Cell culture apparatus
I United States Patent 1 1 1111 3,875,000 Kaneda 1 Apr. 1, 1975 [5 1 CELL CULTURE APPARATUS 3.028.134 4/1962 Means et a1. 123/149 ,04 48 6 B 11 2 259.1 175] lnvemo Kamda Nomsowm P9 3.195 160 7 /136? Kial a l e f a1. 1 95/96 73 A I h ,NHL 3.221.378 12/1965 BIB 210/94 1 ss&#39;gnee Merck Inc Ra way 3,321,086 5/1967 Dews et a1. 210/321 [22] Filed: Apr. 5, 1974 3,338,795 8/1967 McBee 195/127 3.407,l20 10/1968 Weiss et a] .1 195/104 [211 APPl- 1914581425 3,711,329 1/1973 Adams 195/127 [52] U.S. Cl. 195/1.7, 195/127 P im ry x min rAlvin E Tanenholtz 51] lm. Cl C1211 i/00 A rn y. Ag nt. o F r arry esilake. J {58] Field of Search 195/127. 17, 1.8 R lph n n, r-; r n i f [56] References Cited {57] ABSTRACT UNITED STATES PATENTS A tissue cell culture apparatus wherein a culture bot- 827,l48 7/1906 Gordon 11 34/138 tle is connected to supply and collection manifolds via 1 19 6 0/ o p 113/55 lengths of flexible tubing and wherein the bottle is r0- 2998&#39;992 11/1937 Hellbach 195/109 tated in reciprocal fashion so that the flexible tubing 2.686.754 8/[954 MPUOCl 1 .1 195/143 does not become pinched or broken 2952 588 9/1960 Rmderer 1 v 195/143 2996429 8/1961 Toulmin Jr. 19511.7 9 Claims, 4 Drawing Figures PATEHTEUAPR H975 sum 2 u 3 @NVVDV CELL CULTURE APPARATUS This invention relates to the cultivation of tissue cells on surfaces under controlled conditions and more particularly it concerns novel methods and apparatus to maintain those conditions under close control during the cultivation and harvesting operations.  
  Certain virus vaccines and other cell types, are cultured in perfusion devices which essentially comprise one or more glass bottles lying on their sides and rolled continuously about their longitudinal axis while influent nutrients and effluent culture and spent medium passes into and out from the bottles via swivel connectors on their ends. As the bottles rotate, the culture medium is spread smoothly over their inner surfaces in a manner which promotes continuous and efficient production of the tissue cells.  
  It is important of course that the inflowing nutrients and the outflowing culture and spent medium be kept sterile and free from external contamination throughout the entire process. In general, it has not been especially difficult to maintain the perfusion bottles, the supply and discharge lines, the nutrient reservoirs and the collection reservoirs themselves sealed from contamination. A problem arises, however, in maintaining the swivel joints, through which the fluids must pass into and out from the rotating bottles, properly sealed against external contamination.  
  The swivel joints which were used in the past were expensive; and after prolonged periods of use they tended to leak. Also, where two or more fluid passageways were involved, the construction of the swivel joint became quite complicated.  
  The present invention overcomes these problems of the prior art. With the present invention, it is possible to provide a rotating bottle culture apparatus of relatively simple construction which, nevertheless, maintains a positive seal against leakage.  
  According to the present invention, fluids are passed between tissue culture bottles and stationary manifolds via flexible tubes which are sealed at each end to associated conduits on the bottle and manifolds. Means are provided for rotating the bottles reciprocally, first in one direction and then in the opposite direction. This limited rotation permits the culture medium within the bottles to spread itself over the inner surface of the bottles while the reciprocal rotation of the bottles prevents the flexible tubes from becoming pinched due to twisting or kinking.  
  There has thus been outlined rather broadly the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject of the claims appended hereto. Those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other methods and structures for carrying out the several purposes of the invention. It is important, therefore, that the claims be regarded as including such equivalent methods and constructions as do not depart from the spirit and scope of the invention.  
