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Cell Culture Spinner Flasks

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Cell Culture Spinner Flasks Europäisches Patentamt & (19) European Patent Office Office européen des brevets (11) EP 1 736 536 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 27.12.2006 Bulletin 2006/52 C12M 3/04 (2006.01) (21) Application number: 06117829.9 (22) Date of filing: 22.09.2000 (84) Designated Contracting States: • Flickinger, John AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU Beverly MC NL PT SE MA 01915 (US) (30) Priority: 24.09.1999 US 405477 (74) Representative: Gill, Siân Victoria et al Venner Shipley LLP (62) Document number(s) of the earlier application(s) in 20 Little Britain accordance with Art. 76 EPC: London EC1A 7DH (GB) 00963738.0 / 1 222 248 Remarks: (71) Applicant: Cytomatrix, LLC This application was filed on 25-07-2006 as a Woburn, MA 01801 (US) divisional application to the application mentioned under INID code 62. (72) Inventors: • Upton, Todd Chestnut Hill MA 02467 (US) (54) Cell culture spinner flasks (57) The present invention relates to culturing devic- es that are compact, utilize small amounts of cell culture media to establish and maintain cell cultures, and pro- duce a large number of cells in a short period of time when compared to other cell culturing devices and tech- niques. Such devices are useful in the culture of all cell types, but are particularly useful in the culture of cells that are known in the art to be difficult to culture, including cells that lose one or more of their particular attributes/ characteristics (e.g., pluripotentiality), or cells that are difficult to establish cultures of (e.g. primary cells), during culturing in traditional cell culture devices. EP 1 736 536 A2 Printed by Jouve, 75001 PARIS (FR) 1 EP 1 736 536 A2 2 Description [0008] According to one aspect of the invention, an apparatus for culturing cells is provided. The apparatus Field of the Invention comprises a vessel for holding liquid cell culture media. a matrix assembly mounted in the vessel for movement [0001] In general the present invention relates to cell 5 in the media, said matrix assembly including a support culturing, and in particular to improved methods and de- and a plurality of three- dimensional porous matrix mem- vices for culturing cells. In particular aspects, the inven- bers carried by the support for movement therewith, and tion relates to culturing cells in three-dimensional matri- a drive member operatively coupled to the support for ces. moving it with the matrix members through the media. In 10 one embodiment, the support includes a shaft, an out- Background of the Invention wardly extending member from and connected to the shaft, and a holder for carrying at least one three- dimen- [0002] Cell culturing techniques and cell culturing de- sional porous matrix member and mounted on the out- vices are well known in the art. wardly extending member. In one embodiment, the hold- [0003] U.S. Patent 5,888,807, issued to Palsson, et 15 er is removably attached to the outwardly extending al., on March 30, 1999, describes bioreactors in which member. In certain embodiments, the outwardly extend- diverse cell types are simultaneously cultured in the pres- ing member carries a plurality of holders. In further em- ence of appropriate levels of nutrients and growth factors. bodiments, at least one of the holders carries a plurality Such levels are achieved by substantially continuously of three-dimensional porous matrix members. In a still perfusing the cells in the bioreactor while removing un- 20 further embodiment, the holder is permanently attached desirable metabolic products. to the outwardly extending member. [0004] U.S. Patent No. 5,712,154, issued to Mullon, et [0009] In any of the foregoing embodiments, a plurality al., on January 27, 1998, describes a cell culture system of outwardly extending members may extend radially out- comprising liquid nutrient and gas perfusion fibers that wardly from the shaft, each outwardly extending member provide the appropriate culture conditions for cells locat- 25 carrying at least one holder. In important embodiments, ed interstitially between the liquid nutrient and gas fibers. the holder can be detachably connected to the outwardly [0005] U.S. Patent No. 5,320,963, issued Knaack, et extending member, and/or the holder can be mechani- al., on June 14, 1994, describes a bioreactor for perfusion cally coupled to the outwardly extending member for re- culture of cells in suspension. The bioreactor of Knaack leasably retaining the holder on the outwardly extending et al. has an inversely-conical tank which includes a cell 30 member. culture zone and a cell settling zone disposed annularly [0010] In any of the foregoing embodiments, the holder relative