Harry Eagle Renato Dulbecco Theodore T. Puck

Founders of cell culture; successful in vitro growth ofanimal cells and the development of quantitativetechniques in animal virology.

Ross Harrison is considered to be the founder of cellculture techniques. In 1907 he described how he transferred spinal cord tissue to coagulated plasma. The neurons extended neuritesin thisenvironment.

A hanging drop with tissueunderneath a coverslip in coagulated plasma in a hollow glass slide.

Still today – very few cells can be grown in a defined medium, they relyon the addition of serum (usually 5 – 10 %).

Some manufacturers of medium, however, have products for serum-freegrowth of certain cell lines. For example Ultra-MDCK, a serum-freemedium for MDCK-cells.

The problem with serum-free (defined) media is that the manufacturersoften are unvilling to provide information about the contents of themedium. This is information which often is of importance to yourexperimental set-up.

The supplier of Ultra-MDCK has informed us upon request that the onlyprotein/peptide components in this medium are insulin and transferrin.

But looking at the cells, the look somewhat different from MDCK cellsgrown with 5% serum in DMEM.

Small changes in the composition of salts, vitamins etc. in the growthmedium may affect gene expression (protein synthesis), the levels ofenzymes and their substrates, resulting in altered cellular properties.

Transformed cells (cancer cells) are characterized by their reduced communication withneighbouring cells.

Normal cells exhibit mortality in culture: They grow and divide for a characteristic number ofgenerations and then they die…They display the property of contact inhibition: Divide and spreaduntil they reach contact with other cells, stopping to divide when a monolayer is formed.

Transformed cells are immortal: They continue to grow and divide indefinitely and are not inhibited by contact with other cells, but may pile on top of each other. Less demanding growthrequirements than normal cells.

Contamination: Infections in cell cultures:

Bacteria, yeast, fungus, mycoplasma, virus, etc.

All infections, exceptmycoplasma and virus areeasily observed by regular lightmicroscopy by the trained cellculturer.

Mycoplasma are independent, living organisms smaller thanbacteria.

There are ways to test for thepresence of mycoplasma and to remove mycoplasma from thecultures.

Cells from ATCC or other distributors.

Cells from otherresearch groups

Liquid nitrogen: - 196oC

The cells are kept in a small volume of medium with a cryoprotectant, for exampleDMSO: dimetylsulfoksid

Cells can also be stored in freezers at a very lowtemperature (-150oC to – 180oC), but this is more vulnerable because of possible powerfailure. Backup in nitrogen is necessary.

Bacteria might ofcourse also followalong in the tanks.

When an ampulle with cells is takenout of the nitrogen tank, it must be thawed rapidly, at 37oC, withoutcontaminating the cap area. Thenthe cells must be transferred rapidlyto a sterile cell culture bottle withmedium.

Rapidly, because the cells do not have nutrition for a long time in thesmall volume in the ampulle. Transfer to a sterile cell culturebottle is done in the cell culturehood. If the cells adhere to thebottle, this may be observed after 3 – 5 hours.

Suspension cells do not attach..

The air in a cell culture hood is filtered through a HEPA-filter.

A cell culture hood used for mammalian cells must never be used for growth of yeast or bacteria!!!

Yeast cells

Bacterium infected by bacteriophage (virus).

Adherent cells attach to the extracellular matrix:

Some cell types produce their own matrix, while other cell types dependon coating of the bottles or wells with a matrix-like substance in advance. Molecules that attach cells to matrix are for example integrinsand proteoglycans (ex. syndecan). Matrix consists of molecules that aresecreted from cells, for example collagens, proteoglycans, fibronectin, hyaluronic acid, etc.

The cells in the cell culture bottles areplaced in an incubator. An incubatorfor growth of mammalian cells is usually called a CO2-incubator, because the air inside should contain5 % CO2. It should be kept at 37oC and saturated with vapor.

The conditions should be as close as posibleto those mammalian cells areused to in an organism.

Insect cells and fish cells are usuallygrown under other conditions thanmammalian cells.

37oC

5% CO2

vapor

Is 5% CO2 the most relevant condition for all cells?

