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1.
Chinese Journal of Traumatology ; (6): 138-144, 2022.
Article in English | WPRIM | ID: wpr-928493

ABSTRACT

PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.


Subject(s)
Alveolar Epithelial Cells/cytology , Animals , Cell Culture Techniques , Cell Separation/methods , Immunoglobulin G/metabolism , Lung , Magnetic Phenomena , Rats , Rats, Sprague-Dawley
2.
Braz. j. med. biol. res ; 54(2): e10197, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153509

ABSTRACT

Assays based on the flow cytometry technique allow a convenient analysis of multiple cellular parameters; however, their results should be interpreted cautiously due to a strong impact of confounding factors. Different techniques in cell culturing such as either enzymatic or mechanic detachment of adherent cells can heavily influence the structure of the cell membrane or presence of the surface antigens leading to strong false positive signals, and finally, substantial experimental bias. The aim of our study was to assess and compare the impact of cell harvesting methods (both enzymatic and non-enzymatic) on the apoptosis process and on the surface antigen cytometric analyses. We found significant differences in the quality of analysis in terms of the amount of detected surface markers determined by the detachment method. Our results demonstrated clearly how important it is to carefully choose the appropriate detachment method and may help to avoid mistakes in experiment planning. In conclusion, we recommend to adjust the detachment method to the type of analyzed markers (surface antigens or translocated phosphatidylserine).


Subject(s)
Humans , Animals , Cell Separation/methods , Apoptosis , Membrane Proteins , Antigens, Surface , Cell Line, Tumor , Flow Cytometry
3.
Braz. oral res. (Online) ; 34: e033, 2020. graf
Article in English | LILACS | ID: biblio-1089391

ABSTRACT

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.


Subject(s)
Humans , Phenotype , Stem Cells/cytology , Keratinocytes/cytology , Cell Culture Techniques/methods , Epithelial Cells/cytology , Mouth Mucosa/cytology , Receptors, Transferrin/analysis , Biomarkers/analysis , Antigens, CD/analysis , Cell Separation/methods , Reproducibility of Results , Receptors, Nerve Growth Factor/analysis , Flow Cytometry/methods , Nerve Tissue Proteins/analysis
4.
Arq. bras. cardiol ; 113(1): 11-17, July 2019. tab, graf
Article in English | LILACS | ID: biblio-1011228

ABSTRACT

Abstract Background: Pericardium tissue allograft can be used for surgical repair in several procedures. One of the tissue engineering strategies is the process of decellularization. This process decreases immunogenic response, but it may modify the natural extracellular matrix composition and behavior. Objective: The aim of this study was to evaluate the effectiveness of cell removal, maintenance of extracellular matrix properties and mechanical integrity of decellularized human pericardium using a low concentration solution of sodium dodecyl sulfate. Methods: Decellularization was performed with sodium dodecyl sulfate and ethylenediaminetetraacetic acid. Histological analysis, DNA quantification, evaluation of glycosaminoglycans and collagen were performed. Biomechanical assay was performed using tensile test to compare the decellularization effects on tissue properties of tensile strength, elongation and elastic modulus. P < 0.05 was considered significant. Results: There was reduction in visible nuclei present in pericardium tissue after decellularization, but it retained collagen and elastin bundles similar to fresh pericardium. The DNA contents of the decellularized pericardium were significantly reduced to less than 511.23 ± 120.4 ng per mg of dry weight (p < 0.001). The biomechanical assay showed no significant difference for fresh or decellularized tissue. Conclusion: The decellularization process reduces cell content as well as extracellular matrix components without changing its biomechanical properties.


