Chapter 10
Use of Plastics f i r Suspnsion Ctclttcre Vessels ROLAND A. COOK, FREDERICK T. COUNTER, JOYCE K. M C C O L L E Y
AND
Eli Lilly and Company, Indianapolis. Indiana
. . 111. Adaptation of Cell Lines to Growth in Suspension . I. Introduction
.
.
.
.
11. Use of Plastics for Monolayer Cultures
. .
. .
. . . . . . . . .
A. Problems with Glass Suspension Culture Vessels B. Use of Plastic Suspension Culture Vessels. . IV. Toxicity Testing of Plastic Laboratory Ware . . V. Construction of Plastic Suspension Culture Vessels . A. General . . . . . . . B. Small Vessels with Suspended Stirring Bars . C. Five-Gallon Carboy Stirred by Overhead-Drive Motor D. Use of Plastic Tubing and Sheets for Spin-Filter Vessels VI. Conclusions . . . . . . . . References . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . .
113 114 114 114 115 116 117 117
118 119 . 1 2 0 . 122 . 123
I. Introduction The field of suspended culture of mammalian cells is now over 20 years old, but continues to receive impetus from technological advances. Mass suspension culture systems have been devised for the production of virus vaccines and diagnostic reagents; large-scale cultures of lymphoblastoid, or hematopoietic cells-which grow naturally in suspension-have been utilized for the production of antilymphocyte serum and serve as a source of material for analysis of cell membranes and subcellular components. The recent use of DEAE-Sephadex beads (Horng and McLimans, 1975; van Wezel, 1967) and porous silica spherules (Bizzini et al., 1973) as microcarriers may make possible the growth in suspension of many types of functional, differentiated cells, which are normally anchorage dependent. 113
114
R. A. COOK, F. T. COUNTER. AND J. K. MCCOLLEY
Our laboratory has been involved in the adaptation of cell lines derived from normal tissues to growth in continuous suspension culture. Plastic culture vessels were found to facilitate this work, as we have previously reported (Cook et al., 1974a,b; Cook and Counter, 1974). We believe that other workers will appreciate the ease with which plastic materials can be used to construct suspension culture vessels to a given experimental design. In this report we describe our experiences with the use of these vessels and provide detailed information on their construction.
11. Use of Plastics for Monolayer Cultures The use of plastics for monolayer culture of mammalian cells has been well documented. Recent refinements in plastics technology have led to the development of plastic surfaces that are uniform enough for microtitration and microcloning techniques (Fuccillo et al., 1969;Robb, 1970).Amethodof treating plastic petri dishes for use in cloning chick embryo cells has been described (Trager and Rubin, 1966);however, processes used by commercial firms to modify plastic surfaces to enhance cell adhesion have not been published. The advances mentioned above have involved alteration ofthe basic “nonstick’’ qualities shared by most plastics. For certain operations, adhesion of cells to vessels is undesirable and unaltered plastics can beused to advantage. Polystyrene tubes have been used to eliminate cell adhesion in radiation experiments (Walker et al., 1962). Industrial firms have developed a large market for plastic-coated cookware based on the ease with which these utensils can be cleaned.
111. Adaptation of Cell Lines to Growth in Suspension
A. Problems with Glass Suspension Culture Vessels Initial studies were performed with the LLC-MK, cell line-a Lilly Laboratory cell line derived from normal rhesus monkey kidney in R. N. Hull’s laboratory (Hull et al., 1956). It was necessary to maintain suspensions of viable cells for several weeks. During this maintenance period, cells were observed to grow to increasing densities on the walls of glass flasks, with occasional sloughing of clumps of cells back into the culture medium. Calcium has been reported to cause clumping of cells in suspension (Eagle,
10.
115
PLASTICS FOR SUSPENSION CULTURE VESSELS
TABLE I ADHFSION OF CELLS TO G L A S ~ Culture volume Day
1
2 3 6
366 360 354 479 467
Cells in suspension x 106
Cells on flask wallb
183 180 89 144 42
+ +++ +‘
-
70 x
Total cells 183 x 106 Unknown Unknown Unknown 112 x 106 ~
‘From Cook er ol., 1974a. bRelative density of cells in ring on flask wall at meniscus of medium. ‘Increase in media volume resulted in numerouslarge chunks of cells sloughing off ring into medium. dCells on wall of flask removed by trypsinization and counted separately.
1959); however, no calcium was present in our medium formulation (Cook et al., 1974a). Actual growth of suspended cells could not be determined because of these problems. Results shown in Table1weretypical ofproblems encountered with glass vessels. The LLC-MK, cells used for this culture had previously been grown in bottles and were not adapted to suspension growth. At the end of 6 days, only 37.5% of surviving cells were in suspension; the remainder had adhered to the flask wall. Capstick et al. (1962) reported similar difficulties during the early stages of adaptation of the BHK21/13 (baby hamster kidney) cell line to growth in suspension. Conversely, investigators working with HeLa and L cell cultures reported minimal tendencies of these cells to adhere to glass in spinner culture (McLimans et al., 1957). The use of methyl cellulose in culture media (0.12%) and silicone treatment of glass flasks was found to retard but not prevent adhesion of cells to the flask walls.
