Agradecemos a Emeka Ifedigbo de assistência técnica e Shivraj Dr. Tyagi de conhecimentos valiosos. Agradecemos também o Centro NeuroDiscovery Harvard para a assistência com a microscopia. Este trabalho foi financiado em parte por uma Associação Americana do Coração Predoctoral conceder 09PRE2250120 para Hilaire Lam, e concede NIH, R01-HL60234, R01-HL55330, R01-HL079904, concedido a AMK Choi.

O protocolo geral requer dois dias para isolamentos de células do tecido animal, 5-10 dias para a proliferação celular, e um adicional de 10-14 dias para a diferenciação de células no ar-líquido interface. Um dia adicional é necessário para exposições de células e colheita de amostras. 1. Isolamento de rato traqueobrônquica células epiteliais (MTEC). Nota: Todos os procedimentos descritos abaixo foram analisados ​​e aprovados pelo Institutional Animal Care e do Comitê Use em Brigham e do Hospital da Mulher / Área Harvard Medical School. Antes de começar: <ul><li> Prepare F12 Ham mídia contendo antibióticos. F12 a 250 mL Ham da mídia basal (Cellgro) adicionar 2,50 mL de uma 100 X penicilina / estreptomicina (10 2 U/mL-10 2 mg / mL) e solução de 250 mL de uma solução 1,000 Fungizone X. Armazenar a 4 ° C por até 4 semanas. </li><li> Prepare uma solução de pronase 0,15%. Adicionar 15 pronase mg a 10 mL de F12 Ham mídia contendo antibióticos. Faça fresco e manter em gelo até o uso. </li><li> Prepare uma superfície de trabalho limpa e estéril de instrumentos cirúrgicos adequados para a cirurgia de pequenos animais, e uma capa de tecido para cultura lamelar isolamentos celular. Todos os outros equipamentos é considerada padrão para a cultura de células animais, incluindo umidificado incubadoras de CO 2, câmara fria, a célula de mesa centrífugas, microscópio invertido e pipeta de plástico descartáveis ​​e utensílios de cultura. </li><li> Prepare solução de colágeno I (50 mcg / ml em 0,02 N de ácido acético). Pipetar 1,0 mL de solução de colágeno em cada poço de uma placa de 12 poços transwell (Corning). Embrulhe em parafilme e deixe descansar durante a noite em uma superfície plana em temperatura ambiente. </li></ul><ol><li> Usando uma cepa do mouse comercialmente disponíveis (ie, C57BL / 6, machos de 6-8 semanas de idade), euthanize camundongos usando um método aprovado de eutanásia como o CO 2 induzida narcose. Tipicamente 6 ratos irá produzir células suficientes (1,5-2,0 x 10 5 células / mouse) para uma semente bem-12 placa transwell. </li><li> Pulverizar as carcaças de animais com 70% de solução EtOH para esterilizar o campo. </li><li> Com limpa tesoura cirúrgica e bisturi, retire a pele ao redor da área traqueal, e expor a traquéia. Abrir o abdômen, corte ao longo do esterno, e remover a caixa torácica. Remover o tecido até o final da traquéia está exposto. </li><li> Traquéias lugar em um tubo cônico de 50 mL contendo 30 mL Ham F12 mídia contendo antibióticos, no gelo. </li><li> Em uma capela de fluxo laminar estéril, a transferência de tecido traqueal a um prato de 100 milímetros estéril Petri contendo 10 mL Ham F12 mídia contendo antibióticos. </li><li> Suavemente dissecar o tecido conjuntivo com uma pinça e tesouras cirúrgicas estéreis. </li><li> Transferência de tecido traqueal a um 100 milímetros nova placa de Petri contendo 10 mL Ham F12 mídia contendo antibióticos para enxaguar. Corte traquéias longo do eixo vertical para expor lúmen. </li><li> Traquéias transferência para um tubo de 50 mL contendo 10 mL de solução pronase 0,15% e incubar overnight a 4 ° C. </li><li> No segundo dia, ter pronto: <ul><li> Prepare uma solução de DNAse I. A 18 mL de F12 Ham mídia contendo antibióticos adicionar 2 mL de uma 10 mg / mL solução estoque de albumina sérica bovina (BSA), e 10 mg de petróleo pancreático DNAse I. Faça um alíquotas mL e armazenar a -20 ° C (descongeladas em gelo antes de usar). </li><li> Prepare F12 Ham de mídia contendo antibióticos com 20% de soro fetal bovino (FBS). F12 a 200 mL Ham da mídia basal (Invitrogen) adicionar 50 mL de calor FBS inativada, 2,5 mL de uma 100 X penicilina / estreptomicina (10 2 U/mL-10 2 mg / mL) solução, e 250 mL de uma solução 1,000 Fungizone X . </li><li> Prepare MTEC meio básico contendo antibióticos. Para 475,5 mL DMEM/F12 meios básicos (Cellgro) adicionar 7,5 mL de solução 1 M HEPES, 10 mL de solução 200 mM de glutamina, 2 mL de um 7,5% solução de NaHCO 3, 5 mL de uma 100 X penicilina / estreptomicina solução, 500 mL de uma solução 1,000 Fungizone X </li><li> Prepare MTEC% medium/10 FBS. A 45 mL de MTEC meio básico contendo antibióticos, adicionar 5 mL de calor FBS inativado. </li></ul></li><li> Agite delicadamente o tubo (a partir do Passo 1.8) 10-12 vezes, e depois