Resumo Fundameto: O enxerto de pericárdio pode ser usado em muitos procedimentos de correção cirúrgica. Uma das estratégias da engenharia tecidual é o processo de descelularização. No entanto, embora esse processo diminua a resposta imunogênica, a descelularização pode modificar tanto o comportamento como a composição da matriz extracelular natural. Objetivos: Avaliar a eficácia da descelularização usando baixa concentração de dodecil sulfato de sódio na remoção celular, na manutenção das propriedades da matriz extracelular e na integridade mecânica do pericárdio humano descelularizado. Métodos: A descelularização foi realizada com dodecil sulfato de sódio e ácido etilenodiamino tetra-acético. Foi realizada análise histológica, quantificação de DNA, e avaliação de glicosaminoglicanos e colágeno. O estudo biomecânico foi conduzido pelo teste de tração para comparar os efeitos da descelularização sobre as propriedades teciduais de resistência à tração, alongamento e módulo de elasticidade. Foi considerado um valor de p < 0,05 como estatisticamente significativo. Resultados: Observou-se uma redução na quantidade de núcleos presentes no pericárdio após a descelularização, apesar de manter quantidades similares de feixes de elastina e de colágeno. As concentrações de DNA do pericárdio descelularizado foram significativamente reduzidas para menos que 511,23 ± 120,4 ng por mg de peso seco (p < 0,001). O teste biomecânico não apontou diferenças entre os tecidos fresco e descelularizado. Conclusão: A descelularização reduziu a concentração de células bem como os componentes da matriz extracelular sem afetar suas propriedades biomecânicas.


Subject(s)
Humans , Adolescent , Adult , Middle Aged , Young Adult , Pericardium/cytology , Sodium Dodecyl Sulfate/pharmacology , Surface-Active Agents/pharmacology , Cell Separation/methods , Tissue Engineering/methods , Pericardium/drug effects , Biomechanical Phenomena , Regenerative Medicine , Tissue Scaffolds
5.
Biomédica (Bogotá) ; 38(3): 378-387, jul.-set. 2018. tab, graf
Article in Spanish | LILACS | ID: biblio-973991

ABSTRACT

Resumen Introducción. La cuantificación de la inestabilidad cromosómica es un parámetro importante para evaluar la genotoxicidad y la radiosensibilidad. Las técnicas convencionales requieren cultivos celulares o laboriosos análisis microscópicos de cromosomas o núcleos. La citometría de flujo en reticulocitos ha surgido como una alternativa para los estudios in vivo, ya que reduce los tiempos de análisis e incrementa hasta en 20 veces el número de células analizables. Objetivos. Estandarizar los parámetros de citometría de flujo requeridos para seleccionar y cuantificar reticulocitos micronucleados (RET-MN) a partir de muestras de sangre periférica, y cuantificar la frecuencia de esta subpoblación anormal como medida de inestabilidad citogenética en sendas poblaciones de voluntarios sanos (n=25) y pacientes (n=25) recién diagnosticados con gliomas de alto grado antes de iniciar el tratamiento. Materiales y métodos. Las células sanguíneas se marcaron con anti-CD71-PE para reticulocitos, anti- CD61-FITC para la exclusión de plaquetas y yoduro de propidio para detectar el ADN en reticulocitos. La fracción celular MN-RETCD71+ se seleccionó y se cuantificó con un citómetro de flujo automático. Resultados. Se describió detalladamente la estandarización de los parámetros citométricos, con énfasis en la selección y la cuantificación de la subpoblación celular MN-RETCD71+. Se establecieron los niveles basales de MN-RETCD71+ en la población de control y en los pacientes se encontró un incremento de 5,2 veces antes de iniciar el tratamiento (p<0,05). Conclusión. Los resultados evidenciaron la utilidad de la citometría de flujo acoplada a la marcación de las células RETCD71+ como método eficiente para cuantificar la inestabilidad cromosómica in vivo. Se sugieren posibles razones del incremento de micronúcleos en células RETCD71+ de pacientes con gliomas.


Abstract Introduction: The quantification of chromosomal instability is an important parameter to assess genotoxicity and radiosensitivity. Most conventional techniques require cell cultures or laborious microscopic analyses of chromosomes or nuclei. However, a flow cytometry that selects the reticulocytes has been developed as an alternative for in vivo studies, which expedites the analytical procedures and increases up to 20 times the number of target cells to be analyzed. Objectives: To standardize the flow cytometry parameters for selecting and quantifying the micronucleated reticulocytesCD71+ (MN-RET) from freshly drawn peripheral blood and to quantify the frequency of this abnormal cell subpopulation as a measure of cytogenetic instability in populations of healthy volunteers (n =25), and patients (n=25), recently diagnosed with high-grade gliomas before the onset of treatment. Materials and methods: Blood cells were methanol-fixed and labeled with anti-CD-71-PE for reticulocytes, antiCD-61-FITC for platelet exclusion, and propidium iodide for DNA detection in reticulocytes. The MN-RETCD71+ cell fraction was selected and quantified with an automatic flow cytometer. Results: The standardization of cytometry parameters was described in detail, emphasizing the selection and quantification of the MN-RETCD71+ cellular fraction. The micronuclei basal level was established in healthy controls. In patients, a 5.2-fold increase before the onset of treatment was observed (p <0.05). Conclusion: The data showed the usefulness of flow cytometry coupled with anti-CD-71-PE and anti- CD-61-FITC labeling in circulating reticulocytes as an efficient and high resolution method to quantify chromosome instability in vivo. Finally, possible reasons for the higher average of micronuclei in RETCD71+ cells from untreated high-grade glioma patients were discussed.


