Financiado en parte por subvenciones de NIH RO1AI015503. Ratones infectados de schistosome fueron proporcionados por del NIAID esquistosomiasis Resource Center en el Instituto de investigaciones biomédicas (Rockville, MD) a través de NIH NIAID contrato HHSN272201000005I para la distribución a través de recursos del BEI.

Todos los procedimientos experimentales y cuidado de los animales fueron aprobados por el Comité de uso de la Universidad de Wisconsin-Madison bajo protocolo y atención institucional del Animal no. V005717. El siguiente protocolo consiste en trabajar en un centro de bioseguridad nivel 2 (BSL2) para patógenos humanos, aunque ninguna de las etapas de esquistosoma, representadas en este protocolo son infecciosas para los seres humanos o de otros mamíferos. Seguir las políticas institucionales para el manejo de grupo de riesgo 2 (RG2) patógenos humanos.
Nota: Utilizamos mujer ratones Swiss-Webster (6 - semanas de edad) debido a su mayor susceptibilidad a la infección10, produciendo así mayores cargas de huevo. Tucker et al. , describen los protocolos de infección del ratón y caracol utilizados en este modelo de sistema11. La Tabla de materiales enumera todos los equipos específicos, materiales, reactivos y fuentes animales para llevar a cabo este protocolo experimental.
1. proceso de hígados infectados
<ol><li>Eutanasia a ratones, post-infección 6-7 semanas, por asfixia de CO2 (tasa de 10-30% del volumen de la cámara por minuto). Ratones de monitor para el cese de la respiración y del latido del corazón. Nota: Por lo general, a 20 ratones pueden procesarse en un momento dado.</li>
	<li>Después de la transferencia sacrificaron ratones a un gabinete de bioseguridad, poner ratones en sus espaldas y rocíe las superficies ventrales con etanol al 70%. Remojar durante 1 minuto.</li>
	<li>Apriete y tire hacia arriba la piel del abdomen inferior y en toda la base de la piel que mantiene pellizcada más cercana al abdomen con tijeras quirúrgicas estériles. Tire la piel corte anterior (es decir, hacia la cabeza) para revelar el hígado. Tenga en cuenta que el hígado agrandado y manchado debido a una respuesta granulomatosa inducida por huevo (figura 2A).</li>
	<li>Retirar el hígado y colocarlo en un vaso que contiene 200 mL de estéril solución de NaCl del 1,2% que contiene pluma/strep.</li>
	<li>Repita los pasos 1.3 y 1.4 con los ratones restantes. Nota: Veinte ratones generalmente se procesan en una sola cosecha.</li>
	<li>Coloque los hígados en una placa Petri estéril y retirar tejidos de hígado sin grasa/conectivos con pinzas estériles.</li>
	<li>Transferencia ajustado/limpiar hígado a una botella de centrífuga estériles de 250 mL que contiene ~ 200 mL de solución estéril de NaCl de 1.2% con pluma/strep.</li>
	<li>Enérgicamente sacudir la botella que contiene el hígado y, con una pipeta desechable estéril, retire la superficie excesiva espuma/flotación ruina. Poco a poco retirar la solución salina restante en un recipiente o fregadero que contiene 1% cloro (desinfectante).</li>
	<li>Añadir ~ 200 mL de solución salina fresca a los hígados y repita el paso 1.8 tres veces más.</li>
</ol>2. hígado extracción y miracidial aislamiento
<ol><li>Coloque los hígados en una taza pequeña batidora de acero inoxidable estéril y añadir 2 mL de solución de NaCl al 1.2% (figura 2B).</li>
	<li>Cubra la taza con papel de aluminio y una placa de Petri para evitar derrames y mezclar durante 1 minuto alternando baja y alta velocidad (20 s baja velocidad/10 s velocidad; repetición) (figura 2).</li>
	<li>Distribuir la suspensión de hígado mezclada en botellas de centrífuga estériles de 250 mL (para 20 hígados usan 4 botellas).</li>
	<li>Añadir solución salina estéril con antibióticos a ~ 200 mL botella de volumen total. Pesa/balanza botellas antes de centrifugar.</li>
	<li>Centrifugar el hígado mezclado a 290 x g durante 15 min a 4 oC. suavemente retirar el sobrenadante en un recipiente con lejía antes de desecharlos.</li>
	<li>Repita los pasos del 2.4-2.5 una vez. Asegúrese de resuspender completamente el hígado sedimento antes de centrifugar.</li>
	<li>Después de descartar la última solución salina lavar (paso 2.6), agregue 200 mL de agua estéril estanque con pluma/estreptococos al tejido hepático alimentos Peletizado, agitar para Resuspender el sedimento y transferir la suspensión a un matraz aforado estéril de 1 L que ha sido completamente cubierto con aluminio papel de aluminio, excepto en la parte superior 3 cm del cuello (Figura 2D). Nota: Use dos botellas (= 10 hígados) cada frasco de 1 L. Agua de estanque simula el entorno natural de agua dulce necesario para estimular miracidial eclosión de los huevos.</li>
