Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/06—Tubular
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/10—Rotating vessel
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps

Definitions

• This invention relates to an apparatus and method of use for a new culture vessel or bioreactor.
• the vessel is useful for growing cell cultures growing three dimensional cellular aggregates or tissues, for carrying out various cellular processes with tissues and/or organoids and for diagnostic testing and research.
• Bacterial cell culture processes have been developed for the growth of single cell bacteria, yeast and molds which can be characterized as encased with a tough cell wall. Large scale culture of bacterial type cells is highly developed and such culture techniques are less demanding and are not as difficult to cultivate as mammalian cells. Bacterial cells can be grown in large volumes of liquid medium and can be vigorously agitated without any significant damage.
• Mammalian cell culture and tissue generation is much more complex because such cells are more delicate and have a more complex nutrient requirement for development.
• Mammalian cells cannot withstand excessive turbulent action without damage to the cells and must be provided with a complex nutrient medium to support growth. Therefore, bioreactors with internal moving parts or obstructions will subject mammalian cells to high fluid shearing forces that will damage the cells.
• bioreactors that utilize mechanical parts, air or fluid movement as a lift mechanism to achieve particle suspension will likewise cause damage to growing cells and tissues due to fluid shear.
• bioreactors A primary use of bioreactors is in research where large numbers of cells are grown to refine the minute quantities of an active material (e.g. proteins) that the cells might secrete.
• an active material e.g. proteins
• Another use of bioreactors is the scale-up of laboratory cell culture processes for commercial purposes to mass produce the active proteins made by genetically engineered cells and tissues. Because of the need to culture mammalian cells in the laboratory in large quantities, bioreactors and culturing vessels have become an important tool in research and production of cells that produce active proteins.
• a current problem in tissue culturing technology is the unavailability of an effective bioreactor for the in vitro cultivation of cells and explants that allows easy access to the materials contained in the vessel.
• Several devices presently on the market have been used with only limited success since each has limitations which restrict usefulness and versatility. Further, no bioreactor or culture vessel is known that will allow for the unimpeded growth of three dimensional cellular aggregates or tissues.
• Cell culturing devices range upward in complexity from the petri dish to sophisticated computer controlled bioreactors.
• manufacturers have promoted various technologies to culture cells in the laboratory. For instance, simple adaptations of fermentors or stirred tanks used for the culture of bacteria, were marketed previously as the answer to culturing delicate mammahan cells.
• One of the principal factors limiting the performance of these systems is their inability to minimize turbulence due to stirring, i.e., shear due to fluid flow, and hence preventing free form association of cells in three dimensions.
• U.S. Patent Nos. 4,988,623, 5,153,133, and 5,155,034 disclose culture vessels that allow three dimensional cell growth. These vessels are shaped similarly to each other due to a central tubular member that may either be an oxygenator that performs gas exchange through its surface, or it may be a cylindrical filter that allows the passage of an oxygen rich culture medium.
• a central tubular member that may either be an oxygenator that performs gas exchange through its surface, or it may be a cylindrical filter that allows the passage of an oxygen rich culture medium.
• the presence of a centralized tubular oxygenator or filter within a rotating vessel is a significant obstruction that will impede or prevent the growth of larger three- dimensional cellular aggregates.
• U.S. Patent No. 5,153,131 discloses a bioreactor vessel without mixing blades or a central tubular membrane. This apparatus requires transfer of gases into the bioreactor vessel. As shown in the cross sectional view in FIG. 1, air travels through an air inlet passageway, through a support plate member, across a screen, and through a flat disk permeable membrane wedged between the two sides of the vessel housing. The oxygen then diffuses across the membrane into the culture chamber due to the concentration gradient between the two sides of the housing.
• the rate at which oxygen can diftuse across the disk shaped membrane is a significant limitation that restricts the size of the culture chamber.
• Another disadvantage of the flat disk membrane in the '131 patent is that it is designed to flex in order to cause mixing within the culture chamber. This mixing effect is a feature that is described as being critical for the distribution of air throughout the culture media, however, it will also tend to create shear within the chamber. Consequently, an improved apparatus and method for suspending particles
• the preferred dimensions of the vessel described in the '941 patent are limited to between one and six inches in diameter while the width is preferably limited to between one-quarter of one inch and one inch. Such size limitations are not suitable for growing three-dimensional cellular aggregates and tissues.
