Interlinked culture chamber for biologicals Number:7,144,727 from the United States Patent and Trademark Office (PTO) owispatent

Title: Interlinked culture chamber for biologicals

Abstract: The bioreactor system of the present invention has two fluid-filled culture compartments in which cells, tissues and other biologicals are cultured. The two culture compartments are in fluid communication with each other and each culture compartment is transversed by a filter that prevents the exit of the cells from the culture compartment. Both reusable and disposable culture chambers are described for use as the first and/or second culture compartment.

Patent Number: 7,144,727 Issued on 12/05/2006 to Akers,   et al.

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Inventors:	Akers; Roger (Houston, TX), Anderson; William J. (Richmond, TX), Navran, Jr.; Stephen S. (Houston, TX)
Assignee:	Synthecon, Inc. (Houston, TX)
Appl. No.:	10/821,455
Filed:	April 9, 2004

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Current U.S. Class:	435/294.1 ; 210/257.2; 210/321.64; 210/335; 435/297.2; 435/298.2
Current International Class:	C12M 3/06 (20060101)
Field of Search:	435/293.1,293.2,294.1,297.2,297.4,298.2 210/321.64,321.78,321.87,257.2,335

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References Cited [Referenced By]

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U.S. Patent Documents

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	6022733		February 2000		Tam et al.
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	2004/0110273		June 2004		Akers et al.

Foreign Patent Documents

	06134210		May., 1994		JP

Other References

Rai M. et al.; Expression systems for production of heterologous proteins, Current Science, vol. 80, No. 9, May 10, 2001, pp. 1121-1128. cited by other . Verma, R. et al.; Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems, Journal of Immunological Methods, 216, 1998, pp. 165-181. cited by other . Hernaeus Instruments, Inc.; Genetic Engineering News, vol. 14, No. 12, Jun. 15, 1994. cited by other . FiberCell Hollow Fiber Cell Culture Systems, Dec. 1, 2004 printout, Internet at bellcoglass.com. cited by other . Gerecht-Nir, Sharon, et al. Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation, Biotechnology and Bioengineering, Early view online Apr. 8, 2004. cited by other.

Primary Examiner: Beisner; William H.
Attorney, Agent or Firm: Hall; Elizabeth R.

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to pending U.S. patent application Ser. No. 60/462,722 filed Apr. 14, 2003 by inventors Roger Akers, William Anderson, and Stephen Navran, Jr. and entitled "Interlinked Culture Chambers for Biologicals." The entire text of the above-referenced disclosures is incorporated by reference herein.

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Claims

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What is claimed is:

1. A culture system comprising: (a) a fluid inlet; (b) a first culture compartment having a tubular housing; (c) a first end piece attached to the fluid inlet on one side and to a first end of the tubular housing on a second side, (d) a second culture compartment coaxial with the first culture compartment, the second culture compartment having a proximal end and a distal end; (e) a fluid connector having a first side mounted on a second end of the tubular housing and a second side mounted on the proximal end of the second culture compartment, the fluid connector having a through bore passing from the first side to the second side of the fluid connector, wherein the through bore directs a fluid stream from the first culture compartment into the second culture compartment; (f) a connector filter positioned on the first side of the fluid connector to filter a fluid stream passing out of the first culture compartment and into the through bore of the fluid connector; (g) a fluid outlet; (h) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet; and (i) an outlet filter transversing the second culture compartment including: a support cylinder having a first end supported by the fluid connector and a second end supported by the distal end piece, a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the through bore of the fluid connector and the fluid outlet.

2. A culture system comprising: (a) a fluid inlet; (b) a first culture compartment having a tubular housing made of a fluid-impenetrable material, wherein the tubular housing has a first end and an opposed second end; (c) a first end piece attached to the fluid inlet on one side and to the first end of the tubular housing on a second side, (d) a second culture compartment coaxial with the first culture compartment and in fluid communication with the first culture compartment, the second culture compartment having a proximal end and a distal end; (e) a fluid connector having a first side mounted on the second end of the tubular housing and a second side mounted on the, proximal end of the second culture compartment, the fluid connector having a through bore passing from the first side to the second side of the fluid connector; (f) a connector filter having a first end and a second end, wherein the first end is mounted on the first side of the fluid connector, the connector filter positioned to filter a fluid stream passing out of the first culture compartment into the through bore of the fluid connector and into the second culture compartment; (g) a fluid outlet; (h) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet; and (i) an outlet filter having a one end mounted on a proximal side of the distal end piece, wherein the outlet filter is a membrane carrier assembly transversing the second culture compartment wherein the membrane carrier assembly has: a support cylinder; a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the through bore of the fluid connector and the fluid outlet.

3. The culture system of claim 2, wherein the through bore of the fluid connector is intersected by a through bore of a second fluid inlet.

4. The culture system of claim 2, further comprising at least one penetration port extending through a wall of the first or second culture compartment.

5. The culture system of claim 2, further comprising a gas venting means for allowing gas to escape from the first or second culture compartment as the compartment is filled with fluid.

6. The culture system of claim 2, further comprising a fill means for inserting fluids into or removing fluids out of the first or second culture compartment.

7. The culture system of claim 2, wherein the first end, the distal end and the fluid connector are concurrently rotated by a drive assembly.

8. The culture system of claim 2, wherein the second culture compartment has a greater volume than the first culture compartment.