	<li>Usar agua estéril, llenar el frasco a unos 3 cm por encima de la lámina que cubre el cuello. Entonces, sin demora (como miracidia pronto comienzan a eclosionar), quitar la espuma residual y tejido hepático flotando a la superficie mediante pipeteo rápidamente hasta ~ 12 mL de agua del estanque que contenía desechos y descartarlo en un separado deseche el tubo. Vuelva a colocar con agua estéril fresca.</li>
	<li>Repetir limpieza paso 2.8, tres veces, comprobando la presencia de miracidia en el tubo de descarte. Dejar de repetición de la limpieza paso si miracidia aparece en la solución de lavado.</li>
	<li>Después del último lavado, agregue lentamente ~ 12 mL de agua tibia charca estéril (previamente calentado a 30 oC) para crear un gradiente de temperatura con agua caliente limpia y estéril en la parte superior. Nota: Esto se logra mejor descargando lentamente a lo largo de la parte del cuello del frasco para evitar la mezcla.</li>
	<li>Coloque una cubierta de caja de Petri pequeña sobre la apertura del frasco y brillar una luz brillante en la parte superior del matraz por encima de la cubierta de aluminio para iluminar la superficie superior del agua. Nota: Normalmente, dentro de 5-10 minutos, nadando miracidia, atraído por la luz, se concentrará en gran número dentro de los primeros 2-3 cm de agua de los estanques (Figura 3A).</li>
</ol>3. Miracidial cosecha y cultivo
<ol><li>Cuando miracidia han acumulado en la superficie después de 10 min, utilizando una pipeta de transferencia estéril, retire ~ 8 mL de agua del estanque y transfiéralo a un tubo de centrífuga de 15 mL estéril. Coloque el tubo que contienen miracidia en hielo.</li>
	<li>Añadir back 8 mL de agua estéril tibia por el interior del matraz volumétrico y esperar otros 10 minutos.</li>
	<li>Utilizando tubos de nuevo cada vez, repita los pasos 3.1 y 3.2 tres veces más. Después de la colecciónth 4, mantenga el tubo final en hielo durante 10 minutos permitir que el miracidia resolver. Este conjunto de tubos consta de la primera cosecha.</li>
	<li>Tubos de centrífuga que contienen miracidia 290 x g durante 1 min a 4 oC en una centrífuga refrigerada previamente enfriada.</li>
	<li>Retire inmediatamente la mayor parte del sobrenadante (~ 7 mL) teniendo cuidado de no perturbar el sedimento del parásito (figura 3B).</li>
	<li>Piscina todas la miracidia de esta primera cosecha en un tubo, luego lavar todos los tubos originales con agua estéril ~ 1 mL y añada al tubo "agrupado".</li>
	<li>Permite parásitos a reposar en hielo ~ 5 min y otra vez centrifugar los parásitos a 290 x g durante 1 min a 4 oC.</li>
	<li>Cuidadosamente Quite el sobrenadante y añadir que 6-8 mL estéril Chernin equilibrada solución de sal (CBSS +; véase tabla de materiales) que contienen pluma/strep.</li>
	<li>Suspender suavemente las miracidia y alícuota en una placa de cultivo de 24 pozos (1 mL por pozo), seguido por enjuague el tubo en 6-8 mL CBSS + y añadir 1 mL de enjuague a cada pocillo. Incubar los parásitos a 26 oC normoxic condiciones. Concentraciones de miracidia aislado varían, pero generalmente promediarán ~ 7000/mL.</li>
	<li>Si los parásitos se concentran aún en la fuente de luz, repita los pasos 3.1 a 3.9 a continuar cosechando tarde eclosión miracidia pero aumentar el intervalo de tiempo entre colecciones de 15-20 minutos. Nota: Este nuevo conjunto de tubos representa la segunda cosecha. Sin embargo, porque miracidial números por lo general son bajos, las larvas de todos los tubos generalmente son agrupadas en una sola cultura, bien que contiene 2 mL CBSS +.</li>
	<li>En cultivo y poscosecha de 24 h, resuspender suavemente esporoquistes en sus pozos por pipeteo.</li>
	<li>Espere unos minutos para permitir que los parásitos resolver en el fondo de los pozos. Una vez que han colocado, retire con cuidado el sobrenadante (~1.5 mL), que contendrá la mayoría de las placas epidérmicas ciliadas derramada por miracidia durante transformación larvaria. Esta "transformación" sobrenadante puede desechar o incorporada a estudios de seguimiento de productos de secreción de larvas.</li>
	<li>Añadir 1,5 mL de CBSS fresco + a cada bien y suavemente la pipeta para resuspender esporoquistes. Repita el paso 3.12 si se desea más lavar o cambiar a un medio diferente.</li>
</ol>

