Guillain-Barré syndrome / 中华神经科杂志

Guillain-Barré syndrome (GBS) is a rare, but potentially fatal, immune-mediated disease of the peripheral nerves and nerve roots, which is usually triggered by infections. It is characterized by rapidly progressive, symmetrical weakness of the extremities. Some patients develop respiratory insufficiency and many show signs of autonomic dysfunction. Diagnosis can usually be made on clinical grounds, but lumbar puncture and electrophysiological studies can help to substantiate the diagnosis and to differentiate demyelinating from axonal subtypes of GBS. Molecular mimicry of pathogen-borne antigens, leading to generation of crossreactive antibodies that also target gangliosides, is generally accepted pathogenesis of GBS. The treatment of GBS is intravenous immunoglobulin or plasma exchange with general clinical treatment. Most patients have a good prognosis and basically recover within weeks to months. A few patients have persistent neurological dysfunction or even death.

Enrichment and isolation of breast cancer stem cells from breast cancer transplantation tumors in nude mice / 肿瘤研究与临床

Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.

Culture and identification of parietal epithelial cells of Bowman′s capsule / 中华肾脏病杂志

Objective:To explore the isolation and culture methods of mouse parietal epithelial cells (PECs) of Bowman′s capsule, so as to provide a cell tool for further study.Methods:Mouse renal corpuscles were isolated by cell sieving combined with magnetic separation. After primary culture, identified parietal epithelial cells were induced to differentiate into podocytes. Immunofluorescence staining, real-time quantitative PCR and Western blotting were used to detect specific markers of parietal epithelial cells and podocytes.Results:Primary cultured PECs grew like paving stone and expressed Claudin-1 (PECs specific marker), CD133 (stem cell marker) and CD24 (stem cell marker), without the expression of tubular epithelial cell proteins, mesangial cell and podocyte specific proteins. Cultured to 6 generations in vitro, the PECs still expressed Claudin-1, CD133 and CD24. After incubated with differentiation medium, PECs were able to express podocyte markers WT-1 and Synaptopodin. Conclusion:The renal corpuscles are extracted by cell sieving combined with magnetic separation, and the mouse PECs successfully cultured in vitro can be induced to express podocytes′ markers.

Establecimiento e implementación de un protocolo simplificado de expansión y cultivo de Células Madre de Pulpa Dental Humana (DPSCh) / Estabelecimento e implementação de um protocolo simplificado para expansão e cultura de células-tronco da polpa dentária humana (DPSCh) / Establishing and implementing a simplified protocol for the expansion and culture of human dental pulp stem cells (hDPSCs)

Resumen Objetivos: Establecer e implementar un protocolo simplificado de extracción, aislamiento primario y cultivo de células madre derivadas de la pulpa dental humana (DPSCh). Analizar cuantitativamente y cualitativamente las células aisladas. Metodología: 10 terceros molares sanos donados por pacientes que concurrieron a la Facultad de Odontología, UdelaR y otorgaron su consentimiento escrito fueron procesados antes de las 48 hs. Se realizó la fractura de la pieza para la obtención del tejido pulpar y se procesó por el método explante. Se analizó viabilidad celular y expresión de marcadores por citometría de flujo en pasajes 4 y 12 y se corroboró mediante inmunocitoquímica. Resultados: Las células obtenidas presentaron una vitalidad mayor al 90% en todos los pasajes, observándose una morfología característica y expresión de marcadores de células madre mesenquimales CD90, C105, CD73, CD29 y 166 mediante citometría de flujo en ambos pasajes. Conclusiones: Se logró establecer un protocolo de aislamiento y expansión celular, con alta tasa de éxito de una población de DPSCh.

