Growing Cells in Culture

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Growing Cells in Culture

• Part 1 Terminology

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Cell Culture
The maintenance of cells outside of the living
animal (in vitro) for easier experimental
manipulation and regulation of controls.

• Pros
• Use of animals reduced
• Cells from one cell line are homogenous and have
  same growth requirements, optimizing growing
  patterns.
• In vitro models allow for control of the
  extracellular environment
• Able to monitor various elements and secretions
  without interference from other biological
  molecules that occurs in vivo

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• Cons
• Removal of cells from their in vivo environment
  means removing the cells, hormones, support
  structures and various other chemicals that the
  cells interact with in vivo.
• It is nearly impossible to recreate the in vivo
  environment. The artificial conditions could
  cause cells to de-differentiate which will cause
  them to behave differently and produce proteins
  other than it would in vivo.
• Genotype the genetic make-up of the cell
• Phenotype the appearance and behavior of a cell
  as a result of their genotype. Most often,
  scientists are looking at phenotypic changes in
  their analysis of cells in culture

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Classification of Cell Cultures

• Primary Culture
• Cells taken directly from a tissue to a dish
• Secondary Culture
• Cells taken from a primary culture and passed or
  divided in vitro.
• These cells have a limited number of divisions or
  passages. After the limit, they will undergo
  apoptosis.
• Apoptosis is programmed cell death

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Primary culture from Poeciliopsis lucida (the
desert topminnow)
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Making a Primary Culture
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Cell Lines

• Cell Line
• Cells that have undergone a mutation and wont
  undergo apoptosis after a limited number of
  passages. They will grow indefinitely.
• Transformed cell line
• A cell line that has been transformed by a tumor
  inducing virus or chemical. Can cause tumors if
  injected into animal.
• Hybrid cell line (hybridoma)
• Two cell types fused together with
  characteristics of each

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Our Cell Line

• PLHC-1 Poeciliopsis lucida (topminnow)
• Hepatocellular carcinoma
• Originially from the liver so they are
  hepatocytes
• Epithelial cells
• ATCC CRL-2406
• http//www.atcc.org/
• Lawrence E. Hightowers lab, in culture since
  1985.

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Our Cell Line

• An immortal cell line, but not tumorogenic, will
  reach contact inhibited state
• Originally used to study heat shock response
• These cells maintain a number of differentiated
  cell functions of hepatocytes. The cells possess
  inducible and stable cytochrome P450 (CYF)
  activity.
• Not known to harbor an agent known to cause
  disease in humans

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Growing Cells in Culture

• Part 2 Understanding Cell Behavior

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Confluency

• How covered the growing surface appears
• This is usually a guess
• Optimal confluency for moving cells to a new dish
  is 70-80
• too low, cells will be in lag phase and wont
  proliferate
• Too high and cells may undergo unfavorable
  changes and will be difficult to remove from
  plate.

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Contact Inhibition

• When cells contact each other, they cease their
  growth.
• Cells arrest in G0 phase of the cell cycle
• Transformed cells will continue to proliferate
  and pile upon each other

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Anchorage Dependence

• Cells that attach to surfaces in vivo require a
  surface to attach to in vitro.
• Other cells or specially treated plastic or other
  biologically active coatings
• Blood cells are primary exception.
• Transformed cells may not require attachment.

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Passage number

• The number of times the cells have been removed
  (or split) from the plate and re-plated.
• Always write this on your plate or flask as P

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Growing Cells in Culture

• Part 3 Solutions used in cell culture

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Phosphate Buffered Saline - Ca2 Mg2 Free (PBS)

• Used to wash/remove excess serum that inhibits
  the function of Trypsin-EDTA.
• Calcium will also inhibit the function of TRED.
• Must be warmed in the water bath before use so
  cells are not shocked by cold liquid.

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Trypsin EDTA

• An enzyme used to detach the cells from a culture
  dish.
• Trypsin cleaves peptide bonds (LYS or ARG) in
  fibronectin of the extracellular matrix.
• More about fibronectin and the ECM next week
• EDTA chelates calcium ions in the media that
  would normally inhibit trypsin.
• Trypsin will self digest and become ineffective
  if left in water bath more than 20 minutes.
• Trypsinizing cells too long will reduce cell
  viability

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Trypan Blue

• An exclusion dye
• Living cells cannot take up the dye and will
  appear bright and refractile.
• Dead cells with broken membranes will absorb the
  dye and appear blue.
• Usually add 200 ml of trypan blue to 200 ml of
  cell suspension in eppendorf tube

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Bleach

• Used to destroy any remaining cells in dishes and
  tubes before they are tossed in the trash can.
• Add enough to change media to clear,
• wait 5 minutes,
• rinse solution down sink
• throw away the dish/flask/plate in the trash can.

