Cell culture spinner flasks

Abstract

The present invention relates to culturing devices that are compact, utilize small amounts of cell culture media to establish and maintain cell cultures, and produce a large number of cells in a short period of time when compared to other cell culturing devices and techniques. 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.

1 . Apparatus for culturing cells, comprising: 
 a vessel for holding liquid cell culture media,    a matrix assembly mounted in the vessel for movement in the media, said matrix assembly including a support and a plurality of three-dimensional porous matrix members carried by the support for movement therewith, and    a drive member operatively coupled to the support for moving it with the matrix members through the media.    
   
   
       2 . Apparatus as defined in  claim 1 , wherein the support includes a shaft, an outwardly extending member from and connected to the shaft, and a holder for carrying at least one three-dimensional porous matrix member and mounted on the outwardly extending member.  
   
   
       3 . Apparatus as defined in  claim 2 , wherein the holder is removably attached to the outwardly extending member.  
   
   
       4 . Apparatus as defined in  claim 2 , wherein the outwardly extending member carries a plurality of holders.  
   
   
       5 . Apparatus as defined in  claim 3 , wherein the holder carries a plurality of three-dimensional porous matrix members.  
   
   
       6 . Apparatus as defined in  claim 4 , wherein at least one of the holders carries a plurality of three-dimensional porous matrix members.  
   
   
       7 . Apparatus as defined in  claim 2 , wherein a plurality of outwardly extending members extend radially outwardly from the shaft, each outwardly extending member carrying at least one holder.  
   
   
       8 . Apparatus as defined in  claim 3 , wherein the holder is detachably connected to the outwardly extending member.  
   
   
       9 . Apparatus as defined in  claim 3 , wherein the holder is mechanically coupled to the outwardly extending member for releasably retaining the holder on the outwardly extending member.  
   
   
       10 . Apparatus as defined in  claim 3 , wherein the holder is made of rigid plastic material and includes a U-shaped frame having an open and a closed end with a pair of opposed sides, said frame being attached at its closed end to the outwardly extending member, said sides having receptacles for receiving at least one three-dimensional porous matrix member and releasably holding the three-dimensional porous matrix member in place on the holder.  
   
   
       11 . Apparatus as defined in  claim 10 , wherein the shaft is supported in the vessel for rotation about the shaft axis.  
   
   
       12 . Apparatus as defined in  claim 11 , wherein the shaft is supported vertically in the vessel and supported therein from its top end.  
   
   
       13 . Apparatus as defined in  claim 12 , wherein the outwardly extending member is disposed in the vicinity of the lower end of the shaft.  
   
   
       14 . Apparatus for culturing cells, comprising: 
 a relatively rigid vessel for holding liquid cell culture media having an opening for providing access to its interior and a cover for the opening,    a shaft disposed in the vessel and supported for rotation in the vessel by the cover,    an outwardly extending member attached to the shaft extending outwardly from the axis of rotation of the shaft for rotation therewith,    a plurality of holders attached to the outwardly extending member, and    at least one three-dimensional porous matrix member carried by the holders for rotation with the shaft in the media.    
   
   
       15 . Apparatus for culturing cells, comprising: 
 a relatively rigid vessel for holding liquid cell culture media having an opening for providing access to its interior and a cover for the opening,    a shaft supported in the vessel by the cover,    an outwardly extending member attached to the shaft extending outwardly from the axis of rotation of the shaft for rotation therewith,    a plurality of holders attached to the outwardly extending member, and    at least one three-dimensional porous matrix member carried by the holders for rotation with the outwardly extending member in the media.    
   
   
       16 . Apparatus as described in  claim 14 , wherein a motor drive is disposed outside the vessel and magnetically coupled to the shaft for rotating the shaft in media in the vessel.  
   
   
       17 . Apparatus as described in  claim 15 , wherein a motor drive is disposed outside the vessel and magnetically coupled to the member for rotating the holder in media in the vessel.  
   
   
       18 . Apparatus as described in  claim 16 , wherein the holder comprises a pair of substantially parallel arms connected together at one end by a base arm and having an open end at the other, a mounting device connected to the base arm for mounting the holder to a support, and a groove in each of the arms generally facing one another to engage the at least one three-dimensional porous matrix member.  
   
   
       19 . Apparatus as described in  claim 18 , wherein a plurality of outwardly extending members extend outwardly from the shaft, each of said members having at least one station for connection to the base arm of the holder.  
   
   
       20 . Apparatus as described in  claim 19 , wherein the at least one station includes a recess, and the base of the holder is connected to a mounting device that fits into the recess to mechanically keep the holder in place on the outwardly extending member.  
   
   
       21 . Apparatus as described in  claim 18 , wherein the three-dimensional porous matrix member is circular and has an edge that engages the arms.  
   
   
       22 . Apparatus as described in  claim 18 , wherein the three-dimensional porous matrix member is rectangular and has an edge that engages the arms.  
   