• U.S. Patent No. 5,449,617 issued to Falkenberg et al, and entitled "Culture Vessel For Cell Culture” discloses a vessel that is horizontally rotated. The vessel is divided by a dialysis membrane into a cell culturing chamber and a nutrient medium reservoir. Gas permeable materials are used in the vessel walls to provide gas exchange to the cell culturing chamber.
• the vessel is not completely filled with the nutrient medium and a large volume of air is maintained above the fluid medium in both chambers.
• the vessel is not designed to minimize turbulence within the cell culture chamber, rather mixing is recited to be essential to keep the dialysis membrane wetted. Further, the disclosure of the '617 patent does not contemplate using the vessel to grow cellular aggregates or tissues of any kind.
• a cylindrical vessel with first and second end walls defines a cell culture chamber which is rotatable about an approximate horizontal axis.
• the first and second end walls are provided with an inlet and an outlet respectively for introducing an oxygen rich nutrient medium into and removing the spent medium and waste products from the vessel.
• Filters are located near the inlet and the outlet to prevent the passage of cells and cellular aggregates from the culture chamber while allowing the passage of the nutrient medium and cellular metabolic waste.
• the culture chamber is free of internal obstructions and structures that might cause fluid shear or that might otherwise impede the growth and/or suspension of large three dimensional cellular aggregates while the vessel is being rotated.
• a pump provides a constant flow of the nutrient medium through the chamber while the vessel is rotated and a gas exchange device is used to transfer gases into and out of the nutrient medium.
• the operation of the bioreactor system is automated by providing means for controlling the temperature of the vessel and/or nutrient medium.
• sensors monitor the flow rate, content, pH and temperature of the nutrient medium as well as the rate of rotation of the vessel.
• a microprocessing unit records data from such sensors and directs adjustments for maintaining the desired operation conditions within the culture chamber.
• additional filters may be used in the cylindrical vessel intermediate between the end walls to define or subdivide the culture chamber. Subdivision of the chamber is useful for growing different types of cells and cellular aggregates, for carrying out various cellular operations and functions and for conducting diagnostic research within the same cylindrical vessel.
• access ports may be provided along the cylindrical wall of the vessel to provide access to each sub-chamber.
• the filter upstream relative to the direction of flow of nutrient medium through the culture chamber can be cylindrical in shape and located adjacent to but spaced apart from the cylindrical wall of the vessel so that fresh nutrient medium can enter the culture chamber and/or sub-chambers at all points along the length of the vessel.
• the filters of the present invention are made of a variety of materials and constructions and are chosen according to the application for which the vessel is to be used.
• the method includes the steps of filling a rotatable cylindrical vessel having a culture chamber with an unobstructed longitudinal axis with an oxygen rich fluid culture medium and introducing cells, cellular aggregates and/or tissues into the medium.
• a flow of oxygen rich fluid culture medium is maintained through the vessel to provide oxygen and materials to sustain cell respiration and growth and to remove cellular metabolic waste.
• the vessel is rotated about its horizontal longitudinal axis to suspend the growing cells and cellular aggregates in the medium. Periodically, the rotation of the vessel may be interrupted for the purpose of removing bubbles that may have formed in the nutrient medium and/or to remove materials from the culture chamber.
• the method includes the steps of providing a rotatable cylindrical vessel having an interior chamber that is unobstructed along its horizontal longitudinal axis.
• the chamber is filled with an oxygen rich fluid culture medium and tissues and/or organoids are introduced into the medium.
• organoid refers to a mass or aggregate of cells that mimics the structure and/or function of a tissue or organ.
• a flow of oxygen rich fluid culture medium is maintained through the vessel to sustain the organoids and to remove cellular metabolic waste.
• the organoids are suspended in the medium by the rotation of the vessel.
• a fluid containing the waste materials is then passed through the chamber and the organoids remove the waste by various cellular mechanisms.
• Additional cylindrical vessels can be connected in series to form a continuous chain of rotating vessels. Periodically, each vessel is replaced with another vessel containing fresh organoids.
• the features of the bioreactor system of the present invention enable the growth of cells and cellular aggregates in larger volumes of media than was possible with prior art vessels. These larger volumes are particularly useful in the commercial scale production of biological materials.
