Este trabalho foi financiado pelo programa KAMIN do Ministério do Comércio e Trabalho de Israel e pela Qrons Inc., 777 Brickell Avenue Miami, FL 33131, EUA.

O uso de animais neste protocolo foi aprovado pelo Comitê Nacional de Cuidados e Uso de Animais.
NOTA: Esqueletos de corais carbonatados com cálcio devem ser usados na forma cristalina de aragonita. Os tipos de corais testados até agora para culturas neurais são Porites Lutea, Stylophora Pistillata e Trachyphyllia Geoffroyi. Os esqueletos podem ser comprados inteiros ou terrestres.
1. Limpeza das peças do esqueleto do coral
CUIDADO: As seguintes etapas devem ser realizadas em uma coifa química à temperatura ambiente, pois as soluções descritas abaixo são perigosas e podem causar queimaduras e irritações.
<ol><li>Use um martelo para quebrar o esqueleto do coral e dividi-lo em fragmentos de 0,5 a 2 cm. Para dissolver resíduos orgânicos e não orgânicos, mergulhe os fragmentos do esqueleto do coral em solução de hipoclorito de sódio a 10% por 10 minutos e, em seguida, lave uma vez com água bidestilada (DDW).</li>
	<li>Para remover os resíduos orgânicos restantes, mergulhe os fragmentos em uma solução de NaOH 1M por 5 minutos e, em seguida, lave uma vez com DDW. Continue com a remoção dos depósitos orgânicos mergulhando os fragmentos em solução de 30% H 2 O2por 10 min, em seguida, lave os fragmentos 3x com DDW.</li>
	<li>Remova o máximo de DDW em excesso possível e deixe os fragmentos de coral no capô secar (1 a 8 h).</li>
</ol>2. Limpeza das tampas de vidro
<ol><li>Transfira as lamínulas de vidro para uma placa de Petri de vidro de 100 mm. Adicionar 10 mL de etanol 95% por 15 min.</li>
	<li>Retire o etanol e lave com DDW 3x, aguardando 10 min entre cada lavagem. Coloque o prato numa placa de aquecimento pré-aquecida a 80 °C até evaporar o DDW. Mexa suavemente as tampas dentro do prato várias vezes durante a secagem para evitar que grudem umas nas outras.</li>
	<li>Autoclave as lamínulas.</li>
</ol>3. Preparação de grãos de esqueleto de coral
<ol><li>Triture os fragmentos do esqueleto do coral usando uma argamassa e pilão (moagem manual) até a completa quebra. O resultado é uma mistura de grãos com tamanhos que variam de 20 μm a 200 μm.</li>
	<li>Alternativamente, triture os fragmentos do esqueleto do coral usando uma máquina de moagem elétrica a uma velocidade de 1.000 rpm por 30 s (comprimento da lâmina = 6 cm; largura = 0,5 cm – 1,0 cm). O tamanho de grão resultante é semelhante à faixa de tamanho produzida pela moagem manual.</li>
</ol>4. Purificação de grãos de uma faixa de tamanho específica
NOTA: Se o controle sobre o tamanho dos grãos em uma matriz for desejado, use o seguinte procedimento de purificação de grãos baseado em filtração.
<ol><li>Transfira os grãos para um filtro de malha filtrante manual ou elétrico de 40 μm.</li>
	<li>Divida os grãos em duas faixas específicas, peneirando os grãos através do filtro (se estiver usando um filtro elétrico, as seguintes condições são recomendadas: agitação = 600 amplitudes/min, salto = 6/s). Este procedimento produz dois grupos de tamanhos de grãos, um <40 μm e o segundo >40 μm. NOTA: Os dois tamanhos podem ser determinados usando filtros de malhas variadas. Se um intervalo mais restrito for desejado, então peneire novamente cada grupo da etapa 4.2 através de filtros com malhas diferentes.</li>
	<li>Autoclave os grãos.</li>
</ol>5. Preparação de pratos ou lamínulas revestidas com grãos de coral
<ol><li>Para revestimento de frascos, placas ou placas de Petri NOTA: As etapas a seguir devem ser executadas em condições estéreis.<ol><li>Adicionar os grãos do esqueleto do coral em uma solução de poli-D-lisina (PDL) de 20 μg/mL dissolvida em solução de Hanks. A concentração recomendada é de 5 mg/10 mg de grãos por 1 mL de solução de PDL.</li>
		<li>Deite a solução em frascos e pratos (aproximadamente 2 ml/25 cm2) e incube durante a noite a 4 °C. Os grãos afundam e se prendem ao fundo.</li>
