Microscopy Solutions for Cell Culture

Biomedical research on living cells was revolutionized in 1934 when the Dutch physicist Frits Zernike described the concept of phase contrast. Within two years ZEISS was applying Zernike's original design in the first prototype phase contrast microscopes. This contrasting technique, which would earn Zernike the Nobel Prize for physics in 1953, is still the method of choice for many cell biologists today as it is ideal for thin unstained specimens such as culture cells on glass or plastic.

Cell culture studies are important in many research areas, ranging from cell biology, pharma, biotechnology, to cell therapy and regenerative medicine. Cell culture, also called tissue culture, deals with the growth of cells in culture media outside the organism in an artificial environment (in vitro). It encompasses the cultivation of adherent cells, suspension cells, primary cells, stem cells, bacteria, fungi, or plant cells. The latter are sometimes more precisely referred to as microbial culture, fungal culture or plant tissue culture, respectively.

Model organisms and immortalized cell lines are often used for studying the biology of cells or tissues. Prominent examples are summarized in the table.

1857 ZEISS sells his first compound microscope

Prokaryotes

 

Bacteria

Escherichia coli (E. coli), Bacillus subtilis

Eukaryotes

 

Protists

Dictyostelium discoideum

Fungi

Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces pombe (fission yeast)

Plant

Tobacco BY-2 cells, Rice (Oryza sativa), Arabidopsis thaliana

Animal cell lines

CHO (Chinese hamster ovary), COS-7 (green monkey kidney), PC12 (rat adrenal medulla), dog MDCK (Madin-Darby canine kidney)

Human cell lines

HeLa (cervical cancer), U2OS (bone osteosarcoma), HEK 293 (embryonic kidney), MCF-7 (breast cancer), Hep G2 (liver cancer)

Model organisms and cell lines that are often used in cell culture

Such cell lines are cultured in special vessels such as Petri dishes, flasks or multiwell plates. Culture media containing nutrients and optional supplements provide the necessary conditions for optimized cell growth. Depending on the type of cell, a certain temperature, humidity, and CO2 and O2 level must be used to best mimic the in vivo conditions. Most mammalian cell lines are cultured in an incubator at 37° C and 5 % CO2 atmosphere.

Microscope Requirements

Cell culture labs use microscopes on an everyday basis to examine cell growth or cell proliferation as well as cell vitality. This includes checking the cell confluency level, whether the cell morphology looks normal, if a contamination is present, and when the culture medium needs to be exchanged. These tasks most commonly require phase contrast microscopy at 50x – 200x magnification. You will need to be quick to minimize the time outside the incubator. Therefore, your cell culture microscope should be compact to fit inside a laminar flow cabinet or on a lab bench in short distance to the incubator. A simple and user-friendly microscope leads to fast turnaround times and minimizes the strain on the cells. Once the cells reach a certain confluency level, they need to be passaged. Before transferring them to a new culture vessel, use a cell counter or a counting chamber such as a Makler chamber to define the cell number, then calculate an appropriate dilution factor. Good cell culture practice is essential since this provides the basis for meaningful and reproducible results in your research.

Many other microscopy procedures are carried out in cell culture labs. Typical assays include the scratch or wound healing assay, live-dead assay and the transwell or translocation assay. Apart from phase contrast and brightfield observation, fluorescence is often used and is becoming a standard. Proteins in cells or tissues can be labeled with immunofluorescence markers. Various fluorophores – among them, DAPI, Hoechst, GFP, Alexa 488, RFP, Texas Red and Cy3 – let you differentiate and locate the signals using multichannel fluorescence microscopy.

On the other hand, living cells can be transfected (by viral or non-viral transfection) with foreign DNA or RNA to express, for example, fluorescent proteins. This process can be quite tedious if you need a stable transfection so fluorescence microscopy is very useful here. Expression level and transfection efficiency are key indicators during this procedure. Gentle fluorescence visualization and imaging can be achieved best using light-emitting diodes (LEDs). Phototoxic effects originating from unwanted ultraviolet (UV) light are reduced compared to other fluorescence illumination sources such as mercury arc lamps. In addition, LEDs offer a significantly increased lifetime and are maintenance-free.

Applications

HeLa cells transfected with GFP expression plasmid
HeLa cells transfected with GFP expression plasmid
Brightfield – tobacco protoplasts acquired with Axiovert
Brightfield – tobacco protoplasts acquired with Axiovert
U2OS cells imaged in live cell culture, GFP Actin stained.
U2OS cells imaged in live cell culture, GFP Actin stained.
HeLa cells in phase contrast - acquired with Primovert
HeLa cells in phase contrast - acquired with Primovert
Human astrocytes.
Human astrocytes.
Phasecontrast – lactobacillus casei acquired with Axiolab
Phasecontrast – lactobacillus casei acquired with Axiolab
MDCK cells (dog) after short incubation period, acquired with Axio Observer © R. Nitschke, Life Imaging Center, University of Freiburg, Germany
MDCK cells (dog) after short incubation period, acquired with Axio Observer
Downloads

Cell Culture

Technology Note: A Quick Guide to Cytological Staining

A Quick Guide to Cytological Staining

pages: 6
file size: 1109 kB

PALM User Protocols

Cell Culture, non-contact Laser Capture Microdissection (LCM) and Recultivation

pages: 14
file size: 500 kB

Special edition of Imaging & Microscopy

Advanced Live Cell Imaging, collection of white papers, confocal imaging

pages: 56
file size: 7572 kB

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