This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

O protocolo descrito a seguir segue as diretrizes da UIUC comitê de ética em pesquisa humana. 1. Recolha e digestão de amostra de tecido Surgical <ol><li> Recolha da amostra de tecido do tumor após ressecção do cólon direito (Figura 1A e 1B). Recolha de tecido de um site saudável adjacente também. </li><li> Transferir o tecido imediatamente para um frasco de 15 ml contendo 12 ml de solução HBSS. Manter o frasco no gelo dentro de uma caixa de espuma isolante. </li><li> Transportar o tecido contendo frasco para uma cultura capa de tecido para posterior processamento dentro de 45 min. Manter o frasco em um bloco de gelo no interior do exaustor. </li><li> Verter o tecido fornecido em uma placa de 6 poços contendo 6-7 ml de solução de HBSS, utilizando uma pipeta. Nota: A quantidade de tecido por poço não é crítica neste passo de enxaguamento. </li><li> Mantenha a placa de 6 bem em um bloco de gelo. Lave o tecido em solução HBSS duas vezes. </li><li> Corte o tecido de forma limpa em seções separadas com scis estéreissor. Triture tecido em seções menores não superior a 1-2 mm 3 em tamanho, com uma lâmina de bisturi estéril. </li><li> Pesar um frasco rotulado 0,1% de tripsina (contendo 1 mL de 0,1% solução de tripsina) antes de transferir o tecido. Transferir ~ 20 mg de tecido em cada um dos frascos de 0,1% de tripsina com uma pipeta. </li><li> Tentar minimizar a transferência de HBSS para dentro do frasco de tripsina para evitar diluição adicional de tripsina. </li><li> Pesar os frascos com tripsina após a transferência do tecido; documentar a diferença como a massa de tecido. </li><li> Pour ~ 20 mg de tecido e 1 ml de tripsina a cada poço de uma placa de 24 poços. </li><li> Colocar a placa de 24 poços num agitador no frigorífico a 4 ° C durante 16-20 horas. Durante este período de espera ou em uma hora mais cedo, preparar substratos em gel de poliacrilamida e funcionalizar-los com proteínas da MEC, conforme descrito na seção 3. </li></ol> 2. A dissociação enzimática do tecido de amostra em suspensão única célula e cultura de células isoladas no ECM funcionalizados Elastic Substrates e pratos de poliestireno rígidos <ol><li> Após 16-20 horas de incubação em tripsina bem a 4 ° C num agitador, a transferência de tecido de 4 ° C com tripsina a 4 ° C frasco de meio de crescimento completo (contendo 2-3 ml de meio de crescimento completo) utilizando uma pipeta. Garantir a transferência mínima de tripsina em meios de crescimento. Nota: formulação de meio de crescimento completo é como se segue: meio RPMI 1640 de base complementado com soro de cavalo a uma concentração final de 10% e penicilina-estreptomicina a 1% da solução total. </li><li> Inserir o frasco de tecidos contendo meios de comunicação num banho de água quente a 37 ° C durante 12-15 min. </li><li> Transferir o tecido para um frasco de 15 ml contendo solução de HBSS com uma pipeta, agitar suavemente. </li><li> Repetir 2.3. </li><li> Remover o tecido a partir da solução de HBSS e transferência para 0,1% de solução de colagenase (em HBSS) frasco contendo 1 ml de solução. Incubar durante 45 minutos a 37 ° C e 5% de CO 2 ambiente numa incubadora de cultura de células humidificada. Durante o período de espera, growt quenteh mídia em um frasco de 15 ml a 37 ° C em banho-maria. </li><li> Retire todos os fragmentos de tecido em pipeta minimização colagenase transição. Ejetar apenas os fragmentos de tecido para frascos de meios de cultura pré-aquecido. </li><li> Colocar um filtro bem inserção 40 um em cada poço de uma placa de 6 poços. Humedecer o filtro com 1 mL de meio de cultura de células. </li><li> Tritura-se o tecido em meio com uma pipeta de vidro de 10 ml várias vezes até que o tecido está completamente dissociado. </li><li> Manter a ponta da pipeta tão próximo quanto possível para a base do frasco. </li><li> Depositar a solução sobre o filtro para remover os detritos e as partes sem dissociar. </li><li> Centrifugar filtrada suspensão de células no meio a 150 xg durante 5 min à temperatura ambiente. </li><li> Remover o sobrenadante e ressuspender depois pelete de células com 2 ml de meio de cultura fresco. </li><li> Contar as células utilizando um hemocitómetro (se necessário). Semente isolado células primárias na matriz extracelular (ECM) géis funcionalizados e pratos de isopor na concentração desejada.