- Email: [email protected]
A culture chamber for the continuous biochemical and morphological study of living cells in tissue culture
10
A CULTURE BIOCHEMICAL LIVING
CHAMBER FOR THE CONTINUOUS AND MORPHOLOGICAL STUDY OF CELLS IN TISSUE CULTURE1
G. S. CHRISTIANSEN, BETTY DANES, and P. J. LEINFELDER Departments
of
Ophthalmology
and Physiology, College of Medicine, Iowa City, U.S.A.
L. ALLEN
State University
of Iowa,
Received June ‘I, 1952
THE parallel
study of the biochemistry and morphology of living cells has long been the ambition of both biochemists and morphologists. With the recent advent of phase contrast microscopy, coupled with the well developed techniques of tissue culture, this ambition is realized in principle. It is now possible to study living, unfixed cover slip cultures of many types of cells and to observe the intracellular organs of these cells with no question of fixation artefacts. \\‘ith this combination of techniques it should now be possible to study the “pharmacology” of chemical substances on the basis of the type of cell and even the particular part of the cell which is influenced by the compound in question. much can be Conversely, learned of the causal biochemical reactions associated with various cellular processes and structures by administering compounds whose biochemical action is known. However, one further device or technique is required for the complete realization of this biochemical study of cells in tissue culture. A\ method must be developed which meets the rather rigid requirements for optimum growth and cellular activity of the cultures. These requirements are primarily: (a) sterility and (b) optimum conditions of temperature, nutrition and oxygenation. The same method must also meet the optical requirements of phase contrast microscopy, which are: (a) The culture vessel must have optically flat and parallel surfaces. (b) These surfaces must be no more than 1 mm apart2. (c) The culture vessel and the culture must, 1 This work was supported by a grant from the Damon Runyon Memorial Fund for Cancer Research. 2 This requirement is based on the necessity of having the microscope condenser and objective at the critical distance from the cells for adequate phase contrast optics. The sum of these distances depends on the magnification and on the make of the microscope. For Bausch and Lomb oil immersion (97 *) objective this distance is about 1.0 mm.
Cdfure chamber for phase confrasf microscopy
11
of course, be transparent. This includes the requirement that a continuous liquid phase tills the culture chamber during periods of observation. A final requirement of a biochemical-morphological technique is that biochemically interesting substances can be introduced and removed at will. The use of the Gey plate (3) meets in part these requirements. The culture is mounted between two cover slips in a Gey plate and placed under the microscope. In this way it is possible to observe migrated cells with phase contrast optics, but only for relatively short times. The cellular activity is greatly modified under these circumstances, as is evidenced by the relative rarity with which mitosis is observed and by the failure of the cultures to grow or even to survive for more than a few hours. It is possible to introduce liquids between the cover slips, but once introduced they cannot be removed. A more recent modification of the Gey plate (2), the formation of a perfusion chamber between two cover slips, goes further in meeting the requirements for an adequate biochemical-morphological method. The perfusion chamber is formed by paraffin side walls between two cover slips, one of which carries the culture explant. Inlet and outlet tubes pass through the paraffin walls so that solutions can be passed constantly through the culture chamber. The two major disadvantages of this device are its cumbersome yet fragile construction and the obvious difficulty of mounting the two cover slips parallel. The present paper describes the construction and indicates the use of a culture chamber in which tissues can be grown for unlimited time and in which phase contrast observation is possible continuously throughout the culture period. The chamber also provides for the introduction and removal of any desired series of fluids. The device is easily and cheaply constructed and is simple and stable in operation; cultures are easily set up and show growth and cellular activity comparable to conventional hanging drop cultures; and finally, the device is permanent and is easily cleaned and sterilized.
CONSTRUCTION
OF
CULTURE
CHAMBER
a. Plastic Chamber. The tissue culture slide shown in Fig. I is milled from l/4 inch thick acrylic plastic sheet (first grade polished Plexiglass) to the dimensions shown in the working drawings of Fig. 2. The milling and cutting must be done slowly and under water cooling to prevent heating and its consequent permanent distortion of the thin plastic. The dimensions of Fig. 2 are critical for the use of
Fig. 1. Perspective dra~ir~g of plastic culture slide (without top and bottom cover slips).
Side uieu.
End view
Fig. 2. Working drawings of plastic culture slide.
