Multilayered cell culture apparatus

Abstract

A multilayered cell culture apparatus for the culturing of cells is disclosed. The cell culture apparatus is defined as an integral structure having a plurality of cell culture chambers in combination with tracheal space(s). The body of the apparatus has imparted therein gas permeable membranes in combination with tracheal spaces that will allow the free flow of gases between the cell culture chambers and the external environment. The flask body also includes an aperture that will allow access to the cell growth chambers by means of a needle or cannula. The size of the apparatus, and location of an optional neck and cap section, allows for its manipulation by standard automated assay equipment, further making the apparatus ideal for high throughput applications.

1 . (canceled) 
     
     
         2 . A method of culturing cells in a gas permeable multi-shelf cell culture apparatus, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising two or more culture compartments, each compartment including a shelf comprised of gas permeable, liquid impermeable material for cells to reside upon, each shelf connected to an opposing surface, a fluid pathway shared by said culture compartments, and each said shelf is in contact with a gas space, whereby said apparatus is incubated in the presence of ambient gas suitable for cell culture, oriented in a position such that said culture compartments are located one above the other, each said shelf is in a horizontal position with said gas space located below it, cells reside upon at least a portion of each said shelf, said culture compartments include media in contact with said shelf and said opposing surface, and ambient gas resides within each said gas space and is in contact with each shelf.   
     
     
         3 . The method of  claim 2 , wherein a culture compartment support is in contact with a portion of each said shelf. 
     
     
         4 . The method of  claim 3 , wherein said culture compartment support includes projections that make contact with said shelf. 
     
     
         5 . The method of  claim 4 , wherein said projections are dispersed throughout said gas space. 
     
     
         6 . The method of  claim 2 , wherein each said shelf is parallel to its said opposing surface. 
     
     
         7 . The method of  claim 2 , wherein the ratio of the volume of media within said apparatus to the surface area of said gas permeable, liquid impermeable walls is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         8 . A method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising two or more culture compartments, each having a gas permeable, liquid impermeable wall for cells to reside upon and an opposing wall, a portion of each said gas permeable, liquid impermeable wall in contact with a culture compartment support, a manifold that creates a fluid pathway between said culture compartments, at least a portion of each said gas permeable, liquid impermeable wall is in contact with a gas space, and   culturing said cells while said apparatus resides in the presence of ambient gas suitable for cell culture and in a position wherein said culture compartments reside at differing elevations, said culture compartments are full of media, each said gas permeable, liquid impermeable wall is held in a horizontal position by said culture compartment support, cells reside upon at least a portion of each gas permeable, liquid impermeable wall, said ambient gas suitable for cell culture resides in each said gas space and makes contact with each said gas permeable, liquid impermeable wall.   
     
     
         9 . The method of  claim 8 , wherein said culture compartment support includes projections that make contact with said gas permeable, liquid impermeable wall. 
     
     
         10 . The method of  claim 9 , wherein said projections are dispersed throughout said gas space. 
     
     
         11 . The method of  claim 8 , wherein each said gas permeable, liquid impermeable wall is parallel to its said opposing wall. 
     
     
         12 . The method of  claim 8 , wherein the ratio of the volume of media within said apparatus to the surface area of said gas permeable, liquid impermeable material is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         13 . A static method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf cell culture apparatus comprised of more than one culture space, each culture space including a shelf comprised of gas permeable, liquid impermeable material for cells to reside upon, each shelf connected to an opposing surface that is oriented in parallel to the shelf, each culture space is connected to a common fluid pathway, a gas space in contact with at least a portion of said gas permeable, liquid impermeable material of each shelf, a shelf support structure in contact with a portion of each said shelf and acting to maintain the shelf in a planar state, and   culturing said cells when said cell culture apparatus is in a location that includes ambient gas suitable for cell culture and said cell culture apparatus is in a position wherein each said shelf is in a horizontal position and a gas space is located below each said shelf, said shelves oriented one above the other, the space between each shelf and its opposing surface includes media in contact with the shelf and its opposing surface, and ambient gas is in the gas space located below each shelf.   
     
     
         14 . The method of  claim 13 , wherein said shelf support structure includes projections that make contact with said shelf. 
     
     
         15 . The method of  claim 14 , wherein said projections are dispersed throughout said gas space. 
     