When cells have been grown to desired number or density, theexperiments may be carried out. Such experiments must be conductedwith similar results at least 3 times. Therefore reproducibility in thehandling of cells is of great importance.

If there is reason to suspect that the cell culture is infected, it should be discarded. If these cells are particularly ”valuable” the situation is sad, but it is hard to get rid of contaminating organisms by trying to washthem away. Most mammalian cells are grown in the presence ofantibiotics, but bacterial infections may still bloom.

Mycoplasma is not easily seen, but the medium might show signs ofchanges. Changes in the medium colour towards orange and yellowindicates ”extra metabolism.” Mycoplasma can be identified by PCR or fluorescense, and may be removed by commercial kits.

However, one should bear in mind that a negative test for mycoplasmaafter it has been fought is no guarantee that it will not return, because a minor fraction might have survived the treatment.

If you are unable to establish reproducible cell culture conditions, or the cells you grow are contaminated, this will give you large problems regardless the nature of your experiments. Your cells in culture willoften have a reduced lifetime, when they are contaminated. Cells thatappear to survive may also give particular problems with theexperiments.

*Proteomics. Other proteins than those produced by your cells will be present.

*Immunology. Unwanted immune responses will appear.

*DNA/RNA: Foreign nucleic acids and enzymes might disturb/destroythe analysis (for example homologous genes found in bacteria).

*Studies by fluorescence microscopy (including confocal microscopy) need reproducible growth conditions to give reproducible results.

Cells to be studied by conventionalfluorescence are most conveniently platedon glass-coverslips. Glass will let theactual wavelengthsthrough, contrary to cell culture plastic. Coverslips (= dekkglass) areautoclaved in glass-dishes and transferredto sterile wells or dishes in a cellculture hood with a sterile foreceps.

With a confocal microscope the focal planes may be set above the glass, plastic or evenpolycarbonate filter (for epithelial cells), so that the support material is not that important.

Model organisms

Have becomemore popularagain

MANIPULATION OF GENE EXPRESSION IN CELL CULTURE

**Stably transfected cell linesA cell line is added DNA in a suitable vector together with a transfection agent. The DNA in the vector enters the cell and is incorporated in the genomic DNA of the cells where this happens, so thatthe vector-DNA will be copied during the cell cycle. The vector mightcontain one or several genes of interest. The vector also codes a selectionmarker/factor that makes transfected cells resistant to a particular drug. Cells that are not transfected with this vector will thus not survive.

When the selection-drug is added to the medium after transfection, wehave introduced a selection-pressure (seleksjonstrykk). It will take a few days for non-transfected cells to succumb. The surviving cells aresparsely plated, so that each individual surviving cell will give rise to a new colony. These are new transfected cell clones and are harvested and propargated as new cell lines.

Serum-starved cells

Stimulation of Rho: Many actin fibers assemble

Stimulation of Rac: Formation of a large actin ”foot” at the periphery of the cell.

A Rho family member

Many long filopodia protrude from the cell body.

When the cell is on themove, the ATP-producingmitochondria (orange stain) follow towards the leadingedge.

Although microtubules are important during cell division, most ofthem depolymerize. These microtubules are in interphaseengaged in movement and positioning of organelles. However, during cell division, it is important that the two daughter cellsreceive equal shares of the organelles from the mother cell.

In the example, this situation has been mimiced by chemicaldepolymerization of MTs. The dispersal of Golgi vesicles is shown.

One important class arethe keratin filaments.

Different variants ofkeratin are expressedduring development. Genetic defects in theformation of keratins maylead to various skindiseases.

Great tensile (elastisk) strength. Allow cells to withstand mechanicalstress.

Next time we will look at movement of organelles along cytoskeletalelements.

Also microorganisms, like the bacterium Listeriamonocytogenes, take advantage of the same mechanisms and use actin filaments to move towards the cell surface and spread to a neighboring cell.

**Transiently transfected cells

When DNA is added to cells in a vector - at the same time as thetransfection agent and most of the cells in the culture are penetrated, the gene transcription, the mRNA translation and protein expressionmay be studied within hours after transfection.

