Difference between revisions of "SOMS Honours Research Techniques - Tissue Culture"

From CellBiology
 
(65 intermediate revisions by 3 users not shown)
Line 1: Line 1:
 
==Introduction==
 
==Introduction==
[[File:Incubator-with-cultures.jpg|thumb|400px|Incubator with cultures]]
+
[[File:Incubator-with-cultures.jpg|thumb|300px|Incubator with cultures]]
A general tutorial introduction to cell culture techniques in research.
+
A general tutorial introduction to cell culture techniques in research.
 +
 
 +
Dr Mark Hill has used tissue culture (TC) techniques from his own Honours year to today.  
  
 
Before beginning any research:
 
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).
+
You should be aware and clearly understand the '''Legal and Ethical Issues''' related to research practice (See also primary tissue culture).
** [http://www.nhmrc.gov.au/grants/policy/researchprac.htm 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).
+
Australian Code for the Responsible Conduct of Research has two parts:
** [http://medicalsciences.med.unsw.edu.au/SOMSWeb.nsf/page/OHS School of Medical Sciences, Occupational Health and Safety (SOMS OHS)]
+
* '''Part A''' advocates and describes best practice for both institutions and researchers.
** [http://www.hr.unsw.edu.au/ohswc/ohs/ohs_home.html UNSW OHS]
+
** guidance is provided on how to manage research data and materials, how to publish and disseminate research findings, including proper attribution of authorship, how to conduct effective peer review and how to manage conflicts of interest.
** [http://www.hr.unsw.edu.au/ohswc/ohs/ohsms.html UNSW OHS Management System]
+
* '''Part B''' provides a valuable framework for handling breaches of the Code and research misconduct.
** [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook/cell-culture-techniques-3.html#Safety%20Aspects Safety Aspects of Cell Culture]
+
** helps institutions and researchers to understand their responsibilities in the event that a breach of the Code occurs or an allegation of research misconduct is made.  
 +
** guides institutions through the appropriate responses and explains the responsibilities and rights of researchers if they witness research misconduct. 
 +
** designed to ensure there are agreed, clear, fair and effective processes in place in the event of an allegation of research misconduct.
 +
 
 +
:'''Links:''' [http://www.nhmrc.gov.au/guidelines/publications/r39 Australian Code for the Responsible Conduct of Research] | [http://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/r39.pdf PDF]
 +
 
 +
 
 +
You should be aware and clearly understand all '''Health and Safety''' (H&S) issues that relate to your project (your supervisor or PI).
 +
* [http://medicalsciences.med.unsw.edu.au/SOMSWeb.nsf/page/Health+and+Safety School of Medical Sciences, Health and Safety (SOMS H&S)]
 +
 
 +
 
 +
:'''Links:''' [http://www.ohs.unsw.edu.au UNSW OHS] | [http://www.ohs.unsw.edu.au/ohs_hazards/index.html UNSW OHS Management System] | [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook/cell-culture-techniques-3.html#Safety%20Aspects Safety Aspects of Cell Culture]
 +
 
 +
 
 +
You should be aware of the legislation and risks associated with working with '''Genetically Modified Organisms''' (GMOs).
 +
* the Australian Gene Technology Act was first introduced in 2000 and has been recently updated in 2011.
 +
* legislation has requirements for research facility design, record keeping, spills, release, transport and disposal of GMOs.
 +
* researchers should be aware of the risks and hazards associated when working with specific vectors and the genes placed in these vectors.
 +
 
 +
 
 +
:'''Links:''' [http://www.ohs.unsw.edu.au/ohs_hazards/genetech.html UNSW Gene Technology] | [http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/gtr-review Gene Technology Amendment Regulations 2011] | [http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/transport-guide-1 Guidelines for the Transport, Storage and Disposal of GMOs]
 +
 
 +
 
 +
 
 +
{{External Links}}
  
 
== Objectives ==
 
== Objectives ==
Line 20: Line 46:
 
* Brief understanding of cell growth requirements
 
* Brief understanding of cell growth requirements
 
* Brief understanding of analytical techniques
 
* Brief understanding of analytical techniques
 
  
 
==Cell Culture Basics Seminar ==
 
==Cell Culture Basics Seminar ==
{|
 
|-bgcolor="FAF5FF"
 
|
 
'''Thursday 31st March, 10-10:45am'''
 
  
Lowy Cancer Building, Level 4
+
Usually at least once a year commercial presentation to researchers of current technologies in either SOMS or Lowy.
  