		<li>No dia seguinte, lave os frascos e pratos uma vez com DDW estéril. Deixe os frascos e pratos secarem no capô. NOTA: É preferível usar frascos e pratos recém-revestidos. Os frascos e pratos revestidos podem ser utilizados até uma semana após o revestimento, se conservados a 4 °C. No entanto, a eficácia da matriz pode estar comprometida.</li>
	</ol></li>
	<li>Revestimento de lamínulas de vidro (12 mm de diâmetro, 0,17 mm de espessura)<ol><li>Adicione grãos de esqueleto de coral ao DDW em qualquer concentração desejada. As densidades comuns usadas para células neurais são 5 mg/mL e 10 mg/mL. Despeje 40 μL da solução de grão sobre o centro da lamínula (Figura 4).</li>
		<li>Coloque as lamínulas numa placa de aquecimento pré-aquecida a 80 °C e aguarde a evaporação completa (normalmente 15 min). Nessas condições, os grãos aderem à lamínula. Autoclave as lamínulas revestidas. Armazenar em condições estéreis.</li>
		<li>Um dia antes da cultura, coloque as tampas na tampa de uma placa estéril de 24 poços. Adicionar 100 μL de 20 μg/ml de solução de PDL a cada lamínula. Use a ponta para garantir que o líquido cubra toda a região do grão e o restante da superfície da tampa.</li>
		<li>Cubra a tampa com o fundo da placa. Embrulhe as laterais das placas com filme de parafina. Incubar durante a noite a 4 °C.</li>
		<li>No dia seguinte, lave uma vez com DDW e seque no capô. NOTA: É preferível usar lamínulas recém-revestidas.</li>
	</ol></li>
</ol>6. Cultivo de células dissociadas do hipocampo em lamínulas de vidro revestidas com grãos de esqueleto de coral
OBS: O método para cultura de células dissociadas do hipocampo foi modificado a partir de procedimentos previamentepublicados24,27. O preparo da cultura é descrito para quatro filhotes de ratos. O rendimento esperado de cada hipocampo é de 1–1,5 x 106 células.
<ol><li>Configuração<ol><li>Prepare uma placa de Petri de 60 mm vazia. Despeje 2 mL de meio essencial mínimo (MEM) em uma placa de Petri de 60 mm e coloque gelo.</li>
		<li>Despeje 2 mL de MEM em uma placa de 35 mm e coloque-o em uma incubadora a 37 °C, 5–10% CO2. Deite 2 ml de MEM num tubo de 15 ml e mantenha-o a 4 °C.</li>
		<li>Despeje 1 mL de meio de primeiro dia em um tubo de 15 mL e coloque-o em uma incubadora a 37 °C, 5-10% CO2. NOTA: A composição do Meio do Primeiro Dia está descrita na Tabela de Materiais.</li>
		<li>Descongelar 200 μL de uma solução de tripsina a 2,5% e incubar a 37 °C.</li>
		<li>Prepare três pipetas de vidro Pasteur com cabeças de 0,5 mm, 0,75 mm e 1,0 mm de diâmetro usando uma chama.</li>
		<li>Prepare ferramentas cirúrgicas adequadas: uma tesoura grande, duas tesouras pequenas, um tweezer grande, quatro pinças pequenas e um bisturi.</li>
		<li>Prepare um estereomicroscópio no capô.</li>
	</ol></li>
	<li>Usando uma tesoura grande, sacrifique filhotes de ratos Sprague Dawley de 0 a 3 dias separando suas cabeças de seus corpos em uma placa de Petri vazia de 60 mm (passo 6.1.1). Descarte os corpos.</li>
	<li>Pegue a cabeça segurando-a da boca com um grande tweezer. Corte a pele que cobre o crânio com uma pequena tesoura.</li>
	<li>Com uma tesoura limpa, entre por baixo do crânio (do lado do cerebelo) e corte o crânio à direita e à esquerda para permitir sua remoção. Usando um pequeno tweezer, descasque o crânio do cérebro.</li>
	<li>Com um tweezer limpo, separe o cérebro da cabeça e coloque-o numa placa de Petri de 60 mm contendo 2 ml de MEM frio (ver passo 6.1.2).</li>
	<li>Usando duas pinças pequenas, disseque os hipocampos sob um estereomicroscópio. Transferir os hipocampos para o prato preparado de 35 mm (a partir do passo 6.1.2) contendo MEM. NOTA: As etapas 6.3–6.6 devem ser repetidas para cada filhote.</li>
	<li>Cortar os hipocampos em fatias de aproximadamente 1 mm com o bisturi. Adicionar 200 μL da solução de tripsina (diluída até uma concentração final de 0,25%). Misture delicadamente e incube a 37 °C, 5–10% CO2 durante 30 min.</li>