Subject(s)
Female , Humans , Male , Reticulocytes/pathology , Glioblastoma/genetics , Chromosomal Instability , Micronuclei, Chromosome-Defective , Flow Cytometry/methods , Specimen Handling/methods , Cell Separation/methods , Risk Factors , Glioblastoma/blood , Glioblastoma/pathology , Neoplasm Grading
6.
Int. braz. j. urol ; 42(4): 817-824, July-Aug. 2016. tab, graf
Article in English | LILACS | ID: lil-794669

ABSTRACT

ABSTRACT Purpose: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. Materials and Methods: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. Results: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. Conclusion: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.


Subject(s)
Animals , Mice , Neoplastic Stem Cells/cytology , Urinary Bladder Neoplasms/pathology , Trypsin/pharmacology , Cell Adhesion/drug effects , Cell Separation/methods , Cell Culture Techniques/methods , Neoplastic Stem Cells/chemistry , Biomarkers, Tumor , Cell Differentiation , Culture Media, Serum-Free , Cancer Vaccines/immunology , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , Flow Cytometry , Mice, Nude
7.
Arq. bras. oftalmol ; 79(2): 105-110, Mar.-Apr. 2016. tab, graf
Article in English | LILACS | ID: lil-782803

ABSTRACT

ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs) and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL). Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.


RESUMO Objetivo: O objetivo do estudo foi estabelecer um protocolo de cultura primária para o isolamento de células acinares da glândula lacrimal (CAGL) e avaliar a relevância de insulina no meio de cultura. Métodos: CAGL foram isoladas e cultivadas a partir das glândulas lacrimais de ratos Wistar machos. Os parâmetros analisados foram: o número de células, viabilidade e secreção da peroxidase ao longo do tempo e em resposta a três concentrações de insulina (0,5; 5,0 e 50,0 μg/ml). Resultados: Na cultura primária de CAGL as células passaram a se agrupar por volta do dia 3. A secreção de peroxidase em resposta ao carbacol aumentou no dia 6. O período de cultura viável foi limitado à 12 dias. Insulina à 5,0 μg/ml no meio de cultura resultou em viabilidade e capacidade secretora maior. Conclusão: o estudo descreveu um método para simplificar o isolamento e cultivo de CAGL. Os dados apresentados confirmam a importância da insulina na manutenção da cultura de CAGL.


Subject(s)
Animals , Male , Acinar Cells/cytology , Primary Cell Culture/standards , Insulin/pharmacology , Lacrimal Apparatus/cytology , Carbachol/metabolism , Cell Count/methods , Cell Separation/methods , Rats, Wistar , Peroxidase/metabolism , Acinar Cells/drug effects , Acinar Cells/metabolism , Insulin/metabolism , Lacrimal Apparatus/metabolism
8.
Acta cir. bras ; 31(1): 59-66, Jan. 2016. graf
Article in English | LILACS | ID: lil-771849

ABSTRACT

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.


Subject(s)
Animals , Rabbits , Cell Separation/methods , Mesenchymal Stem Cells/cytology , Olfactory Mucosa/cytology , /physiology , /physiology , Thy-1 Antigens/physiology , Cells, Cultured , Colony-Forming Units Assay , Cryopreservation , Cell Differentiation/physiology , Cell Plasticity/physiology , Cell Proliferation/physiology , Ethmoid Bone/cytology , Flow Cytometry , Green Fluorescent Proteins/metabolism , Olfactory Mucosa/growth & development
9.
Arch. endocrinol. metab. (Online) ; 59(2): 161-170, 04/2015. graf
Article in English | LILACS | ID: lil-746460

ABSTRACT

Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with “brittle T1DM”, who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre – Rio Grande do Sul, Brazil.