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Autores no tienen nada que revelar.

Miracidia de S. mansoni, aislados y manipulados como se describe en este documento, puede infectar solamente el anfitrión intermedio del caracol y por lo tanto no representan un riesgo biológico humano durante esta fase de desarrollo larvario. Sin embargo, para evitar la exposición accidental, infección de los caracoles, debe tenerse cuidado para realizar aislamientos miracidial en una ubicación diferente de áreas donde especies de caracoles Biomphalaria susceptibles pueden ser presente o mantenid...

El ser humano sangre platijas Schistosoma spp., son los agentes causativos de la esquistosomiasis, una enfermedad tropical desatendida que afecta a un estimado de 230 millones de personas en todo el mundo1. La especie más difundida, Schistosoma mansoni, se distribuye geográficamente en América del sur, el archipiélago Caribe, Oriente Medio y África subsahariana2. El ciclo de vida de S. mansoniy otros esquistosomas, es complejo, que implica un mamífero hospedador definitivo y los caracoles de agua dulce del género Biomphalaria que sirven como hospederos intermediarios obligados.
S. mansoni macho y hembra adulto del gusano pares viven en las venas mesentéricas posteriores de la reproducción del huésped mamífero, lo que resulta en la liberación de huevos embrionados (ova) que se metieran en las pequeñas vénulas de la mucosa intestinal. Huevos se rompe a través de las paredes del recipiente, migran a través de tejido mucoso y finalmente entrar en la luz intestinal donde son anuladas con las heces. Cuando óvulos entran las larvas nadan libremente y de agua dulce (miracidia) Portilla, deben, en poco tiempo, encontrar un caracol Biomphalaria adecuado para infectar a fin de continuar su ciclo de vida. Este proceso de infección consiste en miracidial accesorio a la superficie de cuerpo exterior de caracol seguida por la penetración activo y la entrada de la larva en el host. Pronto después de la entrada, el miracidium comienza a arrojar sus placas epidérmicas ciliadas externas como forma un sincitio se convertirá en la nueva superficie exterior (tegumento) de la siguiente etapa larval, el sporocyst primario o madre. A través de reproducción asexual, cada sporocyst primario produce y libera una segunda generación de esporoquistes, denominados secundarios o esporoquistes de hija, que a su vez, generaran y liberan grandes cantidades de la fase final de la intramolluscan, la cercaria3 . A escapar de la sede de caracol, cercariae nado libre es capaces de conectar a y penetrar directamente en la piel de un humano o de otro huésped mamífero adecuado. Al entrar en su nuevo host, cercariae transforma a la etapa de parásito esquistosómula y invade el sistema vascular. A su vez, migran a los pulmones y luego a la vena hepática donde maduran a gusanos adultos, forman pares masculinos y femeninos y viajar a las venas mesentéricas para completar su ciclo de vida.
Exitoso intramolluscan o el desarrollo de la "fase Caracol" de esquistosomas larvales es esencial para la continuación del ciclo de transmisión humana de host a host. De importancia crítica es la entrada siguiente períoda del miracidium nadan libremente en el host de caracol y su transformación temprana a la etapa del sporocyst primario. Dependiendo de la compatibilidad fisiológica e inmunológica entre huésped y parásito, es durante esta fase de desarrollo larval inicial éxito o el fracaso para establecer infecciones es determinado4,5,6 . Desarrollo posterior de esporocisto primaria capaz de producir asexualmente esporoquistes secundarios, que luego generan cercariae infeccioso en humanos, además requieren un entorno de host fisiológica permisiva que ofrece para todos los del crecimiento del parásito y necesidades de reproducción.