deixe repousar por 30-60 minutos a 4 ° C. </li><li> Adicionar 10 mL Ham F12 mídia contendo 20% FBS e antibióticos para o tubo e rock 12 vezes. </li><li> Prepare 3 15 mL tubos cônicos contendo 10 mL Ham F12 mídia contendo 20% FBS e antibióticos. </li><li> Remover traquéias de solução pronase, deixando de lado esta solução sobre o gelo. Transferência de traquéias de tubo cônico primeira contendo F12 Ham e inverter tubo de 12 vezes. Repita esse processo mais duas vezes. </li><li> Combine solução pronase com os três sobrenadantes do passo 1,13 em um tubo de 50 mL. Descartar tecido remanescente. </li><li> Centrifugar a 1400 rpm (390 xg; centrífuga Eppendorf 5810R equipado com um rotor A-4-62) por 10 min a 4 ° C, e descartar o sobrenadante. </li><li> Delicadamente ressuspender o sedimento em 1 mL de solução DNAse (100-200 mL / traquéia)e incubar 5 min no gelo. </li><li> Centrifugar a 1400 rpm (390 xg) por 5 min a 4 ° C, e descartar o sobrenadante. </li><li> Ressuspender o pellet celular em 8 mL contendo meio MTEC FBS 10% </li><li> Suspensão placa de célula de Placas Primaria (Falcon). Incubar a 37 ° C em uma atmosfera de ar de 95%, 5% de CO 2 durante 5 horas (Nota: este é um passo seleção negativa para fibroblastos). </li><li> Coletar suspensão de células a partir de chapas e placas de lavar duas vezes com 4 mL MTEC contendo 10% FBS. Suspensão pool de células e lavagens juntos em um tubo cônico de 50 mL. </li><li> Conserve 1 mL para cytospin e contagem das células. Girar em uma centrífuga de mesa por 5 min a 5.000 rpm (Eppendorf 5415D). Remover 500 mL e ressuspenda sedimento em sobrenadante restante. Use de 100 L para contagem de células usando o método de coloração Trypan Blue vital. Conserve quatro alíquotas de 100 mL para análise cytospin </li><li> Girar restantes 15 mL suspensão celular em 1400 rpm (390 xg), a 4 ° C por 10 min. </li></ol> 2. Propagação e Diferenciação de MTEC No Ar Líquido de Interface Preparar soluções estoque ácido retinóico. Solução estoque A: Pesar ácido retinóico no escuro (Mr 300,44 g / mol) para fazer uma solução estoque 5 mM em EtOH 95%. Loja em um tubo de alumínio envolto a -80 ° C. Conforme necessário, prepare Solução B (5 mM), adicionando 50 mL de Stock A, 500 mL de solução BSA (100 mg / mL) e 49,5 mL de Hank solução salina balanceada (HBSS). Armazenar em um tubo de papel alumínio envolto a -80 ° C por até 4 semanas. Prepare MTEC meio de proliferação contendo ácido retinóico. Para 45,7 mL de mídia MTEC basal contendo antibióticos, adicione inativado pelo calor 2,5 mL FBS, 1 mL de ácido retinóico stock B, 250 mL de solução de insulina (2 mg / mL de insulina em 4 mM HCl), 250 mL de solução fator de crescimento epidérmico (5 mg / mL EGF em HBS contendo 1 mg / mL BSA), 200 mL extrato de pituitária bovina (15 mg / mL em HBS contendo 1 mg / mL BSA), 50 mL de solução Transferrina (5 mg / mL de transferrina no HBS contendo 1 mg / mL BSA ), solução de 50 mL da toxina da cólera (100 mg / mL em HBS contendo 1 mg / mL BSA). Filtro de esterilizar a preparação dos meios final e use dentro de 2 dias de preparação. <ol><li> Remoção da solução de colágeno a partir de chapas transwell, deixe repousar sob o capô por 5 min, e lavar com PBS duas vezes. </li><li> Centrifugação seguinte, ressuspender o pellet celular em um volume adequado de proliferação da mídia (500 mL) para facilitar o revestimento de 7,5 x 10 4 -1,0 x10 5 células por poço. Pipetar 500 mL suspensão celular na superfície apical da inserção da membrana transwell policarbonato. Adicione 1,5 mL de mídia proliferação para o compartimento basal do transwell. </li><li> Incubar as culturas MTEC submersa a 37 ° C em uma incubadora umidificada contendo ar de 95%, 5% de CO 2 para 10/07 dias. </li><li> Esperar três dias antes de mudar de mídia pela primeira vez. Mudança de mídia todos os dias. Monitor de culturas por inspeção visual e medição da resistência transepitelial célula usando um eletrodo (EVOM Ohmvoltometer, instrumentos de precisão Mundial, Sarasota, FL). </li><li> Quando as células aparecem confluentes e resistência epiteliais atinge 1.000 Ω / cm 2, as células estão prontos para diferenciar a ALI. </li><li> Prepare MTEC media basal contendo NuSerum 2%. Adicionar NuSerum para MTEC meio básico para uma concentração final de 2% V / V. Adicionar B Stock ácido retinóico para uma concentração final de 1 X 10 -7 M antes de usar. Prepare fresco e usar dentro de 2 dias. </li><li> Permitem diferenciar células por 10-14 dias, através da remoção de mídia apical e substituindo os meios de comunicação basal com 750 mL MTEC media basal contendo 2% de ácido retinóico e Nuserum. Troque a mídia basal e lavar o lado apical com MTEC NuSerum contendo 