Resumo Objetivos: Estabelecer e implementar um protocolo simplificado para a extração, isolamento primário e cultura de células-tronco da polpa dentária humana (DPSCh). Analise as células isoladas quantitativa e qualitativamente. Metodologia: 10 terceiros molares saudáveis ​​doados por pacientes que frequentaram a Faculdade de Odontologia UdelaR e deram consentimento por escrito foram processados ​​antes de 48 horas. A fratura da peça foi realizada para obtenção do tecido pulpar e processada pelo método do explante. A viabilidade celular e a expressão do marcador foram analisadas por citometría de fluxo nas passagens 4 e 12 e confirmadas por inmunocitoquímica. Resultados: As células obtidas apresentaram viabilidade superior a 90% em todas as passagens, observando uma morfologia característica e expressão dos marcadores de células-tronco mesenquimais CD90, C105, CD73, CD29 e 166 por citometría de fluxo em ambas as passagens. Conclusões: Foi possível estabelecer um protocolo de isolamento celular, com alta taxa de sucesso e segurança para isolar o DPSCh.

Abstract Objectives: To establish and implement a simplified protocol for the extraction, primary isolation, and culture of human dental pulp stem cells (hDPSCs). To analyze the isolated cells quantitatively and qualitatively. Methodology: Ten healthy third molars were donated by patients who attended the School of Dentistry, UdelaR, and gave their written consent. The teeth were processed within 48 hours. The teeth were sectioned to obtain the pulp tissue and processed with the explant method. Cell viability and marker expression were analyzed by flow cytometry at passages 4 and 12 and verified by immunocytochemistry. Results: The cells obtained had a vitality greater than 90% in all passages. We found the characteristic morphology and the expression of CD90, C105, CD73, CD29 and 166 mesenchymal stem cell markers by flow cytometry in both passages. Conclusion: It was possible to establish a cell isolation protocol that is highly successful and safe to isolate hDPSC.

Procedimiento para el cultivo e identificación de células madre obtenidas de lipoaspirado humano con fines de investigación / Procedure for culture and identification of stem cells from human lipoaspirate for research purposes

RESUMEN Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.

ABSTRACT Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.

A Method for Isolating Tumor Cells from Large Volume of Malignant Pleural Effusion and Its Efficacy Evaluation / 中国肺癌杂志

BACKGROUND@#Malignant plural effusion (MPE) is one of the most common specimen for liquid biopsy gene detection. This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection.@*METHODS@#Pleural effusions (>500 mL) from 20 advanced lung cancer patients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll. Cell number and purity were calculated. DNA was extracted from the supernatant (etDNA), total cells and isolated tumor cells of pleural effusion (ETC-DNA) to detect the mutation of tumor-related genes by next-generation sequencing.@*RESULTS@#The median number of cells isolated from malignant pleural effusion was 8.50×10⁴ (interquel range: 9.25×10³-3.75×10⁵), 85.50%±5.80% of the cells were identified as tumor cells. The detection rates of epidermal growth factor receptor (EGFR) gene mutation of etDNA, total cell DNA and ETC-DNA were 70.00%, 50.00% and 70.00%, reseparately, while the median EGFR mutation abundance in 3 components was 16.05% (4.78%-43.06%), 1.09% (0.00%-2.39%), and 33.02% (18.50%-76.70%), respectively. ETC-DNA had good consistency with tissue DNA (P>0.999, kappa=1.000) and etDNA (P>0.999, kappa=1.000). ETC-DNA inclined to have higher EGFR mutation than etDNA, but the result was not statistically significant.@*CONCLUSIONS@#Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity. Using ETC-DNA as specimen improves the efficacy of gene detection, thus is worth further study.

Characterization of Decellularized Human Pericardium for Tissue Engineering and Regenerative Medicine Applications / Caracterização do Pericárdio Humano Descelularizado para Engenharia de Tecidos e Aplicações de Medicina Regenerativa

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.

Isolation and identification of exosomes derived from bone marrow mesenchymal stem cells / 基础医学与临床

Objective To develop a method of isolation and identification of exosomes of bone marrow mesenchymal stem cells.Methods The mesenchymal stem cells were cultured by whole bone marrow adherence method.A new reagent -exosomes extraction kit was used to isolate and collecte exosomes.The exosomes were identified by elec-tron microscopy,particle size detection,flow cytometry and Western blot.Results The expression of CD45 on the surface of the third generation bone marrow mesenchymal stem cells was negative,and CD73 and CD105 were posi-tive;exosomes derived from bone marrow mesenchymal stem cells were round or oval,the size is non-uniform,the diameter is 30~100 nm,have a complete membrane structure,and containing low-density substances;Particle size detection particle diameter of the main peak was 61.25 nm, in which the diameter of particles was about 20-200 nm accounted for 72.4%;exosome expressed CD63 and CD81;The expression of CD9 and CD63 from cell cul-ture supernatants was positive.Conclusions The exosomes can be collected in the medium of mesenchymal stem cells.The exosomes derived from bone marrow mesenchymal stem cells can be identified by electron microscopy, particle size detection,flow cytometry and Western blot.

