Types of Cells Grown in Culture
Tissue culture is a term that refers to both organ culture and cell culture. Cell
cultures are derived from either primary tissue explants or cell suspensions.
Primary cell cultures typically will have a finite life span in culture, whereas
continuous cell lines are, by definition, abnormal and are often transformed cell
Work Area and Equipment
Preservation and Storage
- Laminar Flow Hoods: There are 2 types of laminar flow hoods, vertical and
horizontal, and both types of hoods are available in the microbiology
laboratory. The vertical hood is best for working with hazardous organisms,
since the aerosols that are generated in the hood are filtered out before they
are released into the surrounding environment. Horizontal hoods are
designed so that the air flows directly at the operator, hence, they are not
useful for working with hazardous organisms, but are the best protection
for your cultures.
Both types of hoods have continuous displacement of air that passes
through a HEPA (high efficiency particle) filter that removes particulates
from the air. The hoods are equipped with a shortwave UV light that can
be turned on for a few minutes to sterilize the surfaces of the hood, but
be aware that only exposed surfaces will be accessible to the UV light. Do
not put your hands or face near the hood when the UV light is on, as the
shortwave light can cause skin and eye damage. The hoods should be
turned on about 10–20 minutes before being used. Wipe down all surfaces
with ethanol before and after each use.
- Microscopes: Inverted phase contrast microscopes are used for visualizing
the cells. Microscopes should be kept covered and the lights turned down
when not in use. Before using the microscope or whenever an objective is
changed, check that the phase rings are aligned.
- CO2 Incubators: The cells are grown in an atmosphere of 5%–10% CO2,
because the medium used is buffered with sodium bicarbonate/carbonic
acid and the pH must be strictly maintained. Culture flasks should have
loosened caps to allow for sufficient gas exchange. The humidity must also
be maintained for those cells growing in tissue culture dishes, so a pan
of water is kept filled at all times.
- Preservation: Cells are stored in liquid nitrogen.
- Vessels: Anchorage-dependent cells require a nontoxic, biologically inert,
and optically transparent surface that will allow cells to attach and allow
movement for growth. The most convenient vessels are specially-treated
polystyrene plastic that are supplied sterile and are disposable. These
include petri dishes, multiwell plates, microtiter plates, roller bottles, and
is used to preserve tissue culture cells, either in the liquid phase or
in the vapor phase. Freezing can be lethal to cells due to the effects of damage
by ice crystals, alterations in the concentration of electrolytes, dehydration, and
changes in pH. To minimize the effects of freezing, several precautions are
taken. First, a cryoprotective agent that lowers the freezing point, such as
glycerol or DMSO, is added.
The freezing medium is typically 90% serum, 10% DMSO. In addition, it is
best to use healthy cells that are growing in log phase and to replace the
medium 24 hours before freezing. Also, the cells are slowly cooled from room
temperature to –80°C to allow the water to move out of the cells before it freezes.
The optimal rate of cooling is 1°C–3°C per minute. Some labs have fancy
freezing chambers to regulate the freezing at the optimal rate by periodically
pulsing in liquid nitrogen.
The tubes filled with 200 mL of isopropanol at room temperature and the
freezing vials containing the cells are placed in the container. The container is
placed in the −80°C freezer. The effect of the isopropanol is to allow the tubes
to come to the temperature of the freezer slowly, at about 1°C per minute.
Once the container has reached −80°C the vials are removed and immediately
placed in the liquid nitrogen storage tank. Cells are stored at liquid nitrogen
temperatures because the growth of ice crystals is retarded below −130°C.
Cultures should be examined daily, observing the morphology, the color of the
medium, and the density of the cells. A tissue culture log should be maintained.
The log should contain the name of the cell line, the medium components, and
any alterations to the standard medium, the dates on which the cells were split
and/or fed, a calculation of the doubling time of the culture (this should be
done at least once during the semester), and any observations relative to the
- Growth Pattern: Cells will initially go through a quiescent or lag phase that
depends on the cell type, the seeding density, the media components, and
previous handling. The cells will then go into exponential growth where
they have the highest metabolic activity. The cells will then enter into
stationary phase where the number of cells is constant. This is characteristic
of a confluent population (where all growth surfaces are covered).
