CELL CULTURES

Table of contents :

  • culture media
  • culture synchronization
  • culture growth curve
    		•
  • cell counting tools
  • cell viability assays
  • most commonly used cell lines
  • aims of cell culturing
  • Mycoplasma contamination
  • web resources
    		•
  • culture : the propagation of microorganisms or of living tissue cells in special media conducive to their growth.  2. a growth of microorganisms or other living cells.  3. to induce the propagation of microorganisms or living tissue cells in media conducive to their growth
  • viable but nonculturable : organisms that are alive but cannot be cultured on laboratory media. Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. 3 screening methods are currently available for the metagenome approach :
  • colony : a small area of growth that ideally represents the progeny of a single cell, named colony-forming unit (CFU)
  • fur : an area of growth arose from confluence of more colonies
  • pure (axenic) culture : one containing only one kind of microorganism, without any contaminants; it is created by plating (i.e. : Petri dish insemination) through skimming or inclusion
  • mixed culture : one containing 2 or more kinds of microorganisms.
  • osmotic shocks
  • direct culture : a culture of microorganisms made by direct transfer from a natural source to an artificial medium
  • primary culture : a cell or tissue culture made by direct transfer from a natural source to an artificial medium.
  • secondary culture : one derived from a primary culture
  • subculture culture : one derived from an existing culture.
  • type culture : a culture of any species of microorganism usually maintained in a central collection of type or standard cultures.
  • stock culture : a culture of microorganisms maintained in a viable state as a reference strain and subcultured into fresh medium as necessary.
  • sensitized culture : bacterial cells that have been incubated with specific antiserum.
  • attenuated culture : a culture of pathogenic microorganisms whose virulence is weakened or abolished.
  • blood culture : microbiologic examination of a blood sample to check for presence of microorganisms.
  • chorioallantoic culture : the cultivation of microorganisms, cells, or tissues on the chorioallantois of the developing chick.
  • selective culture : one grown on a medium, usually solid, that has been supplemented to encourage the growth of a single species of microorganism. It may also include substances that inhibit the growth of other species.
  • hanging-block culture : one grown on a block of agar medium fastened to a coverglass, which is then inverted over a hollow slide.
  • hanging-drop culture : a culture in which the material to be cultivated is inoculated into a drop of fluid attached to a coverglass, which is inverted over a hollow slide.
  • radioisotopic culture : a bacterial culture in a medium containing 14C-labeled carbohydrate. Metabolism is detected by the release of 14CO2, offering earlier detection of growth than do conventional methods.
  • roll-tube culture : one made by inoculating a tube of molten agar medium and rotating it while it is solidifying, the medium being dispersed in a thin layer on the inner surface of the tube. The method is used for making colony counts, particularly of anaerobic bacteria.

    •  

    Culture medium : a substance used to support the growth of microorganisms or other cells. They can be classified according to ...