B. Use of Plastic Suspension Culture Vessels It seemed possible that cells would be less likely to adhere to plastic surfaces. Polypropylene Erlenmeyer 125-ml flasks (Kimble) were obtained and equipped with magnetic stirring bars suspended from fishing swivels as described by Cherry and Hull (1956). Cultures of LLC-MK, cells did not adhere to the walls of these flasks. Suspended cells grew singly, or in small clumps of 3-4 cells and could be accurately counted. Subsequently, PK-15 cells-which are of normal pig kidney origin-were obtained from the American Type Culture Collection. These cells had previously been grown as monolayers and initially grew as large clumps in
116
R A. COOK, F. T. COUNTER, AND I. K. MCCOLLEY TABLE 11 ADHESION OF PK-15 SUSPENSION CULTURES TO NEWAND PREVIOUSLY USEDPOLYCARBONATE STIRRER FLASKS AFTER20 HOURSSE’ITLING
Temperature (“C) 36 25 4
AT
DIFFERENT TEMPERATURE+’
New flasks
Previously used flasksb
+ t -
“Symbols: +, Cells adhered to flask bottoms-did not detach after shaking. -, Cells readily resuspended. *Previously used flasks are flasks that have been used for several weeks and appear to have surface characteristically altered by this use. CFromCook et al.. 1974a.
suspension. It was necessary to disperse these clumps several times during the adaptation process. These cells also did not adhere to plastic stirrer flasks. In more recent studies, however, PK-15cells were found to adhere to polycarbonate stirrer flasks, which had been used as suspension culture vessels for several months, but not to new polycarbonate flasks (Table 11).
IV. Toxicity Testing of Plastic Laboratory Ware The polypropylene Erlenmeyer flasks which we initially used as suspension culture vessels were not treated or washed prior to use. No evidences of cell toxicity were observed. To determine whether other varieties of plastic might also be suitable, eleven different types were obtained from commercial suppliers and modified for use as suspension culturevessels(Tab1e 111).Some of the materials were not autoclavable, so all were sterilized withp-propiolactone and rinsed with medium prior to inoculation with suspensions of LLC-MK, cells. Glass flasks were used for control cultures. No evidence of gross toxicity could be observed in cultures after 3 days of incubation at 36°C. Cells did not adhere to any of the plastic containers; heavy rings of cells had grown at the meniscus on the glass control flasks. It should be mentioned here that all plastics contain a variety of compounds, such as plasticizers, fillers, and stabilizers. Also, formulas for a given type of plastic may differ from one manufacturer to the next. It is possible that a given formulation might be toxic to cultured cells; however, many of our plastic culture vessels have been in continuous use for several months and toxicity problems have not yet been encountered.
10.
117
PLAsIlCS FOR SUSPENSION CULTURE VESSELS
TABLE 111 PLASTICS OBTAINED FOR TOXICITY TESTING' Type of plastic Polypropylene Polycarbonate Teflon FEP Teflon TFE Linear polyethylene (LPE) Conventional polyethylene (CPE) Polyvinyl chloride (PVC) Polystyrene Polymethylpentene(TPX) Styrene-acrylonitrile Methyl-methacrylate (Lucite)
Item
Supplier
Catalog No.
Bottle, 4-ounce Erlenmeyer flask, 125-ml Bottle, 4-ounce Evaporating dish, 100-ml Bottle, 4-ounce
Kimble Matheson Matheson Star Matheson
53250 24093-25 4356-10 s-3450 4371-20
Bottle, 4-ounce Bottle, 4-ounce Container, 30-dram Graduated cylinder, 250-ml Graduated cylinder, 250-ml
Matheson Matheson Dynalab Matheson Preisser
4335-70 4334-10 2635 18591-30 F28684
Jar, I-quart
LPF
8025
'From Cook et a/., 1974a.
V. Construction of Plastic Suspension Culture Vessels A. General Polycarbonate and polypropylene were found to be most practical for this purpose. Polycarbonate is transparent; polypropylene is translucent. Both types of plastic are nontoxic, autoclavable, relatively inexpensive and readily available in various sizes of flasks and bottles from a number of commercial suppliers. Polycarbonate sheets up to 4 x 8 feet, and tubing up to 4 inches in diameter are available locally (Hyaline Plastics, Indianapolis, Indiana). Plastic culture vessels of various types were adapted to our needs by the simple expedient of heating stainless steel cork borers and melting holes in the plastic where desired(Caution:This operation produces noxious, possibly toxic fumes and should be done underafume hood). Anelectricdrill was used to make holes in thicker materials, such as plastic sheets and tubing. A drill bit smaller than the desired hole was used, followed by larger drill bits. This was necessary to prevent chipping; thin-walled containers could also be drilled with care taken to prevent cracking. Holes drilled in thicker materials were threaded when desired, using conventional taps. Probes, thermometers, breathers, and glass tubing for inlet, outlet, and sample lines were then inserted into the culture vessels where desired-either through rubber stoppers sized to fit the holes melted or drilled in the vessels, or through polypropylene fittings which are available commercially (Matheson Scientific, Elk Grove Village, Illinois).