</li></ol> 3. Preparação e funcionalização de diferentes Rigidez poliacrilamida (PA) Géis e poliestireno Substratos Nota: Para demonstração visual da Seção 3, os telespectadores / leitores são encaminhados para uma recente Journal of Experiments Visualized (Jove), do artigo 17. <ol><li> Adotando protocolos publicados 18, 19, 12 milímetros ativar 2 tampa de vidro desliza quimicamente para garantir a ligação covalente do hidrogel. <ol><li> Primeiro, tampa de vidro deleite desliza com 3- Aminopropyltrymethoxysilane (ATS) para 7 min a RT. </li><li> Remover o ATS completamente com DI lavagem com água e tratamento de cobertura desliza com 0,5% de solução de glutaraldeído, durante 30 min (diluído em PBS a partir de solução stock de glutaraldeído a 70%). </li></ol></li><li> Obter duas soluções de gel kPa por mistura de 5% w solução de N &#39;-methylenebisacrylamide (bis) em HEPES 10 mM-solução salina tamponada com solução a 20 / v acrilamida e 0,05% de N,. Use 8% w / v de acrilamida e 0,13Solução de N &#39;-methylenebisacrylamide (bis) em 10 mM de solução salina tamponada com HEPES durante 10 kPa gel de 20% de N,. <ol><li> Em ambos os casos, usar 1: 200 de persulfato de amónio (10% w / v) e 1: 2000 N, N, N &#39;, N&#39; tetrametiletilenodiamina (TEMED) como o iniciador e o catalisador para o processo de polimerização, respectivamente. </li><li> Além disso, adicionar 100 ul grânulos fluorescentes a 2 solução de gel kPa como marcadores fiduciários 21, 22. </li></ol></li><li> Depositar uma gota de 20 ul da solução gel PA pré-polímero no dia 12 mm2 ativado deslizamento tampa de vidro. Coloque outra 12 milímetros 2 regulares lamela de vidro sobre a queda. Certifique-se de que a gota se espalha entre as lamelas, devido à capilaridade. Inverta a sanduíche para 2 géis kPa para assegurar que a maior parte dos grânulos de se aproximar da superfície da cultura de células. </li><li> Curar o gel de PA durante 45 min a RT. </li><li> Retire o deslizamento tampa superior usando um único razor borda. Nota: Durante peeling, destacamento procede de uma pontado sanduíche. O gel permanece aderente à lâmina de vidro activada. </li><li> Armazenar os géis em solução de PBS antes da utilização. </li><li> Funcionalizar géis PA e vidro com moléculas de ECM, fibronectina humana a uma concentração de 50 ug / ml, após métodos publicados 23. <ol><li> Resumidamente, incuba substratos com 2 ml de hidrato de hidrazina pura S / N. </li><li> Remover hidrato de hidrazina e enxaguar os substratos cuidadosamente com água DI. </li><li> Lavar os substratos com ácido acético a 5% durante 30 min. </li><li> Remover o ácido acético e os substratos enxaguar abundantemente com água desionizada. </li><li> Manter os substratos imersos em água desionizada durante 30 min. </li><li> Incubar os substratos com fibronectina oxidada durante 35 minutos a uma concentração de 50 ug / ml. </li><li> Lavar os substratos com PBS em agitador a baixa rpm para 10 min. </li><li> Incubar todos os substratos a 37 ° C em 2-3 ml de meio de cultura durante 30 min antes do plaqueamento das células. Nota: células da placa em um preenchimento escassamented forma (1.000-3.000 células / cm2). Cada deslizamento de vidro cobertas de gel precisa ser contido em uma placa de Petri 35 milímetros. Permitir que as células aderir completamente antes de qualquer microscopia (pelo menos S / N). </li></ol></li></ol> 4. Microscopia de Força de tracção e de imunofluorescência Microscopia Assays <ol><li> Traction Microscopia de Força Assay <ol><li> Quente EDTA de tripsina-/ 10% de solução de 0,25% de SDS a 37 ° C em banho de água durante 