    Culture synchronization Culture growth curve Cell counting tools


    Cell viability assays
    Method Technological platform Main advantages Main disadvantages Recommendedfor HTS*
    ATP consumption Luminometer Rapid and highly sensitive • Highly sensitive to metabolism- related ATP fluctuations
    • Cannot identify cell death modes    		 1
    Autophagic flux   Luminometer Rapid • Expensive 2
    Fluorescence microscopy Allows real-time monitoring • Different inducers might influence the pH of the lysosomes 2
    Immunoblotting Assessed by routine methods • Time-consuming N/A
    Autophagosome quantification  Electron microscopy Provides precise ultrastructural information • Expensive and time-consuming N/A
    Fluorescence microscopy Rapid and inexpensive • Cannot distinguish between increased autophagy and decreased degradation 1
    Caspase activation (fluorescent substrates or Förster resonance energy transfer (FRET) constructs)   Cytofluorometry Allows automated analyses on a per-cell basis • Caspase activation may occur in cell death-unrelated settings
    • Fluorescent substrates may emit upon unspecific degradation    		 2
    Fluorescence reader Rapid   1
    Fluorescence microscopy Can be coupled with other cell-death markers   1
    Cell detachment Impedance reader Allows real-time monitoring • Unable to discriminate between different cell death modes 2
    mitochondrial transmembrane potential (Δψm) dissipation  Cytofluorometry Rapid, inexpensive and no need for permeabilization • Temporary Δψm dissipation may occur in cell death-unrelated settings
    • Fixable probes are suitable for end-point determinations only    		 2
    Fluorescence microscopy Non-fixable probes allow for real-time Δψm monitoring   1
    Extracellular release of proteins  Absorbance reader Inexpensive • Unable to discriminate between different cell death modes 1
    Fluorescence microscopy Allows the study of late events and is applicable to real-time monitoring • Detection might be aggravated by morphological changes of dying cells 1
    Immunological methods     Cytofluorometry Allows automated analyses on a per-cell basis • Rely heavily on the performance of primary antibodies
    • Poorly expressed antigens may be under-detected or undetected
    • Antigens may get denatured during sample processing    		 2
    Electron microscopy Irreplaceable for precise colocalization studies   N/A
    Fluorescence microscopy Compatible with sample fixation and storage   1
    Immunoblotting Allows the study of early biochemical parameters   N/A
    Light microscopy Allows the detection of early cell death-related events   N/A
    IMS protein release  Fluorescence microscopy Release of fusion proteins can be followed in real-time • Release of more than one intermembrane space (IMS) protein must be assessed to avoid false positive results N/A
    Immunoblotting Subcellular fractionation allows the detection of multiple proteins   1
    microtubule-associated protein 1 light chain 3 (LC3) (also known as MAP1) lipidation Immunoblotting Assessed by routine methods • Expensive and time-consuming N/A
    Mitochondrial swelling Absorbance reader Allows the study of mitochondria in the absence of metabolic interference • Laborious and time-consuming N/A
    Morphological determinations   Electron microscopy Provides precise ultrastructural information • Expensive N/A
    Fluorescence microscopy Inexpensive and fixable dyes available • Morphological alterations can be caused by cell death-independent mechanisms N/A
    Light microscopy Rapid and inexpensive, can be used for monitoring cell cultures • Prone to underestimation, unsuitable for quantitative studies 1
    phosphatidylserine (PS) exposure Cytofluorometry Rapid, specific for an early event of apoptosis execution • PS exposure can occur independently of apoptosis 2
    ROS overgeneration  Cytofluorometry Inexpensive • ROS generation can occur independently of cell death N/A
    Fluorescence microscopy     N/A
    Tetrazolium salt conversion Absorbance reader Rapid and inexpensive • Cannot discriminate between cytotoxic and antiproliferative effects 1
    Translocation of cell death mediators Fluorescence microscopy Applicable for live cell monitoring • Detection might be complicated by morphological changes of dying cells 1
    Detection of DNA-strand breaks   Cytofluorometry Useful in co-staining protocols; compatible with long term sample storage • terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) positivity can derive from sample processing
    • Expensive    		 N/A
    Fluorescence microscopy     N/A
    Light microscopy Seen as the gold standard for detecting cell death in situ (in tissues slides) by IHC   N/A
    Vital dyes  Cytofluorometry Routinely employed in co-staining protocols • Unable to discriminate between different cell death modes 1
    Fluorescence microscopy Facilitates the identification of dead cells by visual inspection   1
    IHC, immunohistochemistry; IMS, intermembrane space; ROS, reactive oxygen species; *1, suitable for primary screening; 2, suitable for secondary screening and validation; N/A, not applicable.


    Most commonly used cell lines : a group of animal cells derived from a primary culture at the time of first subculture; it is considered to be an established cell line when it demonstrates the potential for indefinite subculture in vitro.

