The Effects of Cryopreservation on Different Passages of Fibroblast Cell Culture in Brown Brocket Deer (Mazama gouazoubira).

The brown brocket deer Mazama gouazoubira is 1 of the 10 recognized brocket deer of the Neotropical region. Recently, this species has suffered a population decline due to current threats, mainly poaching and habitat loss. Several studies have shown that some endangered species can benefit from interspecies somatic cell nuclear transfer technology through the use of their somatic cells, such as the fibroblasts. Thus, the aim of this study was to verify the viability and the effect of cryopreservation on fibroblasts after several passages. For this purpose, fibroblast cells were cultured until passages 4, 7, and 10 (cultured control groups) and cryopreserved in cryotubes (frozen/warmed groups). The cellular viability, functionality, and percentage of cells undergoing necrosis and apoptosis were evaluated. The survival rates were always higher than 80% irrespective of the tested group, except for passage 10 in the frozen/warmed group. Population doubling time of cultured cells from passage 10 was significantly higher than that of passages 4 and 7, exhibiting low metabolic activity and a higher percentage of cells in initial apoptosis. In conclusion, the M. gouazoubira fibroblast-derived cell line provides an essential resource for further studies regarding reproductive biotechniques and is likely to be useful as an ex situ conservation strategy.

Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood / Isolamento, cultura e caracterização de células tronco mesenquimais multipotentes provenientes do sangue do cordão umbilical caprino

Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.(AU)

As células tronco mesenquimais (MSC) residem em pequenas quantidades em muitos tecidos e órgãos adultos, desempenhando um papel ativo na homeostase destes locais. O isolamento de MSC já foi demonstrado em amostras de medula óssea, tecido adiposo e fluido amniótico de cabras. O sangue de cordão umbilical é considerado uma fonte importante desse tipo de células. No entanto, até o presente momento, não foi demonstrado o isolamento de MSC provenientes do sangue de cordão umbilical de cabras. Dessa forma, o objetivo do presente estudo foi isolar, cultivar e caracterizar células tronco mesenquimais provenientes do sangue do cordão umbilical caprino. As MSC foram isoladas utilizando o gradiente de densidade Ficoll-Paque e cultivadas em DMEM suplementado com 10% ou 20% de FBS. A caracterização desse tipo celular foi realizada através de análise por citometria de fluxo e diferenciação em linhagens celulares mesodermais. A analise no citômetro de fluxo demonstrou a presença de duas populações distintas, um grupo com células maiores e outro com células menores; observando expressão positiva de CD90, CD44 e CD105, e negativa para CD34 nas células maiores; enquanto que as menores foram positivas para CD90 e CD105, mas negativas para CD44 e CD34. As células isoladas demonstraram capacidade de se diferenciar em condrócitos e osteoblastos quando incubadas com meio de diferenciação específico. O presente estudo demonstrou que células tronco mesenquimais podem ser obtidas com sucesso do sangue do cordão umbilical caprino.(AU)

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro

Abstract Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro.

Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.

Evaluation of pattern-reversal visual evoked potential in patients with neuromyelitis optica.

BACKGROUND: The visual evoked potential (VEP) is used in the evaluation of multiple sclerosis (MS) patients, showing a delay in P100 wave latency with no changes in amplitude in 60-100% of cases. In the last decade, the recurrent form of neuromyelitis optica (NMO) has been recognized, and clinically characterized by acute events of transverse myelitis (TM) and optic neuritis (ON), differing from MS in clinical and laboratory criteria. Despite these differences, so far, the VEP parameters described in MS have been used in the evaluation of patients with NMO. The objective of this study was to investigate VEP responses in NMO. METHODS: Patients with NMO underwent pattern-reversal visual stimulation. Nineteen patients were selected for the study. RESULTS: Among the 38 eyes examined, 18 (47.4%) had no visual evoked responses and 13 (34.2%) had a reduction of P100 wave amplitude with normal latency. Only two (5.3%) had the pattern described in MS and five (13.2%) were normal. CONCLUSION: Evaluation of VEP in patients with definite NMO revealed a pattern that is different from that of MS in 81.6% of eyes examined, characterized by the absence of responses, or decreased amplitude with normal latency.