118
R. A. COOK, F. T. COUNTER. AND J. K. MCCOLLEY
Polycarbonate materials can be cemented together, using Thickened Cement for Plexiglas Acrylic Sheet (Dayton Plastics, Dayton, Ohio). One of the two pieces to be joined was covered with cement, and both pieces were then immediately joined and held together for about 1 minute. Joints were ready for use in less than 1 hour.
B. Small Vessels with Suspended Stirring Bars Three types of small plastic suspension culture vessels are shown in Fig. 1. Each of these is equipped with a plastic-coated magnetic stirring bar suspended by a nichrome wire from a stainless steel, ball-bearing swivel (Eagle Claw, Denver, Colorado). A staple fashioned from this wire was used to secure the swivels to rubber stoppers or plastic lids(stap1eswere heated to penetrate plastic lids, then bradded down on top). Swivels were mounted with the large end up-resembling a child’s top-to shield the ball-bearing mechanism from liquids inadvertently splashed on the swivel. These swivels have proved to be reliable in many months of continuous use. In Fig. 1 a rubber vaccine stopper can be seen inserted in the sidewall of the 125-rnl Erlenmeyer flask. This was done to permit sampling of cultures with a syringe, rather than by opening the culture vessel. Plastic cement (Dekadhese, Donald Tulloch Jr., Chadds Ford, Pennsylvania) was applied to seal rubber stoppers to the plastic vessels after autoclaving. The vaccine stopper becomes hardened after several autoclave cycles and will leak after needle punctures unless replaced periodically. The 500-ml and 1000-ml plastic bottles shown in Fig. 1 are equipped to operate as closed systems. Glass sampling tubes run to the bottom of both
FIG. 1. Plastic suspension culture vessels with suspended strirring bars. Left to right: 125-ml polycarbonate Erlenmeyer flask,500-mlpolycarbonate centrifuge bottle, and 1OOO-ml polypropylene centrifuge bottle.
10. PLASIICS FOR
SUSPENSION CULTURE VESSELS
119
vessels. These tubes must be carefully positioned inside the vessels, so that the suspended stirbars do not hang up on the tubes and become inoperative. A short tube mounted on the sidewall is used for media addition and also to cycle the cell suspension from the sampling tube back into the culture, via an attached syringe-allowing a representative sample of the culture to be taken. A cotton-filled breather is mounted on the vessel wall in a location where it will not become wet during these operations. Surgical latex tubing connects the glass tubing with Luer-type stainless steel fittings (BectonDickinson, Rutherford, New Jersey). Tubing is autoclaved in 0.1 N NaOH for 5 minutes, rinsed, autoclaved in deionized water for 5 minutes and then thoroughly rinsed and dried prior to use.
C. Five-Gallon Carboy Stirred by Overhead-Drive Motor Polypropylene carboys of the type shown in Fig. 2 were used to grow suspension cultures of LLC-MK, and PK-15 cells in volumes of up to 18
FIG.2. Fivegallon polypropylene carboy stirred by overhead-drive motor.
120
R. A. COOK, F. T. COUNTER, AND J. K. MCCOLLEY
liters. This vessel was also operated as a closed-system stirrer and is shown equipped with a Teflon seal assembly, Viton-A O-ring, 24/40 ground-glass joint, glass stirring shaft, and Teflon stirring blade (Cole-Parmer, Chicago, Illinois). The tapered end of the ground-glass joint was cut off and the remainder was mounted in a hole drilled in a No. 12 stopper, which fits the neck of the carboy. This type of vessel has been useful in the preparation of batch cultures.
D. Use of Plastic Tubing and Sheets for Spin-Filter Vessels A section of polycarbonate tubing 3 inches in diameter by 7$ inches in height was cemented to a square of polycarbonate to form the suspension culture vessel shown in Fig. 3. A second, larger polycarbonate square forms the top. These squares sawed out of a large polycarbonate sheet. The top of the vessel was secured to the tubing by horizontally drilling a hole in each of 4 pieces of polycarbonate $ by 1 inch in diameter, $ inch thick, and inserting 2-inch stainless steel screws through the holes. The pieces were then cemented to the tubing. The top was drilled to accept the screws, and wingnuts
FIG 3. Oneliter spin-filter suspension culture vessel made from polycarbonate sheet and tubing.
10.
PLAmCS FOR SUSPENSION CULTURE VESSELS
121
were used to secure the top. A Neoprene rubber gasket was cut so as to slightly overlap the rim of the tubing, and provided an airtight seal. Figure 4 shows an automated cell culture system utilizing a 4-liter polycarbonate jar which has been similarly equipped with a square top fashioned from a polycarbonate sheet. Both of the vessels shown in Figs. 3 and 4 are modified spin-filter suspension culture vessels. The spin-filter concept was originated by Arthur D. Little Co., as reported by Himmelfarb et al. (1969). The principle of the device is that boundary effects at the surface of the spinning filter allow large volumes of spent medium to be withdrawn from a suspension culture without clogging the filter with cells. Spin filter equipment is available commercially from the VirTis Co., Gardiner, New York. A disassembled filter head assembly is shown in Fig. 5. In our hands, it has been necessary to
FIG.4. Automated 4-liter spin-filter suspension culture vessel. Section of polycarbonate sheet forms top for polycarbonatejar; modified stirbar assembly.