    Aims of cell culturing Ex vivo incubation affects the expression levels for many genes in human peripheral blood cells : Mycoplasma contamination : hard to detect, and even harder to eliminate, these simple intracellular bacteria frequently contaminate cell culture lines, wreaking havoc on cell function and metabolism without raising obvious warning flags such as changes in turbidity or pH. Labs often don't test their cell lines adequately for mycoplasma, in part because the methods for detecting contamination historically have been time-consuming and difficult to interpret, and sending cells to a specialized testing service can be prohibitively expensive. But in recent years, kits for easy, rapid mycoplasma detection have become available and dramatically reduce the time and technical expertise required to perform the screening. Members of the class Mollicutes, mycoplasmas are simple, tiny prokaryotes that lack the cell wall found in other bacteria. More than 90 species of mycoplasma have been identified, many of which cause disease in humans and livestock, and > 20 of which have been found in contaminated cell lines. But the vast bulk of mycoplasma contamination is caused by a few usual suspects: Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasmafermentans, Mycoplasmahominis, Mycoplasmahyorhinis, and Mycoplasma orale account for 90–95% of infections. Mycoplasmas generally attach themselves to cell membranes but can sometimes penetrate into the cell's interior, where they are even more difficult to eliminate. Once present in a cell line, mycoplasma contamination can affect everything from metabolism to morphology to protein synthesis. Testing is important, as the effects of mycoplasma infection can affect research parameters in subtle ways. It is a good idea to periodically test a cell line to see if it's contaminated. If they're kind of sickly, that's a time to suspect contamination. But they don't always act sickly, and can yet be contaminated, so it's best to be certain. Contamination commonly stems from sloppy lab technique resulting in cross-contamination, the use of contaminated materials, or direct infection from the researcher. Estimates in the literature of how many continuously cultured cell lines are contaminated with mycoplasma vary widely, ranging from about 15% to an alarming 80%ref. Many cell culture facilities that offer mycoplasma-testing services, including the American Type Culture Collection, still use indirect and direct-culture methods. The FDA requires direct culture for screening products that will be injected into human subjects. But for a lab that does its own mycoplasma testing, he says, the time involved in using the standard methods, and the subjectivity of the results yielded, may be prohibitive. Enter the latest crop of mycoplasma detection kits, which borrow from a variety of toolkits in life sciences to deliver quick and objective results. Once a cell line is contaminated with mycoplasma, the options typically are not good: HeLa cell line infected with Mycoplasma hyorhinis. Scanning electron micrograph of critical point-dried cell culture grown on cover slips. Note the loop- and rod-like mycoplasmas attached to the host cell membrane. Smaller web-like structures on the cell surface represent microvilli of the cell. Original magnification 3,000×.

    HeLa cells stained for actin (red), eukaryotic nuclei (blue), and Mycoplasma fermentans genomic DNA (green, revealed by FISH analysis)

    The culture of animal cells is key to much of basic research today and an important starting point for therapeutic applications. But each cell type has its own quirks. Some cells are happy with most media and protocols, but others can become the bane of a scientist's existence with their seemingly inexplicable needs. Caitlin Smith reports. The RCMW Perfused Flow System showing various culture chamber options. (Courtesy of Synthecon, Inc.) :

