Source: https://patents.google.com/patent/JP2016536998A/en
Timestamp: 2019-10-15 03:59:18
Document Index: 569945671

Matched Legal Cases: ['Application No. 61', 'art 500', 'art 500', 'art 500', 'art 500', 'art 500', 'art 500', 'art 500', 'arts 600', 'art 500', 'arts 600', 'arts 402', 'arts 600', 'art 600', 'art 600', 'art 600', 'art 600', 'art 600', 'art 600', 'art 600', 'art 600', 'art 700', 'art 700', 'art 700', 'art 1412', 'art 1512', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'art 700', 'arts 500', 'arts 500', 'arts 500', 'arts 500']

JP2016536998A - Cell growth in bioreactors - Google Patents
Cell growth in bioreactors Download PDF
JP2016536998A
JP2016536998A JP2016530921A JP2016530921A JP2016536998A JP 2016536998 A JP2016536998 A JP 2016536998A JP 2016530921 A JP2016530921 A JP 2016530921A JP 2016530921 A JP2016530921 A JP 2016530921A JP 2016536998 A JP2016536998 A JP 2016536998A
JP2016530921A
JP2016536998A5 (en
ジェイ． ナンカーヴィス、ブライアン
イー． ジョーンズ、マーク
2013-11-16 Priority to US201361905182P priority Critical
2013-11-16 Priority to US61/905,182 priority
2014-11-14 Application filed by テルモ ビーシーティー、インコーポレーテッド, テルモ ビーシーティー、インコーポレーテッド filed Critical テルモ ビーシーティー、インコーポレーテッド
2014-11-14 Priority to PCT/US2014/065829 priority patent/WO2015073918A1/en
2016-12-01 Publication of JP2016536998A publication Critical patent/JP2016536998A/en
2017-12-21 Publication of JP2016536998A5 publication Critical patent/JP2016536998A5/ja
Embodiments for cell growth in a bioreactor are described. In one embodiment, a method is provided for improving cell growth in a bioreactor by distributing the cells throughout the bioreactor and attaching the cells to specific portions of the bioreactor. Embodiments are implemented in a cell proliferation system that performs cell input, distribution, attachment, and proliferation. [Selection] Figure 13C
This application claims the priority of US Provisional Patent Application No. 61 / 905,182 (Title: Method for loading and distributing cells in a bioreactor of a cell proliferation system) filed on November 16, 2013. The entire provisional US patent application is hereby expressly incorporated herein by this disclosure.
Particular attention has been focused on the potential use of stem cells in various treatments and therapies. By using a cell proliferation system, other types of cells such as stem cells and bone marrow cells can be expanded (eg, grown). By using stem cells grown from donor cells, damaged or defective tissue can be repaired or replaced, and there is a wide range of clinical applications for a wide range of diseases. Recent advances in the field of regenerative medicine have shown that stem cells have properties such as proliferation ability, self-renewal ability, ability to maintain undifferentiated state and ability to differentiate into specialized cells under certain conditions .
A cell growth system has one or more compartments for growing cells, such as a cell growth chamber (also referred to as a “bioreactor”). In order to grow the cells, usually an initial amount of cells is loaded into the bioreactor for distribution. Accordingly, there is a need for a cell input / distribution method in a bioreactor associated with a cell growth system. The present invention has been made to address these and other needs.
Embodiments of the present invention have been made in view of these and others. However, application of the embodiment of the present invention to other problems is not limited by the above-described problems.
This section is intended to describe aspects of some embodiments of the invention in a simplified form and is not intended to identify key or essential elements of the claimed invention, It is not intended to limit the scope of the claims.
The present invention should be understood to include a variety of different embodiments, and this section is not intended to limit the invention or include all of them. In this section, features included in the embodiments are schematically described, but may include more specific descriptions of other features included in other embodiments.
One or more embodiments generally relate to methods and systems for loading and dispensing cells in a bioreactor of a cell growth system. Accordingly, embodiments include a method of adding a plurality of cells to a fluid circulating at a first rate in a bioreactor of a cell growth system. In certain embodiments, the bioreactor may comprise a hollow fiber membrane having a plurality of hollow fibers. Cells and other fluids circulate through the hollow fiber. First, fluid is circulated through the hollow fiber membrane of the bioreactor, and cells are added to the circulating fluid. This fluid circulates at a first predetermined circulation rate. During circulation, the bioreactor is placed in a horizontal position. By adding the cells to the circulating fluid, the cells are circulated throughout the system and uniformly distributed while flowing into and out of the hollow fibers of the hollow fiber membrane after the cells have been introduced. Then, the circulation is stopped. The cells settle under the influence of gravity and adhere to the first part of the hollow fiber of the bioreactor. In certain embodiments, the cells settle for a first predetermined period. In some embodiments, the cells are attached to the first portion of the hollow fiber by selecting a predetermined period of time.
After the first predetermined period, the bioreactor is rotated 180 degrees. After rotation of the bioreactor, the cells in the bioreactor are precipitated again. Then, the cells are allowed to settle for the second predetermined period in the opposite part of the hollow fiber. The second predetermined period is selected so that the cells adhere to the facing portion. After the elapse of the second predetermined period, the bioreactor is returned to its original horizontal position, and a proliferation process is performed on the cells.
In some embodiments, the dosing process has additional steps. In some embodiments, circulation is resumed after the bioreactor is returned to its original horizontal position. The circulation rate at that time is set to a rate lower than the first predetermined circulation rate. Upon circulation, cells that did not adhere to the surface will be distributed again. This circulation occurs for a third predetermined period of time so that unattached cells are evenly distributed throughout the system including the bioreactor. Then, the circulation is stopped, the cells in the bioreactor are precipitated, and the hollow fibers are attached again.
After a fourth predetermined period for reprecipitation of the cells, the bioreactor is rotated 180 degrees. After rotation of the bioreactor, the cells in the bioreactor precipitate again. And a cell settles toward the opposing part of a hollow fiber for the 5th predetermined period set so that it could adhere to the opposing part of a hollow fiber. After the fifth predetermined period, the bioreactor is returned to its original horizontal position.
The above process is repeated by circulating cells in the system to redistribute unattached cells again uniformly. However, every time the circulation is restarted, the circulation rate is lower than the previous circulation rate. Stop circulation and allow cells to settle and adhere. Then, the bioreactor is rotated 180 degrees to precipitate and attach cells. The bioreactor is then returned to its original position. These steps of circulation, precipitation, rotation, precipitation, and rotation are repeated a predetermined number of times, after which the layered cells are grown in the bioreactor.
Other embodiments also relate to methods and systems for loading and dispensing cells in a bioreactor of a cell growth system. Embodiments include a method of adding a plurality of cells to a fluid circulating at a first rate in a bioreactor of a cell growth system. In certain embodiments, the bioreactor may comprise a hollow fiber membrane having a plurality of hollow fibers. Cells and other fluids circulate through the hollow fiber. First, fluid is circulated through the hollow fiber membrane of the bioreactor, and cells are added to the circulating fluid. This fluid circulates at a first predetermined circulation rate. During circulation, the bioreactor is placed in a horizontal position. By adding the cells to the circulating fluid, the cells are circulated throughout the system and uniformly distributed while flowing into and out of the hollow fibers of the hollow fiber membrane after the cells have been introduced. Then, the circulation is stopped. The cells settle under the influence of gravity and adhere to the first part of the hollow fiber of the bioreactor. In certain embodiments, the cells settle for a first predetermined period. In some embodiments, the cells are attached to the first portion of the hollow fiber by selecting a predetermined period of time.
After the first predetermined period, the bioreactor is rotated 180 degrees. After rotation of the bioreactor, the cells undergo a growth process. As will be appreciated, the previously attached cells are at the top of the hollow fiber. When the cells proliferate, the cells are affected by gravity and promote cell growth toward the hollow fiber bottom.
Further effects of the embodiment presented here will be easily understood from the following description and the accompanying drawings.
