THE CELLS OF MANY ANIMAL TISSUES can be kept alive for long periods outside the animal by means of the technique known as tissue culture. In some cases the original cells maintain themselves without dividing; in others they divide repeatedly. Essentially tissue culture consists in transferring cells from an animal to a glass vessel containing the appropriate nutrients at body temperature. One line of cells established in this way has been maintained for almost 20 years, long after the death of the animal from which they came. Such cells maintain their vitality and show no evidence of aging. Tissue culture thus offers insights into some intriguing questions. Do the cultured cells acquire a longevity they lack when they are part of an animal? Or is this longevity an intrinsic property of the cell, one that disappears when the cell functions .IS a member of the highly organized and complex cellular community that constitutes the intact animal?

Until recent years experiments with tissue cultures were all but closed to amateurs. Mastery of the essential procedures required a long apprenticeship. The ingredients of the nutrient mixtures were difficult to obtain and even more difficult to compound. The procedure called for rigorous routines to prevent the infection of cultures by bacteria.

Today ready-made ways of solving such problems are at hand. Nutrient mediums can be bought inexpensively from distributors of biological supplies. Bacterial infections are controlled by antibiotics. Cultures of living cells can also be bought. With such materials Ted M. Fancolli, who attends the American River Junior College in Sacramento, Calif., has developed simplified methods of making tissue cultures. The methods require little more skill than growing bacteria on a plate of nutrient agar. In effect these procedures place in the amateur's hands a powerful tool for looking into such diverse matters as the structure and function of cells, the susceptibility of cells to various bacteria and viruses, the nutrition of cells and the effects of drugs and radiation on cells.

"Cell cultures," Fancolli writes, "have been grouped in three classes according to the origin of the cells and their behavior. Those established directly from animal tissue are known as primary cultures. Examples are cells from the kidneys of rhesus monkeys that are used in the production of poliomyelitis virus for both the Salk and the Sabin vaccines. Primary cultures can be established from almost any kind of tissue, but they must be prepared fresh each time they are used, necessitating a constant source of tissue.

"The cells of some primary cultures can be serially subcultivated. They are then known as cell strains and will usually persist through 40 or more generations before dying out. In form and structure, cell strains do not differ significantly from primary cultures.

"For reasons that remain obscure the cells of some strains continue to reproduce indefinitely. Such cultures are known as cell lines. The oldest culture of this type, called the L line, was established in 1947 by Wilton R. Earle of the National Cancer Institute from tissue taken from a male mouse 100 days old. The oldest culture of malignant origin is the 'HeLa' cell line taken from a human cancer in 1952. It has since become one of the most extensively investigated cell lines. In contrast to primary cultures, cell lines reproduce indefinitely and contain an abnormal number of chromosomes. They grow much faster than cell strains.

"All cell cultures require the same growth factors and nutrients at approximately the same concentrations regardless of the animal from which the tissue is taken. This astonishing uniformity of metabolism is quite different from the requirements of bacteria and other microorganisms, which exhibit varied nutritional needs. Twenty-nine factors appear to be enough for supporting the growth of most cell cultures: 12 amino acids, eight vitamins, glutamine, dextrose or glucose, six inorganic salts and serum protein. Compared with what an animal needs in its diet, a cell culture requires a greater variety of amino acids but fewer vitamins in its diet. The fact that a single medium can be used to grow a wide variety of cell strains and lines enables the experimenter to maintain many different kinds of cell for study.

"Before setting up a tissue culture the beginner should learn the elements of standard bacteriological procedures as previously discussed in this department [March, 1958]. An autoclave is almost indispensable for sterilizing glassware, certain mediums and reagents. A large pressure cooker can serve as the autoclave. Materials placed in the autoclave will be thoroughly sterilized in 15 to 20 minutes at 121 degrees centigrade, the temperature of steam at a pressure of 15 pounds per square inch. Start timing the sterilization after the pressure of the autoclave reaches 15 pounds. When sterilizing apparatus in the autoclave, always loosen the screw caps of bottles so that steam can reach the inside. Cool the autoclave slowly, particularly after sterilizing fluids, to prevent the contents from boiling when returned to atmospheric pressure.

"Particles of dead bacteria, molds and yeasts suspended in sterilized fluids can be removed by filtering the material through either asbestos pads or unglazed porcelain. An inexpensive and convenient apparatus for filtering C011sists of a syringe of the Luer type fitted with a Swinny adapter that holds the asbestos filter. A syringe of this type can be procured from the Fisher Scientific Company, 633 Greenwich Street, New York, N.Y. 10014. The Swinny adapter is available from the Baltimore Biological Laboratories, 2201 Asquith Street, Baltimore, Md. 21218.

