32.2
Cultivo de células
La mayoría de las células de vertebrados crecen in vitro adheridas a un sustrato en forma de monocapa, llamados cultivos adherentes. Los matraces y pl…
El cultivo celular es una técnica de cultivo de células eucariotas en un entorno de laboratorio.
Proporciona un sistema modelo para estudiar la fisiología de las células fuera de su entorno natural.
Cuando las células de una muestra de tejido se cultivan directamente en un medio de cultivo, los cultivos celulares resultantes se denominan cultivos primarios.
Dichas células necesitan condiciones controladas artificialmente para ayudarlas a crecer, como un material de cultivo correcto, dependiendo de si son cultivos adherentes o en suspensión; una temperatura óptima; un medio de crecimiento adecuado que pueda proporcionar nutrientes esenciales, como aminoácidos, vitaminas, sales y factores de crecimiento; y un sistema de amortiguación del pH.
A medida que el cultivo primario de adherencia celular se multiplica, ocupa gradualmente toda la superficie de la placa de cultivo.
En esta etapa, las células deben tratarse con una solución de proteasa y agente quelante, como Tripsina-EDTA, para aflojar las interacciones célula-sustrato y célula-célula para obtener células individuales.
Un pequeño número de estas células se puede subcultivar en una nueva placa que contenga un medio de crecimiento fresco para facilitar el crecimiento continuo. Estas células subcultivadas forman cultivos secundarios.
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Q1: What is the difference between primary and secondary cell cultures?
Primary cultures are eukaryotic cells grown directly from tissue samples in culture medium. As primary adherent cultures multiply and occupy the entire dish surface, cells must be treated with protease and chelating agents like Trypsin-EDTA to obtain single cells. A small number of these cells are then subcultured into fresh medium, forming secondary cultures that continue growing.
Q2: What are the key components of a growth medium for mammalian cell culture?
Growth media contain amino acids, vitamins, inorganic salts, and glucose as a carbon source. Fetal bovine serum is added as a source of growth factors, hormones, lipids, and minerals. Antibiotics prevent microbial contamination, while buffering systems like sodium bicarbonate maintain pH at 7.4. A pH indicator such as phenol red shows pH changes in the culture.
Q3: How do adherent and suspension cultures differ in their growth requirements?
Adherent cultures grow as monolayers attached to chemically treated flasks and plates. Suspension cultures, such as hematopoietic cells, grow in non-treated cultureware and require magnetic stirrers or spinner flasks to agitate the media. Both culture types need optimal temperature, suitable growth medium, and pH buffering systems for proper development.
Q4: What role do plant growth hormones play in plant cell culture?
Plant growth hormones, specifically auxins and cytokinins, control tissue development in plant cell cultures. A balanced ratio of these hormones produces undifferentiated cells called callus. By altering the hormone ratio, the callus can be differentiated into roots or shoots, generating a complete plant from the explant tissue.
Q5: What conditions must be maintained in an incubator for optimal mammalian cell growth?
Mammalian cells require an optimal temperature of 37°C and a CO2 concentration of 5% in the incubator. The growth medium pH is maintained at 7.4 using buffering systems with sodium bicarbonate and exogenous CO2. These controlled conditions, combined with aseptic culturing protocols, ensure proper cell growth and prevent microbial contamination.
Q6: How are plant cell cultures used in agricultural and conservation applications?
Plant cell cultures produce improved hybrid plants and conserve endangered plant species. Explant cultures are also used in large-scale production of plant-derived products. Plant tissue, known as explant, is cultured in nutrient medium containing plant growth hormones, micronutrients, and a carbon source under controlled temperature, light intensity, and photoperiod conditions.
Q7: Why is Trypsin-EDTA treatment necessary when subculturing adherent cells?
Trypsin-EDTA is a protease and chelating agent solution that loosens cell-substrate and cell-cell interactions in adherent cultures. As primary cultures multiply and occupy the entire dish surface, this treatment is essential to obtain single cells for subculturation. The dissociated cells can then be transferred to fresh growth medium to establish secondary cultures.