Tissue Culture Methods
I.
TYPES OF CELLS GROWN IN CULTURE
Tissue
culture is often a generic term that refers to both organ culture and cell
culture and the terms are often used interchangeably. 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 lines.
II.
WORK AREA AND EQUIPMENT
A.
Laminar flow hoods. There are two types of laminar flow hoods, vertical and
horizontal. The vertical hood, also known as a biology safety cabinet, 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 such 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. In a vertical hood, the filtered air
blows down from the top of the cabinet; in a horizontal hood, the filtered air
blows out at the operator in a horizontal fashion. NOTE: these are not fume
hoods and should not be used for volatile or explosive chemicals. They
should also never be used for bacterial or fungal work. The hoods are
equipped with a short-wave 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 short wave 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. Keep the hood as free of
clutter as possible because this will interfere with the laminar flow air
pattern.
B. 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. Cells should be left out of the incubator for
as little time as possible and the incubator doors should not be opened for very
long. 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.
C.
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.
D.
Preservation. Cells are stored in liquid nitrogen (see Section III-
Preservation and storage).
E.
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, multi-well plates, microtiter plates, roller bottles, and screwcap
flasks - T-25, T-75, T-150 (cm2 of surface area). Suspension cells
are either shaken, stirred, or grown in vessels identical to those used for
anchorage-dependent cells.
III.
PRESERVATION AND STORAGE. Liquid N2 is used to preserve tissue
culture cells, either in the liquid phase (-196°C) or in the vapor phase
(-156°C). 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 which lowers the freezing point, such as
glycerol or DMSO, is added. A typical freezing medium is 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°-3°C per minute. Some labs
have fancy freezing chambers to regulate the freezing at the optimal rate by
periodically pulsing in liquid nitrogen. We use a low tech device called a Mr.
Frosty (C#1562 -Nalgene, available from Sigma). The Mr. Frosty is filled with
200 ml of isopropanol at room temperature and the freezing vials containing the
cells are placed in the container and 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 (about 4 hours or, more conveniently, overnight) the vials are
removed from the Mr. Frosty 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. To maximize recovery of the
cells when thawing, the cells are warmed very quickly by placing the tube
directly from the liquid nitrogen container into a 37°C water bath with moderate
shaking. As soon as the last ice crystal is melted, the cells are immediately
diluted into prewarmed medium.
IV.
MAINTENANCE
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 that is
separate from your regular laboratory notebook. 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 morphology, etc.
A. 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).
B.
Harvesting. Cells are harvested when the cells have reached a population
density which suppresses growth. Ideally, cells are harvested when they are in a
semi-confluent 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) three times a week.
1.
Suspension culture. Suspension cultures are fed by dilution into fresh medium.
2. 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 semi-confluent, several methods are used to remove the cells from
the growing surface so that they can be diluted:
-
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.
-
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:
1.
Visually inspect daily
2.
Release cells from monolayer surface
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a. wash once with a buffer solution
b. treat with dissociating agent
c. observe cells under the microscope. Incubate until cells become
rounded and loosen when flask is gently tapped with the side of the
hand.
d. Transfer cells to a culture tube and dilute with medium containing
serum.
e. Spin down cells, remove supernatant and replace with fresh medium.
f. Count the cells in a hemacytometer, and dilute as appropriate into
fresh medium. |
C. Media
and growth requirements
1.
Physiological parameters
-
A.
temperature - 37C for cells from homeother
-
B. pH -
7.2-7.5 and osmolality of medium must be maintained
-
C. humidity
is required
- D. gas phase - bicarbonate conc. and CO2
tension in equilibrium
-
E. 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 as possible;
2. Medium
requirements: (often empirical)
A. Bulk
ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2
B. Trace elements - iron, zinc, selenium
C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. 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 hormone-like growth factors that
promote healthy growth.
H. antibiotics - although not required for cell growth, antibiotics are
often used to control the growth of bacterial and fungal contaminants.
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