9. The culture system of claim 2, further comprising an identifier.

10. The culture system of claim 9, wherein the identifier is a bar code.

11. A culture system comprising: (a) a fluid inlet; (b) a first culture compartment having (i) a fluid-impenetrable tubular sleeve having a first end and an opposed second end, (ii) a growth compartment within the sleeve, and (iii) a first end piece having one side attached to the fluid inlet and a second side attached to a first end of the tubular sleeve; (c) a second culture compartment coaxial with the first culture compartment, the second culture compartment having (i) a fluid-impenetrable housing having a proximal end and a distal end; and (ii) a growth compartment within the housing that is in fluid communication with the growth compartment within the sleeve, (d) a fluid connector having a first side mounted on the second end of the tubular sleeve and a second side mounted on the proximal end of the housing, the fluid connector having a through bore passing from the first side to the second side of the fluid connector wherein the through bore is in fluid communication with the growth compartment of the first and second culture compartment; (e) a connector filter having a one end supported by the first side of the fluid connector; (f) a membrane carrier assembly transversing the second culture compartment comprising (i) a support cylinder, (ii) a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and (iii) a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the through bore of the fluid connector and the growth compartment within the housing; (g) a fluid outlet; and (h) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet.

12. The culture system of claim 11, wherein the connector filter includes a molecular weight cut-off membrane.

13. The culture system of claim 12, wherein the connector filter includes a molecular weight cut-off membrane having a different molecular weight cut-off than the molecular weight cut-off membrane of the membrane carrier assembly.

14. The culture system of claim 12, wherein the molecular weight cut-off membrane of the connector filter is identical to the molecular weight cut-off membrane of the membrane carrier assembly.

15. The culture system of claim 11, wherein the connector filter includes: a cylindrical support transversing the first culture compartment, the support having a first end supported by the first end of the sleeve and a second end supported by the first side of the fluid connector; a molecular weight cut-off membrane secured to an exterior surface of the cylindrical support, and a channel between the exterior surface of the cylindrical support and an interior surface of the membrane, the channel in fluid communication with the through bore of the fluid connector and the growth compartment of the first and second culture compartment.

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Description

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BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a culture chamber for culturing cells, cellular aggregates, particles, tissues and organoids that respond to secretions from other cells, cellular aggregates, particles, tissues and organoids. More particularly, the present invention relates to a culture chamber having a first set of one or more biological culture chambers interconnected with a second set of one or more other biological culture chambers, wherein the secretions of the first set of biological culture chambers is fed into the second set of biological culture chambers.

2. Description of the Related Art

A variety of cell lines, including human embryonic stem (HES) cells require hormones, growth factors or other materials that are secreted from another cell type (commonly referred to as feeder cells). The therapeutic potential of such cells is only beginning to be realized. To keep pace with the ever increasing demand for the potential presented by such cells, new processes and apparatuses are needed that can efficiently provide a first set of cells with the secretions from another set of cells.

One of the major problems in the production of cells and cellular products is the required cleaning, sterilization and validation of the standard stainless steel or glass bioreactors by the customer. Furthermore, none of the currently available culture chambers are designed to have secretions of one culture chamber feed into another culture chamber.

Thus, a need exists for disposable culture chambers having a reduction in the risk of cross contamination and the downtime needed for equipment changeover between production runs.

In addition, a need exists for a simplified, efficient means of interlinking culture chambers such that one can culture one cell and transfer that cell's secretions into the media used to grow another cell type.

SUMMARY OF THE INVENTION

The invention contemplates a culture chamber having a first set of one or more biological culture chambers interconnected with a second set of one or more other biological culture chambers, wherein the secretions of the first set of biological culture chambers is fed into the second set of biological culture chambers. The present invention includes a serially arranged culture chamber system having separate but closely interconnected fluid-filled chambers in which cells, tissues and other biologicals are cultured without intermingling and wherein first, upstream chambers provide growth factors or precursor compounds for the second, downstream chambers.

Each of the upstream culture chambers is either traversed by one or more molecular weight cut-off membranes or provided with outlet filters that serve to separate the cells of an upstream chamber from the other, downstream flowstreams and chambers.

One aspect of the invention is a culture system comprising: (a) a fluid inlet; (b) a first culture compartment having a tubular housing; (c) a first end piece attached to the fluid inlet on one side and to a first end of the tubular housing on a second side, (d) a second culture compartment coaxial with the first culture compartment, the second culture compartment having a proximal end and a distal end; (e) a fluid connector having a first side mounted on a second end of the tubular housing and a second side mounted on the proximal end of the second culture compartment, the fluid connector having a through bore passing from the first side to the second side of the fluid connector; (f) a connector filter positioned on the first side of the fluid connector to filter a fluid stream passing out of the first culture compartment and into the through bore of the fluid connector; (g) a fluid outlet; (h) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet; and (i) an outlet filter supported by the distal end piece.

Another aspect of the invention is a culture system comprising: (a) a fluid inlet; (b) a first culture compartment having (i) a tubular sleeve, (ii) a growth compartment within the sleeve, (iii) a first end piece attached to the fluid inlet on one side and to a first end of the tubular housing on a second side, and (iv) a membrane carrier assembly transversing the growth compartment comprising a support cylinder, a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the fluid inlet and the growth compartment; (c) a second culture compartment coaxial with the first culture compartment, the second culture compartment having a proximal end and a distal end; (d) a fluid connector having a first side mounted on a second end of the tubular sleeve and a second side mounted on the proximal end of the second culture compartment, the fluid connector having a through bore passing from the first side to the second side of the fluid connector wherein the through bore is in fluid communication with the chamber of the membrane carrier assembly and the interior of the second culture compartment; (e) a fluid outlet; (f) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet; and (g) an outlet filter supported by the distal end piece.