This is a much faster procedure than stable transfection, because theselection time is avoided, but new cells must be transfected with everynew experiment. Variability in the transfection efficiency may be a problem, both with respect to how much DNA that enters each cell, and how many cells that are transfected each time.

Some cells are more easily transfected than others, however, and in many cases transient transfection might be the method of choice.

siRNA – technique:This is a technique where theexpression of a protein is knocked outby transfection. It was first performedby adding siRNA to the growth medium of the worm C. elegans. The techniqueis now frequently used to knock downthe expression of particular proteins in cell lines.

Small RNA segments (15-25 basepairs) are transfected into animal cells. Theseform duplexes with endogenous mRNAthat are recognized as foreign/unwantedand degraded. One can buy the siRNAfrom companies that help you to choosethe right target sequence in the mRNA.

Small interfering RNA

HOW DO WE GROW ADHERENT CELLS?

Cells are usually grown to confluency – covering the wholeavailable surface in a bottle/well/dish.

To increase the number of cells, the cells are detached, usuallyby trypsin before transfer to newbottles/wells/dishes/plates. Trypsin is a protease thatdegrades proteins that attach thecells to the growth support. At the same time, matrix proteins will also be degraded. Detachedcells will float in the medium and may be transferred with a pipette.

Cells may be distached from theirgrowth support with a cell scraper(slikkepott). This is when it is necessary to avoid that surfaceproteins are degraded, as happenswhen cells are detached with theprotease (enzyme) trypsin.

Trypsin (in green) attacks extracellular proteines, includingthose that attach the cells to the support material.

One should not leave the cells for too long with trypsin. Then thecells become more vulnerable, and their membranes might brake. Sticky DNA will leak out and mediate clumping of other cells. Someadd DNAse to the trypsin. The trypsin solution also contains someglucose.

TRYPSINATION OF EPITHELIAL CELLS.

In addition to neighbour contact, like for cells like fibroblasts, epithelial cells are ”sealed” together by tight junctions. These are so tight that the protein (enzyme) trypsin is unable to penetrate betweenthe cells to cleave attachment proteins. Tight junctions are, however, dependent on Ca2+-ions, and addition of EDTA to the trypsin resultsin binding of the Ca2+-ions, so that tight junctions open up, and trypsin gets access to the basal cell surface.

Growth of MDCK epitelial cells on filters. Transfer to glass-disheswith 90 ml of medium for establishment of confluent cell layers.

Basolateral medium

Apical medium

The microvilli extendfrom the apicalsurface of an epithelium and areorganized aroundactin filaments in bundles.

Microtubules in epithelial cells

The microtubules ofepithelial cells areorganized in lateral bundles. Themicrotubules do not radiate from a MTOC in mature epithelia.

When a T-lymphocyte recognizes a target cell, there is a reorientation of the MTOC (the Golgi apparatus follows) towards the target cell. Actin polymerization takes place in both cells and contributes to the organization of thecontact site.

Organization of keratin filaments in an epithelial cell. Bridging desmosomes and hemi-desmosomes, importantstructural attachment points.

Usually, cells in a confluent bottle with adherent cells are split on 5-6 new bottles of the same size.

Why is it not recommended to split a bottle on 25 new ones, if that is the number of bottles you need?

Time

Cellnumber

Factors produced by the cells stimulate growth and division.

MICROINJECTION The technique mayalso be carried outwith adherent cells at a large scale with an automated robot.

DNA, antibodies, water soluble inhibitors, etc.

Cloning of animals……….

Transgenic mice, including knock-out mice

Pluripotent stem cellsmay develop intomany different celltypes.

O2 pressure

Differentiation ofblood cells.

*Reprogrammingof cells is possible.

*Brain cells candivide in adults.

http://www.atcc.org/

ATCC is the largest cell bank in the world.

From here you may buy all cell lines that arecommercially available.

ATCC also provides usefulinformation about cell lines and cell culture.

http://www.nccc.com/services.html

Services within cellculture are available ifyou want to do something special and you do not know how to.

Endosomestransported in TNTs (tunneling nanotubes). Actindependence.

First reported in 2004 (Gerdesand co-workers).