Basics of

Tissue Culture
 History of Cell culture

 Harrison, R.G. Observations on the living developing nerve fiber.

Proc. Soc. Exp. Biol. Med. 4:140-143, 1907.

 Fell, H.B. Recent advances in organ culture.

Sci. Prog. 162:212, 1952.

 Eagle, L. Growth characteristics of virus-transformed cells.

J. Exp. Med. 131:863-879, 1970.

 Tissue culture definitions

Organ culture :

Whole organ culture. E.g., Liver, pancreas, etc.

Explant culture :

Culturing a small part of the organ (tissue mass).

Involves placing a piece of tissue into the tissue culture dish
and allowing cells to migrate out from the tissue.

Cell culture :

Isolating or separating the cells from tissue mass

and culturing. Maintenance of cells outside of the living
animal for easier experimental manipulation and regulation of controls.
 PROS
• Use of animal 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 occur in vivo
 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 protiens othr than
it would in vivo.
 Cell culture classification

 Primary cell 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.

 Continuos cell lines

 Cell culture classification

Primary cell culture:

 Tissues collected directly from the organism

 Cells isolated using mechanical or chemical dissociation and cultured

 These cells grow only for a short time period (5-10 divisions)

Continuos cell lines:

 Also isolated from the organism but are manipulated using

physical, chemical and biological methods.
E.g., Radiation, Mutagens, Viruses etc.

 Once established they grow for longer periods (100-200 divisions)

 Making a primary culture
• Remove tissue
• Mince or chop
• Digest with proteolytic enzyme
• Place in cuture
 Cell line
• Cells that have undergone mutation and wont
undergo apoptosis after a limited number of
passages. They will grow indefinetly.
• Transformed cell line: Acell line that has been
transformed by a tumor inducing virus or
chemical. Can cause tumors if injected in to
animal.
• Hybrid cel line (hybridoma): two cell type fuse
together with characteristics of each.
 Cell culture classification

Continuous cell lines are of two types:

Suspension culture

Adhered (Monolayer) culture

Explant culture
Isolation of cells
 Growing cells in culture
(understanding cell behaviour)
• Confluency is the full growth in dish
• This is usually a guess
• Optimal confluency for moving cells to a new dish
is 70-80%.
• Too low, cells will be in lagphase and wont
proliferate
• Too high and cells may undergo unfavourable
changes and will be difficult to remove from plate.
 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.
 Anchorage dependence
• Cells that attach to surfac in vivo require a
surface to attach to in vitro.
• Blood cells are primary exceptions.
• Transformed cells may not require
attachments.
 Passage number
• The number of times cells have been
removed or split from the plate and
replated.
• This is indicated as p#
 Solutions used in culture
• PBS Ca2+ mg2+ free
• Used to wash or remove excess serum that
inhibits the func of trypsin-edta
• TRYPSN –EDTA: detach cells from the
culture dish
• Trypsin cleaves peptide bonds (lys or arg)
in fibronectin of the extracellular matrix.
• EDTA chelates ca ions in the media that would
normally inhibit trypsin.
• Trypsinising cells too long will reduce cell
viability.
• 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.
• Bleach: used to destroy any remaining cells
in dishes and tubes before they are tossed In
the trash can.
• Add enough to chane media to clear, wait 5
minutes, rinse the solution, down sink,
throw away the dish or flask
 Personal Safety

Disinfection:
Work surfaces, culture waste and equipment must be kept clean to
prevent contamination.

Alcohol:
70% ethanol or 60-70% isopropanol

Waste disposal:
Dispose regularly, do not allow to accumulate tissue culture waste
(media, pipettes, flasks, etc.,)
 Introduction

 Work environment and surface

 Plasticware and glassware

 Handling technique

 Sterilization of solutions for maintenance, growth, and treatment

 Methods and Principle

 Freezing of cells

 Thawing / Revival of cells

 Equipment in cell culture room

 Sterile room.

 Biosafety cabinet

 CO2 incubator

 Inverted microscope (equipped with camera)

 Refrigerator dedicated to storage of cell culture solutions

 Water bath

 Deep freezers (-20C and -80C)

 Liquid Nitrogen containers for storing cells

 Mechanical pipetter

 Vacuum pump.
Biosafety cabinet
 CO2 Incubator

Temp : 37C
CO2 : 5%
Humidity : 95
%
 waterbath
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
 Vacuum pump
For permanent
aspiration of
liquids (media, PBS
and TRED).

Use unplugged glass

pasteur pipets, throw
into sharps box
when done.
 Inverted 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
Phase contrast microscopy Light microscopy
Deep freezers
Liquid Nitrogen
 Materials for cell culture

 Complete cell culture medium, appropriate for the cell lines

 Tissue culture flasks and dishes of appropriate sizes

 Sterile pipettes

 Micropipettes and sterile tips

 70% alcohol
 Plastic ware and Glassware

Plasticware:
Pipettes, Flasks, Petridishes, plates etc

Glassware:
Bottles
 Advantages and Disadvantages

Advantages Disadvantages

Single cell type Effects of serum, plasma, etc.

Controlled conditions Poorly represent the in vivo

situation

Easy access Most continuous cell lines retain

little phenotypic differentiation.
 Cell Morphology

Human muscle cells Human endothelial cells

Primary cell
culture

THP Monocytes RIN cells

Continuous cell
line

Suspension Adhered
 Medium formulation

Composition of medium:

 Complement of amino acids, vitamins, organic salts, glucose, phenol red,

antibiotics and serum (source of growth factors, hormones and
attachment factors.