Yet another aspect of the present invention is a culture system comprising: (a) a fluid inlet; (b) a first culture compartment having a tubular housing; (c) a first end piece attached to the fluid inlet on one side and to a first end of the tubular housing on a second side, (d) a second culture compartment coaxial with the first culture compartment, the second culture compartment having a proximal end and a distal end; (e) a fluid connector having a first side mounted on a second end of the tubular housing and a second side mounted on the proximal end of the second culture compartment, the fluid connector having a through bore passing from the first side to the second side of the fluid connector; (f) a connector filter positioned on the first side of the fluid connector to filter a fluid stream passing out of the first culture compartment and into the through bore of the fluid connector; (g) a fluid outlet; (h) a distal end piece mounted on the distal end of the second culture compartment and connected to the fluid outlet; and (i) an outlet filter transversing the second culture compartment including: a support cylinder having a first end supported by the fluid connector and a second end supported by the distal end piece, a molecular weight cut-off membrane secured to an exterior surface of the support cylinder, and a chamber between the exterior surface of the cylinder and an interior surface of the membrane, the chamber in fluid communication with the through bore of the fluid connector and the fluid outlet.

Still yet another aspect of the present invention is a culture system comprising: (a) a fluid inlet; (b) a first culture compartment having a tubular housing; (c) a first end piece attached to the fluid inlet on one side and to a first end of the tubular housing on a second side, (d) a fluid connector having a first side mounted on a second end of the tubular housing, the fluid connector having a through bore passing from the first side to a second side of the fluid connector; (e) a connector filter positioned on the first side of the fluid connector to filter a fluid stream passing out of the first culture compartment and into the through bore of the fluid connector; (f) a culture bag including a flexible external wall having a first end, a second end, an internal side, and an external side, wherein the internal side of the external wall is positioned to face an interior of the culture bag, a first bag end fused to the first end of the external wall and attached to the second side of the fluid connector, a second bag end fused to the second end of the external wall, and an outlet filter supported by the second bag end; and (g) a fluid outlet.

Yet another aspect of the present invention is a culture system comprising: (a) a fluid inlet; (b) a first culture bag having a flexible external wall having a first end, a second end, an internal side, and an external side, wherein the internal side of the external wall is positioned to face an interior of the first culture bag, a first bag end fused to the first end of the external wall and attached to the fluid inlet, a second bag end fused to the second end of the external wall, and a first bag filter positioned on the second bag end to filter a fluid stream passing out of the first culture bag; (c) a fluid connector having a first side mounted on the second bag end, the fluid connector having a through bore passing from the first side to a second side of the fluid connector; (d) a second culture bag including a flexible outer wall having a first end, a second end, an internal side, and an external side, wherein the internal side of the outer wall is positioned to face an interior of the second culture bag, a proximal bag end fused to the first end of the outer wall and attached to the second side of the fluid connector, a distal bag end fused to the second end of the outer wall, and an outlet filter supported by the distal bag end; and (g) a fluid outlet.

The foregoing has outlined several aspects of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiments disclosed might be readily utilized as a basis for modifying or redesigning the method or process for carrying out the same purposes as the invention. It should be realized that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows an oblique view of interlinked culture chambers;

FIG. 2 shows an oblique view of one embodiment of interconnected culture chambers of approximately equal capacity;

FIG. 3 is a longitudinal cross-sectional view of the interconnected culture chambers of FIG. 2;

FIG. 4 shows interconnected culture chambers where the chambers have different capacities and the larger chamber has a single diffusion membrane positioned within its interior;

FIG. 5 shows an oblique view of interconnected culture chambers where the chambers have different capacities and the larger chamber has multiple diffusion membranes positioned within its interior;

FIG. 6 is a longitudinal cross-sectional view of the interconnected culture chambers shown in FIG. 5;

FIG. 7 shows an oblique view of one embodiment of interconnected culture chambers of approximately equal capacity;

FIG. 8 is a longitudinal cross-sectional view of the interconnected culture chambers of FIG. 7;

FIG. 9 is one embodiment of interconnected culture chambers with an oblique view of the first chamber shown and a longitudinal cross-sectional view of the other chambers shown;

FIG. 10 is a longitudinal cross-sectional of a tandem bioreactor system wherein flexible disposable bioreactor chambers are mounted in tandem within a clamshell housing;

FIG. 11 is a longitudinal cross-sectional view of a dual conduit hydraulic flow swivel adapted for conveying two separate fluids into or out of a rotating bioreactor system;

FIG. 12 is a longitudinal cross-sectional of another tandem bioreactor system wherein flexible disposable bioreactor chambers are mounted in tandem within a clamshell housing; and

FIG. 13 is a longitudinal cross-sectional view of another tandem bioreactor system wherein flexible disposable bioreactor chambers are mounted in tandem within a clamshell housing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The interlinked culture chambers of the present invention are designed for culturing cells, cellular aggregates, particles, tissues and organoids that respond to secretions from other cells, cellular aggregates, particles, tissues and organoids. The tandem culture chamber of the present invention has a first set of one or more biological culture chambers interlinked with a second set of one or more other biological culture chambers, wherein the secretions of the first set of biological culture chambers are fed into the second set of biological culture chambers without the intermingling of cells. Both reusable and disposable culture chambers are described for culturing cells, cell aggregates, particles, tissues and organoids.