A simplified method for isolating murine primary pancreatic acinar cells / 中华胰腺病杂志

Objective To explore a simplified and economical method to isolate the murine primary pancreatic acinar cells.Methods The collagenase and trypsin inhibitor dissolving in DMEM solution were used to digest the murine pancreas,and 4％ BSA dissolving in DMEM solution was used to purify and isolate primary pancreatic acinar cells from pancreas.CCK-8 method was applied to check the ability of pancreatic acinar cells to secret amylase.Results After digestion,shaking in the water bath,resuspension,filtration and precipitation,murine primary pancreatic acinar cells could be obtained within 2 hours.Pancreatic acinar cells in good conditions appeared in clusters,and their basolateral domains were round and devoid of blebs,and the cytoplasm appeared clear.Their apical domain were surrounded by hundreds of zymogen granules which looked darker.The nucleus was located in the basal area of the vesicular region.The basal level of amylase release as a percent of total release from pancreatic acinar cells was around 2.5％ in CCK8-unstimulated group.This rate started to increase after CCK-8 stimulation and reached its peak [(12.83 ± 1.04) ％] at a concentration of 50 pmol/L of CCK-8,but the ratio of the amylase level secreted by the pancreatic acinar cells to the total amylase level displayed a decreasing trend with the increase of CCK-8 concentration.Conclusions This optimized method had the advantage of being fast and simple,low technical difficulty and good repetition.It was a new simplified and cheap method for isolating murine pancreatic acinar cells.

Microfluidic technology and its application in the detection of circulating tumor cells / 中华检验医学杂志

In recent years , the potential clinical value of circulating tumor cells ( CTCs ) is more obvious, and a myriad of detection methods for CTCs have been developed .Among them, a series of microfluidic devices are particularly promising as they uniquely offer micro-scale analytical systems that are highlighted by low consumption of samples and reagents , high flexibility to accommodate other cutting-edge technologies , precise and well-defined flow behaviors , and automation capability , presenting themselves significant advantages over the conventional larger systems .Here, a brief review of the recent advances in the CTC detection by microfluidic devices and their challenges in clinical application will be given .

In vitro isolation and cultivation of human axillary dermal papilla cells / 中华皮肤科杂志

Objective To investigate an efficient rapid method for the isolation and cultivation of human axillary dermal papilla cells.Methods Skin specimens with hair follicles were obtained from the axillary area of patients who received bromhidrosis surgery in the Department of Dermatology of the First Affiliated Hospital to Army Medical University from October 2015 to May 2016.The axillary dermal papilla cells were isolated by two-step enzyme digestion method,one-step digestion method and micro-dissection method separately.Then,axillary dermal papilla cells were cultured and identified.Differences in the operative procedure,separation efficiency and adhesion efficiency of dermal papilla cells,cell emigration duration,total operation duration and actual operation duration were compared among the above 3 methods.Results Compared with the one-step digestion method and micro-dissection method,the two-step enzyme digestion method showed simpler operative procedure,more than 30％ separation rate and 96％ adhesion rate of dermal papilla cells after 1 week.Moreover,the cell emigration duration was shortened by 3-4 days by the two-step enzyme digestion method.The two-step enzyme digestion method also showed longer total operation duration,but shorter actual operation duration compared with the one-step digestion method and micro-dissection method,as well as lower contamination rate compared with the micro-dissection method.Cultured axillary dermal papilla cells grew in an aggregative pattern in the early stage,but grew in a nonaggregative pattern after 6 passages.Immunofluorescence assay showed positive staining for laminin and collagen Ⅳ in axillary dermal papilla cells.Conclusion The modified two-step enzyme digestion method is a kind of simple,efficient and rapid method for the isolation of human axillary dermal papilla cells,and axillary dermal papilla cells can be harvested through this method by using a few specimens.