Presented by Naomi Morison, PhD (Cell Culture Technical Specialist, Australasia)
 
  
All Welcome
+
{|
 +
| valign="bottom"|{{Chemotaxis movie 1}}
 +
| valign="bottom"|{{Chemotaxis movie 2}}
 +
|}
  
Refreshments and opportunity to discuss applications will follow the presentation Please RSVP to marc.hogan@lifetech.com for catering purposes  and to request a copy of the Cell Culture Basics Handbook.
 
|}
 
  
==Cell Biology Handbook==
+
The linked online commercial publication (shown below) by ECACC gives a good general introduction to tissue culture techniques.
  
This online publication by ECACC gives a good general introduction to tissue culture techniques. [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook.html 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.
+
:'''Links:''' [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook.html ECACC Handbook]
  
 
==Basic Tissue Culture Equipment==
 
==Basic Tissue Culture Equipment==
Line 47: Line 67:
 
# '''Personal Protective Equipment''' PPE (enclosed shoes, gown, gloves, eye protection)
 
# '''Personal Protective Equipment''' PPE (enclosed shoes, gown, gloves, eye protection)
 
# '''Decontamination''' ([[Media:ChemAlert_-_CHLORINE_BLEACH_-_Summary.pdf|bleach]], [[Media:ChemAlert_-_ETHANOL_DENATURED_-_Summary.pdf|ethanol]], [[Media:ChemAlert - VIRACLEAN- Summary.pdf|Viraclean]])
 
# '''Decontamination''' ([[Media:ChemAlert_-_CHLORINE_BLEACH_-_Summary.pdf|bleach]], [[Media:ChemAlert_-_ETHANOL_DENATURED_-_Summary.pdf|ethanol]], [[Media:ChemAlert - VIRACLEAN- Summary.pdf|Viraclean]])
# '''Autoclave''' (clean, dirty)
+
# '''Autoclave''' (clean, dirty) LOWY
# '''Waste facilities''' (clean, chemical and biowaste)
+
# '''Waste facilities''' (clean, chemical and biowaste) ([http://medicalsciences.med.unsw.edu.au/staff/health-safety/hazardous-waste hazardous waste])
 
# '''Biohood Class 2''' (sterile environment)
 
# '''Biohood Class 2''' (sterile environment)
 
# '''Fridge/freezer''' (4C, -18C, -80C, liquid nitrogen)
 
# '''Fridge/freezer''' (4C, -18C, -80C, liquid nitrogen)
Line 55: Line 75:
 
# '''CO2 Incubator''' (gas regulated environment, CO2 gas)
 
# '''CO2 Incubator''' (gas regulated environment, CO2 gas)
 
# '''Microscope''' (inverted phase, digital camera)
 
# '''Microscope''' (inverted phase, digital camera)
# '''Consumables''' (tissue culture plates, media, buffers, enzymes, antibiotics, pipettes, sterile filters, 15ml and 50 ml tubes, cells ......lots of consumables)
+
# '''Consumables''' (tissue culture plates, media, buffers, enzymes, antibiotics, pipettes, sterile filters, 15ml and 50 ml tubes, cells ......lots of consumables)  
 +
## Note - most of these consumables are now available through Biostore.
  
