Rachel Henry

1
Cell Culture Basics

• Rachel Henry
• rachel.a.henry_at_vanderbilt.edu
• September 22, 2005

2
Learning Objectives

• Discuss basic techniques of tissue culture
• Learn common cultured cell lines
• Discuss common experiments performed using
  cultured cells

3
Cell Culture

• Pros
• Allows for ex vivo testing of molecules
• Can control cells environment (unlike in
  organism)
• Inexpensive compared with mammals
• Cons
• Culture conditions mimic physiology, but dont
  reproduce it -can alter cells
• Cellular structures are lost

4
What to do with Cultured Cells

• Transfection (plasmid) or transduction (virus)
• Protein Expression
• Ex. Large scale antibody preps from hybridoma
  cells rather than goats, rabbits, etc.
• Protein interaction studies (i.e.
  Co-Immunoprecipitation
• Signaling Studies
• Heterologous cell type can be good
• Localization studies
• Viral replication

5
Cultured Cells

• E. Coli
• Cheap, easy, fast, but prokaryotic
• Yeast
• Eukaryotic, yet single celled
• Sf9/insect
• Eukaryotic, from multicellular organism, but
  single-celled and in suspension
• Mammalian
• Immortalized
• Primary

6
Primary culture from solid tissue

• Dissect out tissue
• May need to fractionate cell types
• Explant
• Wash tissue with buffered salt solution
• Dissociate cells w/protease treatment
  (trypsin/collagenase/dispase) or mechanical means
  (needle passage/pipeting)
• Inactivate protease (wash away/add serum)
• Disperse cells (Pass through steel mesh or pipet
  up and down)
• Add media, and plate

7
Why Primary Culture?

• Unmodified
• Will express your protein of interest
  endogenously and most closely mimic its cellular
  surroundings

8
Why Not Primary Culture?

• Can only passage a few times
• May take a lot of work to obtain results
• Cell viability may be an issue
• Number of cells available from tissue can be
  limiting

9
Immortalized Cell Lines

• Immortalized cell lines derived from
• Actual clinical tumors
• Transformed cells
• Viral oncogenes
• SV40 tumor antigens
• Chemical treatments

10
Immortalized Cell Lines

• Pros
• Cheap and simple
• Can transfect and over-express proteins
• Cons
• May retain little of the original in vivo
  characteristics
• Transformation may alter signaling
• Expression of protein of interest may be altered

11
Passaging Immortalized Cells
12
1) Sterilize working surface with 70 Ethanol
13
Spray all items going into hood with 70 EtOH.
Warm Media, trypsin, and sterile wash buffer (1X
PBS) to 37oC in water bath prior to passage
14
Aspirate media from cells
15
Wash with enough PBS to cover dish(5mL here)
16
Add 1-2mL trypsin, let sit at room temperature
in hood 1-2min, then tap side of dish to dislodge
cells
17
Add 5 times media as trypsin to inactivate
trypsin and dilute cells into new dish with more
media
18
Other Methods to Detach Cells from a Surface

• Mechanical
• Cell Scraper
• May damage cells, best for preparing extract (for
  which viability is not so important)
• Proteolytic
• Trypsin, collagenase, or pronase, usually in
  combination with EDTA
• Cleaves cell surface proteins
• Terminate proteolysis by adding serum
• EDTA alone
• Gentler on cells than trypsin
• 2mM EDTA in 1X PBS (without Ca2 or Mg2)