To further clarify the above and other advantages and features of the present invention, the present invention will be described more specifically with reference to specific embodiments in the accompanying drawings. These drawings only depict exemplary embodiments of the invention and therefore do not limit the scope of the invention. Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1A is a diagram of one embodiment of a cell proliferation system (CES). FIG. 1B is a diagram of a second embodiment of the CES. FIG. 1C is a diagram of a third embodiment of the CES. FIG. 1D is a diagram of one embodiment of a rocking device that moves the cell growth chamber in a rotational or lateral direction during a CES operation. FIG. 2A is a side view of one embodiment of a hollow fiber cell growth chamber. 2B is a partially cutaway side view of the embodiment of the hollow fiber cell growth chamber of FIG. 2A. FIG. 3 is a cutaway side view of another embodiment of a bioreactor showing a circulation path. FIG. 4 is a perspective view of a portion of a CES having a removable bioreactor, according to one embodiment. FIG. 5 is a flowchart of a method for growing cells in a CES according to one embodiment. FIG. 6 is a flowchart of a process for cell loading, dispensing, attachment, and growth with steps used in the method of the flowchart shown in FIG. 5 in some embodiments. FIG. 7 is a flowchart of a process for cell loading, dispensing, attachment, and growth, with steps used in the method of the flowchart shown in FIG. 5 in some embodiments. FIG. 8 is a front view of the bioreactor according to the embodiment in the first posture. FIG. 9 is a front view of the bioreactor when the bioreactor is rotated 90 degrees from the state of FIG. FIG. 10 is a front view of the bioreactor when the bioreactor is rotated 180 degrees from the state of FIG. FIG. 11 is a front view of the bioreactor when the bioreactor of FIG. 8 is rotated and returned to the original position shown in FIG. FIG. 12 is a front view of the bioreactor when rotated 90 degrees from the state of FIG. 8 and rotated 180 degrees from the state of FIG. FIGS. 13A-13C illustrate a hollow fiber (perpendicular to the central axis) that is part of a bioreactor that is in progress through the steps of the process of cell distribution, attachment and growth in a bioreactor according to one embodiment. It is a sectional view. 13D-13E are cross-sectional views (parallel to the central axis) of a hollow fiber that is part of a bioreactor that is in progress through the steps of a cell growth process in a bioreactor according to one embodiment. It is. FIGS. 14A-14D show a hollow fiber (relative to the central axis) that is part of a bioreactor that is in progress through the steps of cell distribution, attachment and growth processes in a bioreactor according to another embodiment. FIG. FIGS. 15A-15F illustrate the hollow fiber (relative to the central axis) that is part of a bioreactor that is in progress through the steps of cell distribution, attachment and growth processes in a bioreactor according to yet another embodiment. FIG. FIG. 16 is a cross-sectional view of a bioreactor showing each hollow fiber zone in which a plurality of hollow fibers and cell-containing fluid circulate at different flow rates. FIG. 17 is a block diagram of a basic computer used to implement an embodiment of the present invention.
The principles of the present invention may be further understood with reference to the following detailed description and embodiments illustrated in the accompanying drawings. Although specific features are shown and described in detailed embodiments, it should be understood that the invention is not limited to the embodiments described below. The present disclosure generally relates to a method for distributing a plurality of cells in a bioreactor of a cell growth system. As described below, the method of dispensing cells within the bioreactor may include loading the cell into the bioreactor, rotating the bioreactor, and holding the bioreactor in a particular position.
An example of a cell proliferation system (CES) is shown schematically in FIG. 1A. The CES 10 includes a first fluid circuit 12 and a second fluid circuit 14. The first fluid flow path 16 has at least both ends 18, 20 fluidly associated with a hollow fiber cell growth chamber 24 (also referred to as a “bioreactor”). In particular, end 18 is fluidly associated with first inlet 22 of cell growth chamber 24 and end 20 is fluidly associated with first outlet 28 of cell growth chamber 24. In the first circuit 12, the fluid passes through the hollow fibers of the hollow fiber membrane disposed in the cell growth chamber 24 (the cell growth chamber and the hollow fiber membrane are described in more detail below). The first flow rate controller 30 is operatively connected to the first fluid flow path 16 and controls the flow of fluid in the first circulation path 12.
The second fluid circulation path 14 includes a second fluid flow path 34, a cell growth chamber 24, and a second flow rate controller 32. The second fluid channel 34 has at least both end portions 36 and 38. The end 36 of the second fluid flow path 34 is fluidly associated with the inlet port 40 of the cell growth chamber 24 and the end 38 is fluidly associated with the outlet port 42. The fluid flowing through the cell growth chamber 24 contacts the outside of the hollow fiber membrane of the cell growth chamber 24. The second fluid circulation path 14 is operatively connected to the second flow rate controller 32.
Thus, the first and second fluid circulation paths 12 and 14 are separated in the cell growth chamber 24 by the hollow fiber membrane. The fluid in the first fluid circuit 12 flows through the capillary inner (IC) space of the hollow fiber in the cell growth chamber. Accordingly, the first fluid circulation path 12 is referred to as an “IC loop”. The fluid in the second fluid circuit 14 flows through the capillary outer (EC) space of the hollow fiber in the cell growth chamber. Therefore, the second fluid circulation path 14 is referred to as an “EC loop”. The fluid in the first fluid circuit 12 may flow in either the cocurrent direction or the counterflow direction with respect to the fluid flow in the second fluid circuit 14.
The fluid inlet channel 44 is fluidly associated with the first fluid circuit 12. Fluid is introduced into the first fluid circulation path 12 by the fluid inlet path 44, and fluid is discharged from the CES 10 by the fluid outlet path 46. A third flow controller 48 is operatively associated with the fluid inlet passage 44. Alternatively, the third flow controller 48 may be associated with the fluid outlet path 46.
The flow controller used here can be a pump, a valve, a clamp, or a combination thereof. A plurality of pumps, a plurality of valves, and a plurality of clamps may be arranged in any combination. In various embodiments, the flow controller is or includes a peristaltic pump. In other embodiments, the fluid circuit, the inlet port, and the outlet port may be composed of piping of any material.
Although various members are described as “operably associated”, as used herein, “operably associated” refers to members that are operatively connected to each other. In addition, embodiments in which members are directly connected, embodiments in which another member is disposed between two connected members, and the like are also included. An “operably associated” member can be “fluidically associated”. “Fluidly associated” refers to members that are coupled together so that fluid is movable between the members. The term “fluidly associated” encompasses embodiments in which another member is placed between two fluidly associated members, embodiments in which members are directly connected, and the like. The fluidly associated member may include a member that operates the system by not contacting the fluid but contacting other members (eg, by compressing the outside of the flexible tube to A peristaltic pump that pumps fluid through the
In general, any type of fluid, such as a buffer, a protein-containing fluid, or a cell-containing fluid, can flow through the circulation path, the inlet path, and the outlet path. The terms “fluid”, “medium” and “fluid medium” as used herein are used interchangeably.
FIG. 1B shows the cell growth system 800 in more detail. The CES 800 has a first fluid circuit 802 (also referred to as “capillary inner loop” or “IC loop”) and a second fluid circuit 804 (also referred to as “capillary outer loop” or “EC loop”). The first fluid flow path 806 is fluidly associated with the cell growth chamber 801 via the first fluid circulation path 802. The fluid flows into the cell growth chamber 801 through the IC inlet port 801A, flows out through the hollow fiber in the cell growth chamber 801, and flows out through the IC outlet port 801B. The pressure sensor 810 measures the pressure of the medium leaving the cell growth chamber 801. In addition to pressure, sensor 810 may be a temperature sensor that detects medium pressure and medium temperature during operation. The medium flows through an IC circulation pump 812 that is used to control the medium flow rate (eg, the circulation rate in the IC loop). The IC circulation pump 812 can pump fluid in a first direction or a second direction opposite the first direction. The outlet port 801B can be used as an inlet in the reverse direction. The medium flowing into the IC loop 802 can flow through the valve 814. As can be appreciated by those skilled in the art, additional valves and / or other devices may be placed at various locations to isolate the media and / or measure the properties of the media in certain parts of the fluid path. You can also. Accordingly, the schematic shown is one possible configuration for elements of CES 800 and should be understood to be deformable within the scope of one or more embodiments.