Hasta la fecha, poco se sabe sobre el subyacente fisiológico, bioquímico y moleculares procesos controlar las interacciones de schistosome caracol, especialmente las moléculas y vías implicados en regulación de la reproducción y desarrollo larval, o modulación de respuestas inmunitarias del huésped. Acceso a un gran número de estos estadios larvarios en condiciones en vitro cultura que permiten manipulación experimental facilitaría enormemente el descubrimiento de vías de desarrollo y los mecanismos subyacentes del crecimiento celular, diferenciación y la reproducción. Crítico parásito vías o mecanismos, identificados a través de la experimentación en vitro , podrían utilizarse para interrumpir el crecimiento/reproducción larvaria en el host de caracol, o caracol huésped inmune mecanismos implicados en el reconocimiento y eliminación de miracidial o sporocyst etapas.
En este artículo y el vídeo de presentación, ofrecemos una descripción detallada de un método para aislar gran cantidad de nado libre S. mansoni miracidia para la introducción en cultivo en vitro que luego puede ser sometida a seguimiento experimental manipulación (ver figura 1 para una descripción esquemática del flujo de trabajo de protocolo). Aunque se han descrito procedimientos similares previamente7,8,9, sentimos que un protocolo detallado sería útil a los investigadores que deseen emplear este modelo para abordar preguntas todavía sin respuesta relacionadas con intramolluscan desarrollo larvario, proliferación celular e interacciones inmunes schistosome caracol. Además, este enfoque se podría adaptar fácilmente para el aislamiento de huevos de otros trematodos médicamente importantes tales como vejiga-vivienda S. hematobium, hígado trematodos o tremátodos del pulmón.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chernin&#39;s balanced salt solution (CBSS+)</td>
			<td></td>
			<td></td>
			<td>For 1L of solution</td>
		</tr>
		<tr>
			<td>2.8 g sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Dissolve salts, except calcium chloride, and&#160;</td>
		</tr>
		<tr>
			<td>0.15 g of potassium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>sugars in 800 mL ddH2O</td>
		</tr>
		<tr>
			<td>0.07 g sodium phosphate, dibasic anhydrous</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Dissolve calcium chloride separately in 200&#160;</td>
		</tr>
		<tr>
			<td>0.45 g magnesium sulfate heptahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td>mL ddH2O</td>
		</tr>
		<tr>
			<td>0.53 g calcium chloride dihydrate&#160;</td>
			<td>Mallinckrodt</td>
			<td>4160</td>
			<td>Slowly add calcium soln to the salt/sugar soln with&#160;</td>
		</tr>
		<tr>
			<td>0.05 g sodium bicarbonate&#160;&#160;</td>
			<td>Fisher Scientific</td>
			<td>S233-3</td>
			<td>with constant mixing</td>
		</tr>
		<tr>
			<td>1 g glucose</td>
			<td>MP Biomedicals</td>
			<td>152527</td>
			<td>Adjust to pH 7.2 and filter sterilize using a&#160;</td>
		</tr>
		<tr>
			<td>1 g trehalose</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td>0.22 &#181;m disposable bottle-top filter</td>
		</tr>
		<tr>
			<td>10 mL 100X penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add filtered penicillin and streptomycin soln&#160; 
			prior use</td>
		</tr>
		<tr>
			<td>Incomplete Bge&#160; medium (Ibge)</td>
			<td></td>
			<td></td>
			<td>For 900 mL solution</td>
		</tr>
		<tr>
			<td>220 mL Schneider&#8217;s Drosophila medium modified</td>
			<td>Lonza</td>
			<td>04-351Q</td>
			<td>Mix Schneider&#39;s medium with 680 mL ddH2O</td>
		</tr>
		<tr>
			<td>4.5 g lactalbumin enzymatic hydrolysate</td>
			<td>Sigma-Aldrich</td>
			<td>L9010</td>
			<td>Add lactalbumin hydrolysate and galactose</td>
		</tr>
		<tr>
			<td>1.3 g galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0625</td>
			<td>Adjust to pH 7.2 and filter sterilize using a 0.22 &#181;m 
			pre-sterilized disposable bottle-top filter</td>
		</tr>
		<tr>
			<td>Complete Bge medium (cBge)</td>
			<td></td>
			<td></td>
			<td>For 100 mL of solution</td>
		</tr>
		<tr>
			<td>90 mL Incomplete Bge medium</td>
			<td></td>
			<td></td>