• Further aspects and objects of the present invention include providing a device and method for growing cells and tissues for replacement of defective and damaged tissues in humans and other animals, providing a device and method for growing cells and tissue cultures for the production of biological products such as but not limited to growth factor proteins, enzymes, platelets and genetically engineered materials, and providing a device and method for growing cells and tissue cultures for diagnostic procedures such as identifying and testing chemotherapy and other biochemical agents.
• FIG. 1 is a partial cross sectional view of a reactor vessel known in the prior art showing the size limitation of such devices.
• FIG. 2 is an elevated view of the prior art reactor vessel illustrated in FIG. 1, as mounted on a base having rotation means and gas exchange means.
• FIG. 3 is also a perspective view of a prior art device that has an elongated growth chamber, but which provides oxygen injection through a centrally disposed cylindrical membrane.
• FIG. 4 is a flow diagram illustrating the fluid flow in an apparatus of the present invention.
• FIG. 5 is a cross sectional view of a reactor vessel of the present invention wherein the first filter is incorporated into the inlet.
• FIG. 6, is a cross sectional view of a reactor vessel of the present invention wherein the first filter is attached to the first end wall.
• FIG. 7, is a cross sectional view of a reactor vessel of the present invention wherein the culture chamber is defined by a cylindrical filter adjacent to but spaced apart from the cylindrical wall of the vessel.
• FIG. 8 is a cross sectional view of a reactor vessel of the present invention wherein the culture chamber is defined by a cylindrical filter and is subdivided into sub-chambers by additional filter elements.
• FIG. 9 is a cross sectional view of a reactor vessel of the present invention wherein multiple outlets are located about the periphery of the culture chamber.
• FIG. 10 is a cross sectional view of a reactor vessel of the present invention wherein the downstream filter is adjacent to but spaced apart from the second end wall.
• FIG. 11 is a cross sectional view of a reactor vessel of the present invention having a cylindrical filter along the length of the vessel and a downstream filter that is adjacent to but spaced apart from the second end wall of the vessel.
• FIG. 12 is a cross sectional view of a reactor vessel of the present invention that is particularly useful for filtering waste materials and toxins from a fluid.
• a main fluid flow loop for growing mammalian cells includes a rotating cell culture reactor vessel
• reactor vessel 10 is made of a cylindrical vessel with first and second end walls, multiple filter elements and an unobstructed horizontal longitudinal axis defining an unobstructed culture chamber.
• the vessel has an inlet and an outlet, one or more vessel access ports for transferring materials into and out of the vessel as well as means for removing bubbles from the nutrient medium.
• the bioreactor system will have pump 3 for maintaining a flow of nutrient medium through the vessel, gas exchange device 5 for dissolving gases into and removing waste gases from the nutrient medium, and means for rotating the vessel about its horizontal axis.
• the bioreactor may also be provided with temperature control means, various sensors for monitoring the operational conditions and a microprocessor unit (not shown) for automating the operation of the bioreactor system.
• materials used to construct cylindrical wall 16 of vessel 10 will preferably be a transparent, non-toxic, biocompatible material such as glass or a clear plastic.
• the clear material is a polycarbonate such as LEXAN® (a registered trademark of General Electric).
• the end walls will preferably be a material that is both durable and machines well.
• the end walls will be manufactured from an acetal polymer such as DELRIN® (a registered trademark of E.I. du Pont Nemours & Co., Inc.).
• Cylindrical wall 16 and end walls 12 and 14 may be formed by injection molding, wherein various parts of the bioreactor vessel 10 may be welded, glued or mechanically attached together.
• End walls 12 and 14 and cylindrical wall 16 may also be gas permeable.
• these structures may be made of a variety of materials such as silicone rubber, polytetrafluroethylene, polyethylene, porous plastic coated with a hydrophobic material, mixtures of silicone with other plastics, and silicone rubber coated cloth.
• the cylindrical wall 16 is constructed of a porous plastic coated with a hydrophobic material on the interior surface.
• the cylindrical wall 16 is made of porous hydrophobic Teflon® (a registered trademark of E. I. du Pont Nemours & Co., Inc.) when gas permeability is desired.
