Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2000-11-22
2003-11-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S183000, C435S298200, C435S297100, C435S297200, C435S286500
Reexamination Certificate
active
06642019
ABSTRACT:
FIELD OF THE INVENTION
This present invention relates to culture vessels, sometimes referred to as bioreactors, for growing cells, three dimensional cellular aggregates, “organoids,” tissues or the like (at times referred to below as “particles”) and their method of use for carrying out various cellular processes with tissues and/or organoids and for diagnostic testing and research.
BACKGROUND OF THE INVENTION
This invention relates to improvements in culture vessels for growing or culturing cells, cellular aggregates, tissues, organoids and the like and to methods for using the same, and in particular to improvements in culture vessels as disclosed in U.S. Pat. Nos. 5,989,913 and 6,080,581, issued to Charles D. Anderson on Nov. 23, 1999 and Jun. 27, 2000, respectively, both hereby incorporated herein by reference in their entirety. Both of these patents are not admitted to be prior art with respect to the present invention by their incorporation into this application.
Most prior art culture vessels have been cylindrical in shape with flat, vertical interior end walls and many prior art vessels incorporate other interior obstructions, such as central cylindrical cores, agitators or impellers, which also impact the flow paths of cells, cellular aggregates, “organoids” or tissue particles (see definition within), and are thought to create impact and/or abrasion surfaces impeding toroidal flow. U.S. Pat. No. 6,001,642 to Tsao, entitled “Bioreactor and Cell culturing Process Using the Bioreactor,” discloses an asymmetrical bioreactor having one spherically-shaped wall facing and connecting to a second flat wall. This bioreactor is commonly named the Hydrodynamic Focusing Bioreactor. The center of the flat wall features a spinner type agitator that projects into the middle of the culture chamber and is designed to enhance bubble removal. The bioreactor of the '642 patent does not have a chamber defined by a smooth, non-flat, spherical, oblate spherical or curved wall that is symmetrical about an axis in that one wall of the culture chamber is essentially flat except for a centrally mounted agitator. The front and back walls of the '642 bioreactor join at a very sharp angle that would impede toroidal flow. There is an agitator in the center of the culture chamber of the '642 bioreactor that impedes toroidal flow across the center of the vessel and creates turbulence. Finally, the hydrodynamic focusing bioreactor is not intended to facilitate toroidal flow but is intended to focus the flow within the vessel into a “donut” pattern. The focusing action created by this centrally located spinner agitator causes shear, turbulence, and vessel wall impacts especially against the flat back wall of the device.
The need exists for a continuously renewed culture vessel, rotated over an essentially horizontal axis, that minimizes interior structures that impedes the ability of cells, cellular aggregates or tissues to freely expand in three dimensional growth under minimally disturbed toroidal flow. The vessel would have an inlet and an outlet in fluid communication with a pump and at least one filter in fluid communication with an outlet. There is a need for a version of such a culture vessel or bioreactor that may be operated as a batch reactor. There is also a need for a method of use of such culture vessels or bioreactors for the production of biological products, and for the removal of toxins and biological waste from a fluid using cellular mechanism. The culture vessels or bioreactors of the present invention address these needs.
SUMMARY OF THE INVENTION
The present invention pertains to a culture vessel essentially horizontally rotatable on a horizontal axis, the vessel having at least one chamber. The chamber in one preferred embodiment has spherical, oblate, extended spherical or extended oblate spherical wall portions. Typically, though not necessarily, the chamber would be symmetrical about the longitudinal axis. The chamber has an inlet and outlet in fluid communication with at least one pump. At least one filter in the chamber passes fluid and cellular waste out the outlet while retaining the cells, tissue, organoids or the like. Also the invention relates to methods for using such a culture vessel for growing cells, cellular aggregates, organs, tissues and the like and for the production of biological products or the removing of toxins or biological waste from a fluid using cellular mechanisms.
Testing has confirmed that a culture vessel with a continuously renewing fluid medium therethrough need not be cylindrical, as is the custom in the industry. Testing shows that the chamber is not limited to a cylindrical shape, but a spherical or oblate spherical or extended spherical or extended oblate spherical shape may provide an improved shape for a culture chamber. A spherical or oblate spherical or extended spherical or extended oblate spherical shape for a chamber offers, as viewed at in cross section through a longitudinal axis, rounded corners that facilitate growth or other operative cultures by minimizing impact forces and shearing forces upon particles moving within the chambers. Such lateral movement is described below. It has further been determined that in the simplest embodiments of a culture chamber, only one filter is needed, in fluid communication with an outlet. A chamber can function adequately without the safety feature of a second upstream filter, although the ability to reverse flow between a chamber inlet and outlet would be sacrificed. Upstream valving could be employed, in lieu of an upstream filter to protect against cell migration through an inlet.
As disclosed in the referenced and incorporated patents above, particles continuously descend or sediment through a culture fluid in a vessel horizontally rotated about a longitudinal axis. While it had been thought that particles in such circumstances descend in a linear or two-dimensional flow path and were uniformly distributed in the horizontally rotated culture vessel, it has been observed that distinct or visible bands of particles are formed in such rotated vessels. The flow path is three-dimensional. Particles migrate along toroidal, three dimensional flow paths. Surmise is that such toroidal flow may be due in part to Coriolis forces acting on the particles as they move inside of the rotated vessel.
The phrase, “toroidal flow” indicates that the particles do not simply rotate in a simple circular pattern with the chamber fluid about an essentially horizontal longitudinal chamber axis. If the end point of a particle at the end of a full 360 degree rotation about a longitudinal axis could be plotted, the following would be apparently detected. A cross section taken through the longitudinal axis would show the end point migrating in a small circle over time, in the plane of the cross section, traversing a portion of the circumference of that circle with each 360 degree revolution around the horizontal axis. A cross section taken normal to the longitudinal axis would show the end point migrating in another small circle in the plane of the cross section, traversing a segment of the circumference of that circle, with each 360 degree rotation. The path of a particle would appear to describe a three dimensional sphere or egg shape over time.
Growth of cells, cellular aggregates, organoids or tissues and the like may be increased and enhanced to the extent the cells, cellular aggregates, organoids or tissues are permitted to freely expand in three dimensions while remaining undisturbed in toroidal flow ,as described above.
Observation of particle motion in horizontally rotated bioreactors indicates that natural toroidal flow is disturbed when the particles collide or have impact with interior walls (and/or any other obstructions within a vessel). The violence of the impact affects growth. Reduced shearing forces and/or reduced number of impacts with interior walls facilitates undisturbed motion and results in faster cellular growth, larger sized cellular aggregates or tissues, more efficient removal of
Anderson Charles Daniel
Anderson Mark Stuart
Dodd Charlie W.
Casperson John R.
Ketter James
Synthecan, Inc.
Vaden, III Frank S
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