    Why some cells start to grow and others do not, we do not know : some scientists believe in adding various supplements, but my guess is that if cells start to grow autonomously, they grow with or without these supplements—but people, including scientists, are superstitious. Most scientists would balk at the notion of superstition in their protocols, and yet desperate times often call for 'whatever works' measures. This article will focus on some peculiarities and potential remedies of culturing three cell types that challenge researchers today: primary, stem and hybridoma cells. Most primary cells are particularly difficult to grow. Once established as cell lines, they become much easier to grow, especially transformed (tumorized) lines. So the difficulty in growing them is typically prior to transformation. The primary culture of animal cells has long been a challenge to cell culturists. Thankfully, there are some media additives that seem to benefit most cultures, such as different types of sera and growth factors. Take human skin cells for example : peptide additives (for example, EGF, insulin and bFGF) are important for culturing both keratinocytes and melanocytes. He uses medium with <0.1 mM calcium to prevent differentiation and prolong proliferative growth. Often one must use the 'try-it-and-see' approach for different additives. Whereas they have little trouble growing cultures of melanoma cells from advanced lesions and metastases, they have difficulties growing melanoma cells from very early primary lesions. The problem is that their exact growth factor requirements are simply not known. Further complicating matters is the exasperating fact that not all cell types respond to additives in the same way. Very often the factors that stimulate the normal cells actually inhibit the malignant cells. We need to perform very meticulous testing to identify the components that can support the proliferation of these primary melanoma cells. This is critical for understanding early changes from normal to malignancy. One of the problems that we face is the paradox that in a tumor specimen, the untransformed cell contaminants will outgrow the tumor : almost all malignant cell cultures grow extremely slowly at first, and often our biggest challenges are preventing normal cell overgrowth and/or contamination. To counteract this effect, some groups grow cultures in the absence of fetal calf serum, which is not as big a handicap as it might sound. Serum often favors the growth of normal cells over malignant cells, compounding the problem of normal cells outgrowing malignant cells.
    Manufacturing of Cell Culture Technologies' media at B. Braun Medical AG, Crissier/Lausanne, Switzerland. (Courtesy of Cell Culture Technologies, LLC.)

    Some cells in primary culture grow better when provided with so-called 'solid phase cues', or a three-dimensional (3D) environment that favors cell growth. A future challenge is going from 2D to 3D cultures : there is some beautiful work out there showing that any number of basic cellular properties are altered when the cells are cultured in three versus two dimensions. While it is impossible to know the exact matrix conditions in vivo, many companies offer matrix or chamber products that claim to better replicate the 3D in vivo environment—for example, Chemicon's 3D Cell Culture Kit, 3DM's Puramatrix, and Oligene's Perfusion Chamber System PCS 3c. The importance of '3D biology' has only recently started to be fully understood and appreciated : Synthecon offers the National Aeronautics and Space Administration (NASA)-designed Rotary Cell Culture System, a bioreactor capable of growing 3D cultures. As a type of primary cell, stem cells are in a category all their own. Whereas their pluripotency has earned them fame and controversy, there is a long way to go before their enormous therapeutic potential is realized. Once the stem cells of interest are isolated, they can be quite difficult to establish in culture and to grow thereafter. Sometimes they simply die after isolation, and one must do a protocol optimization to overcome this if it presents too much of a problem. Since isolation protocols are lengthy, the optimization would be as well. Once cultures are established, scientists growing human embryonic stem (ES) cells need to prevent stock cultures from differentiating into the over 200 cell types that they can become. Typically ES cells require a medium supplemented with serum and are grown on a feeder layer of mouse fibroblasts, which helps to keep the stem cells in their undifferentiated state. This arrangement is less than ideal for clinical research purposes, because the presence of animal contaminants in the medium and the feeder layer can make it unsafe for future therapeutic applications in humans. To alleviate the problem, the feeder cell layer is often replaced by a mouse-derived cell matrix extract (such as Matrigel from BD Biosciences) in combination with conditioned medium from a mouse fibroblast culture. Similarly, the animal serum can be replaced by a serum-replacement formulation (such as Knockout Serum Replacement from Invitrogen). But these tricks do not solve the problem of animal contaminants. A substantial challenge in the near future will be the development of serum-, feeder- and protein substrate-free cultures for growing stem and other cell types. An important step in this direction was taken in March when 2 independent groups reported that a specific formulation of growth factors can block differentiation and replace mouse fibroblast conditioned media for the culture of human ES cellsref1, ref2. Whereas this discovery will go a long way toward making the use of ES cell cultures safer for clinical use, it does not mean that the culture media are yet free of animal components: a bovine-derived serum replacement and a mouse-derived matrix gel are still required. After successfully isolating and maintaining your stem cell of interest, you may want to induce differentiation. R&D Systems offers kits to facilitate the differentiation of ES cells into dopaminergic neurons and oligodendrocytes under serum-free conditions. The kits contain supplements to enrich neural stem cell populations; bovine fibronectin as a matrix for cell attachment and spreading; and the basic human fibroblast growth factor, mouse fibroblast growth factor 8b and mouse sonic hedgehog amino-terminal peptide (for dopaminergic neurons), or human epidermal growth factor and human platelet-derived growth factor AA (for oligodendrocytes). R&D Systems estimates that the kit contents are sufficient for the differentiation of 3 x 107 ES cells. Monoclonal antibodies, whose value in basic research is undisputed, are produced from hybridomas, immortalized cell hybrids resulting from the fusion of spleen cells from an immunized mouse with a continuous myeloma cell line. Hybridomas are traditionally grown in medium supplemented with bovine serum, but as with stem cells, a recent challenge has been the need to grow them in serum-free medium with no animal proteins. The issue arises from the fact that monoclonal antibodies are increasingly useful as human therapeutic agents. Manufacturers are beginning to fill this need for serum-free medium. The most important improvement we've seen is the development of better commercially available serum-free media, but a challenge remains to develop robust animal component-free medium that is equivalent to serum-containing medium. Serum is expensive, so animal component-free medium needs to be less expensive yet be suitable for several cell types. Cross-section through hollow fiber culture system showing extracapillary space. (Courtesy of FiberCell Systems, Inc.) :

