Establishment and characterization of two fetal fibroblast cell lines from the yak

Abstract

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an "S"-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n = 60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

“…Ear skin of a newborn female yak was used as to establish the nuclear donor cell cultures as previously described (Liu et al, 2012). SCNT, fusion and activation of reconstructed yak oocytes, and cloned embryo cultures were performed using methods described in previous studies (Su et al, 2012(Su et al, , 2014a.…”

Células Fetais Bovinas De Cultivo Primário Submetidas a Diferentes Pressões Negativas Antes Do Congelamento Em Palhetas

“…A metodologia de criopreservação empregada em nosso estudo, com fibroblastos de origem fetal, proporcionou taxa de sobrevivência similar às obtidas por Léon-Quinto et al (4) , mesmo após a otimização de seu protocolo. Liu et al (9) obtiveram 89,5% de sobrevivência após o congelamento de fibroblastos fetais de Yak, utilizando o equipamento convencional "Mr Frosty", resultado semelhante ao obtido em nosso trabalho. Assim, a efetividade da metodologia empregada em nosso estudo, associada à sua fácil execução, que dispensa equipamentos onerosos, bem como o reduzido volume das palhetas, que permite aumentar a capacidade de armazenamento em botijões criogênicos, possibilitam sua indicação na criação de bancos genéticos, com diferentes propósitos, principalmente para a manutenção da variabilidade genética animal.…”

“…De forma semelhante ao já observado anteriormente (9) , em todos os tratamentos, as curvas de proliferação demonstraram uma fase de latência, em que as células não apresentaram uma proliferação significativa por um período de 48 horas, necessário para adaptação e possível recuperação de sua estrutura, seguida de um crescimento exponencial, até atingirem sua fase estacionária (plateau) no quinto ou sexto dias de cultivo. As células submetidas a 500 mbar de PN três horas antes do congelamento (Gráfico 1C) demoraram mais tempo para atingir a fase de plateau, evidenciando que, provavelmente, o tempo de execução das fases dos check points pode ter sido aumentado em função de algum mecanismo de quiescência (entrada em G0).…”

MicroRNA let-7f-5p regulates PI3K/AKT/COX2 signaling pathway in bacteria-induced pulmonary fibrosis via targeting of PIK3CA in forest musk deer

“…To provide further evidence that the PI3K/AKT/COX2 signaling pathway was regulated by let-7f-5p in FMD lung, we successfully isolated and cultured primary FMD-C1 cell, a detailed procedure of the methods of cell separation can be found in a previous study ( Liu et al, 2012 ). FMD-C1 cells were transfected with let-7f-5p mimics and mimics NC using Lipo8000™ Transfection Reagent.…”