Fig. 3. Crosssection of plastic culture slide with cover slips in place to form culture chamber. The microscope condenser, objective and stage and the incubator are indicated schematically. the slide in a 1 inch by 3 inch stage incubator? and for maximum excursion of the objective over the culture chamber. Two grooves are engraved in the under side of the block, extending from the circular opening to within 20 mm of each end of the block. At these points each groove joins a i/16 inch hole drilled upward and outward through the block. Acrylic tubes are cemented on the upper surface so as to be contirluous with t.hese holes. To complete the slide and form the floor and roof of the culture chamber, two No. 0 cover slips are attached with petrolatum or paraffin as seen in Fig. 3. The bottom cover glass must be wide enough to cover the 17 mm opening and at least 1 Microscopic electrical incubator for oil immersion objectives, C. S. Cy:E., A. S. Aloe Co., St. Louis.
Culture chamber
for
phase contrast microscopy
13
50 mm long to enclose the grooves and holes cut in the plastic block. The top cover slip carries the culture and covers the entire circular opening. For some purposes, contact of the perfusing fluid with plastic b. Glass Chamber. cannot be tolerated. To avoid this complication an all glass chamber wit.h essentially the same principle of design was made. The chamber was made by grinding the holes and grooves in a 1 mm thick glass micro slide. This slide was then ground to a thickness of 0.8 mm. The inlet and outlet tubes were set in thicker glass blocks which were then attached to the slide by sodium silicate cement. USE
OF THE
CULTURE
CHAMBER
The glass chamber is sterilized by autoclaving; the plastic chamber by ultraviolet irradiation (1). The acrylic chamber cannot be sterilized by autoclaving nor in many germicidal solutions (including alcohol) because of damage to the plastic. Usual sterility measures for cover slips, solutions and culture explants are taken. The culture is started by placing the explant on the upper cover slip as in hanging drop cultures, then attaching the cover slip to the top of the slide with paraffin. A larger cover slip is attached with petrolatum to the bottom of the slide, convcrting the central opening to a culture chamber and the grooves to semicircular tubes (Fig. 3). The whole slide is then placed in a stage thermostat on the microscope. Nutrient fluid is introduced from a tuberculin syringe attached by Tygon tuhing to the inlet tube. The outlet is connected to an empty syringe, giving control on both sides of the chamber so that no pressure or vacuum is created when the fluid is introduced or removed. To oxygenate the culture, the fluid is periodically replaced by air (comparable to the roller tube technique). M3rn microscopic obserrations are made, the chamber must be filled with fluid. However, if desired, the fluid can be continuously flowing through the chamber without optical interference. The effect of biochemical agents such as enzyme inhibitors, narcotics, and vital stains, is observed by replacing the simple nutrient fluid with a solution of the agent dissolved in the nutrient. At the end of the observation the upper cover slip carrying the culture is removed and the culture fixed. The bottom cover slip is also removed, exposing all parts for easy and perfect cleaning. OBSERVATIONS
Cultures have been grown continuously in this device and observed at convenient intervals for as long as three days. There is no experimental reason why they cannot be observed for much longer periods. Fig. 4 shows the activity of a single chick heart fibroblast photographed at 1 hour intervals from the 24th to the 32nd hour of culture. The changing shape, activity of the cell membrane, protruding and retracting of pseudopods, changes in shape and movements of the nucleus, and the activity of the nucleoli are apparent. The movements and shape changes of the cytoplasmic in-
14
(;. S. Christiansen, Il. Banes, L. Allen, and P. ,J. Leinjeltler
Fig. 4. Activity of a single chick heart fibroblast culture. Dark contrast phase photomicrographa,
followed from the 21th to the 3211d hour of original magnification 1000 7
Culture chamber for phase contrast microscopy
15
elusions are not so obvious in this series of pictures. These more rapid cellular activities must be recorded by cinematographic methods, for which this culture chamber would be eminently satisfactory. This chamber has been used to study the effect of cyankle and other enzyme inhibitors on these and other cellular activities. Vital stains have been introduced to cultures which have been examined first by phase contrast, then, after supravital staining, by ordinary bright field microscopy. In this way the intracellular organs as observed by phase contrast have been identified with their counterparts in stained cells, In a similar way specific enzyme reagents have been used to localize enzyme activities to will be published in particular intracellular organs. These observations detail elsewhere. SUMMARY
A culture chamber has been described in which continuous phase contrast microscopic observation of growing tissue cultures can be made. The device meets the rigid requirements for optimum culture of tissues and for optical observation. The tissue can be exposed to biochemical reagents at will. Some of the observations made with this device have been described briefly. REFERENCES 1. CARLSON, J. G., HOLLAENDER, A., and GAULDEN, M. 2. Hu, F. N., HOLMES, S. G., POMERAT, C. M., LIVINGOOD, Repts. Biol. and Med., 9, 739 (1951). 3. LEWIS, S. R., POMERAT, C. M., and EZELL, D., Amt.
E., Science, 105, 187 (1947). C. S., and MCCONNELL, K. P., Texxzs
Rec., 104, 487 (1949).