    For example, Cell Culture Technologies offers their TurboDoma media for culturing myeloma and hybridoma cell lines, as well as kits aimed at saving researchers' time. They offer chemically defined, protein- and peptide-free minimal culture media for growing well-established animal cell lines. All their media are made of pharmaceutical-grade small molecules of nonanimal origin. They do not use complex additives such as hydrolysates, yeast extracts, albumins or proteins, not even insulin, to produce our culture media. For scientists wanting a quick start to serum-free culture, Cell Culture Technologies also offers Starter Kits containing a hybridoma cell line grown in the absence of animal proteins, in minimal culture medium free of proteins and peptides, and protocols from the European Collection of Cell Cultures for maintenance and banking of the cell line. Today, too many scientists in academia and industry waste too much time selecting cells for serum-free culture, so we thought that providing the essential tools at once might help scientists to concentrate on their actual research targets instead of wasting time with boring selection procedures. Maximizing antibody production is an important goal in growing hybridomas, and the culture vessel may have an effect on cell growth and monoclonal antibody output. FiberCell Systems claims that hybridomas grow exceptionally well in their proprietary hollow fiber cells. Because of the tremendous amount of surface area offered in such a small volume, and the high gross filtration rate of our proprietary fiber, cells will grow at extremely high densities. This permits easy adaptation to serum-free medium or the reduction of serum to as low as 2% of the total volume of the medium with no problems. Another advantage to their system is that their fiber allows transforming growth factor , a secreted factor inhibitory to hybridoma growth, to diffuse away while retaining the antibodies in the small volume of the extracellular space. Hybridomas grown in their fiber cell systems can produce up to 2 grams of monoclonal antibody per month. FiberCell's hollow fiber cell culture system. (Courtesy of FiberCell Systems, Inc.)

    Whereas great strides have been made in cell culture technology, there remain even greater challenges to the field in the near future. For example, the importance of tracking karyotypic changes has received too little attention. Cultured cells are continuously under selection pressure. Only those with selection advantages will survive and expand. To track and maintain the karyotypes of each specific cell type is vital for the usefulness of the cells in research. In the culture of primary cells, Halaban says that continuing to identify growth factors that stimulate proliferation and differentiation is an important technical challenge. In addition, she suggests the creation of government-supported centers of excellence that distribute cells to researchers for a nominal fee. Growing human cells is very critical for continuing research. It takes specialized skills that cannot be developed by every scientist. Cell culture is transitioning from being merely"a supporting technique to a biotechnology. Despite the current development of new tools in cell culture, she believes that the key to optimizing cell culture is rooted in cell biology. Scientists involved in the optimization of cell culture systems (irrespective of their application) need to focus on understanding the biology of cells in order to achieve their goal.

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