2% a cada dois dias. </li></ol> 3. Aplicação da fumaça do cigarro para cultura de células epiteliais <ol><li> Expor o lado apical das culturas transwell à fumaça do cigarro convencional usando um sistema de exposição de células CS (EMI Instruments, Pittsburgh, PA). Veja a Figura 1 para fotografia do aparelho. Consulte a Tabela de reagentes e equipamentos específicos para as especificações técnicas. Em resumo, o aparelho consiste de uma bomba peristáltica que fuma cada cigarro em ~ 7-8 puffs, desenho na fumaça principal em uma linha de polipropileno que está ligado a uma câmara de exposição ventilados. A câmara de exposição é mantida a 37 ° C por circulação de água, e mantida em uma atmosfera de 5% CO 2 em uma linha de gás. As células podem ser expostas à fumaça usando Kentucky 3R4F pesquisa cigarros de filtros de referência (The Tobacco Research Institute, University of Kentucky, Lexington, KY). Normalmente as células são expostas à fumaça de cigarros 1-2, cedendo 100-200 mg / m 3 de material particulado total (TPM). Culturas controle são expostos ao ar ambiente por um período de exposição equivalente. </li><li> A TPM é medida de acordo com o protocolo do fabricante fornecido com o TE-10c máquina de fumar (Empresas Teague). Em resumo, um filtro (Pallflex) é colocado em um suporte de aço inoxidável embutido em um tubo de amostragem líder do porto de amostragem na câmara de fumo a uma bomba de fluxo constante (unidade amostral). Um tubo de saída conecta a unidade de amostragem para um contador de gás (medidor de gás seco, ONM61, 67, AEM). O filtro é pesado antes e após a amostragem de gás. O total de material depositado sobre o filtro em mg é normalizado para o volume total de ar amostrado. </li><li> As células podem ser colhidas em qualquer ponto do tempo (ou seja, 0-24 horas) pós-exposição para os procedimentos de célula padrão analítico (ou seja, Western immunoblot análise, análise de mRNA, etc) ou microscopia (ie, microscopia confocal ou elétron). Os meios de cultura também pode ser analisado para liberação de citocinas ou LDH para ensaios de citotoxicidade. </li></ol> 4. Resultados representante Isolamento epiteliais sucesso, proliferação e diferenciação no ALI deve render uma monocamada intacta, com uma morfologia de paralelepípedos. Resistência das células Transepithelilal de culturas saudáveis ​​deve ser de aproximadamente 1000-2000 Ω / cm 2. A Figura 2 mostra uma monocamada de células epiteliais respiratórias do rato coradas para células epiteliais e marcadores de cílios em condições controle e após a exposição a fumaça do cigarro. A Figura 3 mostra micrografias eletrônicas representante de culturas saudáveis ​​MTEC, retratando ultraestrutura e morfologia dos cílios. <img src="/files/ftp_upload/2513/2513fig1.jpg" alt="Figura 1" /> Figura 1. Preparação dos rendimentos células epiteliais através de três fases, uma etapa de isolamento, uma fase de propagação, e um estágio de diferenciação no ar-líquido interface (esquema). Células epiteliais são isolados de rato traquéia, semeados em transwells onde eles proliferam em cultura submersa, e depois convertido em ar-líquido interface onde eles totalmente diferenciar. Experimentos são normalmente realizados no dia 24 de de cultura contínua. A imagem retrata um sistema modelo para a exposição de células cultivadas à fumaça de cigarro mainstream. Um sistema de cultura transwell ALI é colocado dentro de um sistema modular personalizado de entrega de cigarros de fumo que é controlado por temperatura, umidade e CO 2. <img src="/files/ftp_upload/2513/2513fig2.jpg" alt="Figura 2" /> Figura 2 culturas de células epiteliais foram fixados e corados para núcleos (Hoechst 33258), F-actina (verde; Alexa Fluor-faloidina 488-conjugados). Cílios eo marcador acetilada α-tubulina (vermelho; anticorpos Cy3 conjugada secundário). Os painéis inferiores mostram imagens equivalentes de células epiteliais tomadas quatro horas após a exposição à fumaça de cigarro (2 cigarros, 150 mg / m 3). (B) liberação de lactato desidrogenase (LDH) foi medido no meio basal 24 horas após a exposição a doses variadas de fumaça de cigarro, conforme indicado. <img src="/files/ftp_upload/2513/2513fig3.jpg" alt="Figura 3" /> Figura 3. Culturas Air-líquido de interface de células epiteliais isoladas do epitélio traqueal de rato se diferenciar em vários subtipos que, por microscopia eletrônica de transmissão (TEM) tem as características de ciliadas, células basais, e não-ciliadas 1. Estas células do epitélio respiratório pseudoestratificado tipificam. Rótulos figura correspondem a: bb: corpo basal, a: axonema, m: mitocôndrias, n: núcleo, s: substrato (membrana de policarbonato), mv: microvili

