Bioreactor and methods for producing synchronous cells

Apparatus and methods are directed to a perfusion culture system in which a rotating bioreactor is used to grow cells in a liquid culture medium, while these cells are attached to an adhesive-treated porous surface. As a result of this arrangement and its rotation, the attached cells divide, with one cell remaining attached to the substrate, while the other cell, a newborn cell is released. These newborn cells are of approximately the same age, that are collected upon leaving the bioreactor. The populations of newborn cells collected are of synchronous and are minimally, if at all, disturbed metabolically.

TECHNICAL FIELD

The present invention relates to apparatus and methods for producing synchronous cells and in particular newborn cells of a uniform culture, and in particular, a rotating bioreactor apparatus that grows cells in liquid culture media.

BACKGROUND

Thousands of biological research and development facilities worldwide deal with some aspect of the growth and division of cells. Suspensions of minimally disturbed newborn cells are valuable for analysis of the cell cycle, including analysis of all mammalian cell types. The newborn cell suspensions can be used to analyze cell cycle gene expression in mammalian cells, such as hematopoietic cells. Growing and dividing cells are used in basic research, drug development and testing, and bio-product manufacturing, for example.

Furthermore, during the past few years there has been a significant expansion in research in the related areas of cell cycle regulation, cell senescence, apoptosis, and cellular differentiation. Studies related to these specific areas require access to, and reliable production of, large quantities of cells that are at a specific, known phase of growth and division, such as a particular stage in the cell cycle, and state of senescence.

Obtaining adequate quantities of synchronous cells whose physiology is minimally disturbed, is not an easy task. This is because contemporary production methods expose cells to metabolic disturbances, such as with the addition of drugs or deprivation of nutrients, in order to obtain an exponentially growing culture of cells, where the population is distributed throughout all phases of the cell cycle, to become growth inhibited at a particular stage in the cell cycle. Even if these methods are successful, it is unclear if these cellular events represent the true “steady state” of the cells, where all biochemical and metabolic processes are in balance or an artifact created by the disturbance.

Attempts have been made to produce devices and methods for manufacturing large quantities of cells that are at specific stages in cell cycles and senescence. For example, U.S. Pat. No. 6,001,642 (Tsao) and U.S. Pat. No. 5,153,131 (Wolf) are directed to bioreactors that grow cells in suspension or are attached to a substrate for three-dimensional tissue or organ growth. However, while these devices generated cells, they did not generate newborn cells and could not continuously generate cells.

SUMMARY

The present invention is directed to systems, apparatus and methods for producing newborn cells from a culture in continuous steady-state growth. Additionally, these newborn cells are produced continuously, automatically and under simulated microgravity conditions. The apparatus is directed to a perfusion culture system in which a rotating bioreactor is used to grow cells in a liquid culture medium. Within this bioreactor, these cells are attached to an adhesive-treated porous surface, that is continuously perfused with culture medium. This attachment allows cells to divide, with one of the cells remaining bound to the porous surface while the other newborn cell moves toward a collection device. The cell that remains has not been changed metabolically, and can continue to divide and produce newborn cells. This results in cell populations of the same age being collected and the populations of these newborn cells collected are of synchronous and not disturbed metabolically.

Additionally, the rotation is typically performed at speeds that subject the cells to averaging of the gravity vector (or simulated microgravity conditions). This rotation, coupled with the continuous perfusion of culture medium on the porous surface, allows the attached cells to divide, releasing newborn cells, that are collected upon leaving the bioreactor. The cells produced in accordance with the invention can be analyzed to determine gravitational impacts on cell growth, the cell cycle, cell differentiation and cell aging. Also, the cells produced could be particularly useful in cancer or aging research, as well as studies of the effects of gravity on cells.

There is disclosed apparatus for producing synchronous cells having a vessel configured for being rotated and a substrate configured for rotating with the vessel, the substrate configured for holding cells while rotating with the vessel. There is also a system for perfusing the substrate with at least one culture medium. This perfusion is typically continuous while the substrate is rotating.

There is disclosed an embodiment of a method (process) for producing synchronous cells. This method includes providing a bioreactor having a substrate configured for holding cells and rotating in the bioreactor, holding the cells at least proximate to said substrate, rotating the substrate, and perfusing the substrate with at least one culture medium.

There is disclosed another embodiment of a method (process) for producing synchronous cells. This method includes providing a bioreactor having a vessel configured for being rotated; and a substrate for holding cells. The substrate is configured for rotating with the vessel. Cells are then attached to the substrate; and the vessel is rotated to rotate the substrate.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1shows an embodiment of the system20. The system20, includes an apparatus20a, that includes a frame21that supports a bioreactor22, rotated by a drive system, typically formed of a belt drive23and pulleys24h,24m, coupled with a motor25and regulator (not shown). Also included is a fluid coupler26, coupled to a rotatable neck27and reservoir28or fluid source, respectively. The fluid coupler26has an exit port30, that typically empties into a collection flask32.

