Os autores gostariam de agradecer financiamento do Ministério da Educação ACRF Tier 1 (RG41) e start-up bolsa da Universidade Tecnológica de Nanyang. Baixo e Tjin são financiados pelo Bolsista de Investigação (RSS) a partir de Nanyang Technological University, Cingapura.

1. Clonagem de plasmídeos que codificam para Recombinante AECM Proteínas <ol><li> Desenhar a sequência de aminoácidos do domínio funcional (por exemplo, o domínio de ligação celular e repete-elastina semelhantes). Locais de restrição que flanqueiam as extremidades desenho dos domínios funcionais para facilitar a sub-clonagem utilizando software livre de acordo com as instruções de software (por exemplo, http://biologylabs.utah.edu/jorgensen/wayned/ape/). Aqui, seleccionar locais de restrição únicos que não estão presentes no domínio funcional para confinar a digestão para os locais pretendidos. Escolha locais de restrição que aparecem no local de clonagem múltipla (MCS) do vector hospedeiro (por exemplo, pET22b (+)) para a sub-clonagem. </li><li> Inverter traduzir a sequência de aminoácidos para a sequência de nucleótidos utilizando sítios livres de acordo com as instruções de software. (Por exemplo, http://www.bioinformatics.org/sms2/rev_trans.html). Garantir códons são otimizados para E. anfitriões coli. </li><li> Gene de interesse pode ser purchasoligonucleótidos de cadeia ed comercialmente como simples (isto é, se os oligonucleótidos &lt;100 pares de bases (pb)) e efectuar o recozimento de ADN (ver passo 1.4), para reduzir o custo. Caso contrário, para genes maiores que 100 pb, eles poderiam ser comprados por intermédio de empresas comerciais. </li><li> ADN recozimento dos oligonucleótidos <ol><li> Recozer os oligómeros de ADN para se obter a sequência de gene pretendida. Dissolve-se oligonucleótidos de ADN num tampão de oligómero de ADN (10 mM de Tris: 2-amino-2- (hidroximetil) -propan-1,3-diol, pH 8,0, filtrada) para uma concentração final de 1 ug / uL. </li><li> Adicionar 4 mL de cada oligómero com 32 ul de tampão de hibridação de ADN (Tris 10 mM, NaCl 100 mM e MgCl2 100 nm) para se obter uma mistura total de 40 ul. </li><li> Ferver num copo de água utilizando uma placa de aquecimento e imergir a mistura durante 5 min, 95 ° C. Retirar o copo e gradualmente resfriar todo o conjunto em uma caixa de isopor O / N. Os oligômeros foram recozido e está pronto para a digestão. </li></ol></li&gt; <li> Adicionar as enzimas de restrição correspondentes de digerir os oligómeros hibridados (ou seja, designado por inserção) e vector hospedeiro (ou seja, pET22b (+)) separadamente. Utilize a seguinte receita (1-2 ug de ADN, 2 ul de cada uma das enzimas de restrição, 5 uL de tampão de enzima de restrição 10x, e água até um total de mistura de 50 ul) durante 3-4 h a 37 ° C. NOTA: O pET22b (+) vector plasmídico é resistente aos antibióticos ampicilina e contem um 6x-His no C-terminus. A-Sua 6x tag presente em pET22b (+) nos permite usar anticorpos marcada com His para identificar a proteína alvo via transferência de western na etapa 7. </li><li> Adicionar corante 6x a cada mistura de digestão. Executar as misturas de digestão incluindo separadamente uma escada de ADN em géis de agarose a 1,2% contendo ADN de um corante de ADN fluorescente à luz ultravioleta durante 1 h a 100 V. visualizar o DNA em gel de agarose a 1,2% com um iluminador de luz UV. </li><li> Cortar o gel para extrair produtos de ADN digeridos, utilizando kits de purificação de gel comercial. Elute utilizando o volume mínimo para a coluna para atingir uma concentração mínima de ADN de 50-100 ng / mL. </li><li> Combinar o gene de interesse sequencialmente por ligação da inserção de ADN digerido (ou seja, repete elastina ou domínios de ligação de células obtidas por hibridação de ADN) no vector de plasmídeo usando ligase de T4 usando a seguinte receita: (2 jil de vector, 1 ul de ligase de T4, 1,5 ul de