Web Guider
Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Protocols / Cellular Biology / Tissue Culture Technque 1, 2, 3, 4.
Tissue Culture Technique 1
by Julie B. Wolf,
UMBC
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.
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.
III. PRESERVATION
AND STORAGE. Liquid N2 is used to preserve tissue culture cells, either in the liquid phase (-196C) or in the vapor phase (-156C).
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¡ãC to allow the water to move out of the cells before it freezes. The optimal rate of cooling
is 1¡ã-3¡ãC 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 -80C freezer. The effect of the isopropanol is to allow the tubes to come to the temperature of the freezer slowly,
at about 1C per minute. Once the container has reached -80C (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 -130C. 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 37C water bath with moderate shaking. As soon as the last ice
crystal is melted, the cells are immediately diluted into prewarmed medium.