- Harvesting: Cells are harvested when the cells have reached a population
density that suppresses growth. Ideally, cells are harvested when they are
in a semiconfluent state and are still in log phase. Cells that are not
passaged and are allowed to grow to a confluent state can sometime lag
for a long period of time, and some may never recover. It is also essential
to keep your cells as happy as possible to maximize the efficiency of
transformation. Most cells are passaged (or at least fed) 3 times a week.
Mechanical: A rubber spatula can be used to physically remove the cells
from the growth surface. This method is quick and easy, but is also disruptive
to the cells and may result in significant cell death. This method is best when
harvesting many different samples of cells for preparing extracts, i.e., when
viability is not important.
- Suspension cultures: Suspension cultures are fed by dilution into a fresh
- Adherent cultures: Adherent cultures that do not need to be divided can
simply be fed by removing the old medium and replacing it with fresh
medium. When the cells become semiconfluent, several methods are
used to remove the cells from the growing surface so that they can be
Proteolytic enzymes:. Trypsin, collagenase, or pronase,
usually in combination
with EDTA, causes cells to detach from the growth surface. This
method is fast
and reliable but can damage the cell surface by digesting exposed
cell surface proteins. The proteolysis reaction can be quickly
terminated by the addition of
complete medium containing serum.
EDTA: EDTA alone can also be used to detach cells and seems to be gentler
on the cells than trypsin.
The standard procedure for detaching adherent cells is as follows:
- Visually inspect daily.
- Release cells from monolayer surface
- Wash once with a buffer solution.
- Treat with dissociating agent.
- Observe cells under the microscope. Incubate until cells become rounded
and loosen when flask is gently tapped with the side of the hand.
- Transfer cells to a culture tube and dilute with medium containing
- Spin down cells, remove supernatant, and replace with a fresh medium.
Count the cells in a hemocytometer, and dilute as appropriate into a
- Media and Growth Requirements
- Physiological parameters
- emperature –37°C for cells from homeotherms.
- pH–7.2–7.5 and osmolality of medium must be maintained.
- Humidity is required.
- Gas phase-bicarbonate concentration and CO2 tension in equilibrium.
- Visible light can have an adverse effect on cells; light-induced
production of toxic compounds can occur in some media; cells
should be cultured in the dark and exposed to room light as little
- Medium requirements: (often empirical)
- Bulk ions—Na, K, Ca, Mg, Cl, P, Bicarb, or CO2.
- Trace elements—iron, zinc, selenium.
- Sugars—glucose is the most common.
- Amino acids—13 essential ones.
- Vitamins—B, etc.
- Serum contains a large number of growth-promoting activities, such
as buffering toxic nutrients by binding them, neutralizes trypsin
and other proteases, has undefined effects on the interaction between
cells and substrate, and contains peptide hormones or hormonelike
growth factors that promote healthy growth.
- Antibiotics, although not required for cell growth, are often used to
control the growth of bacterial and fungal contaminants.
For our purposes, we will use the following media components:
Serum—10% fetal calf
- Glutamine—1%—an essential amino acid that tends to be unstable—
it is typically stored frozen and added separately; its half-life in
medium at 4°C is 3 weeks, at 37°C, 1 week.
- Antibiotic/antimycotic—1% (streptomycin, amphotericin B,
penicillin; spectrum: bacteria, fungi and yeast).
- Feeding—2–3 times/week.
- Measurement of growth and viability. The viability of cells can be
observed visually using an inverted phase contrast microscope.
Live cells are phase bright; suspension cells are typically rounded
and somewhat symmetrical; adherent cells will form projections
when they attach to the growth surface. Viability can also be
assessed using the vital dye, trypan blue, which is excluded by live
cells but accumulates in dead cells. Cell numbers are determined
using a hemocytometer.
Assume all cultures are hazardous since they may harbor latent viruses or other
organisms that are uncharacterized. The following safety precautions should
also be observed:
Tissue Culture Methods
- Pipetting: use pipette aids to prevent ingestion and keep aerosols down to
- No eating, drinking, or smoking.