122
R. A. COOK, F. T. COUNTER, AND J. K. MCCOLLEY
FIG 5. Disassembled filter head assembly for spin-filter device. Left to right: round magnet in plastic housing; 1-pm ceramic filter core with silicone rubber gaskets; top half of center piece-screws onto magnetic drive base; Teflon seal and Won-A O-ring; and pilot tube.
install a rubber O-ring beneath the Teflon seal which serves as a bearing between the glass pilot tube and filter tube. This was done to eliminate air leakage-noted by the presence of air bubbles in the pilot tube during media withdrawal. operations. Leakage has also occurred when medium levels were inadvertently permitted to drop, exposing the Teflon seal and O-ring and resulting in premature wear. During culture operations of several weeks’ duration the effluent medium is periodically checked for the presence of cells; no problems have been encountered when new seals and O-rings were used at the time of culture initiation, and when care was taken to maintain medium levels above the seal. The round magnet in the base of the spin filter was replaced with a 3-inch stirring bar by drilling holes through the plastic housing of the base to provide greater stirring action in the 4-liter vessel shown in Fig. 4.
VI. Conclusions Plastic laboratory ware, tubing, and sheets can be used to construct suspension culture vessels of almost any desired configuration. In designing such vessels, consideration should be given to such variables as head space
10.
PLASIlCS FOR SUSPENSION CULTURE VESSELS
123
and media surface areas, since these are known to affect gas exchange rates and consequently can be limiting factors in cell growth. Minimum culture volumes are determined by the shape of the vessel and the type of stirring device utilized. Elimination of cell adhesion to vessel walls obviates the need for enzymic detachment of cells and facilitates cell counting operations, thus giving better quantitation of cell growth. Further advantages are obtained in routine cell-handling operations. It is possible to change the medium ofmost cultures by allowing cells to settle out for several hours, decanting spent medium, and replenishing the culture with fresh growth medium. LLC-MK, cells handled in this manner did not adhere to the bottoms of plastic flasks after settling out at 37°C for over 18 hours (Cook et al., 1974a). Cultures grown to high population densities may suffer from oxygen deprivation during an extended settling process and are better suited to media replenishment employing the spin-filter system. Continued exposure to culture media and to cells during settling operations apparently modifies the nonstick property of plastic surfaces in some manner, but the relatively low cost of plastic laboratory ware and materials has permitted us to replace culture vessels after extended periods of use. LLC-MK, cells were grown in several types of plastic suspension culture vessels for over 22 months (Cook et al., 1974b). Suspension cultures of other types of cells have been grown in plastic vessels for varying periods of time; we have never encountered growth problems that were attributable to the use of plastic vessels. Plastics thus provide a source of construction materials with which suspension culture vessels can be economically built to fit the needs of the investigator. We have found these vessels to be well suited for virus production: virus titers of parainfluenza, measles, and canine distemper viruses grown in suspension cultures of LLC-MK, cells were equal to, or greater than, those obtained in our laboratories using monolayer cultures of the same cell line (Cook et al., 1974b). It seems likely that plastic suspension culture vessels can be used to advantage in the production of other viruses, viral reagents, and cell metabolites. ACKNOWLEDGMENTS The authors thank Mr. John May of our Photography and Audiovisual Services Department for providing an excellent set of pictures of our equipment.
REFERENCES Bizzini, B., Chermann, J.-C., Jasmin, C., and Raynaud, M. (1973). U.S. Patent 3,717,551. Capstick, P. B., Telling, R. C., Chapman, W. G., and Stewart, D. L. (1962). Nature (London) 195,1163.
124
R. A. COOK. F. T. COUNTER, AND J. K. MCCOLLEY
Cherry, W. R., and Hull, R. N. (1956). Anut. Rec. 124,483. Cook, R. A., and Counter, F. T. (1974). U.S.Patent 3,850,748. Cook, R. A., Counter, F. T., and McColley, J. K. (1974a). In Vitro 9, 318. Cook, R. A., Counter, F. T.,and McColley, J. K. (1974b). In Vitro 9, 323. Eagle, H. (1959). Saence 130,432. Fuccillo, D. A., Catalano, L. W., Jr., Moder, F. L., Debus, D. A., and Sever, J. L. (1969). Appl. Microbiol. 17, 619. Himmelfarb, P.,Thayer, P. S., and Martin, H. E. (1969). Science 164, 555. Horng, C., and McLimans, W. (1975). Biotedmol. Bioeng. 17, 713. Hull, R. N., Johnson, I. S., and Cherry, W. R. (1956). Anut. Rec. 124.490. McLimans, W. F., Davis, E. V., Glover, F. L., and Rake, G. W. (1957). J. Immunol. 79,428. Robb, J. A. (1970). Science 170, 857. Trager, G. W.,and Rubin, H. (1966). Viro/ogy30,275. van Wezel, A. L. (1967). Nature (London) 216,64. Walker, B. A., Brown. B. B., Krohmer, J. S., and Bonk, F. J. (1962). Tex. Rep. Biol. Med. 20,686.