   
       23 - 52 . (canceled)  
   
   
       53 . Apparatus for culturing cells, comprising: 
 a vessel for holding liquid cell culture media,    a matrix assembly mounted in the vessel for movement in the media, said matrix assembly including a support and at least one three-dimensional porous matrix member carried by the support for movement therewith, and    drive means operatively coupled to the support for moving it with the matrix members through the media.    
   
   
       54 . Apparatus as defined in  claim 53 , wherein the support includes a shaft means, means extending away form the shaft means, and means for carrying at least one three-dimensional porous matrix member and mounted on the means extending away form the shaft means.  
   
   
       55 . Apparatus for culturing cells, comprising: 
 a relatively rigid vessel for holding liquid cell culture media having an opening for providing access to its interior and a cover for the opening,    first means disposed in the vessel and supported in the vessel by the cover,    an outwardly extending member attached to the first means and rotatable in the vessel,    a plurality of holders attached to the outwardly extending member, and three-dimensional porous matrix members carried by the holders, and    means for moving the outwardly extending member with the holders in the media.    
   
   
       56 . A matrix assembly, comprising: 
 a holder having means for mounting the holder to a support,    engaging means on the holder for carrying a three-dimensional porous matrix member, and    at least one three-dimensional porous matrix member attached to the holder by the engaging means.    
   
   
       57 . Apparatus as defined in claims  1 , wherein the three-dimensional porous matrix member is an open cell porous matrix member having a percent open space of at least 75%.  
   
   
       58 . Apparatus as defined in  claim 57 , wherein the three-dimensional porous matrix member has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 μm.  
   
   
       59 . Apparatus as defined in  claim 57 , wherein the three-dimensional porous matrix member is a metal-coated reticulated open cell foam of carbon containing material.  
   
   
       60 . Apparatus as defined in  claim 59 , wherein the metal is selected from the group consisting of tantalum, titanium, platinum, niobium, hafnium, tungsten, and combinations thereof, wherein said metal is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, and combinations thereof.  
   
   
       61 . Apparatus as defined in  claim 59 , wherein the metal is tantalum.  
   
   
       62 . A method for in vitro culture of cells, comprising: 
 introducing an amount of cells into a three-dimensional porous matrix having interconnected pores of a pore size sufficient to permit the cells to grow throughout the matrix,    culturing the cells under conditions sufficient to allow the cells to adhere to the three-dimensional porous matrix, and    moving the three-dimensional porous matrix in a liquid cell culture medium under conditions sufficient to promote maintenance, expansion, or differentiation of the cells.    
   
   
       63 . The method of  claim 62 , wherein the cells are selected from the group consisting of mammalian cells, animal cells, plant cells, eukaryotic cells, prokaryotic cells and genetically engineered cells.  
   
   
       64 . The method of  claim 62 , wherein the cells are hematopoietic progenitor cells.  
   
   
       65 . The method of  claim 64 , wherein the hematopoietic progenitor cells are cultured under conditions and for a time sufficient to increase the number of hematopoietic progenitor cells relative to the amount introduced into the three-dimensional porous matrix.  
   
   
       66 . The method of  claim 65 , wherein the conditions exclude an exogenously added agent.  
   
   
       67 . The method of  claim 66 , wherein the exogenously added agent is selected from the group consisting of a hematopoietic growth factor that promotes hematopoietic cell maintenance, expansion and/or differentiation, inoculated stromal cells and stromal cell conditioned medium.  
   
   
       68 . The method of  claim 64 , further comprising: 
 before said introducing step, obtaining said hematopoietic progenitor cells from a blood product.    
   
   
       69 . The method of  claim 68 , wherein said blood-product is unfractionated bone marrow.  
   
   
       70 . The method of  claim 64 , further comprising harvesting hematopoietic cells.  
   
   
       71 . The method of  claim 70 , wherein said harvesting comprises a first harvesting after a first culturing period and at least one additional harvesting after at least one additional culturing period.  
   
   
       72 . The method of claims  62 , wherein the three-dimensional porous matrix is an open cell porous matrix having a percent open space of at least 75%.  
   
   
       73 . The method of  claim 72 , wherein the three-dimensional porous matrix has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 μm.  
   
   
       74 . The method of  claim 73 , wherein the three-dimensional porous matrix is a metal-coated reticulated open cell foam of carbon containing material.  
   
   
       75 . The method of  claim 74 , wherein the metal is selected from the group consisting of tantalum, titanium, platinum, niobium, hafnium, tungsten, and combinations thereof, wherein said metal is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, and combinations thereof.  
   
   
       76 . The method of  claim 74 , wherein the metal is tantalum.  
   
   
       77 . A matrix assembly for culturing cells, comprising: 
 a support comprising a shaft, and an outwardly extending member extending from and connected to the shaft, and    at least one rigid three-dimensional porous solid matrix member carried by the support for movement therewith, wherein the porous solid matrix member is a unitary structure.    
   
   
       78 . A matrix assembly as claimed in  claim 77 , wherein the support further comprises a holder attached to the outwardly extending member.