  
Line 62: Line 83:
  
 
==Types of Cell Culture==
 
==Types of Cell Culture==
 +
 +
{| border=1px
 +
| width="150px"|'''Low risk'''
 +
|
 +
* Non-human/non-primate continuous cell lines and some well characterised human continuous lines.
 +
|-
 +
| '''Medium risk'''
 +
|
 +
* Poorly characterised mammalian cell lines.
 +
|-
 +
| '''High risk'''
 +
|
 +
* Primary cells derived from human/primate tissue or blood.
 +
* Cell lines with endogenous pathogens (the precise categorisation is dependent upon the pathogen).
 +
* Cell lines used following experimental infection where the categorisation is dependent upon the infecting agent (Genetically Modified Organisms)
 +
|}
 +
 
===Cell Lines===
 
===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.  
+
Also called  Continuous Cultures.  
  
====American Type Culture Collection - ATCC====
+
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.
"...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.
 
  
* [http://www.atcc.org/ ATCC] USA
+
Importation of commercial or research lab provided cells need to conform to AQiS guidelines.
* Bacteria, Bacteriophages, Cell Lines and Hybridomas, Filamentous Fungi and Yeast Plant Seeds, Protozoa and Algae, Viruses and Antisera.
 
* [http://www.atcc.org/CulturesandProducts/tabid/167/Default.aspx Cultures and Products]
 
  
==== European Collection of Cell Cultures - ECACC====
+
Note - SOMS holds a school-wide AQIS licence for the importation of a variety of cells and cell products. (contact Anastasia for more details)
"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.
+
===American Type Culture Collection===
* [http://www.hpacultures.org.uk/collections/ecacc.jsp ECACC] Europe
+
 
* [http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp General Cell Collection]
+
American Type Culture Collection (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.
* [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook.html ECACC Handbook]
+
Bacteria, Bacteriophages, Cell Lines and Hybridomas, Filamentous Fungi and Yeast Plant Seeds, Protozoa and Algae, Viruses and Antisera.
 +
 
 +
 
 +
:"...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.
 +
 
 +
 
 +
 
 +
:'''Links:''' [http://www.atcc.org/ ATCC] | [http://www.atcc.org/CulturesandProducts/tabid/167/Default.aspx Cultures and Products]
 +
 
 +
=== European Collection of Cell Cultures - ECACC===
 +
European Collection of Cell Cultures (ECACC), a Health Protection Agency Culture Collection.
 +
 
 +
:"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."
 +
 
 +
 
 +
 
 +
:'''Links:''' [http://www.hpacultures.org.uk/collections/ecacc.jsp ECACC] | [http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp General Cell Collection] | [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook.html ECACC Handbook]
  
 
=== Primary Culture ===
 
=== Primary Culture ===
Line 98: Line 148:
 
** [http://www.gmo.unsw.edu.au/Ethics/HumanEthics/HumanEthics_index.html UNSW Human Ethics]
 
** [http://www.gmo.unsw.edu.au/Ethics/HumanEthics/HumanEthics_index.html UNSW Human Ethics]
 
** [http://www.nhmrc.gov.au/publications/synopses/e72syn.htm National Statement on Ethical Conduct in Human Research (2007)]
 
** [http://www.nhmrc.gov.au/publications/synopses/e72syn.htm National Statement on Ethical Conduct in Human Research (2007)]
 +
 +
===Fluorescence Activated Cell Sorting''' (FACS)===
 +
 +
Uses fluorescent labelled antibody bound to the surface of living cells to identify and sort cells from a mixed population using a laser to detect the fluorescence.
 +
[[Image:Fluorescence Activated Cell Sorting.jpg|400px]]
 +
 +
'''Links:'''  [http://www.ncbi.nlm.nih.gov/books/NBK26851/figure/A1508 MBoC Figure 8-2. A fluorescence-activated cell sorter]
  
 
==Culture Method==
 
==Culture Method==
Line 131: Line 188:
 
* diffracted light passes through the phase ring as well as the nonphase areas and interacting at the image plane
 
* 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
 
* light diffraction and interference and not of the optical path of the sample
 +
**phase contrast microscopy was awarded with the [http://www.nobelprize.org/educational/physics/microscopes/phase Nobel Prize in Physics, 1953].
  