     
         16 . The method of  claim 13 , wherein the ratio of the volume of media within said apparatus to the surface area of said shelves is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         17 . A method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising more than one culture compartment, each having a gas permeable, liquid impermeable shelf and an opposing wall, a gas space in communication with each said gas permeable, liquid impermeable shelf, and a manifold providing said culture compartments a shared fluid pathway, and   placing said apparatus in contact with ambient gas suitable for cell culture in a position such that said culture compartments are oriented one above the other, media is in a static state, each said gas permeable, liquid impermeable shelf resides in a horizontal position with its opposing wall residing above it and ambient gas makes contact with at least a portion of the underside of each said gas permeable, liquid impermeable shelf.   
     
     
         18 . The method of  claim 17 , wherein a portion of each said gas permeable, liquid impermeable shelf is in contact with a culture compartment support. 
     
     
         19 . The method of  claim 18 , wherein said culture compartment support includes projections that make contact with said gas permeable, liquid impermeable shelf. 
     
     
         20 . The method of  claim 19 , wherein said projections are dispersed throughout said gas space. 
     
     
         21 . The method of  claim 17 , wherein each said gas permeable, liquid impermeable shelf is parallel to its said opposing wall. 
     
     
         22 . The method of  claim 21 , wherein the ratio of the volume of media within said apparatus to the surface area of said gas permeable, liquid impermeable shelves is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         23 . A method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising more than one cell compartment, each cell compartment having a shelf for cells to reside upon comprised of a gas permeable, liquid impermeable membrane connected to an opposing wall, a manifold connecting said cell compartments, each said shelf in contact with projections that hold the shelf a planar position, said projections allowing ambient gas to make contact with each said shelf, whereby the cells are cultured within said apparatus when said apparatus is in contact with ambient gas suitable for cell culture and in a position wherein each said shelf is horizontal, said shelves at differing elevations, ambient gas is in contact with the underside of each said shelf, each said cell compartment is full of media, and the cells reside upon each said shelf.   
     
     
         24 . The method of  claim 23 , wherein each said shelf is parallel to its said opposing wall. 
     
     
         25 . The method of  claim 23 , wherein the ratio of the volume of media within said apparatus to the surface area of said shelves is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         26 . A method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising two or more culture spaces, each space having a gas permeable, liquid impermeable surface for cells to reside upon and an opposing surface, projections acting to maintain said gas permeable, liquid impermeable surface in a planar position so that it can be oriented in a horizontal position with said projections acting to allow ambient gas to make contact with the underside said gas permeable, liquid impermeable surface, a fluid pathway in communication with each said culture space, and   culturing said cells while said apparatus is in contact with ambient gas suitable for cell culture, the orientation of said apparatus being such that each said gas permeable, liquid impermeable surface resides at a different elevation than the other said gas permeable, liquid impermeable surfaces, said culture spaces are full of media that is in a static state, each said gas permeable, liquid impermeable surface is in a horizontal position, and ambient gas is in contact with at least a portion of the underside of each said gas permeable, liquid impermeable surface.   
     
     
         27 . The method of  claim 26 , wherein each said gas, permeable liquid impermeable surface is parallel to its said opposing surface. 
     
     
         28 . The method of  claim 26  wherein the ratio of the volume of media within said apparatus to the surface area of said gas permeable, liquid impermeable surfaces is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         29 . A method of culturing cells comprising:
 incubating cells in the presence of ambient gas suitable for cell culture, said cells residing in a gas permeable multi-shelf cell culture apparatus wherein said cells reside upon more than one shelf, each shelf comprised of gas permeable, liquid impermeable material, each shelf connected by a fluid pathway that is integral to said apparatus, a portion of the bottom of each shelf is in contact with a shelf support that maintains the shelf in a horizontal position, said shelves located at differing elevations, said ambient gas is in contact with the bottom of each said shelf, each said shelf has an opposing surface connected to it and residing above it, and media is in static contact with each shelf and its opposing surface.   
     
     
         30 . The method of  claim 29 , wherein each said shelf support includes projections that are in contact with said gas permeable shelf. 
     
     
         31 . The method of  claim 30 , wherein each said gas permeable shelf is parallel to its said opposing surface. 
     
     
         32 . The method of  claim 30 , wherein each said gas permeable shelf is rectangular. 
     