The serially arranged culture chambers of the present invention have fluid-filled culture compartments in which cells, tissues and other biologicals are cultured. The walls of the chambers can be either rigid or a flexible bag-like construction. Normally, the bag construction is used in disposable applications in order to minimize possibilities of cross contamination between culture lots. Examples of suitable disposable bag-like chambers are described in U.S. Pat. No. 6,673,598 B1 issued on Jan. 6, 2004 and entitled "Disposable Culture Bag", which is hereby incorporated herein by reference. Examples of suitable rigid and bag-like chambers are described in U.S. patent application, Ser. No. 10/725,607 filed Dec. 2, 2004 and entitled "Culture Chamber for Biologicals", which is hereby incorporated herein by reference.

In addition, either the rigid chamber or the bag system may use either a controlled molecular weight cut-off membrane structure or a controlled permeability filter to separate the culture media and the cells in the chamber from the outflow fluid. When a molecular weight cutoff membrane is used, certain constituents of the fluids on both sides of the membrane can diffuse across the membrane, so a properly constituted membrane can function to separate components of the biological media within the chamber from its outflow stream. When a suitably constructed outlet filter is positioned at the upstream end of the outlet end piece flow channel for a bioreactor chamber, the filter serves to prevent the cells and/or large particulate constituents of the biological media from passing through the filter into the outflow stream.

Different types of chambers may be used within an individual serially arranged culture system. For example, a rigid cell first chamber may be best suited for the preparation of a growth hormone, required nutrient, or precursor molecule needed for the well being of the cells in a disposable second chamber or vice versa. Interconnectors are described that allow multiple permutations of one or more rigid or flexible disposable culture chambers of a first set of chambers to be interlinked with one or more rigid or flexible disposable culture chambers of a second set of chambers.

The culture chambers of the present invention are generally configured so that they can be slowly rotated and provided with optimal conditions for the promotion of cell growth. The mechanisms for rotation of the bioreactors are not shown herein, but the details of the bioreactor chambers related to providing this rotational capability are shown. Accordingly, an example of an inlet and outlet fluid coupling swivel joint is described.

Interlinked Reusable Bioreactor Systems

The tandem culture chamber of the present invention has a first set of one or more biological culture chambers, hereinafter referred to as feeder cell chambers, interlinked with a second set of one or more other culture chambers. Reusable cell culture chambers are often desired to investigate a variety of systems for the production of a particular product and for optimizing certain growth parameters. Reusable cell culture chambers are typically made of a rigid plastic material that is non-toxic and non-reactive with biological systems, as well as being sterilizable.

The feeder cell chamber of the present invention has a fluid-filled culture compartment in which cells, tissues and other biologicals are cultured. The culture compartment is transversed by one or more molecular weight cut-off membranes attached to a membrane carrier assembly or provided with outlet filters to prevent the cells from leaving the culture compartment. Incoming nutrients and/or biological modifiers are transported through the membrane into the culture compartment and secretions (such as hormones, growth factors, differentiation factors, and metabolic waste products) are transported away from the fluid-filled culture compartment through the membrane and out the chamber outlet.

Referring now to the drawings, and initially to FIG. 1, it is pointed out that like reference characters designate like or similar parts throughout the drawings. The Figures, or drawings, are not intended to be to scale. For example, purely for the sake of greater clarity in the drawings, wall thickness and spacing are not dimensioned, as they actually exist in the assembled embodiment. In the Figures used to describe the interlinked culture chambers the media flows from the left to the right. Thus, the first set of one or more chambers (i.e., the feeder cell chambers) is positioned on the left and the second set of chambers is positioned on the right.

EXAMPLE 1

Interlinked Reusable Culture Chambers

FIG. 1 illustrates the interlinking of a number of reusable culture chambers. Although numerous permutations of such arrangements are possible, FIG. 1 shows an arrangement connecting three separate rigid bioreactor vessels of the type described in detail in pending U.S. patent application, Ser. No. 10/725,607, entitled "Culture Chamber for Biologicals."

The first chamber 120 utilizes a first molecular cutoff membrane 137 and is upstream of the two bioreactors 130a,b of the second chamber set. The first chamber 120 produces a desired growth hormone or other growth mediator for supply to the second chamber set 130a,b. This arrangement is utilized when the cells grown in one bioreactor chamber 120 are able to provide sufficient growth hormone, precursor, or component to contribute to the growth of cells in multiple second bioreactor chambers 130a,b. The second bioreactors 130a,b may have the same cell line or different cell lines.

Each of the bioreactor chambers 120, 130a,b is rotated. A variety of drive assemblies may be used to rotate the culture chamber such as the drive assembly described in U.S. Pat. No. 6,080,581. The arrangement of the rotational equipment can be multiple independent sets of the standard rotators known to those in the industry, or alternatively a gang rotation system could be used. The bioreactor chambers 130a,b are rotated substantially the same to generate a similar, balanced environment for the chambers. The two bioreactors of the second chamber set are similar to each other and use a second and third molecular cutoff membrane 189a,b optimized for the cells being cultured in the bioreactors and for the products being produced by the second chamber set.

All three bioreactors shown in FIG. 1 are constructed of the same components except for their respective membranes and have most components in common with those used in the interconnected culture chambers shown in FIG. 2 and described in more detail in Example 2. Thus, each chamber has an end piece 11 sealingly engaged on each end of a sleeve 24 and also has a membrane carrier assembly 127, located axially within the interior of sleeve 24, thereby forming a bioreactor chamber. The sleeve 24 has multiple radial wall penetration ports 25, as shown in FIG. 3. These penetration ports 25 are provided in the annular wall of sleeve 24 to allow the introduction of one or more fill ports 40 and one or more samplingports 44.