"All reagents must be of the highest available purity. Triple-distilled water should be used in all procedures. The experimenter should anticipate spending much of his time washing apparatus. A11 containers must be rinsed at least four times with tap water followed by a final rinse with triple-distilled water

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"For specimen materials beginners are urged to buy a starter cell culture. This material can be obtained from Difco Laboratories Inc., 920 Henry Street, Detroit, Mich. 48201, or from the Baltimore Biological Laboratories. Information on available cultures, prices and shipping will be sent on request. Alternatively, tissues and organs for culturing can be taken from an animal. This must be done under sterile operating conditions. If the animal must be killed, the method must be one-such as an overdose of chloroform-that does not introduce toxic agents into the animal's system.

"The tissue specimen must be washed immediately in a sterile salt solution, a specially compounded mixture of salts, phosphates, carbonates and dextrose that maximizes tissue survival. The solution can be obtained from the Baltimore Biological Laboratories or from Difco Laboratories. I used TC-Hanks Balanced Salt Solution. (Reagents and mediums preceded by TC are products of Difco Laboratories.) All superfluous membranes or structures must be removed while the tissue specimen is immersed in the balanced salt solution. The remaining tissue is then washed in the balanced salt solution, placed in a sterile container such as a watch glass, minced into fragments about a cubic millimeter in size with scissors or a scalpel and stored in the balanced salt solution.

"The fragments can be cultivated in either of two ways. One is to keep them in a form called 'plasma clot' on microscope slides or in flasks or tubes. The other is to disperse them as separate cells for cultivation in a liquid medium in tubes or bottles, where they grow as a single layer of closely spaced cells that adhere to the glass walls of the container. The choice between these two procedures depend on the use for which the cultures are intended. The plasma clot is excellent for microscopic examination but poor for maintaining cultures because nutrient can be made available to the growing tissue only a drop or two at a time. The nutrient must be replaced frequently. The monolayer type is widely used for perpetuating cultures, for investigating the interaction of cells and viruses and for studying cell lines.

"A plasma-clot culture is prepared by placing one drop of TC-Chicken Plasma in the center of a square of thin glass of the type used for covering microscope slides. To this drop is added one drop of TC-Embryo Extract EE201. (A numerical subscript indicates the percentage of extract or medium in the solution.) Mix the drops with a spatula and spread the fluid over an area about the size of a dime. Add two pieces of minced tissue to the center of the fluid. The specimens are now called explants. Each one should be about a millimeter square. Measure the size carefully. Explants larger than recommended cannot absorb adequate nourishment. Moreover, the initial size must be known so that the rate of subsequent growth can be determined.

"Cover the preparation and set it aside for about an hour, until it clots. An inverted microscope slide that contains a deep depression makes a convenient cover [see Figure 2]. Seal the cover glass to the slide with a ring of yellow petroleum jelly. Incubate the preparation at approximately 37 degrees C. and observe the culture under a microscope every 24 hours. The growing tissue must be transferred to fresh nutrient every two or three days, a procedure known as 'patching.' Simply cut the old slide culture back to a one-millimeter square, transfer it to a freshly prepared cover glass and continue incubation.

"I use a homemade incubator: a pair of nested cardboard boxes insulated with rock wool [see Figure 3]. Controlled heat is provided by a 75-watt lamp bulb regulated by a thermostat of the type used in aquarium tanks. Such thermostats are also available with a built-in heating unit at slightly higher cost. Alternatively, you can improvise your own from a bimetal strip salvaged from the starter switch of a fluorescent lamp [see Figure 4]. The temperature of the incubator should be maintained between 34 and 37 degrees C.

"Cultures of the monolayer type involve several additional operations. The cells grow as individuals. For this reason the term 'cell culture' seems more appropriate than 'tissue culture.' To disperse the cells the fragments of tissue are first placed in a flask containing a few glass beads, which serve as agitators when the flask is swirled, and a sterile saline solution that contains .25 percent trypsin. The trypsin dissolves the cement between the cells to produce a suspension. Diluted trypsin does not affect living cells and can be removed easily from the suspensions.

"To prepare the solution dissolve 25 milligrams of 1:250 trypsin (the number is part of the name and designates the activity of the preparation) in 10 milliliters of calcium-and-magnesium-free phosphate-buffered saline (CMFPBS) Filter-sterilize this preparation through a sterile Swinny filter into half-ounce prescription bottles. Store five milliliters in each bottle.

"To prepare the CMF-PBS solution dissolve 800 milligrams of sodium chloride, 30 milligrams of potassium chloride, eight milligrams of sodium orthophosphate mono-H, two milligrams of orthophosphate di-H and 200 milligrams of dextrose in 100 milliliters of triple-distilled water. Do not autoclave this preparation but keep it frozen until you are ready to use it. For dispersing tissue cells thaw the solution, add five fragments of tissue of the same size as that used in the tissue-clot experiment, let the mixture stand for six hours at 4 degrees C. and then shake it vigorously to make a uniform suspension of cells.