	<li>Após a incubação, adicionar 2 ml de MEM frio (passo 6.1.2) à placa para desativar a tripsina. Transferir o tecido tripsinizado para um tubo de 15 ml contendo 1 ml de meio do primeiro dia pré-aquecido a 37 °C (passo 6.1.3) utilizando a pipeta de vidro Pasteur de maior diâmetro (passo 6.1.5). NOTA: A melhor relação meio/tecido (v:v) para processamento posterior do tecido é de 1 mL/8 hipocampos. Evite ao máximo transferir o meio com o tecido.</li>
	<li>Triture o tecido passando-o através da pipeta de vidro de maior diâmetro 10 a 15 vezes. Em seguida, repita esse processo usando a pipeta de vidro com o diâmetro médio. Continue triturando com a pipeta de menor diâmetro. Evite borbulhar para reduzir a morte celular.</li>
	<li>Deixe os pedaços de tecido restantes afundarem por 2 a 5 minutos e, em seguida, transfira o sobrenadante para outro tubo de 15 mL. Conte as células usando um hemocitômetro. NOTA: A densidade celular preferencial é de 200.000 a 400.000 células/mL. Use o meio do primeiro dia para diluir ou centrifugação a 470 x g para concentrar as células.</li>
	<li>Semear 100 μL de células em cada lamínula de vidro. Durante a semeadura, certifique-se de cobrir toda a tampa com células. Incubar a 37 °C, 5–10% CO2.</li>
	<li>No dia seguinte, adicione 500 μL de Meio de Crescimento Neuronal aos poços. NOTA: A composição do meio de crescimento neuronal está descrita na Tabela de Materiais.</li>
	<li>Transfira suavemente cada lamínula para o poço apropriado usando uma pinça enquanto remove o meio do primeiro dia, inclinando a lamínula. Sucção do meio restante da tampa.</li>
	<li>Incubar a 37 °C, 5–10% CO2. Evite substituir o meio durante toda a incubação. As culturas podem ser mantidas nessas condições até 1 mês. A maior preocupação é a umidade. Portanto, certifique-se de manter a incubadora ao máximo umidificada.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+Marine-Derived+Porous+Scaffolds+for+Bone+Tissue+Engineering.">Synthetic and Marine-Derived Porous Scaffolds for Bone Tissue Engineering.</a> Materials. 11 (9), (2018).</li><li>Ahmad Khalili, A., Ahmad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Cell+Adhesion+Studies+for+Biomedical+and+Biological+Applications.">A Review of Cell Adhesion Studies for Biomedical and Biological Applications.</a> International Journal of Molecular Sciences. 16 (8), 18149-18184 (2015).</li><li>. Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19218486">https://www.ncbi.nlm.nih.gov/pubmed/19218486</a> (2019)</li><li>Drake, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+skeletal+organic+matrix+from+the+stony+coral+Stylophora+pistillata.">Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (10), 3788-3793 (2013).</li><li>Ramos-Silva, P., 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=The+skeletal+proteome+of+the+coral+Acropora+millepora:+the+evolution+of+calcification+by+co-option+and+domain+shuffling.">The skeletal proteome of the coral Acropora millepora: the evolution of calcification by co-option and domain shuffling.</a> Molecular Biology and Evolution. 30 (9), 2099-2112 (2013).</li><li>Peretz, H., Blinder, P., Baranes, D., Vago, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+matrix+as+an+instructive+microenvironment+for+neural+development.">Aragonite crystalline matrix as an instructive microenvironment for neural development.</a> Journal of Tissue Engineering and Regenerative Medicine. 2 (8), 463-471 (2008).</li><li>Morad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CaCO3 Matrix Dictates Astrocytes Transition to Astrogliosis. , (2019).</li><li>Prada, F., 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=Ocean+warming+and+acidification+synergistically+increase+coral+mortality.">Ocean warming and acidification synergistically increase coral mortality.</a> Scientific Reports. 7, (2017).</li></ol>