tampão de ligação de T4, x ul de inserção, 10,5 x ul de água para um total de mistura de 15 uL). Incubar a mistura de ligação à temperatura ambiente durante 2 h. NOTA: A concentração molar de vector para inserção deve ser variada para optimizar a eficiência da ligação. Volume de inserção descrita como x na receita depende da concentração de ADN eluído a partir do passo 1.7. </li><li> Thaw E. coli DH5a células quimicamente competentes (ou qualquer estirpe de clonagem) em gelo. Placas de agar 2xYT Quente (Tabela 1) contendo ampicilina (25 ug / ml) a 37 ° C. </li><li> Transformar as células usandochoque térmico: <ol><li> Alíquota de 50 ul de células competentes para, pré-refrigerada micro-tubos de centrífuga limpo. Pipetar 5 ul (entre 100 pg e 100 ng) da mistura de ligação para as células, pipetar suavemente para cima e para baixo para misturar. Deixar a mistura em gelo durante 20 min. </li><li> Imergir o tubo de micro-centrifugadora contendo a mistura de células num banho de água a 42 C ° durante 2 minutos e voltou para em gelo durante 2 min. Tempo a duração de imersão para minimizar os danos do calor para as células. </li><li> Adicionar 500 ul de meio SOC (Tabela 1) para dentro do tubo de micro-centrífuga e incuba-se a 37 ° C com agitação durante 1 h. </li><li> Espalhe 50 500 uL da mistura de célula / ligadura numa placa de agar 2xYT que foi pré-aquecida até à TA, contendo ampicilina (25 ug / ml) e incubar as placas de cabeça para baixo, a 37 ° CO / N (12-16 h) . </li></ol></li><li> No dia seguinte, escolha colônias de DNA da placa de agar utilizando pontas de pipeta limpas. Crescer as colônias em 5 ml de meio 2YT (Tabela1) contendo ampicilina (25 ug / ml) O / N a 37 ° CO / N (12-16 h) com agitação (225 rpm). </li><li> No dia seguinte, utilizar um kit de isolamento de plasmídeo para extrair os plasmídeos de ADN para cada colónia escolhido de acordo com o protocolo do fabricante. Elui-se o ADN com 50 ul de água. </li><li> Executar um teste de digest com enzimas de restrição usando a seguinte receita: (5 uL de ADN, 0,2 ul de cada uma das enzimas de restrição, 1 ul de tampão de enzima de restrição e 10x superior para cima de água para um total de mistura de 10 ul), incubar durante 2 horas a 37 ° C a tela de colônias que provavelmente contêm a inserção e correr em 1,2% em gel de agarose DNA como no passo 1.6. NOTA: Após a digestão, uma ligação bem sucedida deve resulta em duas bandas, uma para cada vector e inserção que correspondem ao respectivo peso molecular (Figura 1). </li><li> Envie as colônias prováveis ​​para a sequenciação de ADN utilizando o promotor T7 para a frente e primers para sequenciadores comerciais reverter. </li></ol><ol><li> A partir do resultado de sequenciação, seleccionar uma colónia que foi ligado com êxito e usar o plasmídeo de ADN para transformação em E. hospedeiro de expressão E. coli. </li><li> Thaw E. coli estirpe de expressão (células BL21 (DE3) pLysS) em gelo. Enquanto isso, as placas quentes de agar contendo ampicilina (25 ug / ml) e cloranfenicol (34? G / ml) à temperatura ambiente. NOTA: O plasmídeo pLysS presente em bactérias estirpe BL21 (DE3) pLysS contém um gene de resistência ao cloranfenicol. O plasmídeo pLysS contém um gene repressor de T7, que é expresso constitutivamente para limitar a expressão da proteína gotejante MECa. O cloranfenicol é necessário seleccionar as bactérias células que contém pLysS durante a cultura. </li><li> Repita o passo 1.10 para obter transformada E. coli que estão prontos para expressar as proteínas artificiais. Enrole placas de agar em Parafilm e armazenar de cabeça para baixo em 4 6; C, durante até um mês. </li></ol> 3. Expressão Bacteriana de AECM Proteínas <ol><li> Consultar a Figura 2, escolher uma colónia da placa de agar transformada com uma ponta de pipeta e inocular em 10 ml de Terrific Broth estéril (TB) meios (Tabela 1) contendo ampicilina e cloranfenicol