Isolation and culture of mouse bone marrow mesenchymal stem cells and induced differentiation into alveolar epithelial cells / 医学研究生学报

Objective It has traditionally been difficult to isolate and culture mouse bone marrow mesenchymal stem cells (BMSC),which has low success rate.And thus restricts the development of related research to some extent.We aimed to optimize the whole bone marrow adherent method for isolation and culture of mouse bone marrow mesenchymal stem cells and search for an effective method of inducing BMSCs to differentiate into alveolar epithelial cells.Methods Bone marrow contents harvested from the tibia and femur of C57BL/6 mice were cultured based on the whole bone marrow adherent method.The timing and split ratios of passage were determined according to the size and number of cell colonies.After 6 passages,cells were counted to detect cell proliferation ability,surface markers were examined by flow cytometry and Small Airway Epithelial Cell Medium (SAEpiCM) was used to induce the differentiation of BMSCs.Results With the increase of passages and the purity of BMSCs,the proliferation of cells at passages 6 tended to be stable.Flow cytometry showed that they were strongly positive for bone marrow mesenchymal stem cell surface markers CD29 and Sca-1 (99.1％,88.5％),but almost negative for the surface marker of hematopoietic stem cells CD117 (0.008 2％).BMSCs cultured in SA-EpiCM showed an epithelium-like morphological change and expressed surfactant associated protein C,a specific marker of alveolar epithelial cells.Conclusion It is effective to isolate and culture mouse bone marrow mesenchymal stem cells by adjusting the timing and split ratios of passage according to the size and number of the clonal cell colonies,which possessed the potential to differentiate into alveolar epithelial cells.

Research Progress on Microfluidic Chip of Cell Separation Based on Dielectrophoresis / 分析化学

Cell separation technology is an important means for cell sorting and cell-population purification. It is the current international research hot spot in biochemical analysis which has very important applications in many fields such as biology, medicine, agriculture and environment. This review introduces the development status of cell separation using microfluidic chip based on dielectrophoresis. It expounds the working principle of dielectrophoresis and the key factors such as cell size, electrode shape and signals that impacts the dielectrophoresis of cell separation on different types of microfluidic chip. Finally, it forecasts the future development trend of cell separation using microfluidic chip based on dielectrophoresis.

A experimental study on isolation,culture and identification of osteoblasts from neonatal New Zealand rabbit / 国际检验医学杂志

Objective To investigate the experimental methods of isolation ,culture and identification of osteoblasts from neo-natal New Zealand rabbits in vitro .Methods Two-step enzymatic digestion was adopted to isolate osteoblasts from skull tissue of neonatal New Zealand rabbits to conduct primary cultured .Inverted phase contrast microscope was employed to study the cellular morphology ,acridine orange fluorescent staining was used to detect the cell adhesion function ,methyl thiazolyl tetrazolium(MTT) assay was employed to measure their proliferation ,and Alizarin red and tetracycline staining were used to test their mineralization . Results Primary osteoblasts were successfully obtained .Inverted phase contrast microscopy showed non-adherent cells were round ,while adherent cells were irregular fusiform ,triangular or polygonal .Acridine orange staining showed the nuclei of osteo-blasts green fluorescence ,with good adhesion ability .Good mineralization ability was also demonstrated by tetracycline and alizarin red staining .Osteoblasts possessed good proliferation activity .Conclusion Utilization of two-step enzymatic digestion contributes to getting a lot of osteoblasts with typical morphological features and biological activity in a short time .

O uso de recuperador de sangue em cirurgia cardíaca com circulação extracorpórea / The use of cell saver system in cardiac surgery with cardiopulmonary bypass