For IC loop 802, a sample of media is obtained from sample coil 818 during operation. The medium then returns to the IC inlet port 801A and completes the fluid circuit 802. Cells grown / proliferated in cell growth chamber 801 are flushed out of cell growth chamber 801 and enter harvest bag 899 via valve 898 and line 897. Alternatively, if valve 898 is closed, cells are redistributed into chamber 801 (eg, circulated back) and further grown or loaded.
In the second fluid circuit 804, fluid enters the cell growth chamber 801 via the EC inlet port 801C and leaves the cell growth chamber 801 via the EC outlet port 801D. In the EC loop 804, the culture medium contacts the outside of the hollow fiber of the cell growth chamber 801, thereby allowing diffusion of small molecules into and out of the hollow fiber in the chamber 801.
Before the medium enters the EC space of the cell growth chamber 801, the pressure and temperature of the medium are measured by a pressure / temperature sensor 824 disposed in the second fluid circuit 804. After the medium leaves the cell growth chamber 801, the sensor 826 measures the pressure and temperature of the medium in the second fluid circuit 804. For EC loop 804, a sample of media is obtained from sample port 830 or from a sample coil during operation.
After leaving the EC outlet port 801D of the cell growth chamber 801, the fluid in the second fluid circuit 804 passes through the EC circulation pump 828 to the gas transport module 832. The EC circulation pump 828 can pump fluid in both directions. The second fluid flow path 822 is fluidly associated with the gas transport module 832 via the inlet port 832A and the outlet port 832B of the gas transport module 832. In operation, fluid medium flows into gas transport module 832 via inlet port 832A and out of gas transport module 832 via outlet port 832B. The gas transport module 832 adds oxygen to the medium in the CES 800 to remove bubbles. In various embodiments, the medium in the second fluid circuit 804 is in equilibrium with the gas entering the gas transport module 832. The gas transport module 832 may be any device of suitable size useful for oxygen supply or gas transport, as is known in the art. Air or gas flows into the gas transport module 832 through the filter 838 and out of the oxygen supply or gas transport device 832 through the filter 840. Filters 838, 840 reduce or prevent contamination of oxygenator 832 and associated media. Air or gas purged from the CES 800 during part of the priming process can be vented to the atmosphere via the gas transport module 832.
In the configuration shown as CES 800, the fluid medium in the first fluid circuit 802 and the second fluid circuit 804 flows through the cell growth chamber 801 in the same direction (in a co-current configuration). The CES 800 may be configured to flow in a counterflow.
In at least one embodiment, media containing cells (from a cell container, eg, a source such as a bag) is attached to attachment point 862 and fluid media from the media source is attached to attachment point 846. The cells and the medium are introduced into the first fluid circulation path 802 via the first fluid channel 806. Attachment point 862 is fluidly associated with first fluid flow path 806 via valve 864. Attachment point 846 is fluidly associated with first fluid flow path 806 via valve 850. The reagent source may be fluidly associated with point 844 and associated with fluid inlet passage 842 via valve 848, or may be associated with second fluid inlet passage 874 via valves 848 and 872.
An air removal chamber (ARC) 856 is fluidly associated with the first circuit 802. The air removal chamber 856 may have one or more sensors. The sensors include an upper sensor and a lower sensor for detecting air, fluid shortage, and / or gas / fluid boundaries, eg, air / fluid boundaries, at specific measurement points within the air removal chamber 856. It is. For example, near the bottom and / or near the top of the air removal chamber 856, ultrasonic sensors can be used to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from CES 800 during part of the priming process or other protocol can be vented from air valve 860 to the atmosphere through line 858 fluidly associated with air removal chamber 856. It is.
An EC media source is attached to EC media attachment point 868. A cleaning liquid source is attached to the cleaning liquid attachment point 866. Thereby, the EC medium and / or the cleaning liquid is added to the first fluid channel or the second fluid channel. Attachment point 866 is fluidly associated with valve 870. Valve 870 is fluidly associated with first fluid circuit 802 via valve 872 and first fluid inlet channel 842. Also, by opening valve 870 and closing valve 872, attachment point 866 can be fluidly associated with second fluid circuit 804 via second fluid inlet channel 874 and EC inlet channel 884. Similarly, attachment point 868 is fluidly associated with valve 876. Valve 876 is fluidly associated with first fluid circuit 802 via first fluid inlet channel 842 and valve 872. Also, by opening valve 876 and closing valve 872, fluid container 868 can be fluidly associated with second fluid inlet channel 874.
In the IC loop 802, the fluid is first pumped by the IC inlet pump 854. In EC loop 804, fluid is first pumped by EC inlet pump 878. An air detector 880 such as an ultrasonic sensor may be associated with the EC inlet channel 884.
In at least one embodiment, the first and second fluid circulation paths 802, 804 are connected to a waste line 888. When the valve 890 is opened, the IC medium flows through the waste line 888 and reaches the waste bag 886. Similarly, when the valve 892 is opened, the EC medium flows to the waste bag 886.
After the cells grow in the cell growth chamber 801, the cells are harvested via the cell harvesting path 897. The cells from the cell growth chamber 801 are harvested in the cell harvesting bag 899 via the cell harvesting path 897 by pumping the IC medium containing the cells with the valve 898 opened.
Each part of CES 800 is housed in a device or housing 899, such as a cell proliferator, that maintains the cells and media at a predetermined temperature. In certain embodiments, the components of CES 800 may be combined with other CESs such as CES 10 (FIG. 1A) or CES 900 (FIG. 1C). In other embodiments, the CES may include fewer parts than shown in FIGS. 1A-1C within the scope of this disclosure.
FIG. 1C shows another embodiment of CES. The CES 900 includes a first fluid circuit 902 (also referred to as “capillary inner (IC) loop”) and a second fluid circuit 904 (also referred to as “capillary outer loop” or “EC loop”).
The first fluid flow path 906 is fluidly associated with the cell growth chamber 908 via the first fluid circulation path 902. Fluid enters the cell growth chamber 908 through the inlet port 910, passes through the hollow fibers in the cell growth chamber 908, and exits through the outlet port 907. A pressure gauge 917 measures the pressure of the medium leaving the cell growth chamber 908. The culture medium flows through the valve 913 and the pump 911. Valve 913 and pump 911 are used to control the flow rate of the culture medium. A sample of media can be obtained from sample port 905 or sample coil 909 during operation. A medium pressure and a medium temperature during operation can be detected by a pressure / thermometer 915 disposed in the first fluid circulation path 902. The media returns to the inlet port 910 to complete the fluid circuit 902. Cells grown in cell growth chamber 908 are flushed out of cell growth chamber 908 or redistributed into the hollow fiber for further growth.
The second fluid circuit 904 has a second fluid channel 912 that is fluidly associated with the cell growth chamber 908 in a loop. In the second fluid circuit 904, fluid enters the cell growth chamber 908 via the inlet port 914 and leaves the cell growth chamber 908 via the outlet port 916. The medium contacts the outside of the hollow fiber in the cell growth chamber 908, allowing small molecules to diffuse into and out of the hollow fiber.
Before the medium enters the EC space of the cell growth chamber 908, the pressure and temperature of the medium can be measured by a pressure / thermometer 919 disposed in the second circulation path 904. After the medium leaves the cell growth chamber 908, the pressure of the medium can be measured in the second circulation path 904 by the pressure gauge 921.
In the second fluid circuit 904, the fluid leaves the outlet port 916 of the cell growth chamber 908 and then passes through the pump 920 and valve 922 to the oxygen supplier 918. Second fluid flow path 912 is fluidly associated with oxygenator 918 via oxygenator inlet port 924 and oxygenator outlet port 926. In operation, fluid medium flows into the oxygenator 918 through the oxygenator inlet port 924 and out of the oxygenator 918 through the oxygenator outlet port 926.
The oxygen supplier 918 adds oxygen to the CES 900 medium. In various embodiments, the media in the second fluid circuit 904 is in equilibrium with the gas entering the oxygenator 918. The oxygenator can be any oxygenator known in the art. The gas enters the oxygen supplier 918 through the filter 928 and exits the oxygen supplier 918 through the filter 930. Filters 928, 930 reduce or prevent contamination of oxygenator 918 and associated media.