antibióticos num tubo de ensaio. Incubar na cultura inicial a 37 ° CO / N (12-16 h) com agitação a 225 rpm. </li><li> Transferir 10 ml da cultura de arranque em 1 L de TB fresco meios estéreis e suplementadas com os mesmos antibióticos num balão de Erlenmeyer de 3 L. Incubar a cultura a 37 ° C com agitação a 225 rpm durante 2-3 h e observar a densidade óptica da cultura (OD 600) atingiu 0,6-0,8 pipetando 1 ml de cultura para uma cuvete vazio para leitura. Guardar a 1 ml de cultura antes da indução para a caracterização por SDS-PAGE. <ol><li> Para medir DO600 da cultura de células, preparar um frasco de meios TB numa cuvetepara uma medição em branco. Separadamente, transferir 1 mL de cultura do frasco de cultura em um novo cuvete vazio. Medir a densidade óptica da cultura utilizando um espectrofotómetro contra o branco de controlo a uma absorvância de 600 nm. </li><li> Guardar a amostra (para subsequente análise de SDS-PAGE), transferindo as amostras de 1 ml da cultura para um tubo de microcentrífuga de 1,5 ml, de centrifugação a 12.000 xg durante 2 minutos, e decantar o sobrenadante. </li></ol></li><li> Induzir a cultura com isopropilo β-D-1-tiogalactopiranósido (IPTG) para uma concentração final de 1 mM e incuba-se a 37 ° C com agitação a 225 rpm durante mais 4 h. Guardar a 1 ml da cultura no final da indução a 4 horas para a caracterização por SDS-PAGE. </li><li> Colher as células por transferência da cultura para frascos de centrífuga de 1 L e centrifugar a 12000 xg durante 30 min a 4 ° C. Descartar o sobrenadante, os sedimentos celulares pesar e ressuspender em tampão TEN (Tris 1 M, EDTA 0,01 M, NaCl 0,1 M, pH = 8,0) a 0,5 g / ml. </li></ol> 4. A lise das culturas bacterianas <ol><li> Congelar a cultura de células re-suspensas a -80 ° CO / N. Descongelar a cultura de células congelado num banho de água à temperatura ambiente ou em gelo para lisar as células. Adiciona-se 10 ug / ml de desoxirribonuclease I (ADNase I), 10 ug / ml de ribonuclease A (RNase I), e 50 ug / ml de fluoreto de fenilmetilsulfonilo (PMSF) durante a descongelação e homogeneizar a solução, com agitação lenta. </li><li> Depois de todas as células ressuspensas foram descongeladas, ajustar a solução para pH 9,0 para aumentar a solubilidade da proteína em água 12. Adicionar NaOH 6 N gota a gota com agitação sobre gelo para obter uma consistência homogénea. Lyse pela ruptura de ultra-sons durante 20 minutos em gelo usando um ponta plana de diâmetro 2 mm, 5 seg pulso. </li><li> Centrifugar a solução de células a 12.000 xg durante 30 min a 4 ° C. Transfira o sobrenadante para um ambiente limpo, garrafa vazia e armazenar a 4 ° C para posterior purificação. </li><li> Enquanto isso, ressuspender o sedimento de células com tampão TEN novamente e re-congelamento a -80 ° C. Paralise celular completo, repita congelamento / descongelamento e de ultra-sons processo até três vezes. Economize 20 ligado celular ul para caracterização SDS-PAGE. </li></ol> 5. Purificação de Proteínas AECM Usando inversa Transição Bicicleta <ol><li> Agrupar o ligado celular a partir do passo 4.3 e prossiga para purificar as proteínas AECM usando ITC, semelhante ao utilizado para a purificação de proteínas à base de elastina 21. Ciclos vai ser feito com as diferentes temperaturas que são 4 ° C (referido como &quot;fria&quot;) e 37 ° C (referido como &quot;quentes&quot;) ciclos, respectivamente. </li><li> Dividir o lisado celular em 50 ml de frascos de centrifugação, e centrifugar a 40000 xg durante 2 horas a 4 ° C. A aparência do sedimento celular deve ser marrom escuro e procurar a pingar. </li><li> Remover o sobrenadante por pipetagem para obter uma separação limpa a partir do sedimento. Recolhe-se o sobrenadante em frascos de centrífuga limpo e adicionar NaCl para uma concentração final de 1 M (até um máximo de 3 M). Quentea solução para 37 ° C durante 2 horas com agitação a 225 