An axle40, having ends41a,41b, rotated by the motor25, provides rotation to the bioreactor22. A first end41aof the axle40connects to the housing pulley24h, while the second end41bconnects to the bioreactor22, typically at the vessel42. The axle40and rotatable neck27are typically coaxial, along a horizontal axis H.

The bioreactor22is formed from the vessel42, including an outer shell44and a culture chamber45, a porous substrate46, for example, a membrane, a spacer50, and a plate52, typically with a flat surface53. These elements are typically in a concentric alignment, and are typically substantially perpendicular to the axle40and the neck27and substantially parallel to each other. The vessel42typically clamps to the plate52by bolts54(of stainless steel or the like) holding the substrate and spacer50in place. The vessel42, closed by the plate52, defines an effluent chamber56in its interior. The rotation of the axle40and neck27(as rotated by the drive system), typically rotates the bioreactor22(vessel42and substrate46and other interior components) at speeds that will average the gravity vector on the cells. These rotational speeds may be between approximately 10 rpm to approximately 50 rpm, and for example, may be approximately 14 rpm, to average the gravity vector on the cells, as detailed below.

The vessel42is typically formed such that the outer shell44and the culture chamber45are an integral member. However, the outer shell44and culture chamber45can be separate pieces joined together by numerous known techniques and fasteners. This culture chamber45is typically recessed and cylindrical in shape, for holding culture medium or the like. The vessel42includes a single access port63or ports, that receive a line64, through which culture medium is supplied to the culture chamber45from the reservoir28(as detailed below). The vessel42can also be disposable if desired.

The vessel42is typically made of plastic, stainless steel or other sterilizable material. The culture chamber45can also be lined with plastic or other sterilizable material. When separate pieces, the outer shell44and culture chamber45are typically made of plastic, stainless steel or any other sterilizable material. This culture chamber45typically is recessed (to a depth “d”) so as to be formed of rigid walls and configured to have a high vertical diameter “V”. This vertical diameter “V” is larger than the depth “d” to yield maximum quantities of newborn cells, for example, approximately 20 to approximately 30 times larger.

The access port63or ports can be any type of port used with culture or reaction vessels. This includes valves and membranes that can be penetrated by tubing, syringe, pipette or any other sampling devices.

The substrate46is typically a porous membrane that typically accommodates adhesive along its outer surface68, to allow cells to attach to this surface68. This membrane can be, for example, a 0.22 micron pore-size nitrocellulose filter manufactured by Millipore. While the substrate46is typically non-rigid, rigid materials are also suitable, provided that they will bind with adhesives used to attach the cells (as detailed below). The adhesives used are such that they hold cells to the substrate46, allowing these cells to grow and divide without damaging them. The adhesives can include any composition that binds to a cell surface and can be anchored or attached to the substrate46. Exemplary adhesives include proteins, which bind to the specific cell surface receptors, such as lectins (or lectin based compositions) that bind cell surface components, such as Concanavalin A, charged molecules (or charged molecule based compositions), that bind to cell surfaces, such as poly-D-lysine, antibodies (or antibody based compositions) (e.g., antibodies to cell surface components), other adhesives, such as fibronectin (fibronectin based compositions), as well as other substances and compositions that when placed onto the substrate46will bind and/or provide binding sites for the cells thereto.

The spacer50is typically a thin member placed into contact with the plate52. This spacer50creates a uniform space between the cells66attached to the substrate46and the plate52, so that newborn cells can move out of the port70in the plate52rapidly, without getting caught in crevices and the like. The spacer50is for example, a DACRON® mesh, approximately 0.2 mm thick and having openings of about 1 square millimeter each. This results in the volume of medium between the attached cells66and the flat plate52being typically not more than approximately 2.0 milliliters(ml). The space between the substrate46should be as small as possible to enable released newborn cells to exit the bioreactor22as soon as they have been produced. This space is typically approximately 0.1 mm to approximately 0.2 mm.

In alternate embodiments, use of the spacer50is optional, and typically spacers are not employed at all. Rather, in these alternate embodiments, a rigid substrate46is used, and coupled with the rigidity of the plate52, the approximately 0.1 mm to approximately 0.2 mm gap is uniformly maintained between the cells66and the plate52, eliminating the need for the spacer50.

The plate52includes a vent74, that allows for bubbles to be removed from the chamber61. It is typically made of materials, such as Stainless Steel and the like, that are sterilizable. The port70of the plate52, typically centrally positioned therein, interfaces with the fluid coupler26.