In the configuration shown as CES 900, the fluid medium in the first circuit 902 and the second circuit 904 flows through the cell growth chamber 908 in the same direction (cocurrent configuration). One skilled in the art will appreciate that the CES 900 may be configured in a counterflow configuration. Further, those skilled in the art will understand that the inlet port and the outlet port may be disposed at any location in the cell growth chamber 908.
Cells and fluid medium are introduced into the fluid circulation path 902 via the first fluid inlet path 932. The fluid container 934 is fluidly associated with the first fluid inlet passage 932 via the valve 938 and the fluid container 936 via the valve 940, respectively. Similarly, the cell container 942 is fluidly associated with the first fluid circuit 902 via the valve 943. In some embodiments, cells and fluid are introduced into drip chamber 948 through heat exchanger 944 and pump 946. In embodiments where cells from the cell container 942 are passed through the heat exchanger 944, an additional line (not shown) may be used to connect the cell container 942 to the heat exchanger 944. The drip chamber 948 is fluidly associated with the first circuit 902. Overflow from drip chamber 948 flows out of overflow line 950 via valve 952.
From the fluid containers 954 and 956, fluid is added to the first and second fluid circulation paths 902, 904. A fluid container 954 is fluidly associated with the valve 958. Valve 958 is fluidly associated with first fluid circuit 902 via valve 964, flow path 960 and flow path 932. The fluid container 954 is also fluidly associated with the second fluid inlet passage 962. Similarly, fluid container 956 is fluidly associated with valve 966 and valve 966 is fluidly associated with first fluid circuit 902 via first fluid inlet passage 960. The fluid container 956 is also fluidly associated with the second fluid inlet passage 962.
Second fluid inlet passage 962 is configured to flow through heat exchanger 944 and pump 968 before fluid enters drip chamber 970. The second fluid inlet path 962 continues to the second fluid circulation path 904. Fluid overflow passes through valve 974 to overflow container 976 via overflow line 972.
Cells are harvested via cell harvesting path 978. Cells from the cell growth chamber 908 are harvested through the cell harvesting path 978 to the cell harvesting bag 980 by pumping the medium containing the cells with the valve 982 open.
The first and second fluid circulation paths 902 and 904 are connected by a connection path 984. When the valve 986 is opened, the culture medium can flow between the first circulation path 902 and the second circulation path 904 through the connection path 984. Similarly, the pump 990 can pump the medium between the first fluid circuit 902 and the second fluid circuit 904 via another connection 988.
Each component of the CES 900 is accommodated in an incubator 999. The incubator 999 keeps the cells and medium at a constant temperature.
Any number of fluid containers (eg, media bags) can be fluidly associated with the CES 900 in any combination, as will be appreciated by those skilled in the art. Further, the position of the drip chamber 948 or the position of the sensor independent of the drip chamber 948 may be any position before the inlet port 910 in the CES 900.
CES 800 and 900 may include other components. For example, one or more pump loops (not shown) can be added to the position of the peristaltic pump in the CES. The pump loop can be formed, for example, from polyurethane (PU) (commercially available as Tygothane C-210A). Moreover, you may include the cassette for comprising a piping line containing the piping loop for peristaltic pumps as a disposable part.
In some embodiments, a removable flow circuit (also referred to as a “detachable circulation module”) may be provided. The removable flow circuit may be part of a cell growth module that is configured to attach to a permanently secured portion of the CES. Usually, the fixed part of the CES includes a peristaltic pump. In various embodiments, the fixed portion of the CES can include valves and / or clamps.
The removable flow circuit can include a first fluid flow path having at least two ends. The first end of the first fluid channel is configured to be fluidly associated with the first end of the cell growth chamber, and the second end of the first fluid channel is the first end of the cell growth chamber. It is configured to be fluidly associated with the two ends.
Similarly, the removable flow circuit can include a second fluid flow path having at least two ends. A portion of the removable flow circuit can be configured to be fluidly associated with the oxygenator and / or bioreactor. The removable flow circuit can include a second fluid flow path configured to be fluidly associated with the oxygenator and the cell growth chamber.
In various embodiments, the removable flow circuit may be removably and disposablely attached to the flow controller. The removable flow circuit can include a removable fluid tube (eg, a flexible tube) that connects to several parts of the CES.
In other embodiments, the removable flow circuit includes a cell growth chamber, an oxygenator, and a bag containing media and cells. In various embodiments, the components may be connected together or separated. Further, the removable flow circuit can include one or more portions configured to be fitted with a fluid flow controller such as a valve, pump, and combinations thereof. In a configuration using a peristaltic pump, the removable flow module includes a peristaltic loop configured to be mounted around the peristaltic portion of the piping. In other embodiments, the peristaltic loop is configured to be fluidly associated with the circulation path, the inlet path, and the outlet path. The removable flow circuit may be assembled in the form of a kit according to instructions for assembly to a fluid flow controller such as a pump or valve.
In embodiments, various methods are used to introduce cells into the CES bioreactor. As described in detail below, embodiments include methods and systems for distributing cells within a bioreactor to promote steady growth of cells.
According to embodiments, the cells are grown (proliferated) in an IC loop or EC loop. Adherent and non-adherent suspension cells are grown. In one embodiment, the hollow fiber lumen of the cell growth chamber is coated with fibronectin. PBS without divalent cations (eg, without calcium and magnesium) is added to the CES system. Adherent cells are introduced into a cell growth chamber (eg, chambers 24, 801, 908) and then cultured for a sufficient amount of time such that the cells adhere to the hollow fiber. IC and EC media are circulated to ensure that sufficient nutrients are supplied to the cells.
The flow rate of the IC loop and the EC loop is adjusted to a specific value. In various embodiments, the flow rates of the IC loop and EC loop are each independently about 2, about 4, about 6, about 8, about 10, about 15, about 20, about 25, about 30, about 35, about 40. , About 45, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, or about 500 mL / min. In various embodiments, the IC circuit loop flow rate is about 10 to about 20 mL / min and the EC circuit loop flow rate is about 20 to about 30 mL / min (medium can flow through the oxygenator to restore oxygen levels). To a certain extent). To supplement the medium that evaporates through gas exchange modules such as gas exchange / oxygen suppliers 832 and 918, additional medium is added into the CES at a low flow rate (eg, 0.1 mL / min in some embodiments). May be pumped. In various embodiments, the EC loop removes cellular waste and the IC loop contains a growth factor in the medium.
These CES can provide great freedom in various growth conditions and growth criteria. By continuously circulating the medium, the cells can be suspended in the IC loop. Alternatively, cells can be settled by stopping the circulation of the medium. By ultrafiltration, fresh media can be added to the IC loop to adjust the excess volume without removing the cells. EC medium circulation allows exchange of gas, nutrients, and waste, and allows for the addition of new medium without removing cells.
In the art, examples of the cells to be proliferated include adherent cells, non-adherent cells, or cells obtained by co-culturing these cells in any combination. Examples of cells grown in the CES according to the embodiment include, but are not limited to, stem cells (eg, mesenchymal cells, hematopoietic cells, etc.), fibroblasts, keratinocytes, progenitor cells, and other fully differentiated cells. A cell, or a combination thereof.
In embodiments, IC and EC media are replaced with media that does not contain divalent cations (eg, divalent cation-free PBS) to harvest adherent cells. In one embodiment, trypsin is introduced into the first circuit and the adherent cells are cultured for a period of time (in some embodiments from about 5 minutes to about 10 minutes). The trypsin is then discharged from the system. By increasing the flow rate in the cell growth chamber, shear forces are applied to the cells, and adherent cells that are detached from the cell growth chamber are pumped into the cell harvest bag.
When growing non-adherent cells, the cells can be released from the circulating IC circuit. Adherent cells remain in the cell growth chamber while removing non-adherent cells.
Various cell growth methods can be performed using the CES. In one embodiment, the seeded cell population can be expanded. Cells are introduced or seeded into CES. In some situations, the lumen state of the hollow fiber is adjusted so that cell attachment is possible. Cells are then added to the cell growth chamber, adherent cells adhere to the hollow fiber, and non-adherent cells (eg, hematopoietic stem cells or HSCs) do not adhere. Non-adherent cells are released from the system. After culturing for a period of time, adherent cells are detached and harvested.