Use of Plastics f i r Suspnsion Ctclttcre Vessels ROLAND A. COOK, FREDERICK T. COUNTER, JOYCE K. M C C O L L E Y
AND
Eli Lilly and Company, Indianapolis. Indiana
. . 111. Adaptation of Cell Lines to Growth in Suspension . I. Introduction
.
.
.
.
11. Use of Plastics for Monolayer Cultures
. .
. .
. . . . . . . . .
A. Problems with Glass Suspension Culture Vessels B. Use of Plastic Suspension Culture Vessels. . IV. Toxicity Testing of Plastic Laboratory Ware . . V. Construction of Plastic Suspension Culture Vessels . A. General . . . . . . . B. Small Vessels with Suspended Stirring Bars . C. Five-Gallon Carboy Stirred by Overhead-Drive Motor D. Use of Plastic Tubing and Sheets for Spin-Filter Vessels VI. Conclusions . . . . . . . . References . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . .
113 114 114 114 115 116 117 117
118 119 . 1 2 0 . 122 . 123
I. Introduction The field of suspended culture of mammalian cells is now over 20 years old, but continues to receive impetus from technological advances. Mass suspension culture systems have been devised for the production of virus vaccines and diagnostic reagents; large-scale cultures of lymphoblastoid, or hematopoietic cells-which grow naturally in suspension-have been utilized for the production of antilymphocyte serum and serve as a source of material for analysis of cell membranes and subcellular components. The recent use of DEAE-Sephadex beads (Horng and McLimans, 1975; van Wezel, 1967) and porous silica spherules (Bizzini et al., 1973) as microcarriers may make possible the growth in suspension of many types of functional, differentiated cells, which are normally anchorage dependent. 113
114
R. A. COOK, F. T. COUNTER. AND J. K. MCCOLLEY
Our laboratory has been involved in the adaptation of cell lines derived from normal tissues to growth in continuous suspension culture. Plastic culture vessels were found to facilitate this work, as we have previously reported (Cook et al., 1974a,b; Cook and Counter, 1974). We believe that other workers will appreciate the ease with which plastic materials can be used to construct suspension culture vessels to a given experimental design. In this report we describe our experiences with the use of these vessels and provide detailed information on their construction.
11. Use of Plastics for Monolayer Cultures The use of plastics for monolayer culture of mammalian cells has been well documented. Recent refinements in plastics technology have led to the development of plastic surfaces that are uniform enough for microtitration and microcloning techniques (Fuccillo et al., 1969;Robb, 1970).Amethodof treating plastic petri dishes for use in cloning chick embryo cells has been described (Trager and Rubin, 1966);however, processes used by commercial firms to modify plastic surfaces to enhance cell adhesion have not been published. The advances mentioned above have involved alteration ofthe basic “nonstick’’ qualities shared by most plastics. For certain operations, adhesion of cells to vessels is undesirable and unaltered plastics can beused to advantage. Polystyrene tubes have been used to eliminate cell adhesion in radiation experiments (Walker et al., 1962). Industrial firms have developed a large market for plastic-coated cookware based on the ease with which these utensils can be cleaned.
111. Adaptation of Cell Lines to Growth in Suspension
A. Problems with Glass Suspension Culture Vessels Initial studies were performed with the LLC-MK, cell line-a Lilly Laboratory cell line derived from normal rhesus monkey kidney in R. N. Hull’s laboratory (Hull et al., 1956). It was necessary to maintain suspensions of viable cells for several weeks. During this maintenance period, cells were observed to grow to increasing densities on the walls of glass flasks, with occasional sloughing of clumps of cells back into the culture medium. Calcium has been reported to cause clumping of cells in suspension (Eagle,
10.
115
PLASTICS FOR SUSPENSION CULTURE VESSELS
TABLE I ADHFSION OF CELLS TO G L A S ~ Culture volume Day
1
2 3 6
366 360 354 479 467
Cells in suspension x 106
Cells on flask wallb
183 180 89 144 42
+ +++ +‘
-
70 x
Total cells 183 x 106 Unknown Unknown Unknown 112 x 106 ~
‘From Cook er ol., 1974a. bRelative density of cells in ring on flask wall at meniscus of medium. ‘Increase in media volume resulted in numerouslarge chunks of cells sloughing off ring into medium. dCells on wall of flask removed by trypsinization and counted separately.
1959); however, no calcium was present in our medium formulation (Cook et al., 1974a). Actual growth of suspended cells could not be determined because of these problems. Results shown in Table1weretypical ofproblems encountered with glass vessels. The LLC-MK, cells used for this culture had previously been grown in bottles and were not adapted to suspension growth. At the end of 6 days, only 37.5% of surviving cells were in suspension; the remainder had adhered to the flask wall. Capstick et al. (1962) reported similar difficulties during the early stages of adaptation of the BHK21/13 (baby hamster kidney) cell line to growth in suspension. Conversely, investigators working with HeLa and L cell cultures reported minimal tendencies of these cells to adhere to glass in spinner culture (McLimans et al., 1957). The use of methyl cellulose in culture media (0.12%) and silicone treatment of glass flasks was found to retard but not prevent adhesion of cells to the flask walls.