The biologicals being cultured in the rotatable chambers 120 and 130a,b require nutrients, so fluid-conducting swivels 50, stopcock valves 60, intermediate tubings 173a,b and fluid inlet tubing 172 and outlet tubing 171a,b are provided for the system. The system 100 is plumbed so that nutrient fluid enters the first chamber 120 of the system through inlet tubing 172, stopcock 60, and swivel 50. The fluid output from chamber 120 emerges through attachment neck 49 and swivel 50 of the outlet side endpiece 11 of the first chamber 120.

The flow then passes to a fluid conducting cross fitting 151 which has connections to both the two bioreactor chambers 130a,b via identical intermediate tubings 173a,b and which also connects to another stopcock 60. The intermediate tubings 173a,b are identical in order to ensure the balanced flow of nutrients to the second set of chambers. The stopcock 60 on the cross fitting 151 is attached to secondary fluid feed line 174 which can be used to supplement or alter the fluid mixture delivered to the second chambers 130a,b. The outflow from each of the second chambers passes through a swivel 50, stopcock 60, and outlet tubings 171a,b. This arrangement permits full control of the fluid system for the bioreactors of system 100.

EXAMPLE 2

Interconnected Equal Capacity Culture Chambers

FIG. 2 shows two interconnected bioreactors similar to the bioreactors described in Example 1. The particulars of the construction of the components of culture chamber 10 are best understood with reference to FIGS. 2 3. The serially arranged bioreactor system 10 is composed of two individual reusable rigid walled bioreactor chambers 20 and 30 cojoined by a fluid connector or center hub piece 80.

The first bioreactor chamber 20 is located on the inlet side and serves to feed the second bioreactor chamber 30. The media flows from the left to the right in the Figures. The biologicals being cultured in the rotatable chambers 20 and 30 require nutrients, so fluid-conducting swivels 50, stopcock valves 60, and fluid inlet tubing 72 and outlet tubing 71 are provided on the ends of the tandem housing 10 so that the entry and exit of media is controlled.

For first bioreactor chamber 20, an end piece 11 seals to the upstream end of right circular cylindrical tubular sleeve 24 and center hub 80 seals to the downstream end so that a growth compartment 85 is formed within the enclosed space. When in use for culturing cells, cellular aggregates, particles, tissues and organoids, the culture chamber is designed to be supported on and rotated by a roller drive which rotates the chamber about its axis. In this situation, the end pieces 11 and the center hub 80 serve as tires for contact of the assembly 10 with the drive wheels of the drive assembly. A variety of drive assemblies, not shown here, may be used to rotate the culture chamber such as the drive assembly described in U.S. Pat. No. 6,080,581.

Multiple radial wall penetration ports 25 are provided in the annular wall of sleeve 24 to allow the introduction of one or more fill ports 40 and one or more samplingports 44. The swivel 50, the fill port 40, and the samplingport 44 are described in more detail in U.S. Pat. No. 6,673,598 B1 that is incorporated herein by reference.

End piece 11 has a right circular cylindrical central body having a coaxial push-on hose barb attachment neck 49 on its outer face. Axial through hole 15 penetrates through the attachment neck 49 and the rest of the body of end piece 11. Reduced diameter coaxial right circular cylindrical interior projection 12 extends inwardly on the transverse face of the interior end of end piece 11, with a flat-bottomed trepanned groove 14 located on the transverse interior face of end piece 11 immediately exterior of projection 12.

The exterior cylindrical surface of interior projection 12 has annular male O-ring grooves. Elastomeric O-rings 18 are mounted in the O-ring grooves of projection 12. At the interior end of interior projection 12 of end piece 11, flat-bottomed coaxial counterbore 19 intersects through hole 15. An optional lead-in chamfer may be provided at the mouth of counterbore 19 in order to facilitate the stabbing of an O-ring seal with the membrane carrier assembly.

The right circular cylindrical sleeve 24 can be made of a variety of materials such as glass, stainless steel or plastic. Preferably the reusable cell culture chamber is constructed of plastic, typically a transparent plastic such as an acrylic plastic for the cylindrical sleeve 24 and opaque plastics such as Kynar.TM. or Delrin.TM. for the other rigid pieces such as the end pieces 11. Suitable plastics have substantially zero porosity and are impermeable to gases and non-reactive to biological media and its components. Suitable construction materials must also be able to undergo multiple sterilizations by steam, gas, or radiation without deforming, cracking or otherwise being rendered unusable.

Although not shown in FIG. 3, the right circular cylindrical sleeve 24 is preferably provided with a lead-in taper on each of its interior corners to facilitate the stabbing of O-rings 18 over the interior projections 12 and 81 forming the ends of first chamber 20. The interior bore of sleeve 24 is a close sliding fit to the outer diameter of interior projection 12 of end fitting 11, thereby permitting O-rings 18 to sealingly engage the bore of sleeve 24. O-rings 18 also serve to retain sleeve 24 in its desired axial position by virtue to their forceful frictional engagement with sleeve 24.

As previously stated, the sleeve 24 has multiple radial wall penetration ports 25 to allow the mounting of fittings used for inserting fluid into or removing fluids from the growth compartment 85 and for allowing gas to escape from the growth compartment 85 as it is being filled with fluid. At each end of chamber 20, sleeve 24 is stabbed over the interior projections 12 and 81 and bottomed out in the trepanned grooves 14 and 83 at the interior ends of end piece 11 and center hub 80, respectively. The growth compartment 85 is located between the interior bore of the sleeve 24, the membrane carrier assembly 27, the interior end of interior projection 12 of end piece 11, and the interior end of the interior projection 81 of the center hub piece 80.