"Now centrifuge the suspension at 800 revolutions per minute for five minutes. Pour off the solution gently. Add five milliliters of basic salt solution and again shake the container to resuspend the cells. Filter the suspension through sterile gauze into a clean container and centrifuge it again at 800 r.p.m. for five minutes. The washed cells collected at the bottom of the centrifuge tube are ready, after resuspension in basic salt solution, for monolayer culturing [see bottom illustration at right]. Much of this work can be avoided, of course, by buying prepared starter cultures.

"Mediums for making monolayer cultures are available from Difco Laboratories or the Baltimore Biological Laboratories. I use Eagle's Basal Medium (also known as Eagle's HeLa Medium). It is currently priced at $2.50 per 100 milliliters. Glutamine must be added in the proportion of 30 milligrams per 100 milliliters of medium. Glutamine is unstable even at refrigerator temperatures; therefore it must be added as the medium is prepared for use. Make up the glutamine solution by dissolving 30 milligrams of reagent L-glutamine in two milliliters of triple-distilled water. Pass the solution through a Swinny filter into the container of medium. Reagent L-glutamine can be bought from Nutritional Biochemicals Inc., 21010 Miles Avenue, Cleveland, Ohio 44128.

"The medium must also contain serum-5 to 10 percent for maintaining a culture and 15 to 20 percent for encouraging growth. Serum provides growth factors that have not yet been identified. It also appears to encourage the attachment of cells to the glass walls. I use any of three serums: TC-Horse Serum, TC-Fetal Calf Serum or TC-Human Serum.

"To the medium thus completed antibiotics can be added for the control of bacterial infection. I use a combination of antibiotics that is effective against both gram-positive and gram-negative organisms. The combination consists of 100 units of sterile potassium penicillin G and 100 micrograms of dihydrostreptomycin sulfate per milliliter of medium. The antibiotics must be procured in the form of dry powders, without preservatives that might poison the cultures. A 500,000-unit vial of potassium penicillin G and a one-gram vial of dihydrostreptomycin constitute an adequate stock. The drugs can be obtained with the help of a cooperative physician.

"To start a monolayer culture, plant a quarter-milliliter of the cell suspension and one milliliter of the prepared medium in a screw-cap tube about 16 millimeters in diameter and 150 millimeters long. Close the cap tightly and place the tube in the incubator at an angle of about 15 degrees, so that the contents wet most of the wall at the bottom. Within 24 to 36 hours a dense monolayer will form where the fluid wets the glass. I substituted ordinary half-ounce prescription bottles for the screw-cap tubes. Bottles of this type have one flat side. (A druggist let me have six dozen for four cents a bottle.) The bottle is laid on its flat side. The flat inner surface appears to encourage the growth of exceptionally massive cultures.

"The growing culture exhausts the nutrient in about four days. To replenish the spent medium pour off the fluid, refill the container with four milliliters of fresh medium and shake the solution gently until the monolayer disintegrates. Then asceptically transfer two milliliters of the fresh cell suspension to another bottle. This procedure is known as making a 'split.' You now have two cultures.

"The medium as supplied includes phenol red, an ingredient that serves to indicate the acidity or alkalinity of the fluid. The color of a fresh culture ranges from cerise to pink. As the cells metabolize, the solution gradually becomes acid, so that the color changes from pink to yellow. Alkalinity must then be restored by admitting air to the culture. This is done by loosening the cap of the bottle about a quarter-turn and retightening it.

"To stain monolayers for microscopic examination remove from a culture as much of the specimen as can be picked up with a sterilized wire loop approximately three millimeters in diameter [see Figure 6]. Transfer the cells to a clean microscope slide by pressing the loop on the glass. Wash the material gently with three changes of basic salt solution and then fix, or preserve, the cells by a drop of 10 percent formalin in .8 percent saline solution. Wash the material again to remove the formalin and let the specimen dry at room temperature. During the drying the cells become firmly attached to the glass; they can be stained by any of several preparations without becoming dislodged. Wright's stain is particularly easy to use. Flood the dried cells with two drops of a solution composed of .1 milligram of dry certified Wright's stain in 60 milliliters of acetone-free reagent methanol. Let the stain act for two minutes and then add four drops of water. Let the slide stand for five minutes. Rinse off the stain and dry the slide at room temperature.