• the cylindrical wall 16 may be made in any size provided that adequate nutrients and gases can reach the growing cells and cellular aggregates within the culture chamber.
• the size of the vessel is therefore limited only by the flow rate of the fluid nutrient medium into the culture chamber and the contents of that medium.
• the prior art discloses reactor vessels that have a culture chamber with a length of at least 0.25 inches but no more than 1 inch.
• the preferred volumes of these prior art vessel chambers is generally less than 500 ml.
• the length and diameter of cylindrical wall 16 will be much larger than the dimensions of these prior art bioreactors.
• the culture chamber of the present invention has maximum dimensions that are determined by the flow rate and content of the fluid medium that passes through the culture chamber, the culture chamber can have maximum lengths well in excess of 1 inch and can have internal volumes up to 100 liters.
• the end walls of vessel 10 include first end wall 14 and second end wall
• the nutrient medium flows through the vessel generally in only one direction and as such, the ends of the vessel are periodically referred to as upstream and downstream, respectively. However, as noted below, the flow of the nutrient medium may be periodically reversed, and thus, the end walls are more generally referred to as first and second end walls.
• first end wall 14 is connected to the upstream end of cylindrical wall 16 to form the upstream portion of vessel 10.
• the cylindrical wall and the two end walls may be glued, welded or mechanically attached to one another to form vessel 10.
• First end wall 14 is provided with inlet 20 that is coupled via rotative coupling 22 to conduit 24.
• First end wall 14 is shown with only a single inlet 20.
• conduits may be machined into end wall 14 to provide additional inlets into the culture chamber at various locations on the end wall. More specifically, radially oriented bores may machined into the end wall so that a plurality of inlets is arranged about the periphery of end wall 14. Such an arrangement of inlets provides a greater distribution of the oxygen rich nutrient medium as it enters the culture chamber through end wall 14.
• the inlet or inlets of end wall 14 are in fluid communication with conduit 24 which is in fluid communication with gas exchange device 5.
• main pump 3 provides fresh nutrient medium to the gas exchange device wherein the nutrient medium is oxygenated and passed on to vessel 10.
• the return spent nutrient medium from vessel 10 is returned to manifold 1 where it receives a fresh charge of nutrients, acid, base, buffer, or liquid medium, as necessary before recycling.
• Adjustments to the nutrient medium may be made in response to chemical sensors suspended in the medium and/or to electro-chemical sensors located within or down stream of vessel 10.
• the pH of the medium is corrected by controlling carbon dioxide pressures and introducing acids, bases and/or buffers.
• Spent medium may be directed to a waste or drain as it passes from the vessel.
• the cells, cellular aggregates and/or tissues within vessel 10 are synthesizing and/or excreting materials that are to be retained, those materials may be directed to collection device 11.
• first end wall 14 is provided with access port 26 for accessing the interior of the culture chamber.
• the access port 26 provides access to the vessel for the input of medium and cells and for the removal of cultured cells and cellular aggregates.
• the vessel access ports are constructed with valved closure means or a septum- membrane closure.
• the valves preferably are plastic, but may be made of metal or any other material which is non-toxic, capable of being sterilized and is hard enough for machining into an access port.
• access port 26 may be provided with a variety of couplings for connecting with various fittings. Where the culture chamber within vessel 10 is subdivided into sub-chambers, access ports may be provided along the length of the cylindrical wall 16 in order to allow access to each of the various sub-chambers.
• Vessel 10 is also provided with means for trapping bubbles that may develop in the culture chamber and an adjacent port is provided for removing the bubbles from the bubble trapping means.
• bubble removal port 28 is incorporated into first end wall 14.
• bubble trapping means may take a variety forms. For instance, a portion of the inner surface of cylindrical wall 16 may be provided with a recessed structure that is designed to trap bubbles during the rotation of vessel 10. Regardless of the structure that is used to trap bubbles, an access port such as bubble removal port 28 should be located adjacent to such means.
• bubble removal port 28 will have a valve type closure so that a hypodermic syringe may be attached to remove bubbles from the chamber so as to minimize turbulence that might be created during the removal of bubbles.
• a septum membrane type closure may also be employed.
• Second end wall 12 is connected to cylindrical wall 16 to form the downstream portion of vessel 10.