A CULTURE BIOCHEMICAL LIVING
CHAMBER FOR THE CONTINUOUS AND MORPHOLOGICAL STUDY OF CELLS IN TISSUE CULTURE1
G. S. CHRISTIANSEN, BETTY DANES, and P. J. LEINFELDER Departments
of
Ophthalmology
and Physiology, College of Medicine, Iowa City, U.S.A.
L. ALLEN
State University
of Iowa,
Received June ‘I, 1952
THE parallel
study of the biochemistry and morphology of living cells has long been the ambition of both biochemists and morphologists. With the recent advent of phase contrast microscopy, coupled with the well developed techniques of tissue culture, this ambition is realized in principle. It is now possible to study living, unfixed cover slip cultures of many types of cells and to observe the intracellular organs of these cells with no question of fixation artefacts. \\‘ith this combination of techniques it should now be possible to study the “pharmacology” of chemical substances on the basis of the type of cell and even the particular part of the cell which is influenced by the compound in question. much can be Conversely, learned of the causal biochemical reactions associated with various cellular processes and structures by administering compounds whose biochemical action is known. However, one further device or technique is required for the complete realization of this biochemical study of cells in tissue culture. A\ method must be developed which meets the rather rigid requirements for optimum growth and cellular activity of the cultures. These requirements are primarily: (a) sterility and (b) optimum conditions of temperature, nutrition and oxygenation. The same method must also meet the optical requirements of phase contrast microscopy, which are: (a) The culture vessel must have optically flat and parallel surfaces. (b) These surfaces must be no more than 1 mm apart2. (c) The culture vessel and the culture must, 1 This work was supported by a grant from the Damon Runyon Memorial Fund for Cancer Research. 2 This requirement is based on the necessity of having the microscope condenser and objective at the critical distance from the cells for adequate phase contrast optics. The sum of these distances depends on the magnification and on the make of the microscope. For Bausch and Lomb oil immersion (97 *) objective this distance is about 1.0 mm.
Cdfure chamber for phase confrasf microscopy
11
of course, be transparent. This includes the requirement that a continuous liquid phase tills the culture chamber during periods of observation. A final requirement of a biochemical-morphological technique is that biochemically interesting substances can be introduced and removed at will. The use of the Gey plate (3) meets in part these requirements. The culture is mounted between two cover slips in a Gey plate and placed under the microscope. In this way it is possible to observe migrated cells with phase contrast optics, but only for relatively short times. The cellular activity is greatly modified under these circumstances, as is evidenced by the relative rarity with which mitosis is observed and by the failure of the cultures to grow or even to survive for more than a few hours. It is possible to introduce liquids between the cover slips, but once introduced they cannot be removed. A more recent modification of the Gey plate (2), the formation of a perfusion chamber between two cover slips, goes further in meeting the requirements for an adequate biochemical-morphological method. The perfusion chamber is formed by paraffin side walls between two cover slips, one of which carries the culture explant. Inlet and outlet tubes pass through the paraffin walls so that solutions can be passed constantly through the culture chamber. The two major disadvantages of this device are its cumbersome yet fragile construction and the obvious difficulty of mounting the two cover slips parallel. The present paper describes the construction and indicates the use of a culture chamber in which tissues can be grown for unlimited time and in which phase contrast observation is possible continuously throughout the culture period. The chamber also provides for the introduction and removal of any desired series of fluids. The device is easily and cheaply constructed and is simple and stable in operation; cultures are easily set up and show growth and cellular activity comparable to conventional hanging drop cultures; and finally, the device is permanent and is easily cleaned and sterilized.
CONSTRUCTION
OF
CULTURE
CHAMBER
a. Plastic Chamber. The tissue culture slide shown in Fig. I is milled from l/4 inch thick acrylic plastic sheet (first grade polished Plexiglass) to the dimensions shown in the working drawings of Fig. 2. The milling and cutting must be done slowly and under water cooling to prevent heating and its consequent permanent distortion of the thin plastic. The dimensions of Fig. 2 are critical for the use of
Fig. 1. Perspective dra~ir~g of plastic culture slide (without top and bottom cover slips).
Side uieu.
End view
Fig. 2. Working drawings of plastic culture slide.