"An alternative procedure is to stain the cells by basic fuchsin-.5 percent of the stain in a 20 percent solution of methanol in water. Let the preparation act on the cells for five minutes. Then rinse the cells in water or in a 50 percent solution of methanol in water. The diluted alcohol tends to produce slides of greater contrast between the cytoplasm and the nuclei than the water rinse does. On the other hand, the alcohol tends to bleach the dye. The slide must be watched carefully and the action stopped at maximum contrast by rinsing the preparation in water to prevent total destaining.

"As seen under the microscope monolayer cultures can be separated into two broad classes according to their appearance. Those that grow into long, spindly, loosely connected cells are called fibroblastic, because of their similarity to muscle fibroblasts. An example is the widely studied murine L cell line. Cells of the second class grow as closely joined polygonal shapes, called epithelial. The HeLa cell line is an example of this type, as are the great majority of other existing cultures [see Figure 1].

"Because active tissues require frequent changes of medium, biologists sought a method of maintaining cells for long intervals with minimum attention. This was found in the technique of 'agar slant' culture, in which a supply of nutrient is in effect stored in an agar substrate. To establish a culture of this kind prepare a solution of Eagle's Basal Medium of twice the strength used for monolayer cultures. Next make up a 3 percent solution of Noble agar ('Noble' indicates a purified grade of agar) or of ordinary agar washed five times in cold, triple-distilled water and then dried. Autoclave the solution and cool it. Mix equal volumes of the concentrated medium and agar solution, dispense the mixture in four-milliliter amounts in sterile test tubes and cool the tubes in an almost horizontal position so that the agar solidifies as a 'slant' that extends from the bottom of each tube almost to the top. When the agar has cooled, pipette .3 milliliter of Eagle's Basal Medium with 10 percent TC-Horse Serum into the tube. Store the tube upright in the refrigerator. Such slants are satisfactory for six months or more of use.

"In order to establish a tissue culture in the agar slant tubes, harvest cells from a young monolayer (two or three days old) and then wash them once with sterile basic salt solution. Separate the cells from the fluid by centrifuging. Gently pour the solution from the centrifuge tube without disturbing the cells that have settled to the bottom. Add half a milliliter of sterile basic salt solution to the tube and agitate it to make a dense suspension of cells. Using a wire loop that has been sterilized by flame, transfer a loopful of the cell suspension to a prepared agar slant, touching the agar with the loop at several places. (Do not smear the inoculum by dragging the loop across the surface of the agar.) Cap the agar tube tightly and incubate it in an upright position at 37 degrees C. Colonies of cells will appear in about four days and will grow to a diameter of five to 10 millimeters within 10 days. The nutrient solution should be changed at least every three weeks. The culture will live for six weeks or longer. For this reason agar slants are well suited for keeping stock cultures of cell lines and cell strains.

"To establish pure cultures of bacteria one can take advantage of the fact that a single bacterium can give rise to a colony of descendants. A parallel exists in which tissue cultures are used for separating mixed viruses into pure viral strains. The technique is called plaquing. A single virus particle can infect a cell in a monolayer overlaid with agar. The infected cell produces more virus. Neighboring cells become infected and die. A plaque, or pock, appears at the site; it contains the pure viral strain. The procedure is useful, of course, only when the tissue cells are susceptible to infection by the virus under test.

"To prepare an agar overlay for investigating viruses make up the agar solution as for an agar slant, but to each 10 milliliters of the solution add .3 milliliter of a .1 percent autoclaved aqueous solution of 1:30,000 Neutral Red Certified. Then grow the selected cell line or strain on the flat side of a half-ounce prescription bottle, using two milliliters of Eagle's Basal Medium plus 20 percent TC-Horse Serum, until a dense monolayer has formed. This will take about three days.

"With the bottle resting on its flat side wash the monolayer once with sterile basic salt solution, taking care to keep the layer intact. To the bottle add two milliliters of sterile basic salt solution and a specimen of virus that has been suspended in .1 to .5 milliliter of sterile basic salt solution. Incubate the preparation for two hours. During this interval the virus will migrate into the tissue culture. Pour off the surplus fluid and gently flow the prepared agar, at a temperature of 40 to 45 degrees C., into the bottle so that it covers the monolayer. After the agar solidifies incubate the material with the bottle resting on its flat side. Within four days irregular patches of lighter-than-average color will be observed if a cell-destroying virus is present. Each plaque developed from a single virus particle can be cut out and propagated as a pure strain of virus.

"Additional experiments with tissue cultures will be described subsequently in this department. In the meantime I shall be pleased to correspond with amateurs who take up this fascinating hobby. My address is Ted M. Fancolli, 5117 Boyd Drive, Carmichael, Calif. 95608."

Bibliography

THE CULTIVATION OF ANIMAL AND PLANT CELLS. Philip R. White. The Ronald Press Company, 1963.

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