B. Use of Plastic Suspension Culture Vessels It seemed possible that cells would be less likely to adhere to plastic surfaces. Polypropylene Erlenmeyer 125-ml flasks (Kimble) were obtained and equipped with magnetic stirring bars suspended from fishing swivels as described by Cherry and Hull (1956). Cultures of LLC-MK, cells did not adhere to the walls of these flasks. Suspended cells grew singly, or in small clumps of 3-4 cells and could be accurately counted. Subsequently, PK-15 cells-which are of normal pig kidney origin-were obtained from the American Type Culture Collection. These cells had previously been grown as monolayers and initially grew as large clumps in
116
R A. COOK, F. T. COUNTER, AND I. K. MCCOLLEY TABLE 11 ADHESION OF PK-15 SUSPENSION CULTURES TO NEWAND PREVIOUSLY USEDPOLYCARBONATE STIRRER FLASKS AFTER20 HOURSSE’ITLING
Temperature (“C) 36 25 4
AT
DIFFERENT TEMPERATURE+’
New flasks
Previously used flasksb
+ t -
“Symbols: +, Cells adhered to flask bottoms-did not detach after shaking. -, Cells readily resuspended. *Previously used flasks are flasks that have been used for several weeks and appear to have surface characteristically altered by this use. CFromCook et al.. 1974a.
suspension. It was necessary to disperse these clumps several times during the adaptation process. These cells also did not adhere to plastic stirrer flasks. In more recent studies, however, PK-15cells were found to adhere to polycarbonate stirrer flasks, which had been used as suspension culture vessels for several months, but not to new polycarbonate flasks (Table 11).
IV. Toxicity Testing of Plastic Laboratory Ware The polypropylene Erlenmeyer flasks which we initially used as suspension culture vessels were not treated or washed prior to use. No evidences of cell toxicity were observed. To determine whether other varieties of plastic might also be suitable, eleven different types were obtained from commercial suppliers and modified for use as suspension culturevessels(Tab1e 111).Some of the materials were not autoclavable, so all were sterilized withp-propiolactone and rinsed with medium prior to inoculation with suspensions of LLC-MK, cells. Glass flasks were used for control cultures. No evidence of gross toxicity could be observed in cultures after 3 days of incubation at 36°C. Cells did not adhere to any of the plastic containers; heavy rings of cells had grown at the meniscus on the glass control flasks. It should be mentioned here that all plastics contain a variety of compounds, such as plasticizers, fillers, and stabilizers. Also, formulas for a given type of plastic may differ from one manufacturer to the next. It is possible that a given formulation might be toxic to cultured cells; however, many of our plastic culture vessels have been in continuous use for several months and toxicity problems have not yet been encountered.
10.
117
PLAsIlCS FOR SUSPENSION CULTURE VESSELS
TABLE 111 PLASTICS OBTAINED FOR TOXICITY TESTING' Type of plastic Polypropylene Polycarbonate Teflon FEP Teflon TFE Linear polyethylene (LPE) Conventional polyethylene (CPE) Polyvinyl chloride (PVC) Polystyrene Polymethylpentene(TPX) Styrene-acrylonitrile Methyl-methacrylate (Lucite)
Item
Supplier
Catalog No.
Bottle, 4-ounce Erlenmeyer flask, 125-ml Bottle, 4-ounce Evaporating dish, 100-ml Bottle, 4-ounce
Kimble Matheson Matheson Star Matheson
53250 24093-25 4356-10 s-3450 4371-20
Bottle, 4-ounce Bottle, 4-ounce Container, 30-dram Graduated cylinder, 250-ml Graduated cylinder, 250-ml
Matheson Matheson Dynalab Matheson Preisser
4335-70 4334-10 2635 18591-30 F28684
Jar, I-quart
LPF
8025
'From Cook et a/., 1974a.
V. Construction of Plastic Suspension Culture Vessels A. General Polycarbonate and polypropylene were found to be most practical for this purpose. Polycarbonate is transparent; polypropylene is translucent. Both types of plastic are nontoxic, autoclavable, relatively inexpensive and readily available in various sizes of flasks and bottles from a number of commercial suppliers. Polycarbonate sheets up to 4 x 8 feet, and tubing up to 4 inches in diameter are available locally (Hyaline Plastics, Indianapolis, Indiana). Plastic culture vessels of various types were adapted to our needs by the simple expedient of heating stainless steel cork borers and melting holes in the plastic where desired(Caution:This operation produces noxious, possibly toxic fumes and should be done underafume hood). Anelectricdrill was used to make holes in thicker materials, such as plastic sheets and tubing. A drill bit smaller than the desired hole was used, followed by larger drill bits. This was necessary to prevent chipping; thin-walled containers could also be drilled with care taken to prevent cracking. Holes drilled in thicker materials were threaded when desired, using conventional taps. Probes, thermometers, breathers, and glass tubing for inlet, outlet, and sample lines were then inserted into the culture vessels where desired-either through rubber stoppers sized to fit the holes melted or drilled in the vessels, or through polypropylene fittings which are available commercially (Matheson Scientific, Elk Grove Village, Illinois).