Center hub 80 is a structure composed of cylindrical segments and is symmetrical about its transverse midplane. Center hub 80 has an axial through hole 86 that provides communication from one side of the piece to the other, so that center hub 80 not only supports chambers 20 and 30, but also provides a flow connection between the chambers 20 and 30. The interior projection 81 of center hub 80 is identically structured to projection 12 of end piece 11 and carries two male O-ring grooves that hold O-rings 18. The O-rings 18 mounted in the grooves of center hub 80 sealingly mate with and restrain the axial position of cylindrical sleeve 24.

A membrane carrier assembly 27, as shown in FIGS. 2 and 3, traverses the growth compartment 85. An end of the generally cylindrical membrane carrier assembly 27 is mounted in the counterbores 19 and 82 of each of the two end assemblies 11 and 81, respectively, used in chamber 20.

FIG. 3 shows the details of construction of membrane carrier assembly 27. Support cylinder 28 is symmetrical about its transverse midplane. The exterior of each end of cylinder 28 has, sequentially from its end, a lead-in taper to ease blind stabbing into a mounting hole, a first annular male O-ring groove 21 mounting elastomeric O-ring 87, and a second groove 31 configured similarly to an O-ring groove and used to mount a second O-ring 87, wherein the second O-rings serve to sealingly grip their respective ends of the membrane 37 which is deployed around the support cylinder 28. Each end of cylinder 28 also has an axial blind hole 34 with multiple (in this case, four) equispaced coplanar radial cross holes 35 intersecting the inner end of blind hole 34.

A small recessed surface pocket 32 having an arcuate cross-section is located on the exterior of cylinder 28 and is intercepted by each radial cross hole 35. The depth of these surface pockets 32 below the outside cylindrical diameter is largest near the intersection with its cross hole 35 and linearly tapers to zero towards the middle of cylinder 28.

Centrally deployed with a close fit around the exterior of cylinder 28 is a tubular molecular weight cut-off membrane 37. Membrane 37 is flexible with a limited amount of elastic stretch capability. The construction of membrane 37 is very carefully controlled so that the number of molecules, having a molecular weight in excess of the specific limiting molecular weight cut-off value of the membrane 37, diffused through the membrane in either direction is statistically very small and rapidly decreases as a function of increasing molecular weight. Thus, there is essentially no passage of molecules much larger than the molecular weight cut-off value of the membrane through the membrane 37.

The molecular weight cut-off value of the membrane 37 is preselected so that nutrients and growth factors, as well as metabolic waste products, can easily diffuse through the membrane, whereas larger cellular products can be retained. For example, Factor VIII (having a molecular weight of about 350,000 daltons) or IgG monoclonal antibodies (having a molecular weight of about 155,000 daltons) produced by genetically engineered bacteria or cells can be retained by a membrane with a molecular weight cut-off value of about 100,000 daltons; whereas the majority of serum albumin (having a molecular weight of about 67,500 daltons and making up 55% to 62% of serum protein) would be allowed to pass through the membrane. Since the culture chamber 20 is reusable, the membrane carrier assembly 27 can be assembled with membranes 37 having a variety of molecular weight cut-off values depending on the desired secretions being produced. The membrane 37 is selected to allow the user to select a molecular weight cut-off value that would allow the desired protein or other cell secretions to serve as feedstock or stimulant for the cells in the second bioreactor chamber 30.

As shown in FIG. 3, membrane 37 is sealed to the exterior of cylinder 28 at each end by using an O-ring 87 to circumferentially constrict over the exterior of membrane 37, thereby forcing it into sealing engagement with groove 31 on the outside of cylinder 28. In this manner, a small chamber in fluid connection with the radial flow passage 35 is formed between the exterior of surface pockets 32 and the interior of membrane 37. The depth and length of cut for the surface pockets 32 is predetermined to be sufficient to produce a sufficient pressure area so that the elastic resistance of membrane 37 can be overcome. The expansion of the membrane 37 when media is passed through flow passage 35 and along the surface pockets 32 of support cylinder 28 permits a thin flow sheet of media between the membrane 37 and cylinder 28 to be established.

Second chamber 30 is a mirror image of first chamber 20 except that the membrane 89 is used in place of membrane 37 in the assembly of the membrane carrier assembly 27. Membrane 89 is preselected for its molecular weight cut-off value according to desires of the user. The influx of fluid emerging from first chamber 20 and into the membrane carrier assembly 27 has its desired components diffusing across the membrane 89 into volume 88 for reaction with the cells therein. However, the bioreactive fluid within the volume 88 has waste products from the tandem cell arrangement of serial bioreactor 10 transfusing across membrane 89 and into the outflow stream of chamber 30 to emerge through hole 15 in end piece 11 and thence to fluid outlet tubing 71.

The present invention allows the user to select a membrane having a molecular weight cut-off value that would allow the desired protein or cellular product to pass out of the culture chamber with the waste products for collection and purification. However, the present invention also permits the user to select a membrane having a smaller molecular weight cut-off value than the desired protein or cellular product, so that the desired product is retained within the culture chamber and is concentrated with time as the cells multiply and continue to produce the desired product. Since the culture chamber 10 is reusable, the membrane carrier assemblies 27 can be assembled with membranes 37 and 89 having a variety of molecular weight cut-off values depending on the user's needs.

EXAMPLE 3

Interconnected Culture Chambers of Different Capacities

The interconnected culture chambers 10, as shown in FIGS. 2 and 3, may be configured where either chamber is shorter than the other chamber thereby interconnecting culture chambers of different capacities but having a hub 80 that joins chambers of substantially the same internal diameter.

Alternatively, the interconnected culture chambers 300, illustrated in FIG. 4, represent an embodiment of the serially arranged bioreactor system having culture chambers of different capacities. This bioreactor system utilizes basically the same arrangement as is used for the bioreactor system 10, except that the second chamber is a different size either in diameter and/or in length than the first chamber.