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O protocolo descrevendo o isolamento de células epiteliais do mouse traqueal é adaptado dos protocolos de Você et al., 1, 2-3 e outros com modificações. Como acontece com qualquer protocolo de isolamento de células descrevendo, o aspecto mais crítico é o de evitar a contaminação por patógenos, bacteriana ou fúngica, utilizando técnicas de assepsia rigorosa. Um segundo passo fundamental é evitar a contaminação das culturas de fibroblastos, que podem ser evitados por meio de dissecção...

* A máquina de fumar cigarros é um costume projetou e fabricou 14 &quot;x14&quot; x20 &quot;Dual septadas e à prova d&#39;água tonalidade jacketed luz clara 1 / 2&quot; de espessura de policarbonato Lexan câmara de exposição à fumaça do cigarro com sensor de temperatura controlada, nível de água controlado desligar do sistema. Um cigarro / puffing unidade está instalada por um cigarro variável puffing taxas. Quando a unidade estiver em uso, ele imita uma incubadora no sentido de que a temperatura, umidade e dióxido de carbono são controlados no sistema. O sistema inclui: (I) um personalizado unidade de câmara de água / dupla camisa que mantém um ambiente controlado para experimentos de cultura de tecidos. (II) O dever de um pesado digitais, de alta precisão dupla bomba de água do sistema de circulação de temperatura com sensor de nível de água e controle de temperatura (III) A unidade de cigarro fumando com bomba. (IV) Uma bomba de ciclo de sensor de controle de taxa de cycler (IV) A precisão em aço inoxidável de alta-Line no porta-filtro. (V) A tampa destacável montado 02/11 &quot;tamanho axial uniformidade fã fumaça de cigarro de mistura. (VI) Um banho de água de médio porte com suporte de montagem para a circulação de água. (VII) A 1 / 2 &#39;bandeja de acrílico de espessura com suportes para o bomba de sopro e suporte para coletor de cinzas de cigarro. Esta máquina, como descrito pode ser substituído por máquinas similares disponíveis comercialmente fumar tais como o tipo disponível a partir de sistemas TSE ( <a href="http://www.tse-systems.com">www.tse-systems.com</a> ).

isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface

Células epiteliais pulmonares podem ser isolados do trato respiratório de camundongos e cultivadas em ar-líquido interface (ALI) como um modelo de epitélio respiratório diferenciado. Um protocolo é descrito para isolar e expor essas células à fumaça do cigarro convencional (CS), a fim de estudar as respostas das células epiteliais à exposição CS. O protocolo consiste em três partes: o isolamento de células epiteliais das vias aéreas a partir do mouse traquéia, o cultivo dessas células em ar-líquido interface (ALI) como totalmente diferenciada células epiteliais, ea entrega de calibrado principais CS a essas células em cultura. O sistema de cultura ALI permite a cultura do epitélio respiratório sob condições que se assemelham mais a sua configuração fisiológica do que sistemas de cultura comuns líquido. O estudo molecular de pulmão e respostas celulares à exposição CS é um componente crítico de compreensão do impacto da poluição do ar ambiente sobre a saúde humana. Resultados da investigação nesta área pode vir a contribuir para a compreensão da etiologia da doença pulmonar obstrutiva crônica (DPOC), e outras doenças tabaco-relacionadas, que representam grandes problemas de saúde global.

Watch this Scientific Journal Video about Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface at JoVE.com

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Células epiteliais pulmonares podem ser isolados do trato respiratório de camundongos e cultivadas em ar-líquido interface como um modelo de epitélio respiratório diferenciado. Um protocolo é descrito para o isolamento, cultura e expondo essas células à fumaça de cigarro mainstream, a fim de estudar as respostas moleculares para esta toxina ambiental.

Isolamento de rato células epiteliais respiratórias e exposição à fumaça de cigarro Experimental da Air interface líquido

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

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Research

JoVE Journal

Medicine

33.8K visualizações.  Harvard Medical School. Células epiteliais pulmonares podem ser isolados do trato respiratório de camundongos e cultivadas em ar-líquido interface como um modelo de epitélio respiratório diferenciado. Um protocolo é descrito para o isolamento, cultura e expondo essas células à fumaça de cigarro mainstream, a fim de estudar as respostas moleculares para esta toxina ambiental.

Vídeo: Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Transporte pneumocócica experimental humana

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Medicina

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Automáticos de medição de enfisema pulmonar e das vias aéreas pequenas Reformas em cigarro expostas ao fumo Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e Cultura de Expansão de células endoteliais específicos de tumores

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Comparando os efeitos do fumo eletrônico do cigarro e do cigarro em uma novela<em&gt; In Vivo</em&gt; Sistema de Exposição

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Geração de um modelo de doença pulmonar obstrutiva crônica em ratos pela exposição repetida de ozônio

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Avaliação da toxicidade da inalação aguda de partículas transmitidas por expor células pulmonares humanas cultivadas na Interface Ar-Líquido

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Efeito das formulações de lágrimas artificiais na atividade metabólica das células epiteliais da córnea humana após exposição à dessecação

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.