- Wash hands after handling cultures and before leaving the lab.
- Decontaminate work surfaces with disinfectant (before and after).
- Autoclave all waste.
- Use biological safety cabinet (laminar flow hood) when working with
hazardous organisms. The cabinet protects worker by preventing airborne
cells and viruses released during experimental activity from escaping the
cabinet; there is an air barrier at the front opening and exhaust air is
filtered with a HEPA filter.
- Make sure the cabinet is not overloaded and leave exhaust grills in the
front and the back clear (helps to maintain a uniform airflow).
- Use aseptic technique.
- Dispose of all liquid waste after each experiment and treat with bleach.
Each student should maintain his or her own cells throughout the course of the
experiment. These cells should be monitored daily for morphology and growth
characteristics, fed every 2 to 3 days, and subcultured when necessary. A
minimum of two 25-cm2
flasks should be carried for each cell line; these cells
should be expanded as necessary for the transfection experiments. Each time
the cells are subcultured, a viable cell count should be done, the subculture
dilutions should be noted, and after several passages, a doubling time
determined. As soon as you have enough cells, several vials should be frozen
away and stored in liquid N2
. One vial from each freeze down should be
thawed 1–2 weeks after freezing to check for viability. These frozen stocks will
prove to be vital if any of your cultures become contaminated.
- Media preparation: Each student will be responsible for maintaining his or
her own stock of cell culture media; the particular type of media, the sera
type, and concentration, and other supplements will depend on the cell
line. Do not share media with your partner (or anyone else), because if a
culture or a bottle of media gets contaminated, you have no back-up. Most
of the media components will be purchased prepared and sterile. In general,
all you need to do is sterilely combine several sterile solutions. To test
for sterility after adding all components, pipette several mL from each
media bottle into a small sterile petri dish or culture tube and incubate at
37°C for several days. Use only media that has been sterility-tested. For
this reason, you must anticipate your culture needs in advance so you can
prepare the reagents necessary. But, please, try not to waste media. Anticipate
your needs but don’t make more than you need. Tissue culture reagents
are very expensive; for example, bovine fetal calf serum cost ~$200
per 500 mL. Some cell culture additives will be provided in a powdered
form. These should be reconstituted to the appropriate concentration with
double-distilled water (or medium, as appropriate) and filtered (in a sterile
hood) through a 0–22 µm filter.
All media preparation and other cell culture work must be performed in
a laminar flow hood. Before beginning your work, turn on the blower for
several minutes, wipe down all surfaces with 70% ethanol, and ethanolwash
your clean hands. Use only sterile pipettes, disposable test tubes,
and autoclaved pipette tips for cell culture. All culture vessels, test tubes,
pipette tip boxes, stocks of sterile eppendorfs, etc., should be opened only
in the laminar flow hood. If something is opened elsewhere in the lab by
accident, you can probably assume it’s contaminated. If something does
become contaminated, immediately discard the contaminated materials into
the biohazard container and notify the instructor.
- Growth and morphology: Visually inspect cells frequently. Cell culture is
sometimes more an art than a science. Get to know what makes your cells
happy. Frequent feeding is important for maintaining the pH balance of
the medium and for eliminating waste products. Cells do not typically like
to be too confluent, so they should be subcultured when they are in a
semiconfluent state. In general, mammalian cells should be handled gently.
They should not be vortexed, vigorously pipetted or centrifuged at greater
than 1500 g.
- Cell feeding: Use prewarmed media and have cells out of the incubator for
as little time as possible. Use 10–15 mL for T-25s, 25–35 mL for T-75s, and
50–60 mL for T-150s.
- Suspension cultures: Feeding and subculturing suspension cultures are
done simultaneously. About every 2–3 days, dilute the cells into fresh
media. The dilution you use will depend on the density of the cells and
how quickly they divide, which only you can determine. Typically 1:4
to 1:20 dilutions are appropriate for most cell lines.
- Adherent cells: About every 2–3 days, pour off old media from culture
flasks and replace with fresh media. Subculture cells as described below
before confluency is reached.