  A specific embodiment of the invention has been chosen for purposes of illustration and description, and  
 is shown in the accompanying drawings, forming a part of the specification, wherein:  
  FIG. 1 is a perspective view, partially broken away, of a portion of a tissue culture apparatus in which the present invention is embodied;  
  FIG. 2 is an enlarged fragmentary section view taken along line 22 of FIG. 1; and  
  FIGS. 3 and 4 are views similar to FIG. 1 showing different stages in the operation of the apparatus.  
  As shown in FIG. 1 there is provided, as part of a tissue culture apparatus, an elongated glass culture bottle 10, which lies horizontally on, and is supported by, a pair of elongated drive rollers 12. The rollers themselves are mounted in suitable bearing means (not shown for the sake of clarity).  
  Suitable drive means are provided to rotate the rollers about their longitudinal axis. As shown in FIG. 1 this drive means may comprise an electric motor 14, connected through a reciprocating mechanism 16 to a drive gear 18. The drive gear 18 is meshed with a driven gear 20 on the end of one of the rollers 12. The motor 14 operates continuously in one direction; however, the reciprocating mechanism 16 operates to convert the continuous rotational motion supplied by the motor 14 to reciprocal rotary motion of the drive gear I8. Thus the drive gear 18 rotates in one direction over a predetermined distance and then rotates in the opposite direction over the same distance. Reciprocating drive mechanisms for producing reciprocal rotary motion from continuous rotary motion are well known in the art. By way of example the mechanism may comprise a pair of bevel gears keyed to the same shaft as the drive gear 18 and arranged to mesh on opposite sides respectively of a bevel gear on the motor shaft. Alternate corresponding sectors of the pair of bevel gears would have the teeth removed, so that as the motor gear rotates continuously it drives first from one side via one of the pair of gears and then from the opposite side via the other of the pair of gears. This will produce a reciprocal rotation of the shaft of the drive gear 18. Alternatively, cams, crankshafts and eccentrics may be used to obtain the reciprocal rotary motion. The drive gear 18 communicates its reciprocal rotary motion through the drive gears 20 to the drive rollers 12. As shown in FIG. 2 the reciprocal rotary motion of the drive rollers 12 causes a corresponding reciprocal rotary movement of the culture bottle 10 which rests on them.  
  Reverting now to FIG. 1, it will be seen that there are provided a pair of stationary manifolds 22 and 24 through which nutrients and collection fluids may pass. One manifold is used for adding cells, media, seed virus and gases to the bottle while the other is used for drawing off spent media, cells, virus and gases. These manifolds are connected, respectively, to supply and collection reservors (not shown). Additional manifolds may be provided if necessary.  
  Each of the manifolds 22 and 24 is provided with a branch connection 26 and 28 leading to the bottle 10. A length of flexible tubing 30 extends from each manifold branch connection 26 and 28 to a corresponding bottle conduit 32 and 34. The bottle conduits are sealed to and pass through a stopper 36 which tightly closes the end of the bottle. As shown, the bottle 10 contains a liquid substance 38 which lies along the lower side of the bottle while a gaseous substance 40 occupies the space thereabove. The bottle conduits 32 and 34 are arranged within the bottle to communicate with either the liquid or gaseous substance. For example, as shown in FIGS. 1 and 2 the flexible conduit 34 is provided with a weighted mass 42 such as a stainless steel ball at its end to hold it beneath the surface of the liquid 38 toward the neck of the bottle while the other conduit 32, which is fixed or rigid, extends straight through the central region of the bottle in communica tion with the gaseous substance 40. The conduit 34 is, of course, flexible so that it will remain weighted down into the liquid substance 38 as the bottle rotates.  
  The lengths of flexible tubing 30 are tightly sealed to their respective manifold branch connections 26 and 28 and to their respective bottle conduits 32 and 34 by means of clamps 44. Additional clamps 45 may be provided along each of the lengths of flexible tubing 30 to pinch them off, either partially or completely, when desired. This permits control of fluid through the tubing without need for mechanical valves and moving seals.  