118
R. A. COOK, F. T. COUNTER. AND J. K. MCCOLLEY
Polycarbonate materials can be cemented together, using Thickened Cement for Plexiglas Acrylic Sheet (Dayton Plastics, Dayton, Ohio). One of the two pieces to be joined was covered with cement, and both pieces were then immediately joined and held together for about 1 minute. Joints were ready for use in less than 1 hour.
B. Small Vessels with Suspended Stirring Bars Three types of small plastic suspension culture vessels are shown in Fig. 1. Each of these is equipped with a plastic-coated magnetic stirring bar suspended by a nichrome wire from a stainless steel, ball-bearing swivel (Eagle Claw, Denver, Colorado). A staple fashioned from this wire was used to secure the swivels to rubber stoppers or plastic lids(stap1eswere heated to penetrate plastic lids, then bradded down on top). Swivels were mounted with the large end up-resembling a child’s top-to shield the ball-bearing mechanism from liquids inadvertently splashed on the swivel. These swivels have proved to be reliable in many months of continuous use. In Fig. 1 a rubber vaccine stopper can be seen inserted in the sidewall of the 125-rnl Erlenmeyer flask. This was done to permit sampling of cultures with a syringe, rather than by opening the culture vessel. Plastic cement (Dekadhese, Donald Tulloch Jr., Chadds Ford, Pennsylvania) was applied to seal rubber stoppers to the plastic vessels after autoclaving. The vaccine stopper becomes hardened after several autoclave cycles and will leak after needle punctures unless replaced periodically. The 500-ml and 1000-ml plastic bottles shown in Fig. 1 are equipped to operate as closed systems. Glass sampling tubes run to the bottom of both
FIG. 1. Plastic suspension culture vessels with suspended strirring bars. Left to right: 125-ml polycarbonate Erlenmeyer flask,500-mlpolycarbonate centrifuge bottle, and 1OOO-ml polypropylene centrifuge bottle.
10. PLASIICS FOR
SUSPENSION CULTURE VESSELS
119
vessels. These tubes must be carefully positioned inside the vessels, so that the suspended stirbars do not hang up on the tubes and become inoperative. A short tube mounted on the sidewall is used for media addition and also to cycle the cell suspension from the sampling tube back into the culture, via an attached syringe-allowing a representative sample of the culture to be taken. A cotton-filled breather is mounted on the vessel wall in a location where it will not become wet during these operations. Surgical latex tubing connects the glass tubing with Luer-type stainless steel fittings (BectonDickinson, Rutherford, New Jersey). Tubing is autoclaved in 0.1 N NaOH for 5 minutes, rinsed, autoclaved in deionized water for 5 minutes and then thoroughly rinsed and dried prior to use.
C. Five-Gallon Carboy Stirred by Overhead-Drive Motor Polypropylene carboys of the type shown in Fig. 2 were used to grow suspension cultures of LLC-MK, and PK-15 cells in volumes of up to 18
FIG.2. Fivegallon polypropylene carboy stirred by overhead-drive motor.
120
R. A. COOK, F. T. COUNTER, AND J. K. MCCOLLEY
liters. This vessel was also operated as a closed-system stirrer and is shown equipped with a Teflon seal assembly, Viton-A O-ring, 24/40 ground-glass joint, glass stirring shaft, and Teflon stirring blade (Cole-Parmer, Chicago, Illinois). The tapered end of the ground-glass joint was cut off and the remainder was mounted in a hole drilled in a No. 12 stopper, which fits the neck of the carboy. This type of vessel has been useful in the preparation of batch cultures.
D. Use of Plastic Tubing and Sheets for Spin-Filter Vessels A section of polycarbonate tubing 3 inches in diameter by 7$ inches in height was cemented to a square of polycarbonate to form the suspension culture vessel shown in Fig. 3. A second, larger polycarbonate square forms the top. These squares sawed out of a large polycarbonate sheet. The top of the vessel was secured to the tubing by horizontally drilling a hole in each of 4 pieces of polycarbonate $ by 1 inch in diameter, $ inch thick, and inserting 2-inch stainless steel screws through the holes. The pieces were then cemented to the tubing. The top was drilled to accept the screws, and wingnuts
FIG 3. Oneliter spin-filter suspension culture vessel made from polycarbonate sheet and tubing.
10.
PLAmCS FOR SUSPENSION CULTURE VESSELS
121
were used to secure the top. A Neoprene rubber gasket was cut so as to slightly overlap the rim of the tubing, and provided an airtight seal. Figure 4 shows an automated cell culture system utilizing a 4-liter polycarbonate jar which has been similarly equipped with a square top fashioned from a polycarbonate sheet. Both of the vessels shown in Figs. 3 and 4 are modified spin-filter suspension culture vessels. The spin-filter concept was originated by Arthur D. Little Co., as reported by Himmelfarb et al. (1969). The principle of the device is that boundary effects at the surface of the spinning filter allow large volumes of spent medium to be withdrawn from a suspension culture without clogging the filter with cells. Spin filter equipment is available commercially from the VirTis Co., Gardiner, New York. A disassembled filter head assembly is shown in Fig. 5. In our hands, it has been necessary to
FIG.4. Automated 4-liter spin-filter suspension culture vessel. Section of polycarbonate sheet forms top for polycarbonatejar; modified stirbar assembly.
