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[Home](https://www.sigmaaldrich.com/US/en)[Applications](https://www.sigmaaldrich.com/US/en/applications)[Cell Culture & Cell Culture Analysis](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis)[Cell Culture by Technique](https://www.sigmaaldrich.com/US/en/applications/cell-culture-and-cell-culture-analysis/cell-culture-by-technique)Cell Growth & Maintenance
# Cell Growth & Maintenance
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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A1470
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F2442
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Fetal Bovine Serum](https://www.sigmaaldrich.com/US/en/product/sigma/f2442)
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Human Serum](https://www.sigmaaldrich.com/US/en/product/sigma/h4522)
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H5667
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Human Serum](https://www.sigmaaldrich.com/US/en/product/sigma/h5667)
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Stericup® E-GP Funnel-less Sterile Vacuum Filtration System](https://www.sigmaaldrich.com/US/en/product/mm/segpu0538)
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D6421
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Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham](https://www.sigmaaldrich.com/US/en/product/sigma/d6421)
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Corning® Costar® Stripette® serological pipettes, bulk packed](https://www.sigmaaldrich.com/US/en/product/sigma/cls4100)
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Antibiotic Antimycotic Solution (100×), Stabilized](https://www.sigmaaldrich.com/US/en/product/sigma/a5955)
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hEGF](https://www.sigmaaldrich.com/US/en/product/sigma/e9644)
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Trypsin solution from porcine pancreas](https://www.sigmaaldrich.com/US/en/product/sigma/t4674)
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Millicell® Hanging Cell Culture Insert, PET 0.4 μm, 6-well, 48/pk](https://www.sigmaaldrich.com/US/en/product/mm/mcht06h48)
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Z359629
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Bright-Line™ Hemacytometer](https://www.sigmaaldrich.com/US/en/product/sigma/z359629)
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HeLa Cell Line human](https://www.sigmaaldrich.com/US/en/product/sigma/cb_93021013)
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Cancer Cell Line Medium XF](https://www.sigmaaldrich.com/US/en/product/sigma/c28077)
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Choose from a range of optimized cell culture media formulations, including DMEM, RPMI-1640, and serum-free options for diverse cellular needs.
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Enhance cell culture with Millicell® inserts, plates, chamber slides, flasks. Achieve biologically relevant growth, choose hanging or standing inserts.
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Rich in growth-promoting factors, Fetal bovine serum (FBS) or fetal calf serum (FCS) is an ideal supplement for mammalian cell culture.
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Overview
Related Articles & Protocols
Support
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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## Related Articles
- [Why Use Antibiotics in Cell Culture?](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotics-in-cell-culture)
Know when to use antibiotics to prevent bacterial or fungal, mycoplasma, or viral contamination in cell culture and find suitable antibiotics or other biological agents.
- [Antibiotic Kill Curve](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotic-kill-curve)
Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic
- [Poly-lysine Types for Biomedical Applications](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/poly-lysine)
Poly-Lysine enhances cell binding with positively-charged surface ions, optimizing electrostatic interaction on culture surfaces for increased cell attachment.
- [Cell Culture Freezing Tips](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/cell-culture-freezing-tips)
Cell Culture Freezing Tips - Are you following best practices for cryostorage? We have cool tips for protecting your cells.
- [Baseclick EdU Cell Proliferation Detection Kits: Improve Data Quality and Save Time](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/baseclick)
Experimental studies measuring cell proliferation have had implications in cancer biology, immunology, cell biology, and developmental biology.
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## Related Protocols
- [Assessing 2.5D Cell Culture Health Using MILLIPLEX® Cell Health Panel](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/assessing-cell-culture-health-using-milleplex-cell-health-panel)
Learn about using our MILLIPLEX® Cell Health Panel to monitor cell proliferation and stress on cells grown on Millicell® hanging cell culture inserts.
- [CryoStor® Cryopreservation Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/cryostor-cryopreservation-protocol)
Cryopreservation affects post-thaw recovery, viability, and functionality. Stress during freezing and suboptimal media lead to cell death.
- [Fibronectin Coating Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/fibronectin-product-protocols)
Dilute fibronectin for cell attachment, varying per cell type. Coating protocol, products, and FAQs provided.
- [MDCK Cell Culture Protocol Using a 96-well TEER Assay System](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/primary-cell-culture/mdck-cell-culture-protocol-using-a-96-well-assay-system)
Discover an expert protocol for optimizing MDCK-II cell growth on the Millicell® 96-well cell culture assembly with the Millicell® ERS 3.0.
- [Primary Human Hepatic Kupffer Cell Culture Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/primary-cell-culture/primary-human-hepatic-kupffer-cell-culture-protocol)
Discover our collection of primary human hepatic Kupffer cells and protocol for thawing, plating, and growing Kupffer cells. Find the formulation for Kupffer culture maintenance media.
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__Featured Articles__
[Antibiotic Kill Curve](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/antibiotic-kill-curve)
Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic
[Poly-lysine Types for Biomedical Applications](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/poly-lysine)
Poly-Lysine enhances cell binding with positively-charged surface ions, optimizing electrostatic interaction on culture surfaces for increased cell attachment.
[Fibronectin Coating Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/fibronectin-product-protocols)
Dilute fibronectin for cell attachment, varying per cell type. Coating protocol, products, and FAQs provided.
[CryoStor® Cryopreservation Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-growth-and-maintenance/cryostor-cryopreservation-protocol)
Cryopreservation affects post-thaw recovery, viability, and functionality. Stress during freezing and suboptimal media lead to cell death.
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