• Second end wall 12 is provided with central outlet 18 and is connected to drive shaft 32.
• Drive shaft 32 is connected to means for rotating the shaft and the vessel as is discussed below.
• Mean for rotating the vessel may be directly attached to either end wall or the cylindrical wall of the vessel.
• Central outlet 18 is in fluid communication with conduit 58 for directing fluid medium away from the vessel.
• Second end wall 12 may have only central outlet 18 or it may have a plurality of outlets 48. As shown in FIG. 9, outlets 48 may be arranged about the periphery of vessel 10 with fluid communication with central outlet 18 provided via wall bores 60. Plurality of outlets 48 can have a variety of configurations as determined by the particular application of the bioreactor and the ease of manufacture.
• filters are used define and subdivide the culture chamber and to retain the growing cells, cellular aggregates, tissues and/or organoids in the culture chamber. Therefore, the use of filters is preferred over dialysis membranes and the like.
• the filter should be of a size that allows the passage of sufficient oxygen rich nutrient medium to sustain cell respiration and growth while preventing the passage of cells, cellular aggregates tissues and/or organoids. The combination of such a filter with a constant flow of oxygen rich medium will sustain cellular respiration and growth within a much larger vessel than was previously known.
• the filters used in the present invention may be made of a variety of materials having a variety of constructions provided that they have a porosity that allows the nutrient medium and cellular metabolic waste to travel through the filter but that will prevent the passage of cells and cellular aggregates.
• the filters of the present invention may be made of a polycarbonate film that has been irradiated to render it porous, polyester cloth and various woven materials such as a woven fabric of stainless steel.
• Such filters are commercially available from a variety of sources. It is anticipated that filters made of biodegradable materials such as polyglycolic acid may also be used.
• the size, mesh and location of the filters can vary widely.
• the mesh size of the filter material or construction is determined in large part by the application for which the bioreactor system is to be used. If the reactor vessel is used to produce or test white blood cells the mesh size will need to be quite small to prevent the leukocytes from passing. If the vessel is being used to produce a bone marrow, a larger mesh size will be sufficient. Culture work concerning the production or testing of cellular aggregates such as tissues and organoids can utilize still larger mesh sizes.
• a first filter 34 should be used to prevent the cells and cellular aggregates in the culture chamber from travelling upstream from the reactor should the fluid flow through the reactor be reversed.
• filter 34 can be a small filter fixed within inlet 20 or as shown in FIG. 6, can be arranged across the full diameter of the vessel attaching to end wall 14 and cylindrical wall 16 at its periphery.
• the filter elements may be sandwiched between the various components of vessel 10 such as between the end walls and the cylindrical wall as is shown in FIG. 12.
• filter 34 may have additional configurations.
• filter 34 may be attached about its periphery to the cylindrical wall 16 of the vessel, adjacent to but spaced apart from end wall 14 as shown in FIG. 12.
• the nutrient medium enters vessel 10 through inlet 20 and passes across filter 34 at all points on the filter such that an improved distribution of medium is achieved.
• the upstream filter may be formed into cylindrical structure 40 and attached to downstream filter 36 to form a filter enclosed culture chamber 30.
• cylindrical filter 40 is that the nutrient medium passes between cylindrical wall 16 and filter 40 and can thus pass through the filter directly into all parts of culture chamber 30 along the length of the vessel.
• the nutrient medium is not required to pass through a series of chambers and/or filters to reach the downstream portions of culture chamber 30 or downstream sub-chambers.
• the flow of medium may be directed immediately into culture chamber 30 and out through cylindrical filter elements 40 as shown in FIG. 11.
• upstream filter element 20 may be unnecessary in that it is unlikely that the cylindrical filter element 40 and downstream filter element 46 will simultaneously become clogged such that any back flushing would be required.
• FIG. 8 illustrates how the culture chamber may be subdivided into sub- chambers 30a, 30b and on out to 30n, where n is a positive integer greater than 1, by intermediate filter elements 42.
• n is a positive integer greater than 1, by intermediate filter elements 42.
• cylindrical wall 16 can have access ports along its length to provide access to culture chamber 30 and to sub- chambers 30a-30n when the chamber is subdivided by intermediate filter elements 42.
