Este trabalho foi apoiado pelo Deutsche Forschungsgemeinschaft (DFG, KL e KL 1415/3-2 1415/4-2).

1. Preparação <ol><li> Preparar os pratos de cultura <ol><li> Descongelar colagénio Tipo IV O / N a 4 ° C e dissolvem-se em 0,1% de ácido acético estéril. O volume a ser usado depende do tipo e número de pratos em que as células estão a ser semeadas. Use 2 mg / cm 2. </li><li> Incubar pratos, pelo menos durante 1 hora à temperatura ambiente e lava-se duas vezes com água destilada (A. tridest). </li><li> Permitir que os pratos para secar adequadamente. </li></ol></li><li> A suplementação do meio de <ol><li> Aumento do nível de glicose de até 4,5 g / l através da adição de 1,75 g de glicose a 500 ml de meio. Para ajustar o meio a 600 mosmol, adicionar NaCl 100 mM e 100 mM de ureia, para aumentar a osmolaridade de 200 mosmol e 100 mOsmol, respectivamente. </li><li> Adicionar 1% de ácidos aminados não essenciais, 1% ultroser, dbcAMP 500 uM, 20 U / ml nistatina e 0,25 ug / ml de gentamicina (diluição 1:200 de uma solução de estoque com uma concentração de 50 ug / ml) a 600 mosmol meio DMEM frescos no dia da preparação. </li></ol></li&gt; <li> Solução de enzima <ol><li> Adicionar 1 mg / ml de hialuronidase e 2,2 mg / ml de colagenase de DPBS contendo 0,25 ug / ml de gentamicina e nistatina. Prepare 1 ml desta solução de enzimas para a digestão de 2 rim medulas interior. A solução deve ser esterilizada por filtração e armazenada em tubos Falcon de 50 ml. </li></ol></li></ol> 2. Cultivo de Rat primária ducto coletor medular interno (IMCD) Células Todos os procedimentos de manuseio de animais devem ser realizadas em conformidade com a legislação local e federal. <ol><li> Anestesiar ratos em CO 2 (numa caixa saturado com CO 2) e isoflurano (U-400 Anestesia unidade, Univentor Ltd., de Malta, fluxo de ar de 350-400 ml / min, com 2-2,5% de isoflurano). Decapitar os animais para o sangramento, que permite a detecção exata da fronteira entre a esbranquiçada interior ea medula externa mais escura. Além disso, a hemorragia minimiza o número de eritrócitos isolados. Remover e rinslavá-los em DPBS estéril suplementado com gentamicina e nistatina (ver acima) em gelo. Todas as outras etapas devem ser realizadas em condições bancada limpa estéreis. </li><li> Transferir os rins a partir da solução de DPBS para uma compressa esterilizada para remover o líquido em excesso. Retire a cápsula renal graxos com uma pinça e tesouras estéreis. </li><li> Tendo o rim ainda fixado na compressa, cortar ligeiramente ao lado do eixo longitudinal do lado exterior (cortical). Certifique-se de não cortar completamente, mas para deixá-lo em uma peça, conectado na pelve renal. </li><li> Com tesoura curva dissecar cuidadosamente o medulas interior esbranquiçada, que está cercado pela rosé cintilante medula externa. Recolhe-los em DPBS contendo gentamicina e nistatina em uma placa de cultura de 60 milímetros em gelo. </li><li> Uma vez que todos medulas são isoladas e recolhidas em gelo, reduzir o volume de DPBS para um nível em que são cobertos apenas com tampão. Use uma lâmina de barbear estéril para cortar o medulas em cubos de 53 mm. </li><li> Derreta e volta a ponta de uma pipeta Pasteur estéril, segurando-o brevemente em uma chama. Certifique-se que a pipeta é resfriado antes de continuar. </li><li> Transferir a suspensão de tecido para os tubos Falcon de 50 ml contendo solução de enzima esterilizada preparados com hialuronidase e colagenase (ver 1.3.1), usando o arredondado pipeta Pasteur. Fechar hermeticamente a tampa e incubar a 37 ° C, sob agitação contínua (220 rpm) num banho de água de mesa regular para 1,5-2 horas. </li><li> Por trituração da suspensão, pipetar a mistura para cima e para baixo várias vezes, com a pipeta de Pasteur rodada (veja acima) até a suspensão se tornar homogénea turva. </li><li> Centrifugar a suspensão durante 5 min a 300 xg e 16 ° C. Rejeitar o sobrenadante, ressuspender o pellet cuidadosamente em DPBS contendo gentamicina e nistatina e centrifugar novamente. Repetir esta etapa de lavagem uma vez. </li><li> Ressuspender as células em meio totalmente completada e semente-los em cultura pratos e / ou chapas de microtitulação assim. A preparação de 2 medulas (1 animal) irá produzir células suficientes para um prato de cultura de 60 mm. </li><li> Após 24 horas, lave as células duas vezes com 600 mosmol DMEM e adicione meio fresco. Durante a semana seguinte as células deverão ser lavadas 2-3 vezes (Figura 1). 6-8 dias após a sementeira, as células IMCD pode ser utilizado para experiências. Recorde incubar em meio sem dbcAMP e nistatina durante 24 horas antes de começar a experiência. Caso contrário AQP2 será exclusivamente localizado na membrana de plasma. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Nós apresentamos um protocolo detalhado para a preparação e cultivo de células IMCD primárias de rato. A abordagem rende até 21 cm 2 de células de um rato 20. O ensaio requer um equipamento padrão de cultura de células e pode ser realizada por uma única pessoa dentro de aproximadamente 6 horas. Por conseguinte, esta abordagem é adequada como um método padrão de laboratório. IMCD células primárias de rato podem ser semeadas em placas de cultura de diferen...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Na recolha de células renais de condutas principais, a arginina-vasopressina (AVP) controla a reabsorção de água, estimulando a inserção do canal de água aquaporina-2 (AQP2) na membrana plasmática. AVP se liga à proteína acoplada vasopressina tipo-2, receptor de G (V2R) estimular a adenilil ciclase e assim a formação de AMPc. Iniciação desta cascata de sinalização conduz à activação da proteína quinase A (PKA). PKA fosforila AQP2 na serina 256 (S256), que é o factor chave para a sua redistribuição intracelular de vesículas na membrana plasmática. A inserção da membrana facilita a reabsorção de água ao longo de um gradiente osmótico e afina corpo homeostase da água. Desregulação da reabsorção de água AVP-mediada, devido a aberrações da secreção de AVP ou causas sinalização AVP ativados ou está associada a doenças graves. Diminuição da reabsorção de água causada por mutações de V2R ou AQP2 leva a diabetes insípido nefrogênico, enquanto um elevnível AVP plasma sanguíneo decido está associada a reabsorção excessiva de água em doenças cardiovasculares, como insuficiência cardíaca crônica ea síndrome da secreção do hormônio antidiurético inapropriado (SIADH). O significado da reabsorção de água mediada por AVP resulta não só a partir da sua implicação na doença. A translocação AVP induzida por vesículas AQP2-rolamento e fusão com a membrana plasmática representa um processo exocitica estritamente cAMP-dependente, o que atualmente não é bem compreendida. Outros exemplos de um exocitose dependente de AMPc são a secreção de renina a nível renal, secreção de H + no estômago. Portanto, a elucidação dos mecanismos moleculares que regulam a AQP2-translocação em células renais principais não só ajuda a compreender os processos moleculares semelhantes em outros tipos de células, mas também pode abrir o caminho para novas terapias para o tratamento de doenças associadas a distúrbios da reabsorção de água mediada por AVP . Elucidating AQP2 mecanismos de controle requer a coleta principais modelos de células do duto. Por isso, uma série de diferentes linhas de células de rim de mamífero estão disponíveis. No entanto, estes modelos têm vários inconvenientes. Em muitos sistemas de célula o nível de proteína AQP2 é baixa (Tabela 1; M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, outros ectopicamente superexpressam humanos (MCD4, WT10) ou 11,12 rato (CD8) 13 AQP2. Em MCD4 e células COS-7 do receptor V2 não é expresso. Para o nosso conhecimento, não há linha de células permanente como um modelo disponível, que é derivada da recolha da medula renal conduta interior, a parte do rim onde AQP2 expressão é mais abundante, mas a partir da conduta de recolha cortical 1,11,13-16 . Tais modelos de células são por exemplo os mpkCCD imortalizada utilizada (por exemplo, 17) ou os mTERT CCD 14 linhas de células recentemente estabelecidas. Ambas as linhas de células endogenamente AQP2 expressa e V2R, mas uma vez que eles são derivados daduto coletor cortical provavelmente conter um proteoma diferente em relação ao coletor medular interno células do duto (IMCD). Eles devem, por exemplo, expressar diferente Na + sistemas de transporte. Aqui, apresentamos um protocolo de cultura de células primárias IMCD expressando V2R e AQP2 endogenamente. Este modelo, portanto, representa mais de perto a situação fisiológica no ducto coletor renal. Como estabelecer a cultura exige apenas padrão de laboratório, equipamento de outros laboratórios podem facilmente adotar essa abordagem.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
		</tr>
		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
		</tr>
		<tr>
			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
		</tr>
		<tr>
			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
		</tr>
		<tr>
			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
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culturing primary rat inner medullary collecting duct cells