This arrangement basically utilizes two rigid coaxial bioreactor vessels 320 and 330 cojoined in tandem. The first chamber 320 utilizes a first molecular cutoff membrane 37 and is upstream of the bioreactor 330 of the second chamber. One of the features of this embodiment is the large size of second chamber 330 relative to first chamber 320. This arrangement is utilized when the first bioreactor chamber 320 is able to provide sufficient growth hormone or stimulator for a single large second bioreactor chamber 330.

The cojoined bioreactor chambers 320 and 330 are rotated as an unit using standard rotational equipment known to those in the industry, but with three supporting drive rollers which act on the tires of the end pieces 11 and 311 and the center hub 380.

The first chamber 320 produces a growth factor or other mediator for supply to the second chamber 330. The bioreactor of the second chamber uses a second molecular cut-off membrane 337 that is optimized for its cells and the outputs desired from the second chamber.

The first bioreactor chamber 320 of the system embodiment 300 has all of its components including the input fluid system in common with that of the first chamber 20 of the interconnected culture chambers 10 of the present invention, except that a different fluid connector or center hub 380 is used. Thus, first chamber 320 has an end piece 11 sealingly engaged on the inlet end of a sleeve 24 and also has a membrane carrier assembly 27 located axially within the interior of sleeve 24, thereby forming a bioreactor chamber as before. Multiple radial wall penetration ports 25 are provided in the annular wall of sleeve 24 to allow the introduction of one or more fill ports 40 and one or more samplingports 44.

The biologicals being cultured in the rotatable chambers 320 and 330 require nutrients, so fluid-conducting swivels 50, stopcock valves 60, fluid inlet tubing 72, and outlet tubing 71 are provided for the system. The system 300 is plumbed identically to that of the interconnected culture chambers of the bioreactor system 10 so that nutrient fluid enters the first chamber 320 of the system through inlet tubing 72, stopcock 60, and swivel 50. The fluid output from chamber 320 passes through a hole in center hub 380 into the second bioreactor chamber 330. The outflow from the second chamber 330 passes through a swivel 50, stopcock 60, and outlet tubing 71.

The growth compartment 385 of the first chamber 320 is located between the interior bore of the sleeve 24, the membrane carrier assembly 27, the interior end of interior projection of end piece 11, and the interior end of the interior projection 381 of the center hub piece 380.

Center hub 380 is a structure composed of right circular cylindrical segments. Center hub 380 has its first side on the first chamber side of its transverse midplane identical to the corresponding side of center hub 80 of the bioreactor system 10 and likewise identical to the end piece 11 of the first chamber 320 with hose barb attachment neck 49 removed. The first interior projection 381 of center hub 380 carries two male O-ring grooves that hold O-rings 18. The O-rings 18 mounted in the grooves of the interior projection of center hub 380 also sealingly mates with and restrains the axial position of cylindrical sleeve 24. Furthermore, the external cylindrical portion of hub 380 adjacent the interior projection serves as a tire for the rotation of the entire bioreactor assembly 300.

On its first side center hub 380 has a flat-bottomed cylindrical bore for housing the membrane carrier assembly 27 of the first chamber. On its second side obverse to the said first side of center hub 380 and in order from the transverse midplane of center hub 380 is a cylindrical flange 360 and a second projection 312 into the second chamber 330. The flange 360 is the same diameter as, or slightly larger than sleeve 324, used for the second chamber 330. The second interior projection 312 is sized to closely fit to the bore of sleeve 324 and has two male O-ring grooves mounting O-rings 318 to seal therewith.

An axial through hole provides communication from the counterbore on the first side of the hub 380 to a similar axial counterbore on the opposed side of hub 380. Optional lead-in chamfers are provided at the mouth of these counterbores in order to facilitate the stabbing of an O-ring seal with the membrane carrier assembly 327. Thus hub 380 not only supports chambers 320 and 330 but also provides a flow connection for the chambers.

Sleeve 324 is similar in construction to that of sleeve 24 used with the first chamber 320, but differs only in its inner and outer diameters. The inner diameter of sleeve 24 is chosen to be sufficient to accommodate the membrane carrier assembly 327 and provide sufficient volume for chamber 330. The outer diameter is selected for strength and rigidity. As is the case for sleeve 24, sleeve 324 has wall penetrations for the mounting of multiple fill ports 40 and samplingports 44. Sleeve 324 is a close fit to second inner projection 312 of center hub 380.

End piece 311 has a right circular cylindrical central body having the same outer diameter as the enlarged flange having a coaxial push-on hose barb attachment neck 349 on its outer face. A reduced diameter coaxial right circular cylindrical interior projection extends inwardly on the transverse face of the interior end of end piece 311, with a transverse shoulder located on the transverse interior face of end piece 311 immediately exterior of the projection. The exterior cylindrical surface of the interior projection 321 has annular male O-ring grooves. Elastomeric O-rings 318 are mounted in the O-ring grooves of projection 321. An axial through hole penetrates through attachment neck 349 and the rest of end piece 311, where it centrally intercepts a counterbored flat bottom hole to form a flow path for the fluid exiting from the second chamber 330. Optional lead-in chamfers are provided at the mouth of the counterbore in order to facilitate the stabbing of an O-ring seal with the membrane carrier assembly 327.

A membrane carrier assembly 27 as shown in FIG. 4 for this bioreactor system 300 traverses the growth compartment 385. An end of the generally cylindrical membrane carrier assembly 27 is mounted in the counterbores of each of the two assemblies 11 and 380, respectively, forming the end of compartment 385 used in chamber 320.