Subject(s)
Humans , Cell Separation/methods , Diabetes Mellitus, Type 1/therapy , Facility Design and Construction/standards , Islets of Langerhans , Islets of Langerhans Transplantation/trends , Brazil , Insulin/therapeutic use , Islets of Langerhans Transplantation/economics , Islets of Langerhans Transplantation/legislation & jurisprudence , Laboratories/organization & administration
10.
Acta cir. bras ; 29(supl.2): 29-33, 2014. graf
Article in English | LILACS | ID: lil-721375

ABSTRACT

PURPOSE: To investigate the experimental model for obtaining adipose tissue, isolation, characterization of mesenchymal stem cells and evaluation of their distribution in the tram flap in rats. METHODS: Five rats of Wistar were randomly assigned to two groups. In group I, three animals underwent removal of adipose tissue in the groin procedure to establish the experimental model and obtain a cell lineage. The animals of group II (n = 2) underwent surgical flap procedure, and satisfaction injection of mesenchymal stem cells pretreated with marker fluorescente. RESULTS: obtaining adipose tissue of the inguinal region of the rat proved to be possible. The isolated cells were characterized as mesenchymal stem cells and fluorescence microscopy showed the presence of multiple cells arranged around blood vessels and capillaries. CONCLUSION: It was possible to establish an experimental model for obtaining adipose tissue for isolation of mesenchymal stem cells and their distribution in the TRAM flap in rats. .


Subject(s)
Animals , Male , Adipose Tissue/cytology , Cell Separation/methods , Mesenchymal Stem Cells , Models, Animal , Surgical Flaps , Cell Differentiation , Cells, Cultured , Cell Culture Techniques/methods , Immunophenotyping , Microscopy, Fluorescence , Random Allocation , Rats, Wistar , Reproducibility of Results
11.
Korean Journal of Urology ; : 487-492, 2014.
Article in English | WPRIM | ID: wpr-178070

ABSTRACT

PURPOSE: Transforming growth factor-beta1 (TGF-beta1) plays a dual role in apoptosis and in proapoptotic responses in the support of survival in a variety of cells. The aim of this study was to determine the function of TGF-beta1 in bladder cancer cells. MATERIALS AND METHODS: The role of TGF-beta1 in bladder cancer cells was examined by observing cell viability by using the tetrazolium dye (MTT) assay after treating the bladder cancer cell lines 253J, 5637, T24, J82, HT1197, and HT1376 with TGF-beta1. Among these cell lines, the 253J and T24 cell lines were coincubated with TGF-beta1 and the pan anti-TGF-beta antibody. Fluorescence-activated cell sorter (FACS) analysis was performed to determine the mechanism involved after TGF-beta1 treatment in 253J cells. RESULTS: All six cell lines showed inhibited cellular growth after TGF-beta1 treatment. Although the T24 and J82 cell lines also showed inhibited cellular growth, the growth inhibition was less than that observed in the other 4 cell lines. The addition of pan anti-TGF-beta antibodies to the culture media restored the growth properties that had been inhibited by TGF-beta1. FACS analysis was performed in the 253J cells and the 253J cells with TGF-beta1. There were no significant differences in the cell cycle between the two treatments. However, there were more apoptotic cells in the TGF-beta1-treated 253J cells. CONCLUSIONS: TGF-beta1 did not stimulate cellular proliferation but was a growth inhibitory factor in bladder cancer cells. However, the pattern of its effects depended on the cell line. TGF-beta1 achieved growth inhibition by enhancing the level of apoptosis.