to the cell culture zone in the upper region of the can be made of rigid plastic material and includes a U- tank. shaped frame having an open and a closed end with a pair of opposed sides, said frame being attached at its Summary of the Invention 35 closed end to the outwardly extending member, said sides having receptacles for receiving at least one three- [0006] The present invention provides culturing devic- dimensional porous matrix member and releasably hold- es that are compact, utilize small amounts of cell culture ing the three- dimensional porous matrix member in place media to establish and maintain cell cultures, and pro- on the holder. In certain embodiments, the shaft can be duce a large number of cells in a short period of time40 supported in the vessel for rotation about the shaft axis. when compared to other cell culturing devices and tech- In important embodiments, the shaft can be supported niques. Such devices are useful in the culture of all cell vertically in the vessel and be supported therein from its types, but are particularly useful in the culture of cells top end. In further embodiments, the outwardly extending that are known in the art to be difficult to culture, including member can be disposed in the vicinity of the lower end cells that lose one or more of their particular attributes/ 45 of the shaft. characteristics (e.g., pluripotentiality), or cells that are [0011] According to another aspect of the invention, difficult to establish cultures of (e.g., primary cells), during an apparatus for culturing cells is provided. In this aspect culturing in traditional cell culture devices. of the invention the apparatus comprises a relatively rigid [0007] The invention, in one important part, involves vessel for holding liquid cell culture media having an improved methods and devices for culturing cells by pro- 50 opening for providing access to its interior and a cover viding cells an increased access to nutrients. We de- for the opening, a shaft disposed in the vessel and sup- scribe herein a cell culture system that takes advantage ported for rotation in the vessel by the cover, an outwardly of biocompatible, open-pore, three-dimensional matri- extending member attached to the shaft extending out- ces. and uses movement of these matrices in culture wardly from the axis of rotation of the shaft for rotation media to increase accessibility of cells in culture on such 55 therewith, a plurality of holders attached to the outwardly matrices to media. Such culture system provides the ap- extending member, and at least one three-dimensional propriate conditions for the expansion and differentiation porous matrix member carried by the holders for rotation of most cell types. with the shaft in the media. 2 3 EP 1 736 536 A2 4 [0012] In many embodiments, a motor drive is dis- device attached to the base arm for mounting the holder posed outside the vessel and magnetically coupled to to a support, and a closure-cap detachably mounted to the shaft for rotating the shaft in media in the vessel. In the open end of the arms for preventing the three- dimen- a further embodiment, a motor drive is disposed outside sional porous matrix member being withdrawn from be- the vessel and magnetically coupled to the member for 5 tween the arms, said closure-cap having a handle for rotating the holder in media in the vessel. stripping the closure- cap from the holder and for carrying [0013] In any of the foregoing embodiments according the assembly without touching the three-dimensional po- to this aspect of the invention, the holder may comprise rous matrix member. In certain embodiments, the clo- a pair of substantially parallel arms connected together sure-cap has an end wall for spanning the open end at one end by a base arm and having an open end at the 10 space between the arms and a pair of legs for engaging other, a mounting device connected to the base arm for the sides of the arms, and connectors on the arms and mounting the holder to a support, and a groove in each the legs for engaging one another to releasably hold the of the arms generally facing one another to engage the closure-cap in place on the holder. In important embod- at least one three-dimensional porous matrix member. iments, the legs are of unequal length and the handle is [0014] In any of the foregoing embodiments according 15 disposed on the side of the closure- cap nearer the longer to this aspect of the invention, a plurality of outwardly leg. In further important embodiments, the connectors extending members may extend outwardly from the are detents and recesses disposed on arms and the ends shaft, each of said members having at least one station of the legs. In one embodiment, the connectors are dis- for connection to the base arm of the holder.
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