Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium / Comparação das técnicas de cultivo de células epiteliais da córnea para a coleta de meio condicionado

ABSTRACT Purposes: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. Methods: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. Results: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). Conclusion: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

RESUMO Objetivos: Foram estudadas cinco técnicas de cultivo primário de células epiteliais de córnea humana para se determinar o melhor protocolo para a obtenção do maior rendimento de meio de cultivo condicionado para ser utilizado na diferenciação de células tronco mesenquimais para células epiteliais de córnea. Métodos: As técnicas de cultivo estudadas foram: explantes em frascos de cultivo com e sem tratamento hidrofílico de superfície, sobre membrana amniótica, com digestão enzimática e por raspado de córnea. O meio de cultivo condicionado foi coletado e as células tronco mesenquimais induzidas a se diferenciarem em células epiteliais da córnea utilizando o meio de cultivo condicionado. As células foram caracterizadas por citometria de fluxo com pan-citoqueratina e com os marcadores específicos da córnea, citoqueratina 3 e citoqueratina 12. Resultados: A técnica utilizando frascos com o tratamento de superfície apresentou o maior rendimento de meio de cultivo condicionado. Os frascos sem tratamento de superfície levaram a uma taxa de sucesso muito baixa. A digestão enzimática e a raspagem da córnea mostraram contaminação das culturas com fibroblastos de córnea. A cultura sobre membranas amnióticas só permitiu a coleta do meio de cultivo condicionado durante a 1ª confluência celular. A análise de citometria de fluxo confirmou o sucesso da diferenciação celular utilizando o meio de cultivo condicionado coletado, demonstrada pela expressão de citoqueratina 3 (95,3%), citoqueratina 12 (93,4%) e pan-citoqueratina (95,3%). Conclusão: O cultivo de explantes de células tronco mesenquimais em frascos com tratamento hidrofílico de superfície é a melhor técnica para a obtenção de um alto rendimento de meio de cultivo condicionado.

Polimorfismo do gene do receptor de insulina (INSR) em pacientes com síndrome dos ovários policísticos / Polymorphims of the insulin receptor gene (INSR) in patients with polycystic ovary syndrome

OBJETIVO: Avaliar a freqüência do polimorfismo do gene (INSR), localizado no éxon 17 do cromossomo 19, quanto ao seu envolvimento na predisposição genética da SOP e a associação com a sensibilidade à insulina e hiperandrogenismo. MATERIAL E MÉTODOS: Foram estudadas 57 pacientes com SOP e 50 mulheres normais com ciclos menstruais regulares (controle) quanto à frequência de polimorfismos de nucleotídeo único (Single Nucleotide Polymorphisms–SNPs) do gene INSR, através da análise de comprimento do fragmento de restrição (Restriction Lengh Fragment Polymorphisms-RFLPs). Para avaliar a sensibilidade à insulina, utilizou-se os seguintes métodos matemáticos: relação glicemia/insulina de jejum (G/I), Homeostasis Model Assessment (HOMA) e Quantitative Sensitivity Check Index (QUICKI). Foram considerados normais, G/I maior ou igual a 4,5, HOMA menor que 3,8, e QUICKI maior ou igual a 0,34. RESULTADOS: O polimorfismo (C/T, T/T) na SOP não apresentou diferenças significativas com o grupo controle. Quanto a insulinemia, não observamos diferenças entre as pacientes com genótipos (C/T, T/T) e (C/C). CONCLUSÕES: As frequências do polimorfismo do gene INSR, são semelhantes tanto em pacientes com SOP, como em mulheres normais. Esses genótipos (C/T e T/T) não influenciam a sensibilidade à insulina e o hiperandrogeninsmo na SOP

The 3´-untranslated ApaI-Insulin-like Growth Factor II Gene (IGF2) Polymorphism in Women with Uterine Leiomyomas

OBJECTIVE: To explore a possible association betweenthe rare allele A of the single nucleotide polymorphismin the 3´-untranslated region of the insulin-like growthfactor II gene (IGF2), previously described as beingassociated with growth dysregulation, higher body massindex (BMI), and elevated risk of uterine leiomyomas.MATERIALS AND METHODS: A series of 144 women(72 clinically and histologically confirmed uterineleiomyomas and 72 without leiomyomas) was analyzedby a PCR-RFLP (Polimerase Chain Reaction – RestrictionFragment Length Polymorphism) based approach todetect a single nucleotide polymorphism in the 3´-untranslated region of the gene IGF2. Genotypic andallelic frequencies distributions in both groups werecompared with weight, height, and BMI. RESULTS: Nostatistically significant differences in the genotype andallelic frequencies between patients and controls wereobserved. Similarly, the distribution of genotypes andweight, height, and BMI did not differ significantlybetween the two groups although the weight and BMIwere lower in leiomyomas patients homozygous forallele A. CONCLUSIONS: The ApaI polymorphism ofthe IGF2 gene does not produce different risk forleiomyoma development. Our data suggest that thedeviations related in the genotypic frequencies for thispolymorphism among women with uterine leiomyomasis not correlated with BMI and that previous associationswere probably a result of chance statistical samplingpresent in a small studied group.

Clonal chromosome abnormalites found in three non-neoplastic proliferative brain lesions

Chromosome analysis was made of brain lesions from three patients which, according to classical histopathological criteria, did not contain tumor cells. In addition to normal cells, we identified abnormal karyotypes with clonal numerical and structural chromosome alterations in at least two independently originated primary cultures from each lesion. Our data suggest that chromosomal aberrations can exist in vivo in non-neoplastic lesions. Other abnormalities may be due to genetic instability manifested only in vitro (culture artifacts) or may already have been present in brain tissue, reflecting previous chromosome damage (as a result of exposure to chemical treatment or enviromental clastogens).