Subject(s)
Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Cell Separation/methods , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor/methods , Flow Cytometry/methods , Humans , Transforming Growth Factor beta1/administration & dosage , Urinary Bladder Neoplasms/pathology
13.
IJVM-Iranian Journal of Veterinary Medicine. 2013; 7 (2): 83-90
in English | IMEMR | ID: emr-138267

ABSTRACT

Spermatogonial stem cells [SSCs] are infrequent self-renewing cells among the type A spermatogonia within the seminiferous tubules and are the basis of spermatogenesis in mammalian testis. An adequate number of SSCs is a primary requirement for the study of their behavior, regulation, and further biomanipulation. In this paper, we studied the development of the primary co-cultures of type A spermatogonia and prepubertal bovine sertoli cells in the presence of Colony Stimulating Factor 1 [CSF1], a potential contributor in the SSC niche. The effect of different concentrations of CSF1 [0, 10, 50 and 100 ng/mL] on the colonization activity of spermatogonial cells was assessed 4, 7 and 11 days after the beginning of the culture by counting the total number of colonies and measuring their area in each group of the present experiment. Immunofluorescent staining against OCT4 and vimentin led to the confirmation of the nature of both the SSCs and sertoli cells. Results showed that the total number of colonies from day 4 to 11 increased significantly in all groups, independent of CSF1 concentration. In addition, the total number and total area of colonies were higher [not significant] in 10 and 50 ng/mL CSF1 treatments than the control and 100 ng/mL CSF1 groups in all the three evaluations during the experiment. However, this difference was only significant [p<0.05] between the total area of colonies in the control and 10 ng/mLCSF1 groups at day 4 of co-culture. It was concluded that CSF1 can be a suitable growth factor for improving SSCs colonization in vitro, particularly during the first days of culture where accompanying sertoli cells still have not proliferated sufficiently to support the propagating spermatogonial cells


Subject(s)
Animals , Sertoli Cells , Colony-Stimulating Factors , Stem Cells , Spermatogenesis , Cell Separation/methods , Seminiferous Tubules , Macrophage Colony-Stimulating Factor , Microscopy, Electron, Scanning Transmission , Coculture Techniques
14.
Article in English | WPRIM | ID: wpr-152453

ABSTRACT

As the theory of stem cell plasticity was first proposed, we have explored an alternative hypothesis for this phenomenon: namely that adult bone marrow (BM) and umbilical cord blood (UCB) contain more developmentally primitive cells than hematopoietic stem cells (HSCs). In support of this notion, using multiparameter sorting we were able to isolate small Sca1+Lin-CD45- cells and CD133+Lin-CD45- cells from murine BM and human UCB, respectively, which were further enriched for the detection of various early developmental markers such as the SSEA antigen on the surface and the Oct4 and Nanog transcription factors in the nucleus. Similar populations of cells have been found in various organs by our team and others, including the heart, brain and gonads. Owing to their primitive cellular features, such as the high nuclear/cytoplasm ratio and the presence of euchromatin, they are called very small embryonic-like stem cells (VSELs). In the appropriate in vivo models, VSELs differentiate into long-term repopulating HSCs, mesenchymal stem cells (MSCs), lung epithelial cells, cardiomyocytes and gametes. In this review, we discuss the most recent data from our laboratory and other groups regarding the optimal isolation procedures and describe the updated molecular characteristics of VSELs.


Subject(s)
Animals , Cell Lineage , Cell Separation/methods , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Humans , Mesenchymal Stem Cells/cytology , Pluripotent Stem Cells/cytology
15.
Acta cir. bras ; 26(5): 333-338, Sept.-Oct. 2011. ilus
Article in English | LILACS | ID: lil-599633

ABSTRACT

PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70 percent confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80 percent in membrane type 3, 20 percent in type 2, and 10 percent in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.


OBJETIVO: Avaliar três formas de cultivo de células-tronco mesenquimais de ratos; e analisar o potencial do polímero de mamona na forma de membrana como arcabouço para CTMs. MÉTODOS: Foram utilizados quatro ratos machos Wistar, de 20 a 30 dias de idade. Aspirados da medula óssea do fêmur e da tíbia foram colhidos com DMEM alta glicose e heparina. As células foram isoladas de três formas diferentes: cultivo direto e com dois tipos de gradientes de densidade. Após 15 dias, foi feita a 1ª passagem e analisada a viabilidade celular com os marcadores Hoerscht 33342 e Iodeto de Propídio. As CTMs foram então caracterizadas por marcadores de superfície, com o auxílio de citômetro de fluxo. Após, três tipos de membrana à base de óleo de polímero de mamona associadas com as CTMS foram mantidas em cultivo por cinco dias, e analisados por microscópio ótico para confirmar o crescimento e a adesão celular. RESULTADOS: Após 15 dias, Os procedimentos que utilizaram gradientes de densidade permitiram o isolamento das CTMs e favoreceram o crescimento celular com a passagem, sendo obtido 70 por cento de confluência após 15 dias em cultura. O procedimento direto não se mostrou eficaz para o isolamento das células. O crescimento das CTMs foi aproximadamente 80 por cento sobre a membrana tipo 3, 20 por cento na tipo 2 e 10 por cento na membrana tipo 1. CONCLUSÃO: Os dois gradientes de concentração Ficoll-Hypaque permitem isolar CTMs de ratos; e especialmente a membrana de polímero de mamona tipo 3 pode ser usada como um bom arcabouço para as CTMs.