 Serum added to growth medium as supplement usually as 10% fetal

bovine serum or horse serum.

pH of medium:

 Should range between 7.2 to 7.4

 Phenol red used as the indicator dye
 Mostly buffered by NaHCO3
 Medium formulation

 Penicillin

 Streptomycin

 Amphotericin B

 Theory - eliminates, keeps out contamination

 Fact - once contaminated, that is it…

 Fact - it is possible to salvage cultures...

 Medium formulation

Two Forms of medium available - Liquid and Powdered form

Liquid medium:

Ready to use.

Disadvantage: to be used as quick as possible.

Powdered:

Prepared by addition of all the components (powdered medium, glucose,

NaHCO3, antibiotics) and sterilized

 Sterilization of medium and solutions

 Medium is sterilized using a filtration unit with sterile filter with a pore size
of 0.22m

 Medium should be not autoclaved - heat will destroy some of the

components in the medium.

 Sterility check is carried by either incubating prepared medium at 37°C in

CO2 incubator or adding small volume in nutrient broth for 48 hrs

 Storage:

Store medium at 4C
Store in dark place - some components are light sensitive
Fetal bovine or horse serum can be added separately
 Handling techniques

 Bring medium, serum, trypsin to 37°C before adding to cells.

 Keep sterile flasks, bottles, petridishes, etc covered until ready to use.
 Subculture of cell lines in suspension culture

 Dilution

 Total replacement of medium

 Triturate gently to disrupt cell clumps

 Remove 1 ml of cell counting

 Dilute with growth medium to 2 X 105 cells

 Determine cell number by counting, and viability by trypan blue exclusion

 Sub culture of cell line in monolayer culture

 Trypsinize cells

 Resuspend cells in growth medium

 Replate an aliquot of cell suspension in new flasks containing growth medium

 Disperse cells on flasks bottom by slight rotation (cell usually attach with 4 hrs)
 Estimation of cell number

1) Automated method - coulter counting

Amp Readout

2) Hemocytometer counting
 Hemocytometer counting / Neubauer chamber

C = cell concentration
C = N x 10 Cells/ml
4
N = number of cells

N = 1+2+3+4
1 2 4

3 4
 Contamination

Happens to even the best…

Big problem. Can take over laboratories

Fungal - yeast & moulds

Bacterial

Mycoplasma - filterable bacteria which will pass across 0.2 um filter.

Contamination
 Cryopreservation of cells

 Cells should be stored in liquid nitrogen

 Serve as backup for loss from laboratory accident

 Cells that have not undergone genetic drift

 Cells must reach critical freezing points in stepwise fashion to prevent

internal ice crystal formation and water outflow.

 Freezing cells

 Aspirate medium from preconfluent cells trypsinize cells with 2 ml 0.05%

trypsin + 0.25% EDTA return to 37°C for 5 - 10 min, rock solution 4- 5 times
over cells.

 Dislodge cells and resuspend in 10 ml freezing medium - containing 90% FBS

and 10% DMSO.

 Transfer 1 ml cells (~ 1 x 106) into freezing vial labeled with name of cell line,
date and passage number & place in -80C freezer overnight or in controlled
rate freezer

 Transfer into liquid nitrogen chamber

 Thawing of frozen cells

 Remove vial from liquid nitrogen and place it in water bath (37°C) until
fully thawed.

 Remove vial from waterbath when frozen chunks disappear

 Rinse the vial with 70% ethanol to reduce the chances of cross
contamination

 Transfer vial contents aseptically into a T-25 flasks and slowly add growth
medium while mixing.

 Incubate cell at 37°C and within 24 hrs, change medium every day to feed
cells.
 Basic cell culture instructions
• 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.
• 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. Re wipe 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.
• 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.
 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
 Remember
• Some volumes don’t 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.
• 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.
 Troubleshooting Low
Hemacytometer Counts
• 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
• 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)
 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
 Stubborn cells
• cells left on plate a long time (>4 days) will
be more difficult to remove
• very confluent plate will require more
aggressive trypsinization because trypsin
cannot recach plate surface effectively
 Keeping a good lab notebook
• 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.
• 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 partner’s name at the
top of each page.
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.
• 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.
• 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.
• 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 don’t fill an entire page draw a line through
the remaining space, date, and initial.
 Six Essential Calculations
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
 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.
 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--don’t
count it!
 Calculation for total count and
viability count
• 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)
• 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%

Calculate total # of cells in original
suspension
Number of cells per ml x total mls of original suspension

Let’s 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
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
 Growing Cells in Culture: protocol
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?
 What to do with growing cells
• If they are at least 70- •If they are not very confluent
80% confluent •Lift and replace onto same
plate
• Subculture them
•Culture more than 4 days
– Also called passing or old for our cells
splitting •Remove old media, lift
• Remove media, remove cells from plate and
cells, resuspend and resuspend in fresh media
on same plate
transfer some to a new
•“Feed” them
plate •Culture less than 4 days
old
•Remove old media and
replace with fresh, warm
 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
 Some volumes that do not need to be exact—
but 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
“Tissue culture can be a powerful technique if conducted properly and a great

waste of time and money when done sloppily”

Bernice Martin, Ph.D.