In an embodiment, the cell growth chamber of the cell proliferation system has a hollow fiber membrane. The hollow fiber membrane is composed of a plurality of semipermeable hollow fibers, and separates the first fluid circulation path and the second fluid circulation path.
The CES has a device configured to move, or “swing,” the cell growth chamber relative to other parts of the cell proliferation system by attaching to a rotational and / or lateral rocking device. FIG. 1D shows one such device. In the apparatus, the bioreactor 400 is connected to rotate in two rotationally oscillating parts and is further connected to one laterally oscillating part.
The first rotational direction swing device component 402 rotates the bioreactor 400 about the central axis 410 of the bioreactor. The bioreactor 400 is also connected to a lateral swing device 404. A rotational rocker component 402 is associated with the bioreactor 400 for rotation. The rotation direction swing device 402 rotates the bioreactor 400 about the central axis 410 of the bioreactor. The rotation is possible in a clockwise or counterclockwise direction. The bioreactor 400 can be continuously rotated in a single direction (clockwise direction or counterclockwise direction) around the central axis 410. Alternatively, the bioreactor 400 can be rotated alternately about the central axis 410, initially clockwise and then counterclockwise.
The CES also includes a second rotationally oscillating component that rotates the bioreactor 400 about the rotational axis 412. The rotation axis 412 passes through the center point of the bioreactor 400 and is perpendicular to the center axis 410. The bioreactor 400 can continuously rotate in a single direction, either clockwise or counterclockwise, about the rotation axis 412. Alternatively, the bioreactor 400 can be rotated alternately about the axis of rotation 412, initially clockwise and then counterclockwise. In various embodiments, the bioreactor 400 can be rotated about the axis of rotation 412 and can be placed in a horizontal orientation or a vertical orientation with respect to gravity.
A laterally oscillating component 404 is associated with the bioreactor 400 for lateral movement. The plane of the lateral swing component 404 moves in the X and Y directions. Thereby, the precipitation of cells in the bioreactor 400 is reduced with the movement of the cell-containing medium in the hollow fiber.
The rotational and / or lateral movement of the rocking device reduces cell settling in the device and also reduces the possibility of cells being trapped in certain parts of the bioreactor 400. The rate of cell precipitation in a cell growth chamber (eg, bioreactor 400) is proportional to the density difference between the cells and the suspension medium according to the Stokes equation. In one embodiment, as described above, non-adherent red blood cell suspension is maintained by repeating a 180 degree rotation (fast) and rest (total time 30 seconds). In some embodiments, a minimum rotation of about 180 degrees is performed. However, in other embodiments, a rotation of 360 degrees or more can be performed. Different rocking parts may be used separately or in combination. For example, an oscillating component that rotates the bioreactor 400 about the central axis 410 can be combined with an oscillating component that rotates the bioreactor 400 about the axis 412. Similarly, clockwise and counterclockwise rotation about different axes can be independently combined.
The above-described oscillating device and parts of the device can be implemented in the embodiment by using an appropriate structure. For example, in embodiments, one or more motors can be used as an oscillating device or as components of an oscillating device (eg, 402 and 404). In one embodiment, the oscillating device is shown in US Pat. No. 8,339,245 issued on 19 March 2013 (Title: Rotating system for cell growth chamber of cell proliferation system and method of use thereof). It can also be implemented using the embodiment. The entirety of the US patent is incorporated herein by this disclosure.
One embodiment of a cell growth chamber is shown in FIGS. 2B and 2A. A hollow fiber cell growth chamber 200 called a “bioreactor” is shown in a partially cutaway side view. The cell growth chamber 200 is housed in a cell growth chamber housing 202. The cell growth chamber housing 202 has four openings or four ports (inlet port 204, outlet port 206, inlet port 208, outlet port 210).
The fluid in the first circulation path enters the cell growth chamber 200 through the inlet port 204, passes through the capillary inside of the plurality of hollow fibers 212 (also referred to as the capillary inside (IC) or IC space of the hollow fiber membrane), and exits. Exit cell growth chamber 200 through port 206. The terms “hollow fiber”, “hollow fiber capillary”, and “capillary” are interchangeable. The plurality of hollow fibers 212 are collectively called a “membrane”. The fluid in the second circuit enters the cell growth chamber through the inlet port 208, contacts the outside of the hollow fiber 212 (also referred to as the EC side of the membrane or EC space), and passes through the outlet port 210 to the cell growth chamber 200. Get out of. The cells can be included in the first circuit or the second circuit and can be present on either the IC side or the EC side of the membrane.
The cell growth chamber housing 202 is illustrated as being cylindrical, but can be any shape as is known in the art. The cell growth chamber housing 202 can be made from any type of biocompatible polymeric material. The cell growth chamber housing may have different shapes and different sizes.
Although the term “cell growth chamber” is used, those skilled in the art will appreciate that not all cells grown or expanded in CES will be grown in the cell growth chamber. In many embodiments, adherent cells adhere to a membrane placed in a growth chamber or grow in associated tubing. Non-adherent cells (also called “suspension cells”) are similarly grown. Cells can also be grown in other areas within the first or second fluid circuit.
For example, the end of the hollow fiber 212 is potted on the side of the cell growth chamber 200 with a connecting material (also referred to herein as “potting” or “potting material”). If the potting material does not obstruct the flow of the culture medium and the cells into the hollow fiber, and the liquid flowing into the cell growth chamber 200 through the IC inlet port can be made to flow only into the hollow fiber 212, Any material suitable for bundling the hollow fibers 212 may be used. Examples of potting materials include, but are not limited to, polyurethane or other suitable binding materials or adhesives. In various embodiments, the hollow fiber 212 and potting may be cut perpendicular to the central axis of the hollow fiber 212 at each end so that fluid can flow in and out of the IC side. End caps 214 and 216 are located at the end of the cell growth chamber.
Fluid entering the cell growth chamber 200 through the inlet port 208 contacts the outside of the hollow fiber 212. This portion of the hollow fiber cell growth chamber is referred to as the “capillary outer (EC) space”. Small molecules (eg, water, oxygen, lactate, etc.) can diffuse through the hollow fiber 212 from the interior of the hollow fiber to the EC space or from the EC space to the IC space. High molecular weight molecules such as growth factors are usually too large to pass through the hollow fiber 212 and remain in the IC space of the hollow fiber. For embodiments in which cells are grown in IC space, the EC space is used as a medium reservoir for supplying nutrients to the cells and removing cell metabolism byproducts. The medium may be changed if necessary. If necessary, the medium may be circulated through the oxygen supply device to exchange the gas.
In various embodiments, the cells are introduced into the hollow fiber 212 by any method including a syringe. Cells may be introduced into the cell growth chamber 200 from a fluid container such as a bag that is fluidly associated with the cell growth chamber.
The hollow fiber 212 is configured to allow cells to grow within the capillary inner space (ie, within the hollow fiber lumen). The hollow fiber 212 is large enough to allow cells to adhere to the lumen without obstructing the flow of the medium in the hollow fiber lumen. In various embodiments, the inner diameter of the hollow fiber is about 10,000, about 9000, about 8000, about 7000, about 6000, about 5000, about 4000, about 3000, about 2000, about 1000, about 900, about 800, about 700. , About 650, about 600, about 550, about 500, about 450, about 400, about 350, about 300, about 250, about 200, about 150, or about 100 μm or more. Similarly, the outer diameter of the hollow fiber is about 10,000, about 9000, about 8000, about 7000, about 6000, about 5000, about 4000, about 3000, about 2000, about 1000, about 900, about 800, about 700, about 650, about 700, about 650, about 600, about 550, about 500, about 450, about 400, about 350, about 300, about 250, about 200, about 150, or about 100 μm or less. The wall thickness of the hollow fiber should in some embodiments be sufficient to allow small molecules to diffuse.