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Growing Cells in Culture

• Part 4 Equipment

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CO2 incubator

• maintains CO2 level (5-10), humidity and
  temperature (37o C) to simulate in vivo
  conditions.

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Water bath

• To warm media, TRED and PBS before placing on
  cells
• Can harbor fungi and bacteria, spray all items
  with 70 ethanol before placing in the hood.
• Usually takes 10 -15 minutes for media to warm,
  5-10 for TRED to thaw

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Vacuum pump

• For permanent aspiration of liquids (media, PBS
  and TRED).
• Use unplugged glass pasteur pipets, throw into
  sharps box when done.

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Inverted Phase Microscope

• A phase contrast microscope with objectives below
  the specimen.
• A phase plate with an annulus will aid in
  exploiting differences in refractive indices in
  different areas of the cells and surrounding
  areas, creating contrast

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Mechanics of phase microscopy
Shifting of phase by ½ a wavelength Add and
subtract amplitudes to create more contrast
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A comparison
Phase contrast microscopy Light microscopy Can be
used on living cells requires stain, thus killing
cells
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Basic cell culture instructions
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Aseptic Technique

• For best results in tissue culture, we want to
  work to keep microbial (bacteria, yeast and
  molds) contamination to a minimum. To do this,
  there are certain things you must be aware of and
  guidelines to follow.
• Work in a culture hood set-aside for tissue
  culture purposes. Most have filtered air that
  blows across the surface to keep microbes from
  settling in the hood. Turn off the
  UV/antimicrobial light and turn on the hood 30
  minutes prior to entering the hood.
• Wear short sleeves or roll your sleeves up. Turn
  your baseball caps back if you MUST wear them,
  tie long hair back and remove rings and watches.

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• Wash hands with soap and water before beginning
  the procedure and rewash if you touch anything
  that is not sterile or within the hood.
• Spray down your hands, work surface, and anything
  that will go into the hood with 70 ethanol.
  Rewipe at intervals if you are working for a long
  time in the hood. This will reduce the numbers of
  bacteria and mold considerably.
• Do not breathe directly into your cultures,
  bottles of media, etc. This also means to keep
  talking to a minimum. No singing or chewing gum.

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• Work as quickly as you can within limits of your
  coordination. Also, keep bottles and flasks
  closed when you are not working with them. Avoid
  passing your arm or hand over an open bottle.
• Use only sterilized pipets, plates, flasks and
  bottles in the hood for procedures.
• Take special precautions with the sterile pipets.
  Remove them from the package just before use.
  Make certain to set up the numbers on the pipet
  so that they face you. Never mouth-pipet, use the
  pipetting aid. Change pipets for each
  manipulation. If the tip of the pipet touches
  something outside of the flask or bottle, replace
  with a new one. Never use a pipet twice.

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Basic Cell Culture Procedure for Anchorage
Dependent Cells

• View cells using inverted phase microscope
• Aseptically aspirate media
• Rinse media with PBS
• Add Trypsin-EDTA to cells
• Aspirate Trypsin-EDTA
• Incubate cells with layer of Trypsin-EDTA at 37
  C
• Resuspend cells with fresh media
• Take sample and count cells
• Calculate how many cells are needed to add to new
  plate or flask

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Remember

• Some volumes dont need to be exact in cell
  culture
• Rinsing volume of PBS (as long as it fits in the
  dish and is sufficient to rinse the serum).
• Volume of trypsin EDTA as long a bottom of plate
  or flask can be covered.
• Volume of media used to resuspend your cells. The
  same number of cells will be there despite the
  volume of media used.
• Too little resuspension media will result in very
  high cell count and would require more dilution
  (and higher dilution factor). The volume needed
  to seed your next plate would then be very small,
  maybe too small to work with.
• Too much media would result in low cell count/ml
  and you may need a large volume to add to your
  new plate.

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• Volume of cells removed for cell counting.
• You want enough to work with, but not take all of
  your cells from your plate. If you want a
  dilution factor of 2, just add an equal amount of
  trypan blue.
• Exact of cells to be plated
• If you want to plate 2 x 10 5 cells onto your
  plate, but you have 2.1 x 10 5 cells/ml, plating
  1ml will be easier than plating .953 ml.

