Method for in vitro reproduction and growth of cells in culture medium

• US 5,017,490 A
• Filed: 03/10/1989
• Issued: 05/21/1991
• Est. Priority Date: 03/10/1989
• Status: Expired due to Fees

• Current Assignee: Baxter International Inc.
• Original Assignee: Baxter International Inc.
• Inventors: Ventura, Peter, Oakley, Robert V., Taiariol, Van
• Primary Examiner(s): Lacey, David L.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/321,765
• Time in Patent Office: 802 Days
• Field of Search: 435/240.2, 435/240.23, 435/240.241, 435/240.25, 435/243, 435/284-286, 435/296, 435/299-301, 435/311, 435/313, 435/800, 435/818, 435/819, 435/1, 435/2, 422/102, 422/101, 604/410, 206/438, 206/439, 383/2, 383/37, 383/102
• US Class Current: 435/401
• CPC Class Codes: C12M 23/14 Bags
• CPC Class Codes: C12M 23/24 Gas permeable parts
• CPC Class Codes: C12M 23/34 Internal compartments or partitions

Abstract

Method and apparatus for in vitro culture of cells in culture medium to produce a viable cell mass of cell number greater than the initial cell number, wherein a cell mass of particular cell number is cultured in a first oxygen-permeable culture sub-compartment to an increased cell number, and then aseptically transferred directly from the first sub-compartment to a next serial culture sub-compartment providing additional medium and/or culture space for further growth and reproduction to produce a viable cell mass of still higher cell number. Continued transfers are made, if necessary, to successive serial culture sub-compartment until a viable cell mass of desired cell number is attained, particularly one useful for seeding of a production-scale culture system.

Claims

1. A method for the in vitro culturing of animal cells in a culture medium to produce, from an initial viable cell mass of particular cell number, a viable cell mass of larger cell number, comprising the steps of:

(a) providing a series of animal cell culture sub-compartments, each sub-compartment comprising a culture space for animal cells and for culture medium, defined by a flexible material permeable to oxygen, said series further comprising regulating means, operable external of the culture space, for variably permitting or precluding liquid communication between any given sub-compartment in said series and at least a next serial sub-compartment in said series;

(b) arranging said series of animal cell culture sub-compartments in a controlled gaseous environment, comprising oxygen, suitable for in vitro animal cell culture;

(c) introducing culture medium and a viable cell mass of particular cell number into a first culture sub-compartment of said series, isolated by said regulating means from liquid communication with any other sub-compartment of said series;

(d) maintaining in said first culture sub-compartment conditions effective to cause said viable cell mass therein to increase in cell number

(e) thereafter establishing liquid communication, by said regulating means, between said first culture sub-compartment and a next serial culture sub-compartment of said series provided with at least additional culture medium or culture space, and transferring said viable cell mass of increased cell number, and culture medium, from said first culture sub-compartment to said next serial culture sub-compartment;

(f) maintaining in said next serial sub-compartment conditions effective to cause said cell mass therein to further increase in cell number; and

(g) withdrawing from said next serial culture sub-compartment said viable cell mass of further increased cell number.

2. A method according to claim 1 wherein each of the culture sub-compartments of said series has a liquid inlet and a liquid outlet;

wherein said liquid outlet for a given culture sub-compartment is connected by a conduit to the inlet of a next serial sub-compartment of said series; and

wherein said regulating means is arranged at said conduit.

3. A method according to claim 2 wherein said conduit comprises compressible material, and wherein said regulating means comprises a removable compression clamp about the periphery of said compressible material.

4. A method according to claim 1 wherein said series of culture sub-compartments further comprises an elongate common conduit;

and wherein each said sub-compartment further comprises a tube segment, one end of which is in liquid communication with the culture space of said sub-compartment and the other end of which is in liquid communication with said elongate common conduit.

5. A method according to claim 4 wherein said regulating means is arranged at one or more of said tube segments.

6. A method according to claim 4 wherein said regulating means is arranged a one or more locations along said elongate common conduit.

7. A method according to claim 4 wherein said elongate common conduit and said tube segments comprise compressible material, and wherein said regulating means comprises a removable compression clamp about the periphery of said compressible material.

8. A method according to claim 1 wherein said culture sub-compartments of said series comprise segmented portions of a single bag-like enclosure of said flexible material.

9. A method according to claim 8 wherein said segmented portions are formed by removable external segmenting means applied at one or more locations along said single bag-like enclosure, and wherein said regulating means comprise said removable external segmenting means.

10. A method for the in vitro culturing of animal cells in a culture medium to produce, from an initial viable cell mass of particular cell number, a viable cell mass of larger cell number, comprising the steps of:

(a) providing an enclosed overall culture compartment of defined surface area and volume comprising a flexible oxygen-permeable material, said overall culture compartment being divided into two or more culture sub-compartments by removal segmenting means displaced about the periphery of the enclosed culture compartment;

(b) arranging said overall culture compartment in a controlled gaseous environment, comprising oxygen, suitable for in vitro cell culture;

(c) introducing culture medium and a viable cell mass of particular cell number into a first of said sub-compartments;

(d) maintaining in said first sub-compartment conditions effective to cause said viable cell mass therein to increase in cell number;

(e) thereafter removing said segmenting means between said first sub-compartment and a next serial sub-compartment, whereby there is produced a united larger culture space, comprised of said first sub-compartment and said next serial sub-compartment, for culture of the cells from said first sub-compartment; and

(f) maintaining in said united larger culture space conditions effective to cause the cell mass therein to further increase in cell number.

11. A method according to claim 10 wherein each of said culture sub-compartments has culture medium pre-arranged therein.