Any number of hollow fibers can be used in the cell growth chamber as long as the hollow fibers can be fluidly associated with the inlet and outlet ports of the cell growth chamber. In various embodiments, the cell growth chamber has a number of about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 11000, or about 12000 or more. Of hollow fiber. In other embodiments, the cell growth chamber comprises about 12000, about 11000, about 10,000, about 9000, about 8000, about 7000, about 6000, about 5000, about 4000, about 3000, or about 2000 or less hollow fibers. Have In other embodiments, the length of the hollow fiber is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, or about 900 mm or more. In an embodiment, the cell growth chamber has about 9000 hollow fibers having an average length of about 295 mm, an average inner diameter of 215 μm, and an average outer diameter of about 315 μm.
The hollow fiber can be composed of any material that can be sized sufficiently to form a fiber from which liquid can move from the inlet port to the outlet port of the cell growth chamber. In various embodiments, the hollow fiber can be composed of a plastic adhesive material capable of binding certain types of cells, such as adherent stem cells (eg, MSCs). In various embodiments, the hollow fiber is treated with a compound such as fibronectin to form an adhesive surface.
In certain embodiments, the hollow fiber is made from a semi-permeable biocompatible polymeric material. Such polymeric materials include a mixture of polyamide, polyallyl ether sulfone and polyvinyl pyrrolidone (referred to herein as “PA / PAES / PVP”). The semi-permeable membrane allows nutrients, waste, and dissolved gases to move between the EC space and the IC space through the membrane. In various embodiments, the molecular migration characteristics of the hollow fiber membrane allows metabolic waste products to diffuse out of the hollow fiber lumen through the membrane, while at the same time costly reagents (growth required for cell growth). Factors, cytokines, etc.) are selected to minimize loss from the hollow fiber.
In one variation, the outer layer of the PA / PAES / PVP hollow fiber is characterized by a uniform pore structure with a defined surface roughness. The pore openings are sized in the range of about 0.5 μm to about 3 μm, and the number of pores on the outer surface of the hollow fiber is a number in the range of about 10,000 to about 150,000 per square millimeter. . This outer layer has a thickness of about 1 μm to about 10 μm. The next layer is a second layer having a sponge structure, and in one embodiment has a thickness of about 1 μm to about 15 μm. This second layer functions as a support for the outer layer. The third layer next to the second layer has a finger-like structure. This third layer provides mechanical stability and provides a high void volume that reduces the molecular membrane transfer resistance. During use, the finger voids are filled with a fluid, which causes the resistance in diffusion and convection to be lower than in the case of a matrix having a sponge-filled structure with a small void volume. This third layer has a thickness of about 20 μm to about 60 μm.
In other embodiments, the hollow fiber membrane has from about 65% to about 95% by weight of at least one hydrophobic polymer and from about 5% to about 35% by weight of at least one hydrophilic polymer. Hydrophobic polymers include polyamide (PA), polyaramid (PAA), polyallyl ether sulfone (PAES), polyether sulfone (PES), polysulfone (PSU), polyallyl sulfone (PASU), polycarbonate (PC), polyether, It can be selected from the group consisting of a copolymer mixture of any of the above polymers such as polyurethane (PUR), polyetherimide, and polyethersulfone, or a mixture of polyallyl ether sulfone and polyamide. The hydrophilic polymer can be selected from the group consisting of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyglycol monoester, water-soluble cellulose derivative, polysorbate, and polyethylene-polypropylene oxide copolymer.
Depending on the type of cells being grown in the cell growth chamber, the polymeric yarn is treated with a substance such as fibronectin to enhance cell growth and / or cell adhesion to the membrane.
FIG. 3 shows a cutaway side view of another example cell growth chamber, bioreactor 300. Bioreactor 300 has a longitudinal axis LA-LA and comprises a bioreactor housing 304. In at least one embodiment, bioreactor housing 304 has four openings or four ports: IC inlet port 308, IC outlet port 320, EC inlet port 328, EC outlet port 332.
Fluid in the first circuit enters the bioreactor 300 through the IC inlet port 308 at the first longitudinal end 312 of the bioreactor 300 and enters the capillary inside of the plurality of hollow fibers 316 (in various embodiments, The bioreactor enters and passes through the capillary (“IC”) side of the hollow fiber membrane (referred to as the “IC space”) and through the IC outlet port 320 located at the second longitudinal end 324 of the bioreactor 300. Go out of 300. The fluid in the second circuit enters the bioreactor 300 through the EC inlet port 328 and contacts the outside or outside of the capillary of the hollow fiber 316 (referred to as the “EC side” or “EC space” of the membrane). To exit the bioreactor 300 through the EC outlet port 332. Fluid entering the bioreactor through the EC inlet port 328 contacts the outside of the hollow fiber. Small molecules (eg, water, oxygen, lactate, etc.) can diffuse through the hollow fiber from the interior of the hollow fiber to the EC space or from the EC space to the IC space. High molecular weight molecules such as growth factors are usually too large to pass through the hollow fiber and remain in the IC space of the hollow fiber. The medium may be changed if necessary. If necessary, the medium may be circulated through the oxygen supply device to exchange the gas. The cells can be contained in the first circuit and / or the second circuit and can be present on the IC side and / or EC side of the membrane. By way of example and not limitation, in one embodiment, the bioreactor 300 may have about 11520 yarns having an inner diameter (ID) of about 215 × 10 −6 m.
The bioreactor housing 304 is shown as a cylindrical shape, but can take various shapes, such as a rectangular parallelepiped, for example. The bioreactor housing 304 can be made of any type of biocompatible polymeric material that is present in one or more of the hollow fibers 316 or within the bioreactor housing 304. It may be made of a substantially transparent material so that the fluid can be observed. Various other bioreactor housings may have various shapes and sizes.
FIG. 4 is a perspective view showing a part of the CES 430. A portion of CES 430 includes a rear portion 434 of CES 430 body 408. For clarity, the front portion of the body 408 is not shown, but the front portion is attached to the rear portion 434 by, for example, a hinge 438. This allows the front to include a door or hatch that can be opened to access the bioreactor 300 of the CES 430. The bioreactor 300 is provided with a spool 416 for piping and a sampling port 420. The environment around the bioreactor 300 is temperature adjusted so as to be in a condition suitable for cell growth.
FIG. 5 shows a flowchart 500 of one embodiment of a cell growth process using CES. The process includes steps related to loading and dispensing cells into a bioreactor (eg, bioreactor 300), as described below. Although features of CES (eg, CES 430) are described as performing some steps of flowchart 500, the invention is not so limited. Although not described herein (eg, not described in CES 10, 800, 900), other CES with different characteristics may be used in other embodiments. Accordingly, references to features of CES 430 such as bioreactor 300 are for illustration only and flowchart 500 is not limited to use in any particular CES.
Flowchart 500 begins at step 502 and, at step 504, bioreactor 300 and associated piping and associated structures are attached to body 408 to make CES 430 operational. Once connected to the body 408, in step 508, priming is performed on the bioreactor 300, associated piping, and associated structures using a suitable priming fluid such as saline. In step 512, the cells are loaded and dispensed into the bioreactor 300.
In an embodiment, cell input / distribution includes several sub-steps. For example, in some embodiments, step 512 includes sub-step 516, which is an option to place bioreactor 300 in a first direction, and sub-step 520, which is an option to load and dispense cells in bioreactor 300. Have. In optional sub-step 524, the cells are attached to the bioreactor.
After the cells are loaded / distributed into the bioreactor 300, in step 528, cell growth is performed. That is, the cells in the bioreactor 300 are propagated, that is, grown and / or propagated. In step 532, a determination is made as to whether cells need to be added to the bioreactor 300 and / or whether the bioreactor 300 needs to be rotated to distribute the cells within the bioreactor 300. . If the cells need to be added to the bioreactor 300 and / or if the cells need to be dispensed in the bioreactor 300, the flowchart 500 returns to step 512. If no cells need to be added and / or the bioreactor 300 does not need to be rotated, it is determined in step 536 whether the cell growth step 528 has been completed. Here, it is determined that the cell growth process is complete when a sufficient change in cell number and / or cell characteristics is achieved. If cell growth step 528 is complete, in step 540, the cells are harvested. If cell growth step 528 is not complete, cell growth step 528 is continued. The flowchart 500 ends at step 544.