19
Using a Hemacytometer

• Mix cells with trypan blue (dilute based on
  estimated cell number)
• Live cells exclude trypan blue dye
• Dont count dead (blue) cells
• Maximum cell count of 20 to 50 cells per 1-mm
  square is recommended
• Note trypan blue is toxic and a potential
  carcinogen
• Add 10mL mixture into opening between slide
  coverslip

http//www.pharma.ethz.ch/bmm/protocols/endo.html
qc
20
Using a Hemacytometer

• Count 4 squares
• Count cells touching the middle line of the
  triple line on the top and left of the squares
• Do not count cells touching the middle line of
  the triple lines on the bottom or right side of
  the square
• Calculate cell number
• cells/ml average count per square dilution
  factor 104
• total cells cells/ml total original volume of
  cell suspension from which sample was taken

http//www.pharma.ethz.ch/bmm/protocols/endo.html
qc
21
Using a Hemacytometer

• Count 4 squares
• Count cells touching the middle line of the
  triple line on the top and left of the squares
• Do not count cells touching the middle line of
  the triple lines on the bottom or right side of
  the square
• Calculate cell number
• cells/ml average count per square dilution
  factor 104
• total cells cells/ml total original volume of
  cell suspension from which sample was taken

http//www.pharma.ethz.ch/bmm/protocols/endo.html
qc
22
Tissue Cell Culture Media

• Composed of amino acids, glucose, salts,
  vitamins, and other nutrients
• To make complete medium, typically supplement
  with
• L-glutamine
• Unstable amino acid, converts to a form cells
  cannot use upon storage (only half remains after
  9 days at 37?C)
• Antibiotics (typically penicillin and
  streptomycin sulfate, sometimes gentamycin or
  kanamycin)
• Serum
• Indicate percentage of fetal bovine serum (FBS)
  or other serum added, 10 is common
• Media shelf life is 6 weeks at 4ºC once glutamine
  and serum is added

23
Tissue Cell Culture Media

• Sometimes add
• Antimycotics
• Nonessential amino acids
• Growth factors
• Drugs that provide selective growth conditions
• HEPES or bicarbonate buffers
• Optimum pH usually 7.2 to 7.4
• Prevent pH fluctuations that might adversely
  affect cell growth
• HEPES useful for procedures that do not take
  place in a controlled CO2 environment
• Supplements should be added to medium prior to
  sterilization or filtration, or added aseptically
  just before use

24
Basic Types of Media

• Eagle minimal essential medium (MEM)
• Dulbeccos modified Eagle medium (DMEM)
• Glasgow modified Eagle medium (GMEM)
• RPMI 1640
• Ham F10 nutrient mixture

• Current Protocols Online
• Chapter 28 Mammalian Cell Culture
• http//www.mrw.interscience.wiley.com/cp/cpmb/cpmb
  _contents_fs.html
• APPENDIX 3F Techniques for Mammalian Cell Tissue
  Culture
• http//www.mrw.interscience.wiley.com/cp/cpmb/arti
  cles/mba03f/frame.html

25
Cell Preservation

• Cells are stored in liquid nitrogen for long-term
  preservation
• Minimize cell death
• Use healthy, log phase cells
• Freezing media contains DMSO (lowers freezing
  point), which is harmful to cells
• Work quickly
• Only add freezing media to one or two vials of
  cells at a time to ensure rapid transfer into
  freezer

26
Cell Preservation

• Cells are stored in liquid nitrogen for long-term
  preservation
• Minimize cell death
• Slowly cool cells to -80?C, then transfer to
  N2(l)
• Can place vials in room temp. isopropanol in
  -80?C freezer (temp. ? 1?C/min.)
• Allows water to move out of cells before it
  freezes (ice crystals harmful)
• To thaw cells, resuspend cells in media, spin
  them down and remove DMSO-containing supernatant

27
HeLa Cells

• 50-Year old cervical cancer cells
• Flat good for immunofluorescence
• Support growth of wide range of mammalian viruses
• Ex. Adenovirus, measles, poliovirus, echovirus,
  vaccinia, RSV, reoviruse, rhinovirus, Coxsackie

28
HEK 293 Cells

• Human Embryonic kidney cells
• Easy to transfect
• Often used for electrophysiology
• Transformed by adenovirus

29
COS-7 Cells

• African Green Monkey kidney cells
• Transformed with SV40
• Easy to culture/transfect
• Poor traffickers of membrane proteins not good
  for immunolocalization