INTRODUÇÃO: O uso de recuperador de sangue (RS) em cirurgia cardíaca é proposto para diminuir o uso de unidades de concentrado de hemácias estocadas (UCH), que aumenta morbidade, mortalidade e reações inflamatórias. OBJETIVO: O objetivo deste estudo é avaliar se o uso do RS diminui o emprego de UCH, é custo/efetivo e traz benefícios ao paciente. MÉTODOS: Estudo prospectivo realizado entre novembro de 2009 e outubro de 2011, em 100 pacientes consecutivos, submetidos à cirurgia cardiovascular com circulação extracorpórea (CEC), hemodiluição mínima e hemofiltração. Os pacientes foram divididos em grupo 1 (sem RS) e 2 (com RS). Os critérios para a reposição de UCH foram instabilidade hemodinâmica e hemoglobina (Hb) <7-8g/dl. Foram analisados dados demográficos, Hb, hematócrito (Ht), drenagem mediastinal e reposição de UCH, em diversos intervalos, e tempos de CEC, UTI e hospital. RESULTADOS: Nos grupos 1 e 2, a idade média foi de 64,2 e 60,6 anos, com predominância do sexo masculino, o EuroSCORE logístico de 10,3 e 9,6 e a mortalidade de 2% e 4%, não relacionada ao estudo. O grupo 2 apresentou incidência de reoperações superior (12 x 6%), mas o número de UCH usado (4,31x1,25) e o tempo de internamento hospitalar (10,8x7,4) foram menores. Realizada análise uni e multivariada, que não demonstrou valores estatisticamente significativos, exceto no uso de UCH. A relação entre o custo do RS e das UCH foi custo/efetiva e o tempo de internamento, menor. CONCLUSÃO: O uso de RS diminui o número de UCH usadas, não é custo/efetivo e mostrou benefícios ao paciente.

INTRODUCTION: The use of cell saver (CS) in cardiac surgery is proposed to reduce the use of units of packed red blood cells stored (URBC), which increases morbidity, mortality and causes inflammatory reactions. OBJECTIVE: The objective is to evaluate whether the use of CS decreases the use URBC, is cost /effective and beneficial to the patient. METHODS: In a prospective study, between November 2009 and October 2011, 100 consecutive patients who underwent cardiovascular surgery with CPB, hemodilution and hemofiltration, were enrolled. Patients were divided into group 1 (no CS) and 2 (CS). The criteria for the replacement of RBC were hemodynamic instability and hemoglobin (Hb) <7-8g/dl. Demographic data, as well as Hb and hematocrit, mediastinal drainage, number of URBC and CPB, ICU and hospital time, were analysed. RESULTS: In groups 1 and 2 the average age was 64.1 and 60.6 years; predominantly male; the logistic EuroSCORE 10.3 and 9.4; mortality 2% and 4%. Group 2 had a higher incidence of reoperations (12% versus 6%), but the average of URBC used (4.31 versus 1.25) and mean length of hospital stay (10.8 versus 7.4 days) was lower. Univariate and multivariate analysis, were performed, which showed no statistically significant values, except in the use of URBC. The relationship between the CS and the cost of RBC was not cost /effective and length of stay was shorter. CONCLUSION: The use of CS decreases the number of used URBC, is not cost /effective but has shown benefits for patients.

Relation between perfusion speed and acute separation of myocardial cells in New Zealand rabbits / 心血管康复医学杂志

Objective: To establish a simple, stable and high success rate separation method of myocardial cells in New Zealand rabbits. Methods: Compared influence between perfusion speed of 50 r/min (8 ml/min) and 65 r/min (11 ml/min) of Langendorff perfusion device on cell separation during separation course of myocardial cells. Results: Compared with perfusion speed of 50 r/min, there was significant decrease in time to obtain cells [(33±9.10) min vs. (19.29±2.73) min], and significant increase in success rate (50% vs. 91%) and survival rate [(38.26±2.63) % vs. (85.71±2.21) %] by perfusion speed of 65 r/min, P<0.01 all. Conclusion: Perfusion speed is an important factor that affects acute separation of myocardial cells in New Zealand rabbits, and 65r/min perfusion speed may be more stable and successful to obtain single myocardial cell.

Separation of digestive system cancer stem cells / 国际肿瘤学杂志

The separation of digestive system cancer stem cells (CSCs) is the first step of the study of stem cell.At present,there are three methods of separation of the digestive system CSCs:cell phenotype sorting,side population cell sorting and serum-free culture.The separation of digestive system CSCs is based on these three methods or be improved to a higher proportion of CSCs.