'''Links:''' [http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1729 MBoC Figure 9-8. Four types of light microscopy]
+
'''Links:''' [http://www.ncbi.nlm.nih.gov/books/NBK26880/figure/A1729 MBoC Figure 9-8. Four types of light microscopy] | [http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html Nikon - phase microscopy] | [http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dm-il-led phase alternatives]
  
 
==Contamination==
 
==Contamination==
 
+
See also cell growth section.
* [http://www.unclineberger.org/tcf/contaminant_GI.asp Index of Typical Cell Culture Contaminants]
 
 
 
 
===Bacterial===
 
===Bacterial===
 
* sudden change in pH
 
* sudden change in pH
Line 149: Line 205:
  
 
===Mycoplasma===
 
===Mycoplasma===
[[File:Cells infected with mycoplasma.png]]
+
[[File:Cells infected with mycoplasma.png|600px]]
 
* cannot be detected by the naked eye or typical light microscopy
 
* cannot be detected by the naked eye or typical light microscopy
 
* mycoplasma contamination must be detected by testing
 
* mycoplasma contamination must be detected by testing
Line 162: Line 218:
 
* mostly seen as "budding" from other yeast particles (chains of 2,3+)
 
* mostly seen as "budding" from other yeast particles (chains of 2,3+)
 
* in advanced stages can appear as multi-branched chains of particles
 
* in advanced stages can appear as multi-branched chains of particles
 +
 +
 +
:'''Links:''' [http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/biological-contamination/yeast-contamination.html Yeast contaminated culture] | [http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/biological-contamination.html Cell Culture Contamination] | [http://www.unclineberger.org/tissueculture/contaminant Index of Typical Cell Culture Contaminants] | [http://www.sigmaaldrich.com/cellcult/pdfs/antibi.pdf Sigma - Cell Culture Tested Antibiotics]
  
 
==Cell Growth==
 
==Cell Growth==
Line 183: Line 242:
 
===Serum===
 
===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.
+
* '''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).
+
* '''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===
 
===Serum-Free Media===
 
+
* Stem cell and human therapeutic applications.
 
* Allow growth in a fully-defined media.
 
* Allow growth in a fully-defined media.
 
* No batch to batch sera variations.
 
* No batch to batch sera variations.
Line 194: Line 253:
 
===Antibiotics===
 
===Antibiotics===
  
[http://www.sigmaaldrich.com/cellcult/pdfs/antibi.pdf Sigma - Cell Culture Tested Antibiotics]
+
 
 +
:'''Links:''' [http://www.sigmaaldrich.com/cellcult/pdfs/antibi.pdf Sigma - Cell Culture Tested Antibiotics]
  
 
==Cell Maintenance==
 
==Cell Maintenance==
Line 201: Line 261:
  
 
* Rice University [http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html Using a Counting Chamber]
 
* Rice University [http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html Using a Counting Chamber]
 +
* Automated Cell Counting [http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Countess-Automated-Cell-Counter.html? 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."
 +
 +
 +
:Links: [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Cell-Counting-Viability-Cryopreservation/Countess-Automated-Cell-Counter/Countess-Automated-Cell-Counter-Data.html How Countess Works] | [http://www.invitrogen.com/site/us/en/home/Global/videoroom.html?vidNumber=9 Video]
  
 
===Cell Storage===
 
===Cell Storage===
 
# Continuous passage
 
# Continuous passage
# Freezing
+
# Freezing (passage number)
 
* short-term -80C freezer
 
* short-term -80C freezer
* long-term cell storage in liquid nitrogen
+
* long-term cell storage in liquid/gas phase liquid nitrogen.
 +
 
 +
Freezing Cells
 +
* High protein (sera) media (dispensable for some lines)
 +
* DMSO cryoprotectant (variable 10%) toxic to cells.
 +
* Methylcellulose or polyvinyl pyrrolidone can be used for serum free cell lines.
 +
 