10 minutos antes de começar as experiências de tracção. </li><li> Remova um gel da incubadora de cultura de células em um tempo e colocar no palco microscópio fluorescente invertida (ampliação 32X). </li><li> Encontrar uma única célula no campo de visão. Remover a tampa do prato Petri. </li><li> Tomar uma imagem de contraste de fase de células (por exemplo, Figura 3). </li><li> Mude o modo de imagem para fluorescência e seleccionar filtro apropriado. Não mova o estágio do microscópio ou amostra durante este tempo. </li><li> Pegue uma imagem do displac partículas fluorescentesed por tracção celular (Figura 4C). </li><li> Adicionar solução / SDS a 1 ml de tripsina a placa de Petri para separar a célula a partir do gel. Não mova o estágio do microscópio ou amostra durante este tempo. Além disso, tomar uma imagem da célula de controlo para assegurar a remoção completa do gel. </li><li> Tomar uma imagem de referência (força nula) dos grânulos após a célula é removida. </li><li> Repetir passos 4.1.2-4.1.8 para todos os outros géis. </li><li> Faça uma pilha de imagens usando ImageJ das duas imagens adquiridas no passo 4.1.6 e 4.1.8 para cada caso. Para gerar a pilha de imagens, use seguinte seqüência de comandos em ImageJ após a abertura das imagens: Imagem → Pilhas → Imagens de empilhar. </li><li> Para obter o campo de deslocamento e tração, utilize plugins ImageJ seguinte métodos publicados 21, 22 Obter códigos e tutoriais detalhados para esses plugins usando o seguinte link:. <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins. <ol><li> Alinhe as imagens da pilha usando o plugin &#39;Matching Template&#39;. Use seguinte seqüência de comandos em ImageJ após a abertura da pilha de imagens gerado na etapa 4.1.10: Plugins → Template Matching → Alinhar Slices em Stack → OK. Salve a pilha de imagens, consequentemente. Use esta nova pilha de imagens nas etapas seguintes. </li><li> Obter o campo de deslocamento usando o PIV (velocimetria por imagem de partículas) plugin (Figura 4D). Use seguinte seqüência de comandos depois de abrir a pilha de imagens salvou na etapa 4.1.11.1: Plugins → → iterativo PIV PIV (Basic) → OK → Aceitar este PIV e saída → Ok. Salvar a saída PIV no mesmo diretório na pilha de imagem original. Use isso como entrada na próxima etapa. </li><li> Finalmente usar o FTTC (Transformada de Fourier tração citometria) plugin para obter o mapa de tração (Figura 4E). Use seguinte seqüência de comandos: Plugins → FTTC → Inspropriedades do material ert, ou seja, o módulo de Young e proporção de PA gel → OK → de Poisson Selecione o arquivo de saída PIV salvou na etapa 4.1.11.2 → OK. Salvar FTTC resulta no mesmo diretório na pilha de imagem e saída de PIV. </li></ol></li></ol></li><li> Imunofluorescência Microscopia Assays <ol><li> Pegue os substratos a serem imunocoradas para a capa laminar e remova o meio de cultura. </li><li> Lavar os substratos com PBS e fixar as células com paraformaldeído a 4% em PBS durante 20 min à temperatura ambiente. </li><li> Lavar os substratos com PBS durante 3 vezes (5 minutos de cada vez). Por conseguinte, os substratos incubar com 500 uL do sinal potenciador durante 30 minutos e lavar com PBS. </li><li> Incubar as células com anticorpo monoclonal anti-vinculina numa diluição de 1: 250 em PBS durante 45 min à temperatura ambiente. Lavar os substratos com PBS durante 3 vezes (5 minutos de cada vez). </li><li> Incubar as amostras com anticorpo secundário Alexa Fluor 488 anticorpo de cabra anti-IgG de ratinho a uma diluição de 1: 200em PBS à temperatura ambiente durante 30 min. Lavar os substratos com PBS durante 3 vezes (5 minutos de cada vez). </li><li> Para visualizar a estrutura F-actina, incubar as células com TRITC-faloidina conjugados com uma concentração de 50 ug / ml durante 45 minutos à TA. Lavar os substratos com PBS durante 3 vezes (5 minutos de cada vez). </li><li> Imagem das amostras que usam o microscópio confocal de varredura a laser (Figura 5A - 5D). </li></ol></li></ol>