• filter 36 can have a variety of configurations within and without vessel 10. As illustrated in FIGs. 5-6 and 7-8, filter 36 can be provided along the surface of second end wall 12. Alternatively, filter 36 may be arranged within outlet 18 (not shown) or attached about its periphery to cylindrical wall 16 and spaced apart from outlet 18 as illustrated in FIG. 10.
• filter 36 may periodically become clogged with cells and cellular aggregates.
• the direction of the flow of the nutrient medium should be reversed by pump 3 until the clog has been cleared.
• the direction of flow of the nutrient medium may also be reversed in order to cause the suspended cells, cellular aggregates and/or tissues to become attached to a substrate or the filter elements of the vessel.
• the bioreactor system is provided with pump 3 which maintains a flow of fluid nutrient medium through the vessel 10.
• Pump 3 may be a peristaltic pump or similar device that is capable of maintaining a relatively constant flow of fluid medium through the reactor vessel. Pump 3 may be adjusted to a variety of flow rates and is capable of reversing the direction of medium flow through the vessel.
• Gas exchange device 5 may be characterized as an oxygenator, but the device should be capable of maintaining desired gas concentrations for the variety of gases needed to sustain and promote cellular respiration. Oxygen is consumed in the culture chamber and carbon dioxide is given off as a byproduct of cellular respiration. Thus, the gas exchange device must be capable of transferring oxygen into and removing carbon dioxide from the nutrient medium. If not properly balanced, the increasing quantities of carbon dioxide will render the circulating medium acidic.
• gases are transferred into and out of the nutrient medium across a multi-layered cylindrical membrane composed primarily of silicone rubber.
• the cylindrical membrane is located in a cylindrical housing that has upstream openings for receiving the nutrient flow from the pump and downstream openings for passing the oxygen rich medium onto the reactor vessel.
• the housing is provided with a fan attached thereto to maintain a constant flow of air through the housing and across the surfaces of the cylindrical membrane.
• the present invention involves the rotation of reactor vessel 10 about its central horizontal axis.
• the rotational speed of vessel 10 effectively eliminates the velocity gradient at the boundary layer between the fluid and cylindrical wall 16. Thus, shear effects caused between a rotating fluid and stationary wall are significantly reduced or eliminated.
• the clinostat principal involved allows cells or cell aggregates having densities different from the fluid to travel in a nearly circular path and to deviate insignificantly from the fluid path.
• the gravity vector is observed to rotate so that its average time is nearly zero. This allows for suspension of the particles in a carrier medium with low fluid shear and with low interference.
• Cylindrical wall 16 is rotated in order to reduce the adverse fluid velocity gradient through the boundary layer that would otherwise occur at the interface between the moving fluid and the fixed wall.
• the rotation of cylindrical wall 16 is sufficient to cause fluid rotation due to viscosity.
• the operating limits are defined by the sedimentation rate of the particles in the fluid medium and the acceptable centrifugal force due to rotation. Further, it is possible to vary the angular rotation rate in order to induce secondary flow patterns within the vessel which may be useful for distributing nutrients or waste products.
• vessel 10 is rotated about a horizontal axis and the process utilizes zero head space of fluid medium within the vessel.
• the zero head space results in no air bubbles which might cause disruption of the fluid streamlines and thereby subject the culture to adverse shear effects.
• the preferred means for rotation is a motor assembly (not shown).
• the motor assembly is fixed to mounting base and is provided with means for attaching to and rotating vessel 10.
• attachment means may comprise threadably connecting the vessel 10 to the motor assembly 54 through screw threads on drive shaft 32 corresponding to screw threads on end wall 12 of vessel 10. These screw threads are in a direction such that inadvertent loosening of vessel 10 from the motor assembly 54 due to the movement of rotation is avoided.
• a lock nut or similar device may be provided on the drive shaft to prevent unscrewing.
• the attachment means be a series of sprocket gears that cause drive shaft 32, and vessel 10 fixedly attached thereto, to rotate about its horizontal axis.
• the means for rotation in yet another embodiment is a roller mechanism having multiple rollers arranged longitudinally in a horizontal plane. The rollers are rotated simultaneously to rotate a reactor vessel laid between the rollers. Such a roller mechanism may be obtained from Stoval Life Science, Inc. but other roller mechanisms that will provide controlled rotation may also be used.