rpm. NOTA: A adição de NaCl irá disparar a transição do componente de elastina da MECa causando agregação. O sobrenadante ficará turva. Se a concentração de proteína é suficientemente elevada, a proteína espumoso branco pode ser visto com os lados da garrafa centrífuga. </li><li> Centrifugar o sobrenadante do passo de 5,3 a 40000 xg durante 2 horas a 37 ° C. Decantar o sobrenadante. Triturar o sedimento utilizando uma espátula de metal e ressuspender o pellet em pedaços autoclavado água destilada gelada (50 mg / mL) com agitação vigorosa O / N a 4 ° C utilizando uma barra de agitação magnética e placa. O sedimento deve ser completamente dissolvido. </li><li> Repita os passos 5,2-5,4 por mais três a cinco vezes para se obter um grau de pureza mais elevada da proteína. </li><li> Após o ciclo final de purificação, dessalinizar a solução de proteína por diálise contra água destilada a 4 ° C. Dialisar solução de proteína contra a água durante 2-3 dias com as mudanças na água a cada 4hr ou 8 horas para O / N. Guardar a 20 ul da proteína purificada por SDS-PAGE e liofilizar o resto da proteína purificada e armazenar a -80 ° C até à sua utilização. </li></ol> 6. Caracterização de Proteínas AECM utilizando SDS-PAGE A electroforese <ol><li> Preparar um gel de 12% SDS-PAGE (se o peso molecular é grande, utilizam 8% de gel SDS-PAGE para uma melhor separação). Adicionar tampão de carga de SDS 2x a cada amostra, aquecer as amostras durante 10 min a 100 ° C e processar as amostras no gel durante 1 hora a 100 V ou até que a escada proteína correspondendo ao peso molecular da proteína MECa atinge o meio da gel. </li><li> Recuperar o gel da SDS-PAGE configurar e adicionar Coomassie Brilliant mancha azul (Tabela 2), com um volume de submergir o gel durante 1 hora em uma placa de Petri. Enxaguar em gel de uma solução de descoloração (Tabela 2) durante 5 min num agitador rotativo. Mude a solução de descoloração, e continuar a destain o gel com balanço até que o fundo de tele gel torna-se clara. As bandas de proteína deve ser claramente visível. </li><li> Comparar a posição da proteína alvo contra a escada proteína para determinar o peso molecular da proteína alvo. </li><li> Para confirmar ainda mais a presença da proteína alvo, efectuar transferência de western como no passo 7. </li></ol> 7. Caracterização de proteínas utilizando AECM Western Blotting <ol><li> Executar as amostras sobre um gel de 12% SDS-PAGE como na secção 6 com nenhuma coloração. </li><li> Cortar membrana de nitrocelulose para um tamanho ligeiramente maior do que o gel de SDS-PAGE. Cortar quatro peças de papel de filtro para o mesmo tamanho. </li><li> Wet um pedaço de papel de filtro com tampão de transferência Ocidental (20% v / v de metanol, Tris 25 mM, Glicina 190 mM, pH 8,3), colocar o gel de SDS-PAGE no topo do papel de filtro, seguida de uma outra peça de papel de filtro , em seguida, a membrana de nitrocelulose e um final de outros dois pedaços de papel de filtro. Certifique-se de todo o set-up está submersa com tampão de transferência western suficiente. NOTA: O gel e a membrana não deverá estar em contacto neste ponto de tempo como se podia ligar a proteínas da membrana quando em contacto. </li><li> Transferir as proteínas para a membrana de nitrocelulose colocando dois filtros de papel para uma unidade de transferência semi-seca ocidental, seguindo-se a membrana de nitrocelulose, e colocar cuidadosamente o gel de SDS-PAGE dentro e sobre a membrana, por fim, colocar os dois últimos papel de filtro molhado sobre o gel de SDS-PAGE. Executar a transferência ocidental a 45 mA, 30 min. Adicionar tampão se a tensão é maior do que 30 V. NOTA: De um modo preferido colocar o gel de SDS-PAGE sobre a membrana com uma tentativa e evitar a transferência do gel desnecessariamente na membrana como proteínas poderiam ligar-se à membrana quando em contacto. </li><li> Recuperar e bloquear