Os autores declaram não ter interesses financeiros concorrentes.

A técnica aqui apresentada descreve uma maneira de melhorar a manutenção e funcionalidade de células neurais em cultura. Isto é conseguido através da adesão das células a uma matriz feita de grãos de esqueleto de coral que nutre as células e promove o seu crescimento e atividade. O uso dessa técnica aumenta a capacidade do modelo de cultura neural de imitar o ambiente das células no cérebro.
A introdução da matriz como substrato de cultura apresenta diversas vantagens sobre outr...

Culturas de células neurais dissociadas, neste caso células hipocampais, são um valioso modelo experimental para o estudo do crescimento e função neural, proporcionando alto isolamento celular e acessibilidade 1,2,3. Esse tipo de cultura é frequentemente utilizado em neurociências, desenvolvimento de fármacos e engenharia de tecidos devido à grande quantidade de informações que podem ser coletadas, tais como taxas de crescimento e viabilidade, neurotoxicidade, crescimento e rede de neuritos, conectividade e plasticidade sináptica, modificações morfológicas, organização e fiação de neuritos, etc.1,4,5,6,7.
Apesar da importância das culturas, as células cultivadas são geralmente forçadas a crescer sobre lamínulas de vidro em uma monocamada bidimensional. Essas modificações ambientais estritas diminuem significativamente a capacidade de sobrevivência das células neurais ao longo do tempo, pois as lamínulas de vidro são substratos não nutritivos com baixa força de adesão, exibindo menor capacidade de suportar o crescimento celular 8,9,10,11.
Como as células neurais cultivadas são forçadas a crescer em condições desafiadoras, uma abordagem essencial para aumentar sua sobrevivência seria imitar ao máximo seu ambiente natural12,13. Isso poderia ser obtido com o uso de biomateriais que atuarão como matrizes e mimetizarão a matriz extracelular das células, permitindo que elas formem uma estrutura semelhante a um tecido e auxiliem na sua nutrição14.
O uso de biomateriais é uma abordagem promissora no aprimoramento de culturas celulares, pois atuam como arcabouços biocompatíveis, proporcionando estabilidade mecânica e melhorando uma variedade de propriedades celulares, incluindo adesão, sobrevivência, proliferação, migração, morfogênese e diferenciação15,16,17. Vários tipos de biomateriais são utilizados para melhorar as condições das células in vitro. Entre eles estão os biopolímeros, ou componentes biológicos que geralmente fazem parte da matriz extracelular das células. Esses biomateriais são utilizados principalmente como forma de agentes polimerizados de revestimento ou hidrogéis18,19,20. Por um lado, as matrizes citadas acima conferem às células um ambiente 3D familiar para crescer, estimulam sua adesão ao prato e lhes dão suporte mecânico21,22. Por outro lado, sua forma polimerizada e o confinamento das células dentro de hidrogéis perturbam o acesso das células aos componentes nutritivos presentes nos meios de crescimento e também dificultam o acompanhamento das células por métodos microscópicos23.
Exoesqueletos de corais são matrizes biológicas de origem marinha. Eles são feitos de carbonato de cálcio, têm estabilidade mecânica e são biodegradáveis. Estudos prévios utilizando o esqueleto de coral como matriz para o crescimento de células neurais em cultura mostraram adesão muito maior, em comparação com lamínulas de vidro24,25. Além disso, células neurais cultivadas no esqueleto do coral demonstraram sua capacidade de ingerir o cálcio do esqueleto composto, o que protege as células neurais em condições de privação de nutrientes26. Além disso, o esqueleto do coral é uma matriz de suporte e nutrição que aumenta a sobrevivência das células neurais, estimula a formação de redes neurais, eleva a taxa de conectividade sináptica e possibilita a formação de estruturas semelhantes a tecidos27,28. Estudos recentes também têm demonstrado que a topografia superficial da matriz do esqueleto coral desempenha um papel crucial na distribuição e ativação das célulasgliais8,29. Além disso, o esqueleto de coral é eficaz como matriz para o cultivo de outros tipos celulares, como osteócitos30,31, hepatócitos e cardiomiócitos em cultura (dados não publicados).
Assim, o esqueleto de corais é uma matriz promissora para o cultivo de células in vitro. Assim, o protocolo detalhado a seguir descreve a técnica de cultivo de células neurais no esqueleto de corais para a produção de culturas neurais mais estáveis e prósperas do que aquelas alcançadas pelos métodos existentes. Esse protocolo também pode ser útil para o cultivo de cardiomiócitos, hepatócitos e outros tipos celulares.