Dimensionamiento del sistema de microfiltración para la producción de la Vacuna Pertussis elaborada en el Instituto Nacional de Higiene “Rafael Rangel” / Microfiltration sistem dimension for using in Pertussis Vaccine toxin production in the Instituto Nacional de Higiene "Rafael Rangel"

Se evaluó el uso de la tecnología de Flujo de Filtración Tangencial (FFT), para obtener la Vacuna Pertussis Celular a partir de cultivos de la bacteria Bordetella pertussis, usando el proceso de Microfiltración (MF) a objeto de recuperar el paquete celular. Se determinaron las características de los filtros, condiciones de trabajo y el dimensionamiento del equipo a adquirir para la nueva producción in dustrial de Vacuna Pertussis Celular. Se evaluaron el flujo y tiempo de proceso, rendimiento y las características del producto obtenido. Utilizando cultivos con Vacuna Pertus sis en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Se seleccionó las membranas tipo cassettes, formato Suspended Screen, porosidad 0,2 μm, como las adecuadas para el proceso de MF, ya que mostraron un 100% de recuperación del paquete celular sin transmisión de células al filtrado y con un flujo promedio de filtrado de 54.00 L/m2h. Estos resultaron permitieron dimensionar, considerando las variables a utilizar en la nueva producción industrial (Volumen 650 Litros, Tiempo de Procesos, 3 a 4 horas), el área de filtración del equipo de MF a adquirir, estimado en 20 m² .

Tangential Flow Filtration (TFF) technology was evaluated to process Whole Cell Pertussis Vaccine which is produced by Bordetella pertussis bacterium. Microfiltration (MF) is used to recovery cells to produ ce the vaccine. MF pro - cesses was 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 con taining the Whole Cell Pertussis was processed using a laboratory scale TFF system designed to pro duct the TFF effect. The evaluation demonstrated that a cassette in suspended screen format and membrane with 0.2μm pore is the right selection for the MF step. It showed 100% of cell recovery without cell transmission to the filtrate and average process flux of 54.00 L/m2h. These results were used to scale-up the application to process the industrial volume of 650 liters in 3 hours of processing time. Membrane area sizing of MF to be acquired is estimated in 20 m².

Uso de fluorescência em um método de dissector modificado para estimar o número de miócitos no tecido cardíaco / Use of fluorescence in a modified disector method to estimate the number of myocytes in cardiac tissue / Uso de fluorescencia en un método de disector modificado para estimar el número de miocitos en el tejido cardíaco

FUNDAMENTO: Métodos convencionais de dissector atualmente requerem consideráveis custos financeiros, técnicos e operacionais para estimar o número de células, incluindo cardiomiócitos, em uma área de 3D. OBJETIVO: Usar a microscopia de fluorescência em um método de dissector modificado para determinar o número de miócitos no tecido cardíaco em condições normais e patológicas. MÉTODOS: O estudo empregou camundongos Wistar machos com quatro meses de idade e peso de 366,25 ± 88,21 g randomizados em grupos controles (GC, n = 8) e infectados (GI, n = 8). Os animais do GI foram inoculados com cepa Y de T. cruzi (300.000 tripomastigotas/50 g). Após oito semanas, os animais foram pesados e sacrificados. Os Ventrículos Esquerdos (VE) foram removidos para análise estereológica da densidade numérica de cardiomiócitos (Nv [c]) e o número total dessas células no VE (N [c]). Esses parâmetros foram estimados usando um dissector fluorescente (DF) e comparados com os métodos convencionais de dissector óptico (DO) e dissector físico (DFi). RESULTADOS: Em ambos os métodos de dissector, os animais do GI apresentaram queda significativa de Nv[c] e N[c] em comparação com os animais do GC (P > 0,05). Uma correlação forte, igual ou superior a 96%, foi obtida entre DF, DO e DFi. CONCLUSÃO: O método DF parece ser igualmente confiável para determinar Nv[c] e N[c] em condições normais e patológicas, apresentando algumas vantagens em relação aos métodos convencionais de dissector: redução de cortes histológicos e imagens na análise estereológica, redução do tempo de análise das imagens, a construção de DF em microscópios simples, utilizando o modo de epifluorescência, distinção de planos de dissector em ampliações inferiores.