Fig. 3. Crosssection of plastic culture slide with cover slips in place to form culture chamber. The microscope condenser, objective and stage and the incubator are indicated schematically. the slide in a 1 inch by 3 inch stage incubator? and for maximum excursion of the objective over the culture chamber. Two grooves are engraved in the under side of the block, extending from the circular opening to within 20 mm of each end of the block. At these points each groove joins a i/16 inch hole drilled upward and outward through the block. Acrylic tubes are cemented on the upper surface so as to be contirluous with t.hese holes. To complete the slide and form the floor and roof of the culture chamber, two No. 0 cover slips are attached with petrolatum or paraffin as seen in Fig. 3. The bottom cover glass must be wide enough to cover the 17 mm opening and at least 1 Microscopic electrical incubator for oil immersion objectives, C. S. Cy:E., A. S. Aloe Co., St. Louis.
Culture chamber
for
phase contrast microscopy
13
50 mm long to enclose the grooves and holes cut in the plastic block. The top cover slip carries the culture and covers the entire circular opening. For some purposes, contact of the perfusing fluid with plastic b. Glass Chamber. cannot be tolerated. To avoid this complication an all glass chamber wit.h essentially the same principle of design was made. The chamber was made by grinding the holes and grooves in a 1 mm thick glass micro slide. This slide was then ground to a thickness of 0.8 mm. The inlet and outlet tubes were set in thicker glass blocks which were then attached to the slide by sodium silicate cement. USE
OF THE
CULTURE
CHAMBER
The glass chamber is sterilized by autoclaving; the plastic chamber by ultraviolet irradiation (1). The acrylic chamber cannot be sterilized by autoclaving nor in many germicidal solutions (including alcohol) because of damage to the plastic. Usual sterility measures for cover slips, solutions and culture explants are taken. The culture is started by placing the explant on the upper cover slip as in hanging drop cultures, then attaching the cover slip to the top of the slide with paraffin. A larger cover slip is attached with petrolatum to the bottom of the slide, convcrting the central opening to a culture chamber and the grooves to semicircular tubes (Fig. 3). The whole slide is then placed in a stage thermostat on the microscope. Nutrient fluid is introduced from a tuberculin syringe attached by Tygon tuhing to the inlet tube. The outlet is connected to an empty syringe, giving control on both sides of the chamber so that no pressure or vacuum is created when the fluid is introduced or removed. To oxygenate the culture, the fluid is periodically replaced by air (comparable to the roller tube technique). M3rn microscopic obserrations are made, the chamber must be filled with fluid. However, if desired, the fluid can be continuously flowing through the chamber without optical interference. The effect of biochemical agents such as enzyme inhibitors, narcotics, and vital stains, is observed by replacing the simple nutrient fluid with a solution of the agent dissolved in the nutrient. At the end of the observation the upper cover slip carrying the culture is removed and the culture fixed. The bottom cover slip is also removed, exposing all parts for easy and perfect cleaning. OBSERVATIONS
Cultures have been grown continuously in this device and observed at convenient intervals for as long as three days. There is no experimental reason why they cannot be observed for much longer periods. Fig. 4 shows the activity of a single chick heart fibroblast photographed at 1 hour intervals from the 24th to the 32nd hour of culture. The changing shape, activity of the cell membrane, protruding and retracting of pseudopods, changes in shape and movements of the nucleus, and the activity of the nucleoli are apparent. The movements and shape changes of the cytoplasmic in-
14
(;. S. Christiansen, Il. Banes, L. Allen, and P. ,J. Leinjeltler
Fig. 4. Activity of a single chick heart fibroblast culture. Dark contrast phase photomicrographa,
followed from the 21th to the 3211d hour of original magnification 1000 7
Culture chamber for phase contrast microscopy
15
elusions are not so obvious in this series of pictures. These more rapid cellular activities must be recorded by cinematographic methods, for which this culture chamber would be eminently satisfactory. This chamber has been used to study the effect of cyankle and other enzyme inhibitors on these and other cellular activities. Vital stains have been introduced to cultures which have been examined first by phase contrast, then, after supravital staining, by ordinary bright field microscopy. In this way the intracellular organs as observed by phase contrast have been identified with their counterparts in stained cells, In a similar way specific enzyme reagents have been used to localize enzyme activities to will be published in particular intracellular organs. These observations detail elsewhere. SUMMARY
A culture chamber has been described in which continuous phase contrast microscopic observation of growing tissue cultures can be made. The device meets the rigid requirements for optimum culture of tissues and for optical observation. The tissue can be exposed to biochemical reagents at will. Some of the observations made with this device have been described briefly. REFERENCES 1. CARLSON, J. G., HOLLAENDER, A., and GAULDEN, M. 2. Hu, F. N., HOLMES, S. G., POMERAT, C. M., LIVINGOOD, Repts. Biol. and Med., 9, 739 (1951). 3. LEWIS, S. R., POMERAT, C. M., and EZELL, D., Amt.
E., Science, 105, 187 (1947). C. S., and MCCONNELL, K. P., Texxzs
Rec., 104, 487 (1949).