• the preferred speed of rotation is in the range of about 2.0 revolutions per minute (rpm) to about 45 rpm.
• the desired speed of rotation is dependent on the specific dimensions of the vessel 10 and the particular application. For example, for a bioreactor of about 3 to about 5 inches in diameter, with a width of about 0.25 inches, growing BHK-21 cells in a microcarrier culture, the preferred speed of rotation is about 24 rpm. However, in vessels having larger dimensions, the preferred speed of rotation will be decreased to perhaps about 10 to about 15 rpm. It is to be anticipated that the speed of rotation must be adjusted to balance the gravitational force with the centrifugal force caused by that rotation, particularly as larger diameter vessels are used. Further, as cellular masses suspended in the culture medium increase in density with cellular growth, increased rotation rates will be required to maintain those masses in a suspended state.
• While the rotation of the vessel 10 may take place by rotating the vessel about the substantially longitudinal central axis in a substantially horizontal plane, it may also take place by rotating the vessel in a plane inclined no more than about
• Vessel 10 may be provided with temperature control means so as to control the temperature of medium within the vessel and the temperature of the medium within the reservoirs 13, 15 and 17 as shown in FIG 4.
• the entire bioreactor system may be operated within an enlarged incubator to maintain all of the bioreactor system elements at a desired operating temperature.
• the desired temperature will be determined by the particular application of vessel 10 and the types of materials being grown therein.
• the bioreactor system of the present invention may also be constructed with means for attaching vessel 10 to additional similar vessels, thereby creating a chain or series of bioreactors.
• the vessels in such a chain are connected to one another by attachment means located on their respective end walls and/or drive shafts.
• the chain When a chain of bioreactors is formed in this manner, the chain may be attached to a means for rotation at one of its ends for rotation. If a motor assembly is used for rotation of the chain of reactor vessels, access ports 26 should be located on the cylindrical wall 16 of the vessels for easier access. However, if the chain of bioreactors is to be laid on a roller mechanism for rotation, the vessel access ports 26 should be located on the end walls of the vessels.
• Another aspect of the present invention is a method for growing cells, cellular aggregates and/or tissues in a bioreactor system comprising filling a vessel, having an unobstructed horizontal longitudinal axis, with a liquid culture medium. Cells, cellular aggregates and/or tissues are suspended in the liquid culture medium and the vessel is rotated about its horizontal longitudinal axis at a rate that suspends the cells in the liquid nutrient medium. A flow of oxygenated nutrient medium is maintained through the vessel to sustain cellular respiration and growth. The rotation of the vessel and the flow of medium is maintained for a period of time to attain desired cell and/or tissue growth.
• vessel 10 is filled with a fluid nutrient medium, such as those commonly known in the art for growing cells and cellular aggregates, and cells.
• the nutrient medium may include a variety of materials to sustain the cells, to promote the growth of certain cells, and/or to promote the production or excretion of certain substances by the cells and/or tissues. These materials may include fetal bovine serum, regulatory proteins, salts, sugars, dissolved gases and other materials that combine to form a fluid nutrient medium that approximates blood plasma. Substrate particles such as collagen coated beads and the like may be added to the medium if desired. Further, tissue explant material may be diced and added to the medium either as a substrate for growing other cellular materials or as the as the culturing material for further cell growth.
• the cells and/or tissues are introduced into the medium and the vessel is rotated as described above.
• the rate of rotation will depend on the volume of the culture chamber of the vessel and the density of the growing mass of cells and/or tissues.
• the volume of culture vessel 10 may range from 55 ml. to about 500 mis.
• the volume of the culture vessel may range from about 100 ml to as much as 100 1. As the density of the growing cells will increase with growth, the rate of rotation will likely need to be periodically adjusted to compensate for such changes.
• cylindrical wall 16 As the materials of cylindrical wall 16 are preferably transparent, the growth of the cells and/or cellular aggregates may be visually monitored.
• the length of time over which the bioreactor system is operated will vary greatly depending on the application to which the system is being used.
• vessel 10 When the system is used for diagnostic purposes, vessel 10 may be rotated for only a matter of hours. However, when tissues and large cell masses are to be grown or the secretions of such tissues and/or masses is to be produced, a vessel may be operated over a period of days, weeks or even months.
