adesão inicial foi obtida após 24 horas de incubação com colagénio e gelatina comercial 0,2% em 60% dos casos. As monocamadas foram obtidos em 40% após 5 ± 1 dias de incubação com o potencial de migração. As subculturas média foi de 3 ± 1. Este estudo nos permitiu padronizar culturas primárias de CRCC são verificados quanto à conservação da fenotipagem lipogenic, conseguindo desta forma um importante e útil para o estudo da biologia do tumor e teste de nova ferramenta terapêutica

Spermatogonial stem cells as a therapeutic alternative for fertility preservation of prepubertal boys / Espermatogônias-tronco como uma alternativa para a preservação da fertilidade de meninos pré-púberes

ABSTRACT Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients.

RESUMO As espermatogônias-tronco, presentes nos testículos desde o nascimento, são as células progenitoras dos gametas masculinos, e, desse modo, críticas para o processo de espermatogênese. Antes da puberdade, essas células não são capazes de produzir espermatozoides maduros, o que só ocorrerá após o estímulo hormonal. Essa característica do sistema reprodutivo limita a possibilidade de preservação da fertilidade apenas para homens capazes de produzir um ejaculado. Tal fato coloca em evidência o aumento nas taxas de sobrevivência de crianças com câncer nas últimas décadas, devido principalmente à melhora no diagnóstico e ao tratamento dos pacientes pediátricos. Dessa forma, destaca-se um dos mais importantes desafios relativos à preservação da fertilidade masculina, que é o efeito tóxico das terapias anticâncer para o sistema reprodutivo, especialmente a espermatogênese. Tendo isso em vista, a alternativa experimental atualmente estudada para a preservação da fertilidade de pacientes pré-púberes é a criopreservação de tecido testicular para futuro isolamento e transplante de espermatogônias-tronco, a fim de restabelecer a espermatogênese. Apresentamos aqui uma breve revisão sobre isolamento, caracterização e condições de cultivo para a proliferação de espermatogônias-tronco, bem como as futuras perspectivas, como alternativa para preservação da fertilidade de meninos pré-púberes. A possibilidade de restabelecer a fertilidade masculina é uma ferramenta de pesquisa com potencial enorme de uso na pesquisa básica e aplicada. O desenvolvimento dessas técnicas pode fornecer uma esperança futura de preservação de fertilidade nos casos em que não há nenhuma outra opção, como para os pacientes pediátricos de câncer.

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

BACKGROUND: Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, ß-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, ß-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. RESULTS: Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 µg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, ß and γ tocopherols (145.61, 25.52 and 18.56 µg/100 g) were detected in the cell cultures collected at day 6. CONCLUSIONS: As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.

Induced maturation of hepatic progenitor cells in vitro

Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.

Análisis de patrones morfológicos y anatómicos en la embriogénesis somática del banano Williams (AAA) / Analysis of the morphological and anatomical patterns in the somatic embryogenesis of Williams banana (AAA)

La embriogénesis somática representa una herramienta esencial en el mejoramiento genético y en la micropropagación clonal masiva de bananos mejorados. En el presente trabajo se analizaron los patrones morfológicos y anatómicos que ocurren durante la embriogénesis somática del banano Williams, dirigidos a conocer y mejorar este proceso. En la investigación se establecieron suspensiones celulares embriogénicas (SCE) a partir de callo embriogénico obtenido de manos florales inmaduras masculinas, las cuales originaron abundantes embriones que regeneraron plantas. Hacia los tres meses de cultivo se detectaron embriones somáticos (ES) primarios color blanco-crema en las manos florales de los nudos nueve a doce, contados a partir del ápice floral. Al cuarto mes estos ES primarios dieron origen al callo embriogénico, de color blanco crema, estructura granular, con abundantes ES torpedo en su periferia y con una organización celular en tres diferentes zonas. De este callo se cultivaron porciones pequeñas con ES torpedo en medio de multiplicación durante dos meses, dando origen a la SCE I. La misma se tamizó (250 µm) para establecer la SCE II. El sedimento de células y los agregados celulares embriogénicos de ambas SCE se trasladó a medio de maduración. Transcurridos dos meses los embriones maduros se transfirieron a medio de conversión de embriones, lográndose regenerar plantas completas a partir de las dos semanas. Las SCE produjeron numerosos embriones somáticos maduros y mostraron una buena conversión de embriones a plantas y regeneración de plantas. Este sistema de embriogénesis somática permitió la obtención de plantas funcionales en nueve meses.

Somatic embryogenesis represents an essential tool for the genetic improvement and for the mass clonal micropropagation of the improved banana plant. In this present work morphological and anatomical patterns were analyzed in the somatic embryogenesis of Williams banana, to know and enhance this process. In the investigation embryogenic cell suspensions (ECS) were established from embryogenic callus obtained from floral immature male hands, which gave rise to many somatic embryos that regenerated plants. Towards the three months of culture white-cream primary somatic embryos (SE) were detected in the floral hands of the nodes nine to twelve, counted from the floral apex. At the fourth month this primary SE gave origin to a creamy-white embryogenic callus, with granular structure and abundant SE torpedo on its periphery. Cell organization with three different zones was observed in callus. Small portions of this callus were cultivated in the multiplication medium for two months, to originate ECS I. This ECS was filtered through a mesh (250 µm pore size) to establish the ECS II. The sediment of embryogenic cells and cell clusters of the ECS were moved to maturation media. After two months the mature embryos were transferred to conversion medium, and two weeks later, whole plants were developed. The ECS produced numerous mature SE, which showed good conversion of embryos into plants and plant regeneration. This system of somatic embryogenesis permitted the mass production of functional plants in nine months.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.