In some embodiments, a more detailed description is given of the processes (eg, step 512, step 528 (FIG. 5)) used to load, distribute, and grow cells in the bioreactor and CES. 6 and 7 show a flowchart of the process used to load, dispense, attach and grow cells. These processes are performed as part of the process of flowchart 500, for example, as a sub-step in the steps described above (eg, step 512, step 528). In other embodiments, the processes described in flowcharts 600, 700 may be performed independently of the steps of flowchart 500. Further, as shown below, the steps of flowcharts 600 and 700 include parts (eg, motors used as oscillating parts 402, 404) and bioreactors (eg, bioreactors 24, 300, 400, 801, 908). Alternatively, it may be performed by or on a CES that includes a portion of a bioreactor or a portion thereof (eg, CES 10, 800, 900). The description herein is not intended to be limited to flowcharts 600 and 700. There may be steps performed by or on other systems, devices, components, features, etc.
Flow chart 600 begins at step 604, where a fluid with cells is circulated in a bioreactor such as bioreactor 300 (see FIGS. 3 and 8-12). In some embodiments, at step 608, one or more pumps are activated to circulate fluid within the bioreactor 300. For example, an IC circulation pump (eg, 812 or 911) may be activated to cause fluid to circulate on the IC side of the bioreactor 300 at a first circulation flow rate. In at least one embodiment, the fluid carrying the cells moves from the IC side to the EC side via the hollow fiber of the bioreactor 300. In other embodiments, the cells are loaded into the EC side of the bioreactor 300 and flow fluid that carries the cells from the EC side to the IC side. In these embodiments, an EC circulation pump (eg, 828, 974) may be activated and circulated on the EC side of the bioreactor 300 at a first circulation flow rate.
In some embodiments, step 608 comprises rotating the bioreactor 300 in a particular sequence to facilitate cell distribution in the bioreactor 300 and a CES circuit that is fluidly associated with the bioreactor 300. With. An example of an embodiment in which the bioreactor 300 is rotated in a particular sequence to facilitate cell distribution in circulation or input is described in US patent application Ser. No. 12 / 968,483, filed Dec. 15, 2010. (Title: Method for loading and distributing cells in a bioreactor of a cell growth system). The entirety of the US patent application is incorporated herein by this disclosure. In other embodiments, the circulation step 608 may involve rotating the bioreactor 300 for a period of time and further allowing the bioreactor 300 to be stationary for a period of time.
After step 608, in step 612, the fluid circulation rate is decreased. The circulation rate may be reduced to approximately zero (0) ml / min, and in other embodiments, zero (0) ml / min or more, but the cells are bioreactor 300 (eg, hollow fiber 316 of bioreactor 300). May be reduced to a rate that allows precipitation and adhesion on the inner surface). In some embodiments, in step 612, one or more pumps used in step 608 may be stopped to circulate fluid.
The flowchart proceeds from step 612 to optional step 616. In this step, a step of positioning a bioreactor (eg, bioreactor 300) in the initial direction is performed. In certain embodiments, the bioreactor may already be in the initial direction. In that case, step 616 is unnecessary. Step 616 can be performed by one or more motors.
Referring to FIGS. 8-12, FIG. 8 shows a bioreactor 300 in an initial direction. As part of optional step 616, position bioreactor 300 such that bioreactor longitudinal axis LA-LA is aligned with the starting direction (eg, a first horizontal direction as shown in FIG. 8). It may be.
The flowchart proceeds from step 616 to step 620. In this step, the bioreactor is maintained in the first direction and the cells are allowed to settle and attach to the first portion of the bioreactor 300. Step 620 is performed for a first predetermined period.
13A to 13C show cross-sectional views of one hollow fiber 1300 (perpendicular to the central axis of the hollow fiber 1300 and the central axis of the bioreactor 300) of one of the plurality of hollow fibers 316 of the bioreactor 300. These drawings illustrate the placement of cells within hollow fiber 316 that may occur during certain steps of flowchart 600. As shown in FIG. 13A, before reducing the circulation rate in step 612, the cells in each hollow fiber 1300 are evenly distributed throughout the volume of the hollow fiber 1300. When the circulation rate is decreased, the cells begin to settle due to the influence of gravity 1304.
In certain embodiments, when the bioreactor 300 is in the first horizontal direction (FIG. 8), the cells in the bioreactor 300 settle to the first portion of the bioreactor. As shown in FIG. 13B, the first portion of bioreactor 300 has at least one portion 1308 of hollow fiber 1300. The cells are not only settled, but are allowed to settle for a first predetermined period of time (step 620 of flowchart 600) selected to adhere to the portion 1308 of the hollow fiber 1300.
In some embodiments, the first predetermined period may be long enough for the cells to settle and attach to the portion 1308. In these embodiments, the cells need only travel the distance of the inner diameter of the hollow fiber 1308. For example, for embodiments where the inner diameter of the hollow fiber is from about 150 μm to about 300 μm, the first predetermined period may be less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes. In other embodiments, the first predetermined period may be longer than about 1 minute, longer than about 2 minutes, longer than about 3 minutes, or longer than about 4 minutes. In one embodiment, the first predetermined period may be between about 3 minutes and about 8 minutes (eg, about 5 minutes).
In another embodiment, the first predetermined period may be a length of time that allows the attached cells to grow as well as the cells settle and adhere to the hollow fiber. In these embodiments, the cells grow laterally because the lateral resistance is the smallest. That is, if the cells on portion 1308 attempt to grow upward from the hollow fiber wall, the cells will grow at least in the early stages, because the cells will grow against gravity 1304. It is thought to grow laterally. In these embodiments, when cells are grown after attachment, the first predetermined period is greater than about 5 hours, greater than about 10 hours, greater than about 15 hours, greater than about 20 hours, or about 24 hours. It may be longer than time. In other embodiments, the first predetermined period may be less than about 60 hours, less than about 55 hours, less than about 50 hours, or less than about 45 hours. In one embodiment, the first predetermined period may be between about 10 hours and about 48 hours.
Returning to FIG. 6, in some embodiments, after step 620, the flowchart continues to step 640. There, the bioreactor 300 is rotated in a second horizontal direction rotated about 180 degrees from the first horizontal direction. As shown in FIGS. 8 to 10, the bioreactor is first rotated from the first horizontal direction (FIG. 8) to a first vertical direction rotated about 90 degrees from the first horizontal direction (for example, LA -Make the LA axis vertical (FIG. 9)). Then, the bioreactor 300 is further rotated 90 degrees (FIG. 10) to complete the rotation in the second horizontal direction.
In some embodiments, after the second horizontal rotation, the flowchart 600 proceeds to step 644. In this step, cell growth is performed in the bioreactor 300 in the second horizontal direction. FIG. 13C shows a state in which the cells attached to the hollow fiber 1300 are located on the inner top of the hollow fiber 1300 in the second horizontal direction. Step 644 may include a plurality of sub-steps such as circulating fluid in the bioreactor to provide nutrients to cells attached within the bioreactor. As will be appreciated, step 644 may include a sub-step of supplying oxygen to the cells for growth. By controlling several other parameters in the bioreactor, proliferation, i.e. cell growth, can be optimized. In some embodiments, in step 644, circulating the fluid to nourish the cells may be performed for about 24 hours or about 36 hours or about 48 hours or about 60 hours or about 72 hours. In some embodiments, the step of nourishing the cells as part of step 644 is for a period of less than about 120 hours or a period of less than about 108 hours or a period of less than about 96 hours or a period of less than about 84 hours or about It may be performed for a period shorter than 72 hours. Flowchart 600 then ends at step 648.
Without being bound by theory, it is believed that cell proliferation is improved when cells are grown under the influence of gravity as shown in FIG. 13C. The cells grow downward in the hollow fiber 1300 toward the portion of the hollow fiber where no cells are present. The cells are considered to grow toward a portion having the least resistance, such as a portion of the hollow fiber located below the top portion 1308 (see FIG. 13C). Growth under the influence of gravity improves cell yield and reduces cell doubling time when compared to conventional processes.
In other embodiments, the flowchart 600 may include other steps. For example, in some embodiments, after step 620, the flowchart 600 proceeds to step 624, where the bioreactor 628 is rotated vertically. For example, the bioreactor 300 is rotated in the first vertical direction as shown in FIG. After step 624, the flowchart proceeds to step 628, where the bioreactor is maintained in the first vertical direction for a second predetermined period.
