Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2002-10-29
2004-01-06
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S297200, C435S297300, C435S297500
Reexamination Certificate
active
06673598
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a disposable culture bag for culturing cells, cellular aggregates, particles, tissues and organoids. More particularly, the present invention relates to a flexible, disposable culture bag having an inlet and outlet for the introduction and removal of media under sterile conditions.
2. Description of the Related Art
The expense of producing biologicals in aseptic bioreactors is exacerbated by the required cleaning, sterilization and validation of the standard stainless steel or glass bioreactors by the customer. Attempts have been made to solve this problem with the development of presterilized disposables that need not be cleaned, sterilized or validated by end users. The use of such disposables would provide savings of thousands of dollars and multiple hours of labor per run. Furthermore, plastics are lightweight, easy to transport, and require less room than stainless steel or glass vessels.
For example, U.S. Pat. No. 6,245,555 B1 describes a bioreactor having a support housing. The interior chamber of the support housing is lined with a disposable liner and sealed with a head plate attached to the liner to form a sealed chamber. Since the liner is open at the top, it must be used in a vertically oriented bioreactor to prevent the contamination of the head plate. Although this system provides a disposable liner, the head plate and the interior chamber will still require cleaning and sterilization. In addition, oxygen transfer is limited by the air-liquid surface. As the vessel becomes larger, the air-liquid interface becomes smaller relative to the process volume.
Another solution has been to develop flexible, disposable plastic vessels that do not require cleaning or sterilization and require only minimal validation efforts. For example, U.S. Pat. No. 5,523,228 describes a flexible, disposable, and gas permeable cell culture chamber that is horizontally rotated. The cell culture chamber is made of two sheets of plastic fused together. The edges of the chamber, beyond the seams, serve as points of attachment to a horizontally rotating drive means. The cell culture chamber is made of gas permeable material and is mounted on a horizontally rotating disk drive that will support the flexible cell culture chamber without blocking airflow over the membrane surfaces. Thus, the cell culture chamber is placed in an incubator and oxygen transfer controlled by controlling the gas pressure in the incubator according to the permeability coefficient of the bag. The rotation of the bag assists in mixing the contents of the bag and enhances gas transfer throughout the bag. However, the cell culture chamber is limited to use within a controlled gas environment. Furthermore, the cell culture chamber has no support apparatus and is therefore limited to small volumes. The described cell culture chamber is actually a batch culture device in that it does not provide an inlet and an outlet for media to be constantly pumped into and out of the chamber during rotation.
Wave Biotech (Bridgewater, N.J.) has also developed a range of presterile, disposable bioreactors that do not require cleaning or sterilizing. The Wave Bioreactor® is made of sheets of flexible, gas impermeable material. The bag is partially filled with media and then inflated with air that continually passes through the bag's headspace. The media is mixed and aerated by rocking the bags up to 40 times a minute to increase the air-liquid interface. However, since a solid housing does not support the bags, the bags become unwieldy and difficult to handle as they increase in size. Furthermore, the wave action within the rocking bag creates damaging turbulent forces. Certain cell cultures, particularly human cell cultures, thrive better under more gentle conditions.
There is a continuing need to develop lightweight, presterilized, disposable bioreactors with simple connections to existing equipment that require little training to operate, yet provide the necessary gas transfer and nutrient mixing required for successful cell cultures.
SUMMARY OF THE INVENTION
The invention contemplates a cell culture bag for culturing cells, cellular aggregates, particles, tissues and organoids. The cell culture bag is a flexible and disposable culture bag that fits within a support chamber of a bioreactor. The cell culture bag has an inlet means and an outlet means for the introduction and removal of media.
One aspect of the present invention is a culture bag having: (a) at least one sheet having a first end, a second end, an internal side, and an external side, where the internal side of the sheet is positioned to face an interior of the culture bag; (b) a seam formed by fusing overlapped sections of one or more sheets; (c) an inlet means fused to the first end of one or more sheet; (d) an outlet means fused to the second end of one or more sheet; and (e) at least one outer perfusion tube, wherein a proximal end of the outer perfusion tube is in fluid communication with the inlet means, a central section of an external surface of the outer perfusion tube is fused to an interior surface of the seam, and a distal end of the outer perfusion tube is closed.
Another aspect of the present invention is a culture bag having: (a) a bag of flexible material having a first end, a second end, an internal side, an external side and two opposed edges, wherein one edge of each sheet is fused with one edge of an adjacent sheet to form a seam, and wherein the internal sides of said fused sheets are positioned within an interior of the bag; (b) an inlet means fused to the first end of at least one sheet, wherein the inlet means includes an inlet end piece and an inlet fluid coupling swivel joint; and (c) an outlet means fused to the second end of at least one sheet, wherein the outlet means comprises an outlet end piece and an outlet fluid coupling swivel joint.
Yet another aspect of the present invention is a culture bag having: A culture bag comprising: (a) at least one sheet having a first end, a second end, an internal side, and an external side, wherein the internal side of said sheet is positioned to face an interior of the culture bag; (b) a seam formed by fusing overlapped sections of one or more sheets; (c) an inlet means fused to the first end of one or more sheet; (d) an outlet means fused to the second end of one or more sheet; (e) at least one outer perfusion tube, wherein a proximal end of the outer perfusion tube is in fluid communication with the inlet means, a central section of an external surface of the outer perfusion tube is fused to an interior surface of the seam, and a closed distal end, said central section of the outer perfusion tube being perforated to allow fluid communication between the lumen of the outer perfusion tube and the interior of the culture bag; and (f) at least one internal perfusion tube, wherein a proximal end of the internal perfusion tube is in fluid communication with the inlet means, a perforated central section allow fluid communication between the lumen of the internal perfusion tube and the interior of the culture bag, and a closed distal end.
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Akers Roger
Anderson William J.
Dinges, Jr. Adrian F.
Navran, Jr. Stephen S.
Hall Elizabeth R.
Redding David A.
Synthecon, Inc.
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