<table>
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Greiner</td>
			<td>#60-662160</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>#17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>#A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>D &#8211; glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Minimal Essential Eagle (DMEM)</td>
			<td>Sigma</td>
			<td>#D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical sieve</td>
			<td>Ari Levy</td>
			<td>#3700</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serun (FBS)</td>
			<td>Biological Industries</td>
			<td>#04-007-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>First Day Medium</td>
			<td></td>
			<td></td>
			<td>85.1% Minimum Essential Eagle&#8217;s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.</td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td>Greiner</td>
			<td>#60-690160</td>
			<td>25cm^2, Tissue culture treated</td>
		</tr>
		<tr>
			<td>Fluoro-deoxy-uridine</td>
			<td>Sigma</td>
			<td>#F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Menzel-Glaser</td>
			<td>#BNCB00120RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Romical</td>
			<td>#007130-72-19</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Sigma</td>
			<td>#N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>HANK&#39;S solution</td>
			<td>Sigma</td>
			<td>#H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma</td>
			<td>#K3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L - glutamine</td>
			<td>Sigma</td>
			<td>#G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual strainer (40&#181;m)</td>
			<td>VWR</td>
			<td>#10199-654</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimun Essential Eagle (MEM)</td>
			<td>Sigma</td>
			<td>#M2279</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>De-Groot</td>
			<td>4-P090</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO (Sodium Hypochlorite)</td>
			<td>Sigma</td>
			<td>#425044</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>#S8045</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Neuronal Growth Medium</td>
			<td></td>
			<td></td>
			<td>45% MEM, 40% Dulbecco&#39;s modified eagle&#39;s medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>#60-628160, #60-627160</td>
			<td>60mm, 35mm, respectively.</td>
		</tr>
		<tr>
			<td>Poly D &#8211; Lysine</td>
			<td>Sigma</td>
			<td>#P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart Dentin Grinder</td>
			<td>KometaBio</td>
			<td>#GR101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>#15-090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>#U3750</td>
			<td></td>
		</tr>
	</tbody>
</table>

increasing durability of dissociated neural cell cultures using biologically active coralline matrix

