Fundamentos del Cultivo Celular

106 comentarios / blog, Español /

El cultivo celular es una de las herramientas más importantes utilizadas en biología celular y molecular, ya que proporciona excelentes sistemas modelo, para estudiar la fisiología y bioquímica normal de las células (p. Ej., Estudios metabólicos, envejecimiento), los efectos de los medicamentos y compuestos tóxicos en las células, mutagénesis y carcinogénesis. También es utilizado en la detección y desarrollo de fármacos y en la fabricación a gran escala de compuestos biológicos (por ejemplo, vacunas, proteínas terapéuticas). La principal ventaja de usar cultivo celular para cualquiera de estas aplicaciones es la consistencia y reproducibilidad de los resultados que se pueden obtener al usar un lote de células clonables.
 
El cultivo celular se refiere a la eliminación de células de un animal o planta y su crecimiento subsecuente en un entorno artificial favorable. Las células pueden eliminarse del tejido directamente y desglosarse por medios enzimáticos o mecánicos antes del cultivo, o pueden derivarse de una línea celular o cepa celular que ya se ha establecido.
 

Cultivo primario

El cultivo primario se refiere a la etapa del cultivo después de que las células se aíslan del tejido y proliferan en las condiciones apropiadas hasta que ocupan todo el sustrato disponible (es decir, alcanzan la confluencia). En esta etapa, las células deben ser sub cultivadas (es decir, pasarse) transfiriéndolas a un nuevo recipiente con medio de crecimiento fresco para proporcionar más espacio para el crecimiento continuo.

Línea celular

Después del primer subcultivo, el cultivo primario se conoce como una línea celular o sub-clon. Las líneas celulares derivadas de cultivos primarios tienen una vida útil limitada (es decir, son finitas) y a medida que se pasan, predominan las células con la mayor capacidad de crecimiento, lo que resulta en un grado de uniformidad genotípica y fenotípica en la población.

Cepa celular

Si una subpoblación de una línea celular se selecciona positivamente del cultivo a través de la clonación o algún otro método, esta línea celular se convierte en una cepa celular. Una cepa celular a menudo adquiere cambios genéticos adicionales posteriores al inicio de la línea parental.

Líneas celulares finitas vs continuas

Las células normales generalmente se dividen un número limitado de veces antes de perder su capacidad de proliferar, un evento genéticamente conocido como senescencia. Estas líneas celulares se conocen como finitas. Sin embargo, algunas líneas celulares se vuelven inmortales a través de un proceso llamado transformación, que puede ocurrir espontáneamente o puede inducirse química o viralmente. Cuando una línea celular finita se transforma y adquiere la capacidad de dividirse indefinidamente, se convierte en una línea celular continua.

Las líneas celulares en cultivo continuo son propensas a la deriva genética. Las líneas celulares finitas están destinadas a la senescencia. Todos los cultivos celulares son susceptibles a la contaminación microbiana, e incluso los mejores laboratorios pueden experimentar fallas en los equipos. Debido a que una línea celular establecida es un recurso valioso, su reemplazo es costoso y requiere de mucho tiempo, es de vital importancia que se congelen y conserven para el almacenamiento a largo plazo.

Tan pronto como se disponga de un pequeño excedente de células de subcultivo, deben congelarse como inventario de semillas, protegerse y no estar disponibles para uso general de laboratorio. El inventario de trabajo se puede preparar y reponer a partir de existencias de semillas congeladas. Si las reservas de semillas se agotan, las reservas de trabajo crio-preservadas pueden servir como fuente para preparar una reserva de semillas frescas con un aumento mínimo en el número de generación desde la congelación inicial.

El mejor método para crio preservar células cultivadas es almacenarlas en nitrógeno líquido en medio completo en presencia de un agente crio protector como dimetilsulfóxido (DMSO) o glicerol. Los agentes crio-protectores reducen el punto de congelación del medio y también permiten una velocidad de enfriamiento más lenta, lo que reduce en gran medida el riesgo de formación de cristales de hielo, que pueden dañar las células y causar la muerte celular.

DMSO es conocido por facilitar la entrada de moléculas orgánicas en los tejidos. Manejar los reactivos que contienen DMSO utilizando equipos y prácticas apropiadas para los riesgos que plantean dichos materiales. Desechando los reactivos de acuerdo con las regulaciones locales.

Cultivo celular de una reserva celular congelada

  • Asegúrese de completar el registro del banco celular.
  • Determine el número de pasaje, ej. Si las células previas se congelaron, se pasaron tres veces, el proceso de descongelamiento sería el cuarto paso.
  • Agregue los medios de crecimiento (Medios básicos, suero de ternera y antibióticos) a un matraz T75 etiquetado con la línea celular, el número de pasaje y la fecha.
  • Coloque el matraz horizontalmente en una incubadora de CO2 a 37 ° C y 5% de CO2 durante al menos 15 minutos. Esto permitirá que el medio se caliente y alcance su pH normal (7.0-7.6)
  • Descongele el vial congelado con la línea celular mediante agitación suave en un baño de agua a 37 ° C. Para reducir el riesgo de contaminación, mantenga la junta tórica y la tapa por encima del nivel del agua (2-5 minutos).
  • Coloque el vial en el gabinete de bioseguridad (BSL-2). Para evitar la contaminación, antes de abrir el vial, límpielo con etanol al 70%.
  • Transfiera el contenido del vial de células congeladas al matraz T75 que contiene medios de crecimiento. Mezcle suavemente y mueva el matraz para distribuir las células de manera uniforme en la superficie del matraz.
  • Incubar el matraz durante la noche en una incubadora de CO2 a 37 ° C, 5% de CO2. Permitir que las células se unan durante la noche.
  • Cambiar los medios de crecimiento.
  • Incubar el matraz a 37 ° C, 5% de CO2 durante 2-4 días adicionales y controlar hasta el crecimiento celular.
  • Una vez que las células han alcanzado aproximadamente el 90% de confluencia, las reservas celulares se pueden expandir utilizando matraces T150.

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