Cell Growth & Maintenance

Proliferating cells in the eye of E4 chick embryo using BrdU staining

Cell culture is a fundamental technique used in life science research to establish relevant biological models or to produce recombinant proteins, virus particles, or biological therapies. Growth and maintenance of cultured cells like bacteria, yeast and mammalian cells is performed in a biosafety cabinet (often called a cell or tissue culture hood) using appropriate sterile technique to prevent microbial and chemical contamination.   

Types of Cell Culture

Two of the most prominent approaches to mammalian cell culture are primary culture and continuous culture. Primary cultures are derived directly from human or animal tissue and have a lifespan in culture that is limited by cellular senescence. Continuous cultures are considered ‘immortal’ in culture, as they  are often derived from patient cancer tissue. Cell lines may also be created by immortalizing cells and can be serially propagated or passaged for numerous cell division cycles, or indefinitely.

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Maintenance and proliferation of cells in culture

Cells can be cultured in suspension or as a 2D monolayer that attaches to the tissue culture flask or multiwell plate. The culture method is determined by the cells’ tissue of origin; cells derived from blood generally grow in suspension, while those derived from solid tissues typically grow in monolayers.

3D cell culture models (organoids and spheroids) are generally considered to more closely mimic the cell’s in vivo environment than cells grown on 2D surfaces. Spheroids are often formed from cancer cell lines or tumor biopsies (patient-derived xenografts, or PDX) as free-floating cell aggregates in ultra-low attachment plates, while organoids are commonly derived from tissue stem cells embedded and then differentiated within an ECM hydrogel matrix.

Cell Growth Phases

Ensuring adequate cell growth is critical to collecting accurate data from cell culture studies. Cell counts can be determined using a hemocytometer or an automated cell counter, which offers more precise cell counts.

Cell growth in culture generally happens in four phases:

  1. Lag phase – Cells are adjusting to culture conditions and do not divide.
  2. Log (Logarithmic) Growth Phase – Cells are actively dividing, so this is the best stage during which to assess population growth or collect data. Late log phase is the best time to passage (subculture) cells.
  3. Plateau (Stationary) Phase – Growth slows as cells approach 100% confluence, and cells are most susceptible to stress or injury as cellular waste accumulates and resources are depleted.
  4. Decline Phase – The population of live cells declines as cell death predominates.

Cell Culture Media, Supplements and Reagents

Cultured cells require a supply of nutrients for growth. Mammalian cell culture media must maintain physiological pH, in addition to providing balanced salts, carbohydrates, amino acids, vitamins, fatty acids and lipids, proteins and peptides, trace elements, and growth factors. Fetal bovine serum (FBS) is the most widely used growth supplement for mammalian cell culture, as it contains many of these essential cellular nutrients and has been demonstrated to support the growth of cells and tissues in culture. For applications requiring defined media or reduced animal components, xeno-free media formulations provide animal-free formulations of known composition.

Because media and supplements often provide rich nutrients for growth of opportunistic microbes, successful cell culture requires aseptic technique and regular observation to ensure the absence of contaminants which can cause cell death or confound in vivo-like growth. For common microbial contaminants that cannot be observed microscopically, routine screening with mycoplasma detection reagents protects cultures and tissue propagation environments.  

Yeast cultures, like S. cerevisiae and P. pastoris (Pichia), are commonly used in research for recombinant protein expression and gene function studies. Critical nutrients typically included in a yeast culture medium formulation are peptone, yeast extract, and dextrose or glucose.

Cell Passaging Tips and Tricks

Passaging cells is a fundamental step in general cell culture to maintain cell health and optimal cell growth. This tutorial explains the process of cell passaging, including the importance of confluency and cell density. The protocol for cell passaging involves processes such as monitoring and counting cells, trypsinization or dissociation of cells, and reseeding of the cells into new cultureware.

Cell culture environments and cultureware

Cultured cells are grown and handled using single-use, sterile plasticware, including tissue culture flasks and multiwell plates, serological pipettes, sterile bottle-top filters, and sterile syringe filters. Plastic tissue culture flasks and plates are usually treated to provide a hydrophilic surface to facilitate attachment for adherent cells. As an alternative to 2D surfaces, microporous membrane-based plates provide a more physiological growth environment for complex cell assays, such as cell migration, cell-cell communication and cell polarization.

Cultured cells must be maintained at the temperature and gaseous environment that recreates these parameters in the organism from which they are derived. Cultureware containing cells and media are typically maintained in incubation appliances that provide precise control of temperature and gas mixtures. However, small-scale benchtop incubation systems that employ microfluidics and facilitate imaging of cells in uninterrupted culture can provide the most authentic environments for predictive in vitro models.

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