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The authors have nothing to disclose.

Estresse tração celular surgiu recentemente como um potencial indicador biofísico de estado metastático 13. No entanto, não há dados tração experimental com células tumorais primárias existe na literatura até o momento. Além disso, a cultura de células do cólon diretamente primário isoladas em diferentes géis de poliacrilamida rigidez não é relatado ainda. Por isso, nós estabelecemos um optimizados de cólon primário condições de cultura de células em géis e poliestireno (Figur...

Nos últimos anos tornou-se cada vez mais evidente que as micro-ambiente mecânico, além de factores bioquímicos, desempenha um papel importante na regulação funcionalidades celulares. As células podem detectar e responder à rigidez substrato sobre o qual eles são respeitadas (como em cultura 2D) ou rodeado por (como em cultura 3D) 3-7. Ao fazer isso, as células são capazes de modular a sua diferenciação 3, 4 morfologia, a migração / motilidade 5, propriedades biofísicas 6, crescimento de 7, e outros processos. As células cancerosas também responder à rigidez matriz 2D e 3D utilizando, uma combinação coordenado hierárquica mecano-químico de receptores de adesão e as proteínas de membrana associadas a transdução de sinal, a arquitectura do citoesqueleto, motores moleculares e 1, 2. Por exemplo, células epiteliais mamarias (MECs) formar parênquima acinar normal quando cultivadas em substratos de 150 Pa, que é semelhante à rigidezde tecido mamário saudável. Curiosamente, eles apresentam as características de um tumor em desenvolvimento, tanto estruturais como de transcrição, quando cultivadas em substratos mais rígidos (&gt; 5000 Pa) que imitam a rigidez do estroma do tumor 8. Além disso, uma outra experiência mostra que a tumorigénese da mama é acompanhado por reticulação do colagénio e de ECM de reforço 9. Experiências recentes mostram que o carcinoma do cólon humano (HCT-8), as células exibem metástases como fenótipo (MLP), quando eles são cultivadas em substratos 2D possuindo rigidez fisiologicamente relevante (20-47 kPa), mas não em muito dura (3,6 GPa) substratos 10- 12 .Estes células aglomerados de células tumorais como primeira forma e, em seguida, dissociar-se um do outro, a partir da periferia. Como este epitelial ao arredondado morfológica (E a ​​transição R) a mudança ocorre, eles proliferam, reduzir célula-célula e célula-ECM adesão, e tornar-se migratório. HCT-8 células cultivadas em substratos de poliestireno muito duros não apresentam estestraços malignos. Assim, foi levantada a hipótese de que HCT-8 células se tornam metastático devido à sua exposição a micro-ambiente adequado. Vale a pena notar que estas experiências são realizadas com linhas celulares de cancro imortalizadas ou murino derivadas de células primárias, não com células cancerosas humanas primárias. Um estudo recente sugere que o stress de tracção aumentada celular pode ser usada como uma potencial assinatura biofísico de células metastáticas 13 .O estudo envolve a medição de força de tracção para diferentes linhas de células cancerosas humanas em géis de poliacrilamida. Verificou-se que as células de cancro metastático pode exercer esforço de tracção significativamente maior em comparação com as células não metastáticas em todos os casos 13. No entanto, estes resultados contradizem diretamente os achados anteriormente publicados sobre murino derivadas linhas celulares de cancro da mama 14. Além disso, um estudo recente destaca as diferenças notáveis ​​entre as células humanas imortalizadas e primários na sua remodelação do citoesqueleto proprofiling tein e sobrevivência celular a expressão da proteína 15. Por isso, é importante rever muitos dos ensaios de tracção para biofísicas, incluindo células de cancro humano primárias. Isto irá resolver a questão de saber se as células primárias recapitular imortalizada linhas celulares de cancro de tração tendência. O protocolo aqui descrito está optimizado para o isolamento de células primárias do cólon humanos (ambos saudáveis ​​e cancerosas), e para a sua cultura em substratos macios (hidrogeles de poliacrilamida), bem como em placas de Petri. O protocolo é baseado na digestão e consequente dissociação enzimática da amostra de tecido cirúrgico em suspensão única célula 16. Para nosso conhecimento, esta é a primeira demonstração de cultura isolado do tumor de cólon primário e células normais directamente sobre substratos de hidrogel macias com microesferas fluorescentes incorporadas para citometria de tracção. Substratos gel transparente também permitir a imunocoloração. Este ensaio revelou diferenças na organização F-actina eadesões focais em células de cólon humano primários como mudanças de rigidez do substrato. Esta plataforma de cultura de células abre-se a possibilidade de explorar diferentes propriedades biofísicas das células primárias humanas, tais como a rigidez de tracção e célula como parâmetros para o prognóstico do cancro.