Culturas de células neuronais e gliais do hipocampo dissociadas são um modelo experimental valioso para estudar o crescimento e a função neural, proporcionando alto isolamento celular e um ambiente controlado. No entanto, a sobrevivência das células do hipocampo in vitro está comprometida: a maioria das células morre durante a primeira semana de cultivo. Portanto, é de grande importância identificar maneiras de aumentar a durabilidade das células neurais em cultura.
O carbonato de cálcio na forma de aragonita cristalina derivada do esqueleto de corais pode ser usado como uma matriz ativa superior para culturas neurais. Ao nutrir, proteger e ativar as células gliais, o esqueleto do coral aumenta a sobrevivência e o crescimento dessas células in vitro melhor do que outras matrizes.
Este protocolo descreve um método para cultivar células hipocampais em uma matriz coralina. Essa matriz é gerada pela fixação de grãos de esqueletos de corais em pratos de cultura, frascos e lamínulas de vidro. Os grãos ajudam a melhorar o ambiente das células, introduzindo-as em um ambiente tridimensional fino (3D) para crescer e formar estruturas semelhantes a tecidos. O ambiente 3D introduzido pelo esqueleto do coral pode ser otimizado para as células por moagem, o que permite o controle sobre o tamanho e a densidade dos grãos (ou seja, a rugosidade da matriz), uma propriedade que foi encontrada para influenciar a atividade das células gliais. Além disso, o uso de grãos facilita a observação e análise das culturas, principalmente quando se utiliza microscopia de luz. Assim, o protocolo inclui procedimentos para geração e otimização da matriz coralina como ferramenta para melhorar a manutenção e funcionalidade das células neurais in vitro.

Para preparar a matriz do esqueleto do coral, todo o esqueleto do coral (Figura 1A) foi quebrado em fragmentos de 0,5 a 2 cm com martelo (Figura 1B) e completamente limpo dos resíduos orgânicos em três etapas (etapa 1 do protocolo) com solução de hipoclorito a 10%, solução de NaOH 1M e solução de H 2 O2 a 30% (Figura 1C). Os fragmentos de corais foram bem limpos quando a cor do esqueleto mudou de marro...

Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

A cultura de células dissociadas do hipocampo é uma ferramenta experimental fundamental em neurociência. A sobrevivência e a função das células neurais em cultura são aumentadas quando esqueletos coralinos são usados como matrizes, devido ao seu papel neuroprotetor e neuromodulador. Assim, as células neurais cultivadas em matriz coralina apresentam maior durabilidade e, portanto, são mais adequadas para o cultivo.

Aumentando a Durabilidade de Culturas de Células Neurais Dissociadas Usando Matriz Coralina Biologicamente Ativa

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

increasing-durability-dissociated-neural-cell-cultures-using

Research

JoVE Journal

Bioengineering

1.2K visualizações.  Ariel University. A cultura de células dissociadas do hipocampo é uma ferramenta experimental fundamental em neurociência. A sobrevivência e a função das células neurais em cultura são aumentadas quando esqueletos coralinos são usados como matrizes, devido ao seu papel neuroprotetor e neuromodulador. Assim, as células neurais cultivadas em matriz coralina apresentam maior durabilidade e, portanto, são mais adequadas para o cultivo.

Vídeo: Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengenharia

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Forma Polímeros Memória da Cultura célula ativa

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Aumentar os rendimentos cDNA Single-cell quantidades de mRNA em Laboratório Padrão Reações transcriptase reversa usando Microstreaming Acústico

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

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, ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Choque aplicação de ondas de culturas de células

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Melhorado o hidrogel 3D culturas de células gliais primárias para In Vitro modelagem de Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Uma nova técnica de sutura tenorrhaphy com enxerto de colágeno projetado por tecido para reparar defeitos grandes do tendão

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

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 ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Produção de fibras da matriz extracelular através de cultura de células de membrana de fibra oca Sacrificial

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...