2011 Lab 9

Tissue Culture 1

Introduction

Incubator with cultures

A general tutorial introduction to cell culture techniques in research.

1. PC1 - Wallace Wurth G20/G22
2. PC2 - Wallace Wurth G29 (OGTR certified).

Before beginning any research:

• You should be aware and clearly understand the Legal and Ethical Issues related to research practice (See also primary tissue culture).
  • Joint NHMRC / AVCC Statement and Guidelines on Research Practice (1997)
• You should be aware and clearly understand all Occupational Health and Safety (OHS) issues that relate to your project (your supervisor or PI).
  • School of Medical Sciences, Occupational Health and Safety (SOMS OHS)
  • UNSW OHS
  • UNSW OHS Management System
  • Safety Aspects of Cell Culture

Objectives

• Understand the use and limitations of in vitro cell analysis
• Understand the basic requirements for a tissue culture laboratory
• Understand the basic techniques of tissue culture, sterile technique, contamination analysis, bio-waste, cell storage
• Understanding the differences between primary and cell line cultures
• Brief understanding of cell growth requirements
• Brief understanding of analytical techniques

Cell Biology Handbook

This online publication by ECACC gives a good general introduction to tissue culture techniques. ECACC Handbook

I have also provided a brief 4 page handout to introduce the topic giving a broad historic background and some relevant sections from cell biology texts.

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."

• European Collection of Cell Cultures (ECACC), a Health Protection Agency Culture Collection.
• ECACC Europe
• General Cell Collection
• ECACC Handbook

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).
  • UNSW Animal Care and Ethics Committee

• All primary cell culture experiments using human material need Human Ethics approval.
  • UNSW Human Ethics
  • National Statement on Ethical Conduct in Human Research (2007)

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

Contamination

• Index of Typical Cell Culture Contaminants

Bacterial

• sudden change in pH
• cloudiness in the medium
• slight whiteish film on the cell surface of plates, dishes, bottom of bottles of medium (dissipates when vessel is moved)

Fungal

• thin filamentous mycelia and denser clumps of spores
• fungi are usually slow growing
• in advanced stages a fuzzy growth (either whitish or black) seen without a microscope

Mycoplasma

• cannot be detected by the naked eye or typical light microscopy
• mycoplasma contamination must be detected by testing

• • Infection can: alter of growth rate, induce of morphological changes, lead to chromosomal aberrations, alter cell metabolism and decrease viability upon resuscitation of frozen ampoule. EEACC Mycoplasma Testing
  • Detected by PCR, DNA Stain or Culture Isolation.
  • ATCC Mycoplasma
  • Persistent Exposure to Mycoplasma Induces Malignant Transformation of Human Prostate Cells

Yeasts

• appear as round or ovoid particles smaller than mammalian cells
• mostly seen as "budding" from other yeast particles (chains of 2,3+)
• in advanced stages can appear as multi-branched chains of particles

Cell Growth

Cell Media

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

• Neuronal cells - High glucose (4500 mg/L) Dulbecco's Modified Eagle Medium (D-MEM) (1X) liquid (high glucose)
• Non-neuronal cells - low glucose (1000 mg/L) Dulbecco's Modified Eagle Medium (D-MEM) (1X) liquid (low glucose)

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

Typical Cell Lines

C2C12 Myoblast

• Mouse (Mus musculus) muscle cell line

• Original Reference Blau HM, et al. Plasticity of the differentiated state. Science 230: 758-766, 1985. PubMed: 2414846

• ATCC ATCC C2C12 | ATCC C2C12 images

• Blau Lab Protocol Blau Lab - C2 Mouse Myoblast Culture

PC-12 Pheochromocytoma

• Rat (Rattus norvegicus), adrenal gland cancer

• Original Reference Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424-2428, 1976. PubMed: 1065897

• ATCC ATCC PC12 | ATCC PC12 images

3T3 Fibroblast

• Mouse, Swiss albino (Mus musculus)

• Original Reference Todaro GJ, Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17: 299-313, 1963. PubMed: 13985244

• ATCC ATCC 3T3 | ATCC 3T3 images

HeLa Epithelial

• Human (Homo sapiens) cervical adenocarcinoma

• HeLa cells were named for Henrietta Lacks, who died in 1952 from cervical adenocarcinoma and her physician Margaret Gey then began working with these cells used throughout the world for medical research.

• ATCC ATCC HeLa | ATCC HeLa images

Cell Analysis

Cells can be analysed in many different experimental techniques

• Living or Fixed
• Microscopically
  • Time lapse
  • Confocal microscopy
  • Immunohistochemistry
  • In Situ hybridization
• Biochemically
  • DNA (Southern)
  • mRNA (Northern)
  • Protein (Western)
  • Microarray
• Flow cytometry

Practical Methods Video

• Sigma - Aseptic Techniques

• Sigma - The Micropipette

• Journal of Visualized Experiments
  • Counting and Determining the Viability of Cultured Cells
  • Trypsinizing and Subculturing Mammalian Cells
  • Cell Capture Using a Microfluidic Device

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

• Some Landmarks in the Development of Tissue and Cell Culture
• Cells Can Be Grown in a Culture Dish
• Serum-free, Chemically Defined Media Permit Identification of Specific Growth Factors

Molecular Cell Biology

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

• Growth of Animal Cells in Culture
• Figure 6-5. Stages in the establishment of a cell culture

The Cell- A Molecular Approach

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

• Growth of Animal Cells in Culture

Search Online Textbooks

• "cell+culture" Bookshelf

PubMed

• PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed

• PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC

• Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM

• Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

• "tissue culture" Entrez all databases
• "cell culture" Entrez all databases

Reviews

• PMID: 18365235

Books

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

Supplier Information

Sigma

learning-center

Sigma Cell Culture Manual (on line)

Cell Culture Books

ECACC Handbook Cell Culture Virtual Stockroom

GIBCO Media/Serum Cross Reference -¬EU GIBCO Media/Serum Cross Reference - US

Media Expert Media Formulations Mediatech - product cross reference chart MSDS/COA Search On-Site Stocking/Inventory Management

Poster Gallery Serum Planner

Videos cell-culture-video micropipette video

Invitrogen

Invitrogen Mammalian-Cell-Culture Primary Cell Culture

3D Cell Culture 3D-Cell-Culture Collagen I, Rat Tail AlgiMatrix Reduced Growth Factor Basement Membrane Matrix Stem Cell Research Stem-Cell-Research

Other

• BD Biosciences

• Promega

• Serotec

Links

• Protocol Online - Cell Culture
• UMBC general TC method description
• Wikipedia Tissue culture

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 (aseptic) technique - carrying out tissue culture procedures without introducing contaminating microorganisms from the environment.

2012 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2012 Revision | Development

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis

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Dr Mark Hill 2013, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G