30
NIH-3T3 Cells

• Mouse embryonic fibroblast cells
• Can be differentiated into adipocytes
• Good for metabolism studies
• Easy to transfect
• Useful for cell biology studies
• Support growth of wide range of mammalian viruses
• Ex. MLV, MSV, VSV, vaccinia, HSV, and N-tropic
  oncornaviruses C

31
CHO cells

• Chinese Hamster Ovary Cells
• Fibroblast and epithelial-like
• Easy to culture and transfect
• Good for
• Immunofluorescence
• Biosynthetic pathway studies
• Genetics
• Gene Expression
• Toxicity screening

32
PC-12 cells

• Pheochromocytoma12
• Can be differentiated into neuron-like cells
• Difficult to transfect
• Adhere poorly to plastic

33
MDCK Cells

• From canine kidney
• Become polarized in culture
• Good for trafficking studies
• Not so easily transfected/passaged
• Support growth of wide range of mammalian viruses
• Ex., SVEV, VEV, Vaccinia, adeno and reoviruses,
  and Coxsackie B5

34
The Good
Ref. 1
Ref. 1
Jurkat Cells (Human T cell line)
HEK 293 Cells (Human embryonic kidney cell line)
HeLa Cells (Human cervical cancer cell line)
Ref. 2
1. http//www.iclc.it/Listanuova.html 2.
http//www.ambion.com/techlib/misc/rnai_healthy_ce
lls.html
35
The Bad
HeLa Cells (Human cervical cancer cell line)
http//www.ambion.com/techlib/misc/rnai_healthy_ce
lls.html
36
And the Ugly
mycoplasma
fungus/mold
http//www.unc.edu/depts/tcf/badbug_index.htm UNC
Lineberger Comprehensive Cancer Center
37
Keeping the Tissue Culture Hood Sterile

• Wear gloves/lab coat
• If hood is off, wait 10-20 min. for laminar flow
  to be established after turning on (i.e. for air
  to be sterile) and spray surface with 70 ethanol
• Everything that goes in the hood should be
  sprayed down with ethanol-including your hands
  arms!

38
Keeping Your Cultures Sterile

• Once a pipet tip has touched ANYTHING, treat as
  contaminated
• Do not leave media bottles, culture flasks, etc.
  open any longer than necessary
• Work with one cell line at a time to avoid
  cross-contamination

39
More Hood Etiquette

• This is NOT a fume hood, and is not meant for
  volatile chemicals
• Do NOT work with fungal or bacterial cultures in
  the hoodor even anywhere NEAR the hood
• Keep hood free of clutter- this interrupts
  laminar flow

40
Why Should You Be Sterile?

• Contamination will affect cell viability and
  ability to be transected
• Chances are, you are not the only person using
  the hoods, incubators, etc.
• Contamination can be very difficult to get rid of
• This will not make you popular in the lab

41
Safety

• Assume all cultures are hazardous since they may
  harbor latent viruses or other organisms that are
  uncharacterized
• Autoclave all waste
• Treat liquid waste from experiment with bleach
  (at least 2h) before discarding

42
Pretend you are the PIwhat would you tell this
student not to do?
43
(No Transcript)
44
Some Quickies
45
Yesterday I thawed my cells, today they look
dead.What could I have done wrong?(2
explanations)
X X
46
I trypsinized my cells yesterday. When I looked
at them today, they werent adhering to the
dish.What could I have done wrong? (2
explanations)
47
Tissue Cell Culture Resources
Current Protocols Online (Table of contents)
http//www.mrw.interscience.wiley.com/cp/cpmb/cpmb
_contents_fs.html
Cell Line Data Base (CLDB) http//www.biotech.ist.
unige.it/interlab/cldb.html -click on cell line
name index to look up cell line information
American Type Culture Collection (ATCC)
http//www.atcc.org/
Sigmas TC Handbook http//www.sigmaaldrich.com/
Area_of_Interest/Life_Science/Cell_Culture/Helpful
_Resources/Cell_Culture_Handbook.html
QH 585.2 .M355 1997 (library call number)
Mammalian cell culture essential techniques
Doyle, A. (Alan) Chichester Wiley, c1997. Held
by Science Library