Cell Culture

From CellBiology

Introduction

Page under development.


Cell Culture: 3T3 | B35 | C2C12 | C17-2 | HT4 | IGR3 | NT2 | PC12 | RGC5

Basic Tissue Culture Equipment

Biohood
  1. Personal Protective Equipment PPE (enclosed shoes, gown, gloves, eye protection)
  2. Decontamination (bleach, ethanol, Viraclean)
  3. Autoclave (clean, dirty)
  4. Waste facilities (clean, chemical and biowaste)
  5. Biohood Class 2 (sterile environment)
  6. Fridge/freezer (4C, -18C, -80C, liquid nitrogen)
  7. Water bath (37C, warming solutions)
  8. Centrifuge (bench-top,low speed, capped carriers, 15 and 50 ml tubes)
  9. CO2 Incubator (gas regulated environment, CO2 gas)
  10. Microscope (inverted phase, digital camera)
  11. Consumables (tissue culture plates, media, buffers, enzymes, antibiotics, pipettes, sterile filters, 15ml and 50 ml tubes, cells ......lots of consumables)


A Grant......

Types of Cell Culture

Cell Lines

Also called Continuous Cultures. Cell lines may be established or generated by individual research groups or available from a larger cell supplier (bank). There are two main suppliers worldwide of cell lines, ATCC and ECACC. Different cell lines will have different growth properties, passage-ability and characterization.

American Type Culture Collection - ATCC

"...global nonprofit bioresource center that provides biological products, technical services, and educational programs to private industry, government, and academic organizations around the world. Our mission is to acquire, authenticate, preserve, develop, and distribute biological materials, information, technology, intellectual property, and standards for the advancement, validation, and application of scientific knowledge.

  • ATCC was established in 1925 when a committee of scientists recognized a need for a central collection of microorganisms that would serve scientists all over the world. The early years were spent at the McCormick Institute in Chicago until the organization moved to Georgetown University in Washington, DC in 1937. As research in the biosciences expanded, ATCC began to diversify its holdings, and as the collections grew ATCC occupied a series of sites, each providing more storage space. ATCC moved to its current state-of-the-art building in 1998.
  • ATCC USA
  • Bacteria, Bacteriophages, Cell Lines and Hybridomas, Filamentous Fungi and Yeast Plant Seeds, Protozoa and Algae, Viruses and Antisera.
  • Cultures and Products

European Collection of Cell Cultures - ECACC

"The European Collection of Cell Cultures (ECACC) was established in 1984 as a cell culture collection to service the research community and provide an International Depository Authority recognised patent depository for Europe." "The collections currently hold over 40,000 cell lines representing 45 different species, 50 tissue types, 300 HLA types, 450 monoclonal antibodies and at least 800 genetic disorders."

Primary Culture

  • Primary cell cultures are derived from rat, mouse and human in the Lab. The majority of cultures focus on the growth of either neurons and/or glia. Cultures can be generated from embryonic or adult: cortex, retina, spinal cord, dorsal root ganglia, sympathetic ganglia. Depending on the preparation technique either neurons, glia or neurons and glial cultures can be generated.
    • primary cell culture refers to the cells the first time they are placed in culture.
    • Once these cells have been subcultured are no longer primaries and should not be described as primary culture.
  • The advantages of primary cultures are that the cells have not been "modified" in any way (other than enzymatic or physical dissociation).
  • The disadvantages of primary cultures are the mixed nature of each preparation, limited lifespan of the culture and the potential contamination problems. In some cases these cells can also be stored frozen for future use.
    • Remember that in the adult (except for olefactory and ventricular) neurons are post-mitotic and will not proliferate unless transformed.
  • All primary cell culture experiments using animals need Animal Ethics approval and must comply with Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, National Health and Medical Research, Council (6th edn, 1997).

Culture Method

Tissue Cell Culture Dishes

Adherent Cultures

  • cells grow until they cover the surface area available or the medium is depleted of nutrients
  • some cells are "contact inhibited"
    • once a monolayer is formed and cells contact each other, they cease proliferation
  • these cells will need to be separated from the substrate
    • mechanically (scraper)
    • enzymatically (trypsinization)
    • both enzymatic and mechanical

Semi-Adherent Cultures

  • mixed population of cells some adherent and some in suspension
  • both cell populations need to be preserved in passaging
  • not a common culture

Suspension Cultures

  • cell cultures derived from blood (e.g. lymphocytes)
  • grow in suspension, not adherent to a sustratum
  • some cell lines may grow as single cell suspensions or in clumps (e.g. EBV transformed lymphoblastoid cell lines)
    • clumping cells may require centrifugation and resuspension by pipetting generate a single cell suspension (for counting)

Cell Observation

Inverted Biological Microscope

Phase Contrast Microscopy

Phase contrast optical pathway
  • refractive index differences within cellular components and between cells and their surrounding aqueous medium
  • enhances contrast in transparent specimens
  • “phase halo” - can be either bright around dark objects or dark surrounding bright objects
  • diffracted light passes through the phase ring as well as the nonphase areas and interacting at the image plane
  • light diffraction and interference and not of the optical path of the sample

Links: MBoC Figure 9-8. Four types of light microscopy


Cell Growth

Cell Media

Media-bottle-icon.jpg

Basic Constituents of media, inorganic salts, buffering systems, carbohydrates, vitamins, proteins/peptides, fatty acids/lipids, trace elements.

  • cells require pH conditions in the range 7.2 - 7.4 and close control of pH.
    • phenol red acts as a pH indicator, culture medium should be changed if the color turns yellow (acid) or purple (alkali).
  • bicarbonate/CO2 buffering systems need to be maintained in incubator atmosphere of 5-10% CO2.


Tissue culture flasks

Different Cell Metabolic requirements

Serum

  • Heat inactivation - involves heating at 56°C for 1 hour to inactivate complement components and prevent the occurrence of complement mediated lysis in cell cultures.
  • Safety testing - different between countries: No safety testing required (USA/Canada, New Zealand, Finland and Denmark), Safety testing may be required (Australia, Mexico, Central America).

Serum-Free Media

  • Allow growth in a fully-defined media.
  • No batch to batch sera variations.
  • Eliminate cross-species contamination/interactions.

Antibiotics

Sigma - Cell Culture Tested Antibiotics

Cell Maintenance

Cell Counting

  • Rice University Using a Counting Chamber
  • Automated Cell Counting Countess - Automated Cell Counter "The uses trypan blue staining combined with image analysis algorithm to produce accurate cell and viability counts in just 30 seconds. The algorithm also measures average cell size of live, dead, and total cells."

Cell Storage

  1. Continuous passage
  2. Freezing
  • short-term -80C freezer
  • long-term cell storage in liquid nitrogen


Practical Methods Video

References

Textbooks

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Growth Factor Model Image: MBoC Ch18 Fig 45 MCB Figure 6-5. Stages in the establishment of a cell culture. Image: MBoC Ch18 Fig 46/47

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

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Books

Reviews

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Books

  • Animal Cell Culture: A Practical Approach ed R.I Freshney

Terms

  • polyethyleneimine - attachment factor for weakly anchoring cell lines and primary cells. Used in lipofection protocols, more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells.
  • hibitane - commercial name for Chlorhexidine gluconate (5%), an antiseptic effective against a wide range of bacteria, yeasts, some fungi and viruses. Disinfection (removal of surface bacteria) of the skin.
  • sterile technique - (aseptic) carrying out tissue culture procedures without introducing contaminating microorganisms from the environment.

External Links

Supplier Information

Collagen I, Rat Tail AlgiMatrix Reduced Growth Factor Basement Membrane Matrix