     
         33 . The method of  claim 29 , wherein the ratio of the volume of media within said apparatus to the surface area of said shelves is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         34 . A method of culturing cells consisting essentially of:
 adding cells and media into a gas permeable, liquid impermeable cell culture apparatus; and   incubating said apparatus in contact with ambient gas suitable for cell culture, wherein said cells reside upon more than one shelf, each shelf comprised of gas permeable, liquid impermeable material, a portion of the bottom of each said shelf is in contact with a shelf support that maintains the shelf in a horizontal position and at a different elevation relative to other shelves, said ambient gas is in contact with the bottom of each said shelf, each said shelf is connected to an opposing surface residing above it, the space between each said shelf and its opposing surface is full of media, and the space between each said shelf and its opposing surface is in communication with a common fluid pathway.   
     
     
         35 . The method of  claim 34 , wherein each said shelf support includes projections that make contact with said shelf. 
     
     
         36 . The method of  claim 34 , wherein said common fluid pathway is full of said media. 
     
     
         37 . The method of  claim 34 , wherein said ambient gas suitable for cell culture is located within a cell culture incubator that includes carbon dioxide. 
     
     
         38 . The method of  claim 34 , wherein said shelf is parallel to its opposing surface. 
     
     
         39 . The method of  claim 34 , wherein the ratio of the volume of media within said apparatus to the surface area of said shelves is in the range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         40 . A method of culturing cells, the method comprising:
 adding cells and media into a gas permeable multi-shelf apparatus comprising more than one culture compartment comprised at least in part of gas permeable material, each culture compartment having a gas permeable, liquid impermeable planar shelf for cells to reside upon connected to an opposing surface, a shared fluid pathway connecting said culture compartments, a gas space in contact with at least a portion of each gas permeable, liquid impermeable shelf, each gas permeable, liquid impermeable shelf in contact with a culture compartment support, and   placing said apparatus within a cell culture incubator that includes ambient gas suitable for cell culture, said apparatus oriented in a culture position within said cell culture incubator such that said culture compartments reside one above the other, at least a portion of said ambient gas enters each said culture compartment by way of its said shelf, at least a portion of said cells are located upon said shelves, said culture compartments are full of media, and said shelves are horizontal.   
     
     
         41 . A method of culturing cells, the method comprising:
 a first step of adding cells and media into a gas permeable multi-shelf apparatus comprising two or more culture spaces, each having a gas permeable, liquid impermeable shelf for cells to reside upon and an opposing surface, a portion of each said gas permeable, liquid impermeable shelf in contact with a culture compartment support that exposes at least a portion of each said gas permeable, liquid impermeable shelf to ambient gas and maintains said gas permeable, liquid impermeable shelf in a flat position when said gas permeable, liquid impermeable shelf is oriented in a horizontal position with media residing upon it, and a manifold that creates a fluid pathway between said culture compartments; and   a second step consisting essentially of placing said gas permeable multi-shelf apparatus in a cell culture incubator wherein said apparatus is in a position such that said culture compartments reside one above the other, each said gas permeable, liquid impermeable shelf is in a horizontal position, cells reside upon at least a portion of each gas permeable, liquid impermeable shelf and said culture compartments include media.   
     
     
         42 . The method of  claim 41 , wherein said cell culture incubator includes carbon dioxide. 
     
     
         43 . The method of  claim 41 , wherein the ratio of the volume of media within said apparatus to the surface area of said gas permeable, liquid impermeable shelves is in a range of 0.25 ml/cm 2  to 0.4 ml/cm 2 . 
     
     
         44 . The method of  claim 41 , wherein each said gas permeable, liquid impermeable shelf for cells to reside upon is parallel to its opposing surface. 
     
     
         45 . A method of culturing cell comprising:
 incubating cells in the presence of ambient gas suitable for cell culture, said cells residing in a gas permeable multi-shelf cell culture apparatus wherein said cells reside upon more than one shelf, each shelf comprised of gas permeable, liquid impermeable material, each shelf open to only one shared fluid pathway, said shared fluid pathway is located within said apparatus, a portion of the bottom of each shelf is in contact with a shelf support that maintains the shelf in a horizontal position, said ambient gas is in contact with the bottom of each said shelf, each said shelf has an opposing wall connected to it and residing above it, and said apparatus contains media in a static state.