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Troubleshooting Low Hemacytometer Counts
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Trypsinization not complete

• Trypsin is ineffective
• too cold, be sure to warm sufficiently
• self digested or expired check date, don't warm
  too long
• too much serum left on plate rinse
  plate thoroughly with PBS

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Trypsinization technique

• Trypsin doesn't coat plate, completely add full 2
  mls, lay flask down, count to 10, then remove
• trypsin left on plate too long and then
  aspirated...cells removed along with trypsin
• not left long enough in incubator depends on cell
  line 3T3-L1 can go 1-5 minutes
• flask may need to be tapped or slapped to
  facilitate cell removal(this varies by cell
  line, but ok for 3T3s)

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Resuspension technique

• too much media added more media results in low
  cell/ml, but overall cells on plate should remain
  the same
• cells not sprayed off surface properly
• media and cells not pipetted (gently) up and down
  3-4 times to break up clumps
• too long of time before retrieving sample from
  flask (cells may settle). After mixing with
  trypan, don't wait too long before loading
  hemacytometer.  Get hemacytometer ready while
  trypsinizing cells in incubator

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Stubborn cells

• cells left on plate a long time (gt4 days) will be
  more difficult to remove
• very confluent plate will require more aggressive
  trypsinization because trypsin cannot recach
  plate surface effectively

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Keeping a good lab notebook
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• Lab notebooks provide a convenient place for you
  to keep all of your procedures, data and
  observations in one place.
• If written well, a lab notebook should contain
  everything you need to know to allow you or
  someone else to repeat any experiment you have
  ever performed.
• It can be useful in finding the source of errors
  and unexpected results when problems arise.
• Should your work ever be disputed, a lab notebook
  will provide testimony to your research.
• By following the simple guidelines below, you
  will learn how to keep a good lab notebook.

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• The notebook should be bound (no spiral
  notebooks, please).
• The pages should be numbered either by hand or
  preprinted before using the book.
• Use only permanent ink.
• Write your name, contact information, and dates
  the notebook covers on the first page.
• Skip the next 2-3 pages for a Table of Contents.
  Fill in the experiment name and page numbers as
  they are completed.
• Write the date, experiment title, and partners
  name at the top of each page.

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The first time you use a procedure

• Write the whole procedure in your own words into
  the notebook OR tape in the typed version
• Include a reference to the lab manual page or the
  published procedure.
• Note any changes made to the original procedure.
• Do not just copy the lab manual or procedure word
  for word restate each step simply and clearly.
• If you repeat this procedure later, reference the
  page where it was first performed and write down
  any changes made.

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• All data and observations should be written in
  your notebook at the time you took the
  measurement. Do not write on scratch paper to be
  copied later into your notebook little pieces
  of paper may be lost and data forever lost.
• Remember your lab notebook is extemporaneous
  writing. Keep it neat but do not waste too much
  time making it perfect. Errors should be crossed
  out with a single line (example). Do not scribble
  out mistakes.

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• Write down all calculations, no matter how
  simple, in your notebook. For example, every time
  you perform a cell count, cell viability must be
  calculated and recorded.
• Permanently attach (glue or tape) images,
  computer print outs, and other data in your
  notebook. Date and initial over the corner of the
  attachment. Be sure to label the image with any
  pertinent information. For example, if you place
  a Western Blot image into your notebook, label
  the lanes with what was in each, and the gel
  composition. If the lysates were prepared on a
  date different from the date the gel was run make
  a reference to the page that contains information
  on how the lysates were made. Partners may
  photocopy original data for inclusion in the lab
  notebook.

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• Including complete chemical equations,
  statistical equations, sample calculations, and
  sketches or block diagrams of any apparatus used
  is also good practice.
• Record start and stop times.
• Include conclusions from this data. What does it
  mean and did it work as expected? If unexpected
  results occur, explain why. Include expected
  values (with reference) where appropriate.
• Do not skip pages. Use every page of the
  notebook. If you need to rewrite a page, draw a
  large X through the page, date, initial, and
  start over on the next page. The same applies if
  you dont fill an entire page draw a line through
  the remaining space, date, and initial.