BACKGROUND: Conventional disector methods currently require considerable financial, technical and operational costs to estimate the number of cells, including cardyomyocytes, in a 3D area. OBJECTIVE: To use fluorescence microscopy in a modified disector method to determine the number of myocytes in cardiac tissue in normal and pathological conditions. METHODS: The study employed four-month-old male Wistar rats with weight of 366.25 ± 88.21g randomized in control (CG, n=8) and infected (IG, n=8) groups. IG animals were inoculated with T. cruzi Y strain (300,000 trypomastigotes/50g wt). After eight weeks, the animals were weighted and euthanized. The left ventricles (LV) were removed for stereological analysis of numerical density of cardiomyocytes (Nv[c]) and total number of these cells in the LV (N[c]). These parameters were estimated using a fluorescent disector (FD) and compared with the conventional optical (OD) and physical (PD) disector methods. RESULTS: In both disector methods, IG animals presented significant decrease of Nv[c] and N[c] compared to CG animals (P< 0.05). There was no significant difference in these variables despite the disector method applied in CG and IG animals (P> 0.05). A strong correlation, equal or above 96%, was obtained between FD, OD and PD. CONCLUSION: The FD method seems to be equally reliable to determine Nv[c] and N[c] in normal and pathological conditions and presents some advantages compared to conventional disector methods: reduction of histological slices and images in the stereological analysis, reduction of time to analyze the images, construction of FD in simple microscopes using the epifluorescence mode, distinction of disector planes in lower magnifications.

FUNDAMENTO: Métodos convencionales de disector actualmente requieren considerables costos financieros, técnicos y operativos para estimar el número de células, incluyendo cardiomiocitos, en un área de 3D. OBJETIVO: Usar la microscopia de fluorescencia en un método de disector modificado para determinar el número de miocitos en el tejido cardíaco en condiciones normales y patológicas. MÉTODOS: El estudio empleó ratones Wistar machos de cuatro meses de edad y peso de 366,25 ± 88,21 g randomizados en grupos controles (GC, n = 8) e infectados (GI, n = 8). Los animales del GI fueron inoculados con cepa Y de T. cruzi (300.000 tripomastigotas/50 g). Después de ocho semanas, los animales fueron pesados y sacrificados. Los Ventrículos Izquierdos (VI) fueron removidos para análisis estereológico de la densidad numérica de cardiomiocitos (Nv [c]) y el número total de esas células en el VI (N [c]). Esos parámetros fueron estimados usando un disector fluorescente (FD) y comparados con los métodos convencionales de disector óptico (OD) y disector físico (PD). RESULTADOS: En ambos métodos de disector, los animales del GI presentaron caída significativa de Nv[c] y N[c] en comparación con los animales del GC (P > 0,05). Una correlación fuerte, igual o superior a 96%, fue obtenida entre FD, OD y PD. CONCLUSIÓN: El método FD parece ser igualmente confiable para determinar Nv[c] y N[c] en condiciones normales y patológicas, presentando algunas ventajas en relación a los métodos convencionales de disector: reducción de cortes histológicos e imágenes en el análisis estereológico, reducción del tiempo de análisis de las imágenes, la construcción de FD en microscopios simples, utilizando el modo de epifluorescencia, distinción de planos de disector en ampliaciones inferiores.

Acute isolation of rat superior cervical ganglion cells and identification of the electrophysiological properties / 中华麻醉学杂志

Objective To establish a method for acute isolation of rat superior cervical ganglion (SCG) cells and identify the electrophysiological properties.Methods Sprague-Dawley rats of both sexes,aged 5-12 days,were decapitated.The SCGs were removed quickly,and the single SCG cell was enzymatically isolated from the SCGs.When the holding potential was - 60 mV,100 μmol/L acetylcholine was applied and the nicotinic acetylcholine receptor currents were recorded by whole-cell patch-clamp technique.When the holding potential was 760 mV,65 mmol/L KCl was applied and quantal release of catecholamines was detected by using carbon fiber electrodes.Results SCG cells with normal electrophysiological properties were isolated.Typical nicotinic acetylcholine receptor currents and quantal release of catecholamines were recorded successfully.Conclusion The cells suitable for patch-clamp experiments can be obtained by using the method for acute isolation of rat SCG cells.