122
R. A. COOK, F. T. COUNTER, AND J. K. MCCOLLEY
FIG 5. Disassembled filter head assembly for spin-filter device. Left to right: round magnet in plastic housing; 1-pm ceramic filter core with silicone rubber gaskets; top half of center piece-screws onto magnetic drive base; Teflon seal and Won-A O-ring; and pilot tube.
install a rubber O-ring beneath the Teflon seal which serves as a bearing between the glass pilot tube and filter tube. This was done to eliminate air leakage-noted by the presence of air bubbles in the pilot tube during media withdrawal. operations. Leakage has also occurred when medium levels were inadvertently permitted to drop, exposing the Teflon seal and O-ring and resulting in premature wear. During culture operations of several weeks’ duration the effluent medium is periodically checked for the presence of cells; no problems have been encountered when new seals and O-rings were used at the time of culture initiation, and when care was taken to maintain medium levels above the seal. The round magnet in the base of the spin filter was replaced with a 3-inch stirring bar by drilling holes through the plastic housing of the base to provide greater stirring action in the 4-liter vessel shown in Fig. 4.
VI. Conclusions Plastic laboratory ware, tubing, and sheets can be used to construct suspension culture vessels of almost any desired configuration. In designing such vessels, consideration should be given to such variables as head space
10.
PLASIlCS FOR SUSPENSION CULTURE VESSELS
123
and media surface areas, since these are known to affect gas exchange rates and consequently can be limiting factors in cell growth. Minimum culture volumes are determined by the shape of the vessel and the type of stirring device utilized. Elimination of cell adhesion to vessel walls obviates the need for enzymic detachment of cells and facilitates cell counting operations, thus giving better quantitation of cell growth. Further advantages are obtained in routine cell-handling operations. It is possible to change the medium ofmost cultures by allowing cells to settle out for several hours, decanting spent medium, and replenishing the culture with fresh growth medium. LLC-MK, cells handled in this manner did not adhere to the bottoms of plastic flasks after settling out at 37°C for over 18 hours (Cook et al., 1974a). Cultures grown to high population densities may suffer from oxygen deprivation during an extended settling process and are better suited to media replenishment employing the spin-filter system. Continued exposure to culture media and to cells during settling operations apparently modifies the nonstick property of plastic surfaces in some manner, but the relatively low cost of plastic laboratory ware and materials has permitted us to replace culture vessels after extended periods of use. LLC-MK, cells were grown in several types of plastic suspension culture vessels for over 22 months (Cook et al., 1974b). Suspension cultures of other types of cells have been grown in plastic vessels for varying periods of time; we have never encountered growth problems that were attributable to the use of plastic vessels. Plastics thus provide a source of construction materials with which suspension culture vessels can be economically built to fit the needs of the investigator. We have found these vessels to be well suited for virus production: virus titers of parainfluenza, measles, and canine distemper viruses grown in suspension cultures of LLC-MK, cells were equal to, or greater than, those obtained in our laboratories using monolayer cultures of the same cell line (Cook et al., 1974b). It seems likely that plastic suspension culture vessels can be used to advantage in the production of other viruses, viral reagents, and cell metabolites. ACKNOWLEDGMENTS The authors thank Mr. John May of our Photography and Audiovisual Services Department for providing an excellent set of pictures of our equipment.
REFERENCES Bizzini, B., Chermann, J.-C., Jasmin, C., and Raynaud, M. (1973). U.S. Patent 3,717,551. Capstick, P. B., Telling, R. C., Chapman, W. G., and Stewart, D. L. (1962). Nature (London) 195,1163.
124
R. A. COOK. F. T. COUNTER, AND J. K. MCCOLLEY
Cherry, W. R., and Hull, R. N. (1956). Anut. Rec. 124,483. Cook, R. A., and Counter, F. T. (1974). U.S.Patent 3,850,748. Cook, R. A., Counter, F. T., and McColley, J. K. (1974a). In Vitro 9, 318. Cook, R. A., Counter, F. T.,and McColley, J. K. (1974b). In Vitro 9, 323. Eagle, H. (1959). Saence 130,432. Fuccillo, D. A., Catalano, L. W., Jr., Moder, F. L., Debus, D. A., and Sever, J. L. (1969). Appl. Microbiol. 17, 619. Himmelfarb, P.,Thayer, P. S., and Martin, H. E. (1969). Science 164, 555. Horng, C., and McLimans, W. (1975). Biotedmol. Bioeng. 17, 713. Hull, R. N., Johnson, I. S., and Cherry, W. R. (1956). Anut. Rec. 124.490. McLimans, W. F., Davis, E. V., Glover, F. L., and Rake, G. W. (1957). J. Immunol. 79,428. Robb, J. A. (1970). Science 170, 857. Trager, G. W.,and Rubin, H. (1966). Viro/ogy30,275. van Wezel, A. L. (1967). Nature (London) 216,64. Walker, B. A., Brown. B. B., Krohmer, J. S., and Bonk, F. J. (1962). Tex. Rep. Biol. Med. 20,686.