• a continuous flow of oxygen rich nutrient medium is supplied to vessel 10 while the cell, cellular aggregates, tissues and/or organoids are suspended by the rotation of the vessel.
• the flow rate of the nutrient medium is critical since the culturing of cells and tissues requires a minimum supply of nutrients and oxygen to sustain respiration.
• the flow rate of the medium depends on the size, and thus, the volume of the culture chamber within the vessel. A higher flow rate being required to provide sufficient oxygen and nutrients to a larger chamber. It is anticipated that the flow of medium into the vessel may be as high as 10 ml/min. However, at this flow rate the filter elements of the vessel are likely to clog and periodic reversing the direction of medium flow may be required to back flush the clog from the filters. In the preferred method, the flow rate will vary between 2 and 3 ml/min.
• Temperature control is also essential to maintaining culture chamber 30 at an optimum temperature for cell growth.
• the desired temperature will be maintained by operating the entire bioreactor system within an incubator.
• temperature control means may be utilized in the medium reservoirs and within vessel 10. The temperature will preferably range from about 35 °C to about 40 °C for mammalian cells.
• vessel 10 When the desired level of growth or production has been reached, rotation is stopped, the vessel access ports are opened and the cellular materials removed. If vessel 10 is made of sterilizable materials the vessel may be emptied and sterilized for future use. If disposable, the vessel and any undesirable contents are destroyed.
• Another aspect of the present invention is to provide a method for filtering waste materials from fluids.
• organoids of liver, kidney, pancreatic and other tissues and/or cells may be suspended in the rotating culture chamber 30.
• a fluid containing waste materials or toxins is added to the fluid nutrient medium passing into the reactor vessel and through filter element 34.
• the organoids suspended in the chamber filter the waste material from the fluid utilizing various cellular mechanisms.
• the cellular mechanisms carried out by these organoids are the same as or similar to the mechanisms used by human organs to remove toxins and other waste materials from the human body.
• the bioreactor system will have multiple vessels arranged in series so that periodically, a vessel containing depleted organoids may be removed and replaced with a vessel containing fresh organoids.
• the fluid communication between the vessels in such a series allows for more efficient filtering and further allows the filtering process to continue without significant interruption when the organoids in a vessel need to be replaced.
• the reactor vessel of the present invention may be used as a method of carrying out various diagnostic procedures.
• the reactor vessel of the present invention may be used to test new chemical agents for treating various diseases or cancers.
• a reactor vessel that has been subdivided into a number of sub-chambers may contain diseased or cancerous tissues in one sub-chamber while liver, kidney and other tissues may be suspended in the other sub-chambers.
• the fluid medium containing a chemical agent for treating the diseased tissues is circulated through the vessel for a desired period of time to determine the affects the agent.
• the significance of this procedure is that the action of the agent on the diseased or cancerous tissue may be monitored in the presence of various organ tissues that may counteract the affects of the agent or that may be adversely affected by the agent.
• the bioreactor system of the present invention can be used for a variety of applications.
• the reactor vessel may be used in researching cancer, HIV, tissue modeling, genetics, tissue maintenance, virology, extracellular matrix interactions, signal transduction and protein discoveries.
• the reactor vessel can be used to generate bone marrow, liver, pancreas, skin, heart, nerve, cartilage, kidney, blood and blood vessel tissues.
• the reactor vessel may be used to produce various tissues, pharmaceuticals, diagnostic agents, vaccines, cellular aggregates and organoids.
• Cells and tissues that can be grown in the bioreactor system of the present invention include human keratinocytes, epithelial and fibroblast cells of small intestine, lymphocytes, melanocytes, embryonic cells, osteoblasts, hepatocytes, bone marrow and bone marrow stem cells.
• Various cancers that can be produced in the reactor vessel of the present invention include neuroblastoma, breast, prostate, lung, melanoma, kidney, and ovarian cancers and adenocarcinoma.
• viruses that can be grown in the reactor vessel of the present invention include AIDS HIV, ebola, HHV8-Kaposi's Sarcoma, influenza, Epstine Barr virus, Monkey pox and Norwalk.

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• 1999
  • 1999-10-05 EP EP99953053A patent/EP1220891A4/de not_active Withdrawn
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