A membrane carrier assembly 327 as shown in FIG. 4 traverses the growth compartment 395 of the second chamber 330. The membrane carrier 327 is substantially identical to the unit used in the first chamber 385 and the prior embodiments 10 and 100 of the present invention, but membrane carrier 327 is much larger and mounts a different molecular cutoff membrane 337 that is optimized for the cell growth in the second chamber 330. An end of the generally cylindrical membrane carrier assembly 327 is mounted in the counterbores of each of the two chamber end pieces 311 and 380, respectively, forming the end of compartment 395 used in chamber 330.

EXAMPLE 4

Alternative Embodiment of Interconnected Culture Chambers of Different Capacities

The bioreactor system 200 has interconnected culture chambers arranged as shown in both FIGS. 5 and 6. This arrangement basically utilizes two rigid coaxial culture chambers 220 and 230 cojoined in tandem, and utilizes bioreactor vessel types substantially similar to those disclosed in U.S. patent application entitled "Culture Chamber for Biologicals", Ser. No. 10/725,607.

The first chamber 220 utilizes a first molecular cutoff membrane 37 and is upstream of the second chamber 230. One of the features of this embodiment is the very large size of second chamber 230 relative to first chamber 220. The second chamber 230 is designed to provide more molecular cutoff membrane active surface area than is present in the first chamber 220. This arrangement is utilized when the first bioreactor chamber 220 is able to provide sufficient secretions to stimulate and/or nurture the cells in a larger second chamber 230.

The cojoined bioreactor chambers 220 and 230 are rotated as an unit using standard rotational equipment known to those in the industry, but with three supporting drive rollers which act on the tires of the end pieces 11 and 211 and the center hub 280.

The first chamber 220 produces a desired growth factor or mediator for supply to the second chamber 230. The bioreactor of the second chamber 230 uses a second molecular cutoff membrane 237 that is optimized for the cells and the outputs desired from the second chamber 230.

The first bioreactor chamber 220 of the bioreactor system 200 has all of its components including the input fluid system in common with that of the first chamber 20 of the bioreactor system 10 of the present invention, except that a different fluid connector or center hub 280 is used. Thus, first chamber 220 has an end piece 11 sealingly engaged on the inlet end of a sleeve 24 and also has a membrane carrier assembly 27 located axially within the interior of sleeve 24, thereby forming a bioreactor chamber as before. Multiple radial wall penetration ports 25 are provided in the annular wall of sleeve 24 to allow the introduction of one or more fill ports 40 and one or more samplingports 44.

The biologicals being cultured in the rotatable chambers 220 and 230 require nutrients, so fluid-conducting swivels 50, stopcock valves 60, fluid inlet tubing 72, and outlet tubing 71 are provided for the system. The system 200 is plumbed similarly to that of serially arranged bioreactor system 300 so that nutrient fluid enters the first chamber 220 of the system through inlet tubing 72, stopcock 60, and swivel 50. The fluid output from chamber 220 passes through hole 286 in center hub 280 into the second bioreactor chamber 230. The outflow from the second chamber 230 passes through a swivel 50, stopcock 60, and outlet tubing 71.

The growth compartment 285 of the first chamber 220 is located between the interior bore of the sleeve 24, the membrane carrier assembly 27, the interior end of interior projection 12 of end piece 11, and the interior end of the interior projection 281 of the center hub piece 280.

Center hub 280 is a structure composed of right circular cylindrical segments. The first side of the center hub 280 facing the first chamber 220 is basically the same about its transverse midplane as the corresponding side of center hub 80 of the bioreactor system 10 and likewise identical to the end piece 11 of the first chamber 220 with hose barb attachment neck 49 removed.

The external cylindrical portion of hub 280 adjacent the interior projection 281 serves as a tire for the rotation of the entire bioreactor assembly 200. The first interior projection 281 of center hub 280 also carries two male O-ring grooves that hold O-rings 18. The O-rings 18 mounted in the grooves of projection 281 of center hub 280 sealingly mate with and restrain the axial position of cylindrical sleeve 24. On its first side center hub 280 has a flat-bottomed cylindrical bore 282 for housing the membrane carrier assembly 27 of the first chamber.

The second side of the center hub 280, obverse to the first side of center hub 280, has in order from the transverse midplane of center hub 280 a cylindrical flange 260 and second interior projection 261. Flange 260 is the same diameter as or slightly larger than sleeve 224 used for the second chamber 230. Second interior projection 261 is sized to closely fit to the bore of sleeve 224 and has two male O-ring grooves mounting O-rings 218 to seal therewith.

An axial hole 286 provides communication from the counterbore 282 on the first side of the hub 280 to approximately the middle of hub 280. Axial through hole 286 penetrates through interior projection 281 and approximately midway into the body of center hub 280 and is there branched into a number of radial holes 264. Offset from the axis of center of hub 280 are parallel holes 265, each of which centrally intercepts a counterbored flat bottom hole 263 and a corresponding radial hole 264 in order to form a flow path for the fluid entering the second chamber 230. The multiple counterbored holes 263 are positioned in a regular pattern on the transverse face 266 of the reduced diameter end 261 of center hub 280. Optional lead-in chamfers are provided at the mouth of counterbores 263 in order to facilitate the stabbing of an O-ring seal 229 with the membrane carrier assembly 227. Thus hub 280 not only supports chambers 220 and 230 but also provides a flow connection between the chambers.

Sleeve 224 is similar in construction to that of sleeve 24 used with the first chamber 220, but dif