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Six Essential Calculations
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Hemacytometer

• Specialized chamber with etched grid used to
  count the number of cells in a sample.
• use of trypan blue allows differentiation between
  living and dead cells

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Using the Hemacytometer

• Remove the hemacytometer and coverslip
  (carefully) from EtOH and dry thoroughly with a
  kimwipe.
• Center coverslip on hemacytometer
• Barely fill the grid under the coverslip via the
  divet with your cell suspension.
• Count cells in ten squares (5 on each side) by
  following diagram at station.

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Looking at the grid under the phase contrast
microscope
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How the cells will appear

• Bright refractile spheres are living cells,
• Blue cells about the same size as the other cells
  are dead.
• Keep a differential count of blue vs. clear for
  viability determination.
• Sometimes there will be serum debris, and this
  will look red or blue and stringy or
  gloppy--dont count it!

These are blood cells, You will not have this many
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Count 10 squares Any 10 will do but we will
follow convention Watch for stringy,
reddish materialthose arent cells!
serum
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Top group Count cells that touch top and left
lines
DO NOT Count cells that touch bottom and right
lines
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Bottom Group
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Calculate your cells/ml

• Calculate the number of total cells in one ml of
  your suspension.
• Total cells counted x (dilution factor) x
  (10,000)
• number of squares
• Here, dilution factor is 2 and of squares is 10
• (our example 62/10 x2 x104 1.24 x 105)

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Determine your percent viability

• Viability is a measure how many of your cells
  survived your cell culture technique.
• of viable (living) cells x 100
• total number of cells counted
• Our example 54/62 x 100 87.09

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Calculate total of cells in original suspension

• Number of cells per ml x total mls of original
  suspension
• Lets assume 10ml original suspension
• 1.24 x105 x 10 1.24 x 106 cell total
• Total of viable cells available in original
  suspension
• Total number of cells in original suspension x
  viability
• 1.24x106 x 87 1.08x 106 viable cells in the
  original suspension

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Determine the number of cells you need to add to
your flask

• You want the cells to grow happily without
  overcrowding (or being too sparse) before the
  next time you come into class.
• Using the calculation on the next slide, figure
  out the number of cells needed for the size of
  vessel being used
• You need to take into account
• length of time cells are to be grown.
• the size of the cells (not directly in the
  formula)
• their doubling time

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An Exercise

• You will be using a T-25 flask and using cells
  that have a doubling time of 18 hours
• X is the number of cells you want by the time you
  return to passage them (right column of table,
  next slide)
• X0 is the number of cells that were seeded (we
  want to solve for this right now)
• t is the time since plating (hours until the next
  passaging)
• td is the doubling time of the cell line.

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Vessel 3T3-L1 final count 18 hour doubling rate
3.5cm or 6 well plate 1x106
6cm dish or T25 flask 2 x106
10cm dish 5 x 106
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Determine how many mls of cell suspension much to
add to your flask

• of cells needed
• cells/ml

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Determine total mls fresh media you will need
to add to dish or flask

• Use table in VISTA to see how many mls will fit
  in your flask (or we will tell you).
• Volume flask will hold mls suspension to
  you plan to add

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Growing Cells in Culture

• Part 5 The protocol

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Observing cells in culture

• Check color of media
• Healthy growth usually leaves media slightly
  orange
• Too yellow means bacterial growth
• Too purple means low carbon dioxide, cells dead
• Observe cells under phase microscope
• Spread out or rounded?
• How confluent?

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What to do with growing cells

• If they are at least 70-80 confluent
• Subculture them
• Also called passing or splitting
• Remove media, remove cells, resuspend and
  transfer some to a new plate

• If they are not very confluent
• Lift and replace onto same plate
• Culture more than 4 days old for our cells
• Remove old media, lift cells from plate and
  resuspend in fresh media on same plate
• Feed them
• Culture less than 4 days old
• Remove old media and replace with fresh, warm
  media

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Brief subculturing preview

• Remove media, lift cells from plate
• Resuspend cells in fresh media
• Count cells and determine viability
• Seed new plates with appropriate of cells and
  volume of media

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Some volumes that do not need to be exactbut
follow our recommendations until you are
comfortable

• Rinsing volume of PBS
• Volume of trypsin EDTA
• Volume of media to resuspend cells
• Record how much
• Volume of cells removed for counting
• Exact of cells to be plated

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You will need to return to take care of your cells

• Thursday or Friday is an in between point before
  next week.
• First time through may require up to an hour
• If one member cannot make the return time, that
  person should work in hood tonight.
• Choose times that will be consistent each week