The fluid coupler26is stationary, and includes an inlet port80for receiving culture medium (along a line76) from the reservoir28. Within the axle40is a passage84, terminating in an exit port86, to which the line64is attached for transporting culture medium to the vessel42. A pump88, typically a peristaltic pump or the like, typically located along the line76between the reservoir28and the fluid coupler26, provides the forces for delivering the culture medium, in the direction of arrows AA, and forms a system or circuit for delivery of culture medium, from the reservoir28to the vessel42(also in the direction of sub arrows BB), and to the substrate46. This pumping perfuses the substrate46with culture medium, allowing the cells to grow and divide, as detailed herein. Pumping forces are, for example, approximately 1 ml/minute to approximately 3 ml/minute for moving culture medium throughout the bioreactor22.

The bioreactor22can be operated at any temperature or in any environment, for example, ambient or gaseous environments. The specific temperature and environment is dependent on the desired process or the specific cells being used.

In an exemplary operation of the system20, should the substrate46not be fixed in the bioreactor22, the substrate46is coated with a cell adhesive by passing the cell adhesive through it. This is followed by a wash solution of water or phosphate buffered saline. Growing cells are then applied to the substrate46. The substrate46is clamped to the vessel42while being held vertically. The bioreactor22is then turned to the normal horizontal operating position, and the pump28is activated to fill the vessel42with culture medium. These substrate46preparation procedures are performed under gentle vacuum pressure, such that the flow rate is approximately 1 ml/sec.

Alternately, the substrate46can be prepared with adhesive as above, except that the cells have not been added while outside of the bioreactor22. Here, once the prepared substrate46is in the bioreactor22, cells are introduced to the substrate46by pumping cell culture medium into the vessel42in a direction opposite of arrows AA. Once the cells are attached, the pump28direction is reversed (to the direction of arrows AA) to begin pushing fresh culture medium through the bioreactor22.

Still alternately, should the substrate46be fixed in the bioreactor22(the bioreactor22closed), adhesive, cells, wash solutions and cell culture medium are introduced to the bioreactor22by being pumped therein through the exit port30in the fluid coupler26. This pumping is in reverse to the flow direction described herein (the direction opposite arrows AA of FIG.2).

In all three situations above, with fresh culture medium being pumped through the bioreactor22, rotation of the bioreactor22now begins. For example, fresh culture medium can be pumped into the culture chamber45of the vessel42, to keep it between approximately one quarter to approximately full during the course of operation, with an approximately half full, or approximately half full to full, culture chamber45preferred. The vessel42and substrate46are rotated, for example, at speeds of approximately 14 rpm. At this same time, culture medium is continuously perfused into the culture chamber45of the vessel42. The rotation of the vessel42and substrate46coupled with the perfusion of culture medium results in the release of newborn (newly divided) cells, from the mother cells on the substrate46. These newborn cells are moved in the effluent stream through the exit port70in the plate52and leave the system20through the exit port30in the fluid coupler26, whereby they are collected in the flask32.

EXAMPLE

Approximately 5×107L I210 lymphocytic leukemia were attached to a 100 square centimeter nitrocellulose membrane filter coated with Concanavalin A (the substrate) under vacuum at 1 ml/sec. The substrate was now placed into a system having a bioreactor as described above, as the substrate was clamped into the bioreactor. The culture chamber of the vessel was continuously perfused with fresh culture medium, at a flow rate of approximately 2 ml/minute. The bioreactor was rotated at speeds of approximately 14 rpm.

Each division of an attached cell resulted in the release of one newborn daughter cell from the vessel, while the other daughter cell remained attached to the surface of the substrate. Accordingly, the culture vessel maintained a constant cell number, i.e., enabled long-term continuous culture, while releasing 5×107newborn cells each 10 hour generation time.

The process was continued for many generations, and the released newborn cells grew synchronously through the cell cycle. The cell cycle properties analyzed during rotation include mitotic cycle phase durations, cell sizes and DNA distributions in the cell cycle.

The newborn cells increased in size by approximately ten percent during the first generation (e.g., 10-12 hours) of elution from the culture vessel. During the second generation of growth in the vessel, the newborn cells decreased in size, returning to the initial size at the start of culture in the vessel. From this time forward, the size of the newborn cells released from the bioreactor remained constant. Once the cells adapted to the continuous supply of fresh medium, steady state growth ensued and continued for at least eight generations of growth.

The concentration of released cells remained essentially constant at 7.5×104/min. The released cells were at least 95 percent pure newborn cells, based on their size distributions and G1content of DNA. The purity of newborn cells released from the vessel was maximal when the vessel was half filled with medium, here, up to the exit port in the flat plate. During subsequent culture, the cells progressed through the cycle normally.

There has been shown and described at least one preferred embodiment of cell culture device and system. It is apparent to those skilled in the art, however, that many changes, variations, modifications, and other uses and applications for the aforementioned device, system and its components are possible, and also such changes, variations, modifications, and other uses and applications which do not depart from the spirit and scope of the invention are deemed to be covered by the invention, which is limited only by the claims which follow.