Subject(s)
Animals , Male , Rats , Castor Oil , Cell Separation/methods , Mesenchymal Stem Cells , Polyurethanes , Tissue Scaffolds , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Survival/drug effects , Flow Cytometry , Materials Testing , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Rats, Wistar , Surface Properties
16.
Acta cir. bras ; 26(4): 267-273, July-Aug. 2011. ilus
Article in English | LILACS | ID: lil-594345

ABSTRACT

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.


OBJETIVO: Testar diferentes protocolos para o isolamento de células tronco a partir de sangue de cordão umbilical e tecido adiposo de ovinos. MÉTODOS: Foram utilizadas cinco amostras de sangue de cordão umbilical e cinco amostras de tecido adiposo perirrenal de 10 fêmeas de ovelha. A coleta das amostras foi realizada através de procedimento cirúrgico para coleta do material de forma mais asséptica possível. Foram realizados três protocolos de isolamento e cultivo das células-tronco do cordão umbilical e quatro protocolos para o isolamento e cultivo das células-tronco de gordura de ovinos RESULTADOS: Somente um dos protocolos utilizados para o isolamento das células-tronco de cordão umbilical foi efetivo. Dos quatro protocolos utilizados para isolamento das células-tronco de gordura, da mesma forma, apenas um obteve sucesso. Foi realizado o ensaio de unidades formadoras de colônias destas células, sendo contadas 58 colônias ao final de sete dias. Na citometria de fluxo essas células mostraram-se positivas para CD44 e negativas para CD38, CD45, CD41/61. Estas células apresentaram curva de crescimento com fases de LOG, LAG e PLATEAU bem definidas, características das curvas de crescimento das células-tronco de origem mesenquimal. CONCLUSÕES: O isolamento e cultivo das células-tronco mesenquimais do cordão umbilical de ovinos é de difícil realização, exigindo maiores ensaios e estudos profundos. Células tronco do tecido adiposo de ovelhas demonstraram características mesenquimais, de acordo com a curva de crescimento, habilidade de formação de colônias, células com morfologia fibroblastóide e reação positiva ao anticorpo CD44.


Subject(s)
Animals , Female , Adipose Tissue/cytology , Cell Separation/methods , Fetal Blood/cytology , Mesenchymal Stem Cells , Biomarkers , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cell Line/cytology , Reproducibility of Results , Sheep , Time Factors
17.
Rev. Inst. Nac. Hig ; 42(2): 25-32, jul. 2011. ilus, graf, tab
Article in Spanish | LILACS, LIVECS | ID: lil-631801

ABSTRACT

Se evaluó el uso de la tecnología de Flujo de Filtración Tan gencial (FFT), para obtener la toxina tetánica a partir de cultivos de la bacteria Clostridium tetani, usando el proceso de Micro filtración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la Toxina Tetánica usando el proceso de Ultrafiltración (UF). Se determinaron las características de los filtros, condiciones de trabajo y el dimensionamiento de los equipos a adquirir para la nueva producción industrial de Toxina Tetánica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido. Utilizando cultivos con Toxina Tetánica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Se seleccionó las membranas tipo cassettes, formato Suspended Screen, porosidad 0,2μm, como las adecuadas para el proceso de MF, ya que mostraron un 100% de transmisión de la Toxina Tetánica, ausencia de restos celulares y flujo promedio de filtrado de 73.30 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 50 y 70 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (106,7 y 104,4 L/m2h, respectivamente). Estos resultados permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 litros, tiempo de procesos, 3 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.