Arginina-vasopressina (AVP), facilita a reabsorção de água por renal coletando células principais do duto e, assim, afina corpo homeostase da água. AVP se liga aos receptores de vasopressina V2 (V2R) na superfície das células e, assim, induz a síntese de cAMP. Este estimula a processos de sinalização intracelular que conduzem a alterações na fosforilação do canal de água aquaporina-2 (AQP2). Proteína cinase A phoshorylates AQP2 gerando assim a translocação de AQP2 de vesículas intracelulares para a membrana plasmática facilitar a reabsorção de água a partir da urina primária. Aberrações de libertação da AVP sinalização pituitária ou AVP-activado em células principais podem causar insipidus nefrogénica centrais ou diabetes, respectivamente, um nível elevado de AVP plasma sanguíneo está associada com doenças cardiovasculares, tais como insuficiência cardíaca crónica e síndroma de secreção inapropriada de hormona antidiurética. Aqui, apresentamos um protocolo para cultivatino de rato principal coletor medular interno células do duto (IMCD), que expressa V2R e AQP2 endogenamente. As células são adequados para a elucidação dos mecanismos moleculares subjacentes ao controlo das AQP2 e, portanto, para descobrir novos alvos terapêuticos para o tratamento de doenças associadas com a desregulação da reabsorção de água mediada por AVP. IMCD células são obtidas a partir de ratos medulas interior renal e são utilizados em experiências de seis a oito dias após a semeadura. IMCD células podem ser cultivadas em placas de cultura de células normais, frascos e placas de micro-titulação de formatos diferentes, o procedimento requer apenas algumas horas, e é apropriado para laboratórios de cultura de células padrão.

O sucesso do cultivo de células primárias de rato IMCD resultará numa monocamada confluente de 6-8 dias após a sementeira (Figura 2). Por 60 milímetros prato de cultura há aproximadamente 6 x 10 6 células. As células firmemente aderir às placas de cultura, uma vez que estes foram revestidos com colagénio do tipo IV, um componente da membrana basal 18. Portanto, as células IMCD não irá separar mesmo durante vários procedimentos de lavagem cuidadosa. Até 80% das célul...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginina-vasopressina (AVP) controla o ajuste fino da homeostase de água do corpo, facilitando a reabsorção de água pelas células principais renais. Aqui, apresentamos um protocolo para o cultivo de rato principal coletor medular interno células do duto adequados para a elucidação dos mecanismos moleculares subjacentes à reabsorção de água AVP-mediada.

Cultura primária Rat coletor medular interno células dos ductos

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K visualizações.  Max-Delbrück-Center for Molecular Medicine. Arginina-vasopressina (AVP) controla o ajuste fino da homeostase de água do corpo, facilitando a reabsorção de água pelas células principais renais. Aqui, apresentamos um protocolo para o cultivo de rato principal coletor medular interno células do duto adequados para a elucidação dos mecanismos moleculares subjacentes à reabsorção de água AVP-mediada.

Vídeo: Culturing Primary Rat Inner Medullary Collecting Duct Cells

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

Protocolo para Neurônios Cultivar Sympathetic de Gânglios Superior Rat Cervical (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Biologia

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultura primária de Miócitos Coração Adulto Rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Primárias células epiteliais brônquicas humanas Grown a partir de explantes

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolamento de células basais e células Duct submucosa Glândulas de Rato Traquéia

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e cultura primária de células hepáticas de ratos

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolamento de células imunes de tumores primários

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

A cultura de células epiteliais nasais Humano na interface ar líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Um Método para o cultivo embrionário<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Cultura primária de células Adrenocortical rato e ensaios das funções reguladora

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...