- Subculturing adherent cells: When adherent cells become semiconfluent,
subculture using 2 mm EDTA or trypsin/EDTA.
- Remove medium from culture dish and wash cells in a balanced salt
solution without Ca++ or Mg++. Remove the wash solution.
- Add enough trypsin-EDTA solution to cover the bottom of the culture
vessel and then pour off the excess.
- Place culture in the 37°C incubator for 2 minutes.
- Monitor cells under microscope. Cells are beginning to detach when
they appear rounded.
- As soon as cells are in suspension, immediately add the culture medium
containing serum. Wash cells once with serum-containing medium and
dilute as appropriate (generally 4- to 20-fold).
- Prepare a 2 mm EDTA solution in a balanced salt solution (i.e., PBS
without Ca++ or Mg++).
- Remove the medium from the culture vessel by aspiration and wash the
monolayer to remove all traces of serum. Remove salt solution by aspiration.
- Dispense enough EDTA solution into culture vessels to completely cover
the monolayer of cells.
- The coated cells are allowed to incubate until cells detach from the
surface. Progress can be checked by examination with an inverted
microscope. Cells can be gently nudged by banging the side of the flask
against the palm of the hand.
- Dilute cells with fresh medium and transfer to a sterile centrifuge tube.
- Spin cells down, remove supernatant, and resuspend in culture medium
(or freezing medium if cells are to be frozen). Dilute as appropriate into
- Thawing frozen cells
- Remove cells from frozen storage and quickly thaw in a 37°C waterbath
by gently agitating vial.
- As soon as the ice crystals melt, pipette gently into a culture flask
containing prewarmed growth medium.
- Log out cells in the “Liquid Nitrogen Freezer Log” Book.
- Freezing cells
- Harvest cells as usual and wash once with complete medium.
- Resuspend cells in complete medium and determine cell count/viability.
- Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/
10% DMSO, at 106–107; cells/mL. Keep cells on ice.
- Transfer 1-mL aliquots to freezer vials on ice.
- Place in a Mr. Frosty container that is at room temperature and has
- Place the Mr. Frosty in the –70°C freezer overnight. Note: Cells should
be exposed to freezing medium for as little time as possible prior to
- The next day, transfer to liquid nitrogen (DON’T FORGET) and log in
the “Liquid Nitrogen Freezer Log” Book.
- Viable cell counts: Use a hemocytometer to determine total cell counts and
viable cell numbers.
Trypan blue is one of several stains recommended for use in dye exclusion
procedures for viable cell counting. This method is based on the principle that
live cells do not take up certain dyes, whereas dead cells do.
- Prepare a cell suspension, either directly from a cell culture or from a
concentrated or diluted suspension (depending on the cell density) and
combine 20 µL of cells with 20 µL of trypan blue suspension (0.4%). Mix
thoroughly and allow to stand for 5–15 minutes.
- With the cover slip in place, transfer a small amount of trypan blue-cell
suspension to both chambers of the hemocytometer by carefully touching
the edge of the cover slip with the pipette tip and allowing each chamber
to fill by capillary action. Do not overfill or underfill the chambers.
- Starting with 1 chamber of the hemocytometer, count all the cells in the
1-mm center square and each of the four 1-mm corner squares. Keep a
separate count of viable and nonviable cells.
- If there are too many or too few cells to count, repeat the procedure, either
concentrating or diluting the original suspension as appropriate.
- The circle indicates the approximate area covered at 100X microscope
magnification (10X ocular and 10X objective). Include cells on top and left,
touching the middle line. Do not count cells touching the middle line at
the bottom and right. Count 4 corner squares and the middle square in
both chambers and calculate the average.
- Each large square of the hemocytometer, with cover slip in place, represents
a total volume of 0.1 mm3 or 10−4 cm3. Since 1 cm3 is equivalent to
approximately 1 mL, the total number of cells per mL will be determined
using the following calculations:
Cells/mL = average cell count per square × dilution factor × 104;
Total cells = cells/mL × the original volume of fluid from which the cell
sample was removed; % Cell viability = total viable cells (unstained)/total
cells × 100.
|Plant Growth Regulators Commonly Used in Plant Tissue Culture