 +
 
 +
Links: [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/d2650pis.Par.0001.File.tmp/d2650pis.pdf DMSO Cell Culture Tested] | [http://tools.invitrogen.com/content/sfs/manuals/synth_a_freeze_cts_man.pdf Commercial freezing media]
  
 
==Typical Cell Lines==
 
==Typical Cell Lines==
Line 265: Line 337:
  
 
* [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/cell-culture-videos/the-micropipette.html Sigma - The Micropipette]
 
* [http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/cell-culture-videos/the-micropipette.html Sigma - The Micropipette]
 +
 +
===Journal of Visualized Experiments===
 +
  
 
* [http://www.jove.com/ Journal of Visualized Experiments]
 
* [http://www.jove.com/ Journal of Visualized Experiments]
Line 280: Line 355:
 
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
 
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
  
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mboc4.table.1516 Some Landmarks in the Development of Tissue and Cell Culture]
+
* [http://www.ncbi.nlm.nih.gov/books/NBK26851/table/A1516 Some Landmarks in the Development of Tissue and Cell Culture]
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mboc4.section.1506#1510 Cells Can Be Grown in a Culture Dish]
+
* [http://www.ncbi.nlm.nih.gov/books/NBK26851 Serum-free, Chemically Defined Media Permit Identification of Specific Growth Factors]
* [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?db=Books&rid=mboc4.section.1506#1512 Serum-free, Chemically Defined Media Permit Identification of Specific Growth Factors]
 
  
 
====Molecular Cell Biology====
 
====Molecular Cell Biology====
Line 299: Line 373:
 
* "cell+culture" [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=cell+culture Bookshelf]
 
* "cell+culture" [http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books&cmd=search&term=cell+culture Bookshelf]
  
===Books===
+
===JoVE Videos===
 +
 
 +
====Neural Culture====
 +
 
 +
* [http://www.jove.com/details.stp?id=1773 Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord]
 +
* [http://www.jove.com/details.stp?id=2462 Organotypic Hippocampal Slice Cultures]
 +
* [http://www.jove.com/details.stp?id=2106 The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury]
 +
* [http://www.jove.com/details.stp?id=2316 Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice]
 +
* [http://www.jove.com/details.stp?id=2373 Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons]
 +
* [http://www.jove.com/details.stp?id=562 Preparation of Dissociated Mouse Cortical Neuron Cultures]
 +
* [http://www.jove.com/details.stp?id=990 Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons]
 +
 
  
  
Line 331: Line 416:
 
[http://www.sigmaaldrich.com/life-science/cell-culture/learning-center.html learning-center]
 
[http://www.sigmaaldrich.com/life-science/cell-culture/learning-center.html learning-center]
  
[http://sigma-aldrich.dirxion.com/WebProject.asp?BookCode=cel08pfp# Sigma Cell Culture Manual (on line)]
+
[http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/cell-culture-manual.html Sigma Cell Culture Manual (on line)]
  
 
[http://www.sigmaaldrich.com/catalog/search/TablePage/14573948 Cell Culture Books]
 
[http://www.sigmaaldrich.com/catalog/search/TablePage/14573948 Cell Culture Books]

Latest revision as of 09:59, 7 February 2014

Introduction

Incubator with cultures

A general tutorial introduction to cell culture techniques in research.

Dr Mark Hill has used tissue culture (TC) techniques from his own Honours year to today.

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).

Australian Code for the Responsible Conduct of Research has two parts:

  • Part A advocates and describes best practice for both institutions and researchers.
    • guidance is provided on how to manage research data and materials, how to publish and disseminate research findings, including proper attribution of authorship, how to conduct effective peer review and how to manage conflicts of interest.
  • Part B provides a valuable framework for handling breaches of the Code and research misconduct.
    • helps institutions and researchers to understand their responsibilities in the event that a breach of the Code occurs or an allegation of research misconduct is made.
    • guides institutions through the appropriate responses and explains the responsibilities and rights of researchers if they witness research misconduct.
    • designed to ensure there are agreed, clear, fair and effective processes in place in the event of an allegation of research misconduct.
Links: Australian Code for the Responsible Conduct of Research | PDF


You should be aware and clearly understand all Health and Safety (H&S) issues that relate to your project (your supervisor or PI).