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isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

As células cancerosas responder a matriz de rigidez mecânica de uma forma complexa utilizando um, hierárquica sistema coordenado mecano-química composta de receptores de adesão e as proteínas de membrana associadas a transdução de sinal, a arquitectura do citoesqueleto, motores moleculares e 1, 2. Mecanosensibilidade de células cancerosas in vitro são diferentes investigou principalmente com linhas de células imortalizadas ou murino derivadas de células primárias, não com células cancerosas humanas primárias. Assim, pouco é conhecido sobre o mecanosensibilidade de células de cólon humano primários in vitro. Aqui, um protocolo optimizado é desenvolvido que descreve o isolamento de células de cólon humano primários a partir de amostras de tecidos humanos saudáveis ​​cirúrgicos e cancerosos. Células de cólon isolados são então cultivadas com sucesso em mole (2 kPa rigidez) e rígida (10 kPa rigidez) hidrogeles de poliacrilamida e poliestireno rígida (~ 3,6 GPa rigidez) substratos funcionalizados por uma matriz extracelular (fibronectinaneste caso). Microesferas fluorescentes são incorporados em cápsulas gelatinosas perto da superfície da cultura de células e ensaio de tracção é realizado para avaliar tensões contrãcteis celulares usando software de acesso livre e gratuito. Além disso, a microscopia de imunofluorescência em diferentes substratos de rigidez proporciona informação útil sobre a morfologia da célula primária, organização do citoesqueleto e vinculina contendo adesões focais em função da rigidez do substrato.

O protocolo descrito acima é empregada com sucesso para várias amostras de tecido (n = 12) de quatro pacientes diferentes sob as diretrizes do conselho de revisão institucional. Figura 1A ilustra um tumor colorretal representante logo após a cirurgia a partir do qual são obtidos os cortes de tecido para culturas de células. Uma secção de tecido típico em solução de HBSS após a transferência para a capa laminar para processamento adicional é mostrado na Figura 1B. Um esquem...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento de células do cólon tumor primário Humanos a partir de tecidos cirúrgicos e cultivando-as Diretamente macias elásticas Substratos para Traction Citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K visualizações.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

Vídeo: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Isolamento rápido de células tumorais viáveis ​​circulantes de amostras de sangue de doentes

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bioengenharia

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometria de massa: Protocolo para Ajuste Diário e amostras de células em execução em um citômetro de Massa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Padronização celular em photolithographically Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Substratos

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Recuperação cirúrgica, isolamento e<em&gt; In vitro</em&gt; Expansão de humanos anteriores Cruciate células do ligamento derivados para aplicações em engenharia de tecidos

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caracterização biomecânica de tecidos moles Humanos Usando recuo e testes de tração

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Isolamento de células de gânglio retiniano murino primário (RGCs) por citometria de fluxo

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Medição da rigidez de substratos de Silicone macio para estudos de Mechanobiology, usando um microscópio de fluorescência Widefield

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.