Tangential Flow Filtration (TFF) technology was evaluated to process tetanus toxin which is produced by Clostridium tetani bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is co - llected and further processed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Dia filtration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow ra te, processing time, yield and product attributes were characterized. The cell harvest containing the tetanus toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in sus pended screen format and membrane with 0.2μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 73.30 L/m2h. The UF step was conducted using the same system with cassettes in me dium screen format with pores of 50 and 70kDa. It showed 100% retention of the toxin with a process flux of 106,7 and 104,4L/m2h, respectively. To maximise product retention during UF, the 50 kDa membrane was selected. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters in 3 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20m2 and 5m2, respectively.


Subject(s)
Humans , Male , Female , Tetanus Toxin/analysis , Bacterial Infections/complications , Ultrafiltration/instrumentation , Proteins/metabolism , Cell Separation/methods , Microstraining , Public Health
18.
Rev. Inst. Nac. Hig ; 42(1): 27-34, jun. 2011. ilus, graf, tab
Article in Spanish | LILACS, LIVECS | ID: lil-631790

ABSTRACT

Se evaluó el uso de la tecnología de Flujo de Filtración Tangencial (FFT), para obtener la toxina diftérica a partir de cultivos de la bacteria Corynebacterium diphtheriae, usando el proceso de Microfiltración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la toxina diftérica usando el proceso de Ultrafiltración (UF). Se determinaron características de los filtros, condiciones de trabajo y dimensionamiento de los equipos a adquirir para la producción industrial de Toxina Diftérica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido, utilizando cultivos con Toxina Diftérica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Seseleccionó las membranas tipo cassettes, formato Médium Screen, porosidad 0,2 μm, como las adecuadas para el proceso de MF, ya que mostraron 100% de transmisión de la Toxina Diftérica, ausencia de restos celulares y flujo promedio de filtrado de 9.16 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 10 y 30 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (97,5 y 125,9 L/m2h, respectivamente), Estos resultaron permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 Litros, Tiempo de Procesos, 3 a 5 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.


Tangential Flow Filtration (TFF) technology was evaluated to process diphtheria toxin which is produced by Cory ne - bacterium diphtheriae bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is collected and further pro - cessed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Diafiltration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow rate, processing time, yield and product attributes were characterized. The cell harvest containing the diphtheria toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in medium screen format and membrane with 0.2 μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 9.16 L/m2h. The UF step was conducted using the same laboratory equipment with cassettes in medium screen format with pores of 10 and 30 kD. It showed 100% retention of the toxin with a process flux of 97,5 and 125,9 L/m2h, respectively. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters between 3 to 5 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20 m2 and 5m2, respectively.


Subject(s)
Humans , Male , Female , Ultrafiltration/instrumentation , Cell Separation/methods , Microstraining/methods , Diphtheria Toxin/toxicity , Proteins/metabolism , Public Health
19.
Biocell ; 35(1): 35-36, Apr. 2011.
Article in English | LILACS | ID: lil-595003

ABSTRACT

E. canis infection of the canine cell line DH82 is a routine in studies with this bacteria. A protocol for isolation of host cell free bacteria was developed based on the use of glass beads. Improvement of infection with E. canis isolated by this method was detected by real-time PCR.


Subject(s)
Humans , Animals , Dogs , DNA, Bacterial/analysis , Ehrlichia canis/genetics , Ehrlichia canis/isolation & purification , Dog Diseases/microbiology , Cell Separation/instrumentation , Cell Separation/methods , Cell Line , Fluorescent Dyes/metabolism , Organic Chemicals/metabolism , Ehrlichiosis/microbiology , Glass , Polymerase Chain Reaction/methods
20.
Electron. j. biotechnol ; 14(2): 8-8, Mar. 2011. ilus, tab
Article in English | LILACS | ID: lil-591938

ABSTRACT

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 10(5) clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003 percent of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of < 1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones...


Subject(s)
Antibodies, Monoclonal, Humanized/metabolism , Cell Line/metabolism , Cell Separation/methods , Biopharmaceutics , Cell Culture Techniques , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Fluorescence , Recombinant Proteins , Time Factors , Transfection
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