Links: UNSW OHS | UNSW OHS Management System | Safety Aspects of Cell Culture


You should be aware of the legislation and risks associated with working with Genetically Modified Organisms (GMOs).

  • the Australian Gene Technology Act was first introduced in 2000 and has been recently updated in 2011.
  • legislation has requirements for research facility design, record keeping, spills, release, transport and disposal of GMOs.
  • researchers should be aware of the risks and hazards associated when working with specific vectors and the genes placed in these vectors.


Links: UNSW Gene Technology | Gene Technology Amendment Regulations 2011 | Guidelines for the Transport, Storage and Disposal of GMOs


External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.

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 Culture Basics Seminar

Usually at least once a year commercial presentation to researchers of current technologies in either SOMS or Lowy.


Cells.jpg
 ‎‎Chemotaxis
Page | Play
Melanoma chemotaxis movie.jpg
 ‎‎Chemotaxis
Page | Play


The linked online commercial publication (shown below) by ECACC gives a good general introduction to tissue culture techniques.


Links: ECACC Handbook

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) LOWY
  4. Waste facilities (clean, chemical and biowaste) (hazardous waste)
  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)
    1. Note - most of these consumables are now available through Biostore.


A Grant......

Types of Cell Culture

Low risk
  • Non-human/non-primate continuous cell lines and some well characterised human continuous lines.
Medium risk
  • Poorly characterised mammalian cell lines.
High risk
  • Primary cells derived from human/primate tissue or blood.
  • Cell lines with endogenous pathogens (the precise categorisation is dependent upon the pathogen).
  • Cell lines used following experimental infection where the categorisation is dependent upon the infecting agent (Genetically Modified Organisms)

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.

Importation of commercial or research lab provided cells need to conform to AQiS guidelines.

Note - SOMS holds a school-wide AQIS licence for the importation of a variety of cells and cell products. (contact Anastasia for more details)

American Type Culture Collection

American Type Culture Collection (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. Bacteria, Bacteriophages, Cell Lines and Hybridomas, Filamentous Fungi and Yeast Plant Seeds, Protozoa and Algae, Viruses and Antisera.


"...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.


Links: ATCC | Cultures and Products

European Collection of Cell Cultures - ECACC

European Collection of Cell Cultures (ECACC), a Health Protection Agency Culture Collection.

"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."


Links: ECACC | 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).

Fluorescence Activated Cell Sorting (FACS)

Uses fluorescent labelled antibody bound to the surface of living cells to identify and sort cells from a mixed population using a laser to detect the fluorescence. Fluorescence Activated Cell Sorting.jpg

Links: MBoC Figure 8-2. A fluorescence-activated cell sorter

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 | Nikon - phase microscopy | phase alternatives

Contamination

See also cell growth section.

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

Cells infected with mycoplasma.png

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

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


Links: Yeast contaminated culture | Cell Culture Contamination | Index of Typical Cell Culture Contaminants | Sigma - Cell Culture Tested Antibiotics

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

  • Stem cell and human therapeutic applications.
  • Allow growth in a fully-defined media.
  • No batch to batch sera variations.
  • Eliminate cross-species contamination/interactions.

Antibiotics

Links: 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."


Links: How Countess Works | Video

Cell Storage

  1. Continuous passage
  2. Freezing (passage number)
  • short-term -80C freezer
  • long-term cell storage in liquid/gas phase liquid nitrogen.

Freezing Cells

  • High protein (sera) media (dispensable for some lines)
  • DMSO cryoprotectant (variable 10%) toxic to cells.
  • Methylcellulose or polyvinyl pyrrolidone can be used for serum free cell lines.


Links: DMSO Cell Culture Tested | Commercial freezing media

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

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

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

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.

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

Journal of Visualized Experiments

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

Search Online Textbooks

JoVE Videos

Neural Culture


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


Reviews

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

Links

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.