			<td>To heat-inactivate FBS: Incubate thawed FBS in&#160;</td>
		</tr>
		<tr>
			<td>9 mL heat-inact. fetal bovine serum (FBS) (Optima)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td>waterbath at 60&#176;C for 1 hr while gently&#160;</td>
		</tr>
		<tr>
			<td>1 mL 100X penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>swirling the bottle every 10 min 
			Aliquot heat-inactivated FBS into 15-mL tubes&#160; 
			and store at -20&#176;C.&#160; Mix medium + FBS and 
			filter sterilize using a 0.22 &#181;m pre-sterilized&#160; 
			disposable bottle-top filter&#160; 
			Add penicillin and streptomycin prior to use</td>
		</tr>
		<tr>
			<td>Pond water (stock solution)</td>
			<td></td>
			<td></td>
			<td>1L of stock solution</td>
		</tr>
		<tr>
			<td>12.5 g calcium carbonate</td>
			<td>Fisher Scientific</td>
			<td>C64-500</td>
			<td>Mix all salts in 1L of ddH2O</td>
		</tr>
		<tr>
			<td>1.25 g magnesium carbonate</td>
			<td>Fisher Scientific</td>
			<td>M27-500</td>
			<td>Note that the salts will not have completely&#160;</td>
		</tr>
		<tr>
			<td>1.25 g&#160; sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>dissolved.&#160; Shake vigorously to suspend&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>0.25 g&#160; potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>salts prior to making the working soln&#160;&#160;</td>
		</tr>
		<tr>
			<td>Pond water (working solution)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</td>
		</tr>
		<tr>
			<td>0.8 mL stock solution pond water (shake prior use) in&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td>Mix stock to ddH2O</td>
		</tr>
		<tr>
			<td>1500 mL of ddH2O</td>
			<td></td>
			<td></td>
			<td>Sterilize pond water by autoclaving (slow&#160;cycle)</td>
		</tr>
		<tr>
			<td>1.5 mL of 100X penicillin/streptomycin&#160;</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add penicillin and streptomycin prior use</td>
		</tr>
		<tr>
			<td>Saline solution (1.2% NaCl)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</td>
		</tr>
		<tr>
			<td>18 g sodium chloride in 1500 mL of ddH2O</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Autoclave saline solution to sterilize</td>
		</tr>
		<tr>
			<td>1.5 mL of 100X penicillin/ streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add penicillin and streptomycin prior use</td>
		</tr>
		<tr>
			<td>Additional equipment and material:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-L mouse euthanizing chamber</td>
			<td></td>
			<td></td>
			<td>Following approved IACUC protocol no. V001551</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Taconic Biosciences</td>
			<td></td>
			<td>Swiss-Webster, female, 6-wk old, murine&#160;&#160;</td>
		</tr>
		<tr>
			<td>CO2 tank and regulator</td>
			<td></td>
			<td></td>
			<td>pathogen-free</td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>TPP</td>
			<td>92424</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L volumetric flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light source (150W)</td>
			<td>Chiu Tech Corp</td>
			<td>Model F0-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, refrigerated, swinging bucket</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles (250 mL)</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>430053</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable transfer pipets&#160;</td>
			<td>Fisher Scientific</td>
			<td>1371120</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m pre-sterilized disposable bottle-top filter&#160;</td>
			<td>EMD Millipore</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel blender</td>
			<td>Waring Commercial</td>
			<td>Model 51BL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender cup, 100 mL capacity</td>
			<td>Waring Commercial</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted compound microscope</td>
			<td>Nikon Instruments&#160;</td>
			<td>Eclipse TE300</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni

Humanas sangre trematodos, Schistosoma spp., tienen un ciclo de vida complejo que comprende las fases del desarrollo asexuales y sexuales dentro de un caracol intermedio y mamífero huésped definitivo, respectivamente. La capacidad para aislar y sostener las etapas del ciclo de vida diferente bajo condiciones de cultivo en vitro ha facilitado enormemente las investigaciones de los mecanismos celulares, bioquímicos y moleculares de regulación de host, el desarrollo y crecimiento del parásito interacciones. Transmisión de la esquistosomiasis requiere reproducción asexual y el desarrollo de múltiples etapas larvales en el host de caracol; desde el miracidium contagiosa, a través de esporocisto primaria y secundaria, a la etapa final de cercarial que es contagioso a los seres humanos. En este trabajo presentamos un protocolo paso a paso para la eclosión total y aislamiento del mansoni del schistosoma miracidia de huevos obtenidos de hígados de ratones infectados y su posterior introducción en cultivo en vitro . Se prevé que el protocolo detallado anime a nuevos investigadores y ampliar este importante campo de investigación de esquistosoma.

La gran mayoría de miracidia normalmente habrán sido recogida en las dos primeras "cosechas". Incubación de miracidia aislado en CEMB + desencadena el proceso de transformación de miracidio-sporocyst (Figura 4A- C). En la primera hora siguiente traslado a cultura pozos que contienen CBSS +, la mayoría de miracidia deje nadar (Figura 4A). A las 6 h en cultura, miracidia estamos activamente vertimiento sus pla...

Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Este artículo describe un protocolo para el aislamiento axénico a gran escala y la colección de miracidia nadan de la humana platija de la sangre mansoni de Schistosoma y su posterior procesado para la introducción en cultivo en vitro .

Total aislamiento y cultivo In Vitro de Intramolluscan etapas de la humano platija de la sangre Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

mass-isolation-vitro-cultivation-intramolluscan-stages-human-blood

Research

JoVE Journal

Developmental Biology

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

9.1K vistas.  University of Wisconsin, School of Veterinary Medicine. Este artículo describe un protocolo para el aislamiento axénico a gran escala y la colección de miracidia nadan de la humana platija de la sangre mansoni de Schistosoma y su posterior procesado para la introducción en cultivo en vitro .

Video: Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

El aislamiento de células madre mesenquimales humanas y su cultivo en la matriz del hueso poroso

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Investigación

Biología del Desarrollo

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Cultivo de óvulos para la proyección de imagen de cigoto polarización de Live-celular y patrones del embrión en Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Aislamiento de células endoteliales progenitoras de la sangre de cordón Umbilical humano

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imágenes en vivo de la fusión secundaria del paladar del ratón

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Disección y tinción inmunofluorescente de seta del cuerpo y las neuronas fotorreceptoras en cerebros de adultos de Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Célula de ensayos de agregación para evaluar el atascamiento de la muesca de Drosophila con Trans-ligandos y su inhibición por Cis-ligandos

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

Aislamiento de células Stromal Endometrial humanas In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imágenes y análisis de la orientación del tejido y la dinámica del crecimiento en el desarrollo de la Drosophila Epithelia durante las etapas pupales

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generación de un chip de barrera hematoencefálica basada en iPSC humana

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...