  Turning now to FIGS. 3 and 4, it will be seen that the lengths of flexible tubing 30 twist around each other as the bottle is rotated in each direction. Each time the bottle rotation is reversed, the lengths of tubing 30 untwist and then retwist in the opposite direction. In order to fully bathe and nourish the cells adhering to the inner surface of the bottle, the bottle should be rotated approximately 360 so that the liquid therein will flow over its entire surface. Lesser amounts of rotation will suffice where the liquid levels in the bottle are higher. For example, where the pool of liquid in the bottle covers a sector of 40, a total rotation of about 320 will suffice to bathe the entire surface. This total rotation may be carried out by rotating the bottle alternately in opposite directions by half the total amount, e.g., about 180, from its normal position shown in FIG. 1. Thus, as shown in FIG. 3, the bottle 10 has been rotated approximately 180 in a clockwise direction, while in FIG. 4 the bottle 10 is shown to have been rotated by approximately 180 in the counterclockwise direction. The total rotation of the bottle, however, is approximately 360.  
  As can further be seen in FIGS. 3 and 4, the described manner of rotating the bottle 10 through a total sector of approximately 360 imposes only a half twist on the lengths of flexible tubing 30 connecting the bottle to the manifolds 22 and 24. Because of this the possibility of the tubing 30 becoming pinched or cracked is minimized. At the same time a perfect seal is main tained from the manifolds to the inventor of the bottle without need or rotary seals with moving parts.  
  It will be appreciated that while two manifolds are shown to be connected to the bottle, a greater number may be similarly connected. For example, a third line may be provided to extend from a corresponding manifold and into the bottle just beyond the stopper to provide limit control of the total amount of gases and liquids in the bottle. Also, the degree of bottle rotation between rotation reversals may be increased or decreased to maintain the effective cell sheet surface bathed in the fluids, so long as the amount of rotation in any one direction is less than that which would close off or damage the flexible tubing.  
  It will also be appreciated that this apparatus can be used to harvest or charge fluids, either by slow continuous flow, or completely, at programmed intervals.  
  Although a particular embodiment of the invention is herein disclosed for purposes of explanation, various modifications thereof, after study of this specification, will be apparent to those skilled in the art to which the invention pertains.  
  What is claimed and desired to be secured by Letters Patent is:  
  1. Tissue culture apparatus comprising a tissue culture bottle, support and drive means supporting said bottle with its longitudinal axis horizontal, said support and drive means being operative to rotate said bottle about its longitudinal axis reciprocally first in one direction and then in the opposite direction, fluid conduit means for effecting transfer of fluids between the interior of the bottle and an exterior manifold during rotation of said bottle, said fluid conduit means comprising at least one flexible tube extending between one end of said bottle and said manifold, said flexible tube being fixedly secured at each end, respectively, to said bottle and to said manifold, said tube being twistable while remaining open, over the rotational movement of said bottle.  
  2. Tissue culture apparatus according to claim 1 wherein said support and drive means is operative to rotate said bottle through a total rotational sector of approximately 360 in each direction.  
  3. Tissue culture apparatus according to claim 1 wherein said support and drive means is arranged to rotate said bottle through sectors of approximately 180 in each direction from a neutral position with said flexible tube extending in non-twisted condition between said bottle and said manifold.  
  4. Tissue culture apparatus according to claim 1 wherein said support and drive means comprises a pair of elongated rollers extending parallel to each other and supporting the bottle on them.  
  5. Tissue culture apparatus according to claim 1 wherein at least two flexible tubes are provided between associated stationary manifolds and corresponding conduits extending into said bottle.  
  6. In a method for culturing tissue cells, the steps of supplying nutrients and gases to a bottle containing culture medium and tissue cells, withdrawing fluids from the bottle and passing said fluids through flexible conduits extending between the bottle and stationary manifolds, while supporting said bottle with its longitudinal axis in a horizontal direction and reciprocally rotating said bottle about said axis, first in one direction and then in the opposite direction.  
  7. A method according to claim 6 wherein said bottle is rotated by causing at least one roller, on which it is supported, to rotate first in one direction and then in the opposite direction.  
  8. a method according to claim 6 wherein said bottle is rotated in each direction over a total of approximately 360.  
  9. A method according to claim 6 wherein said bottle is rotated in each direction a total of approximately 180 from a neutral position where said flexible conduits extend between said bottle and said manifolds in a non-twisted condition.  
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