13D and 13E show a cross-sectional view of a hollow fiber 1300 that is one of a plurality of hollow fibers 316 of the bioreactor 300 (parallel to the central axis of the hollow fiber 1300 and the central axis of the bioreactor 300). 13D and 13E show the hollow fiber 1300 after step 620. FIG. There, the cells settle and adhere to certain parts of the hollow fiber 1300. As shown in FIG. 13D, when the bioreactor 300 rotates in the first vertical direction, the first end 1312 of the hollow fiber 1300 is positioned above the second end 1316.
As described above, without being bound by theory, the cells attached to the hollow fiber 1300 are affected by gravity 1304 and begin to grow or proliferate in the longitudinal direction toward the end 1316. Accordingly, step 628 (maintaining in the first vertical direction) is performed for a second predetermined period of time that is long enough for the cells to grow in the longitudinal direction. In some embodiments, the second predetermined period may be greater than about 5 hours, greater than about 10 hours, greater than about 15 hours, greater than about 20 hours, or greater than about 24 hours. In other embodiments, the second predetermined period may be less than about 60 hours, or less than about 55 hours, or less than about 50 hours, or less than about 45 hours. In one embodiment, the second predetermined period may be between about 10 hours and about 48 hours.
After step 628, the flowchart continues to step 632. In this step, the bioreactor is rotated in the second vertical direction. An example of a bioreactor 300 in the second vertical direction is shown in FIG. After step 624, the process proceeds to step 636. In this step, the bioreactor is maintained in the second vertical direction for a third predetermined period.
FIG. 13E shows the hollow fiber 1300 after step 632. There, the cells settle and adhere to a portion of the hollow fiber 1300, and the bioreactor 300 is rotated from the first vertical direction to the second vertical direction and maintained in the second vertical direction. As shown in FIG. 13E, when the bioreactor 300 rotates in the second vertical direction, the first end 1312 of the hollow fiber 1300 is positioned below the second end 1316.
Similar to step 628 (maintaining in the first vertical direction), cells attached to the hollow fiber 1300 are affected by gravity 1304 and are considered to begin growing, ie, proliferating in the longitudinal direction toward the end 1312. Step 636 (maintaining in the second vertical direction) is performed. Step 636 is performed for a third predetermined period of time that is long enough for the cells to grow longitudinally toward end 1312 as shown in FIG. 13E. In some embodiments, the third predetermined period may be longer than about 5 hours, longer than about 10 hours, longer than about 15 hours, longer than about 20 hours, or longer than about 24 hours. In other embodiments, the third predetermined period may be less than about 60 hours, or less than about 55 hours, or less than about 50 hours, or less than about 45 hours. In one embodiment, the third predetermined period may be between about 10 hours and about 48 hours.
Returning to flowchart 600, after step 636, control proceeds to step 640. In this step, as described above, the bioreactor is rotated to the second horizontal position as shown in FIG. As described above, the flowchart proceeds from step 640 to step 644 where the cells are propagated or propagated. Then, the flowchart ends at step 648.
Turning to FIG. 7, the flowchart 700 begins at step 704 and proceeds to step 708. In this step, a fluid containing cells is circulated in a bioreactor such as bioreactor 300 (see FIGS. 3, 8-12). In some embodiments, in step 708, one or more pumps are activated to circulate fluid within the bioreactor 300. For example, an IC circulation pump (eg, 812 or 911) may be activated to cause fluid to circulate on the IC side of the bioreactor 300 at a first circulation flow rate. In at least one embodiment, the fluid carrying the cells moves from the IC side to the EC side via the hollow fiber of the bioreactor 300. In other embodiments, the cells are loaded into the EC side of the bioreactor 300 and flow fluid that carries the cells from the EC side to the IC side. In these embodiments, an EC circulation pump (eg, 828, 974) may be activated and circulated on the EC side of the bioreactor 300 at a first circulation flow rate.
In some embodiments, the first circulation flow rate may be a relatively high flow rate. In certain embodiments, the first circulation flow rate may be less than about 500 ml / min, less than about 400 ml / min, or less than about 300 ml / min. In other embodiments, the first circulation flow rate may be greater than about 50 ml / min, greater than about 100 ml / min, or greater than about 150 ml / min. In one embodiment, the first circulation flow rate is between about 100 ml / min and about 300 ml / min, such as about 200 ml / min.
In some embodiments, step 708 comprises rotating the bioreactor 300 in a particular sequence to facilitate cell distribution in the bioreactor 300 and a CES circuit that is fluidly associated with the bioreactor 300. Accompanied by. In other embodiments, the circulation step 708 may involve rotating the bioreactor 300 for a period of time and further allowing the bioreactor 300 to be stationary for a period of time.
After step 708, in step 712, the fluid circulation rate is decreased. The circulation rate may be reduced to approximately zero (0) ml / min, and in other embodiments, zero (0) ml / min or more, but the cells are bioreactor 300 (eg, hollow fiber 316 of bioreactor 300). May be reduced to a rate that allows precipitation and adhesion on the inner surface). In some embodiments, in step 712, one or more pumps used in step 708 may be stopped to circulate fluid.
The flowchart proceeds from step 712 to optional step 716. In this step, a step of positioning a bioreactor (eg, bioreactor 300) in the initial direction is performed. In certain embodiments, the bioreactor may already be in the initial direction. In that case, step 716 is unnecessary. In some embodiments, performing step 716 can be performed by one or more motors.
Referring to FIGS. 8-12, FIG. 8 shows a bioreactor 300 in an initial direction. As part of optional step 716, position bioreactor 300 such that bioreactor longitudinal axis LA-LA is aligned with the starting direction (eg, a first horizontal direction as shown in FIG. 8). It may be.
The flowchart proceeds from step 716 to step 720. In this step, the bioreactor is maintained in the first direction and the cells are allowed to settle and attach to the first portion of the bioreactor 300. Step 820 is performed for a first predetermined period.
14A to 14D and FIGS. 15A to 15F are cross-sections (perpendicular to the central axis of the hollow fiber 1400 and the central axis of the bioreactor 300) of one of the hollow fibers 316 of the bioreactor 300. The figure is shown. These drawings illustrate the placement of cells within hollow fiber 316 that may occur during certain steps of flowchart 700. As shown in FIG. 14A, the cells in each hollow fiber 1400 are evenly distributed throughout the volume of the hollow fiber 1400 before the circulation rate is reduced in step 712. When the circulation rate is decreased, the cells begin to settle due to the influence of gravity 1404. FIG. 15A also shows a similar situation for hollow fiber 1500 and gravity 1504.
In certain embodiments, when the bioreactor 300 is in the first horizontal direction (FIG. 8), the cells in the bioreactor 300 settle to the first portion of the bioreactor. As shown in FIGS. 14B and 15B, the first portion of bioreactor 300 has at least a portion 1408 of hollow fiber 1400 and / or at least a portion 1508 of hollow fiber 1500. In certain embodiments, the cells are not only settled, but are allowed to settle for a first predetermined period of time selected to attach to the portion 1408 of the hollow fiber 1400 (and the portion 1508 of the hollow fiber 1500).
In some embodiments, the first predetermined period may be a period of time that allows the cells to settle and attach to the portions 1408 and 1508. In these embodiments, the cells need only travel the distance of the inner diameter of the hollow fiber 1400 or 1500. For example, for embodiments where the inner diameter of the hollow fiber is from about 150 μm to about 300 μm, the first predetermined period may be less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes. In other embodiments, the first predetermined period may be longer than about 1 minute, longer than about 2 minutes, longer than about 3 minutes, or longer than about 4 minutes. In one embodiment, the first predetermined period may be between about 3 minutes and about 8 minutes (eg, about 5 minutes).
After step 720, the flowchart proceeds to step 724. There, the bioreactor 300 is rotated in a second horizontal direction rotated about 180 degrees from the first horizontal direction. As shown in FIGS. 8 to 10, the bioreactor is first rotated from the first horizontal direction (FIG. 8) to a first vertical direction rotated about 90 degrees from the first horizontal direction (for example, LA -Make the LA axis vertical (FIG