a membrana de nitrocelulose com solução de bloqueio (5% de leite desnatado em PBS pH 7,4, filtrada com papel de filtro) durante 2 h à TA. Descarte de tampão de bloqueio e adicionar PBS para lavar. NOTA: Mudança para novas luvas para evitar a contaminação da membrana com prote indesejadoins. </li><li> Incubar anti His-anticorpo primário com diluição em PBS a 1: 1000 em TA durante 1 h com agitação. Prepara-se uma mistura em um volume que pode submergir toda a membrana, por exemplo, com a razão de diluição 1: 1000, adicionar 1 ml de anticorpo (concentração: 1 mg / ml) a 999 ul de PBS para uma mistura total de 1 ml. </li><li> Lavar a membrana com 5 mL de PBST (PBS com 0,1% de Tween 20). </li><li> Incubar com anticorpo secundário conjugado com peroxidase de rábano (HRP) com diluição em PBS a 1: 5000 em TA durante 1 h com agitação. Prepara-se uma mistura em um volume que pode submergir toda a membrana, por exemplo, com a razão de diluição 1: 5000, adicionar 1 ml de anticorpo (concentração: 1 mg / ml) para 4999 uL de PBS para uma mistura total de 5 ml. </li><li> Lava-se a membrana com 5 mL de PBST e repetir duas vezes. </li><li> Aplicar o substrato quimioluminescente para a membrana com um volume para cobrir a membrana e incubar, seguindo as instruções para o substrato utilizado. NOTA: A sensibilidade da substrato para detecção de proteína pode variar, recomendamos um substrato com sensibilidade máxima para sinais muito baixos, no caso o aumento da concentração de anticorpos não aumenta sinais. </li><li> Capturar os sinais quimioluminescentes que utilizam um gerador de imagens CCD com base. Ajuste a razão de diluição de anticorpo primário e secundário, se os sinais são fracos e não-específica. Incubar mais em solução de bloqueio se o sinal de fundo é elevado. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Engenharia de proteína recombinante é uma técnica versátil para criar novos materiais de proteína usando uma abordagem bottom-up. Os materiais à base de proteínas podem ser concebidos para ter várias funcionalidades, adaptado de acordo com a aplicação de interesse. Devido ao aumento do avanço nas tecnologias de clonagem e de expressão de proteínas, tornou-se relativamente simples (e de baixo custo) para criar uma variedade de proteínas artificiais de uma maneira reprodutível e escalonável. O domínio sem...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Na concepção de proteínas de fusão que contêm repetições de elastina semelhante, é importante para manter um teor de elastina geral, bastante grande fracção da proteína de fusão 18. Isto é para assegurar que a construção de proteína de fusão retém as suas características elásticas do tipo, a fim de utilizar CIT para purificação. A concepção e as sequências descritas nesta secção foram especificamente proteínas MECa retirado do trabalho por Tjin et ai. 14. Neste ...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Design e Construção de Matriz Extracelular Artificial (MECa) Proteínas de<em&gt; Escherichia coli</em&gt; Para engenharia de tecidos da pele

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K visualizações.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Vídeo: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Bioengenharia

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Engenharia de Tecidos: Construção de um multicelular 3D andaime para a entrega de camadas celulares Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Repovoamento celular epitelial e Preparação de Rodent Matriz Extracelular Andaimes para o desenvolvimento do tecido renal

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparação de Matriz Extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabricação de espumas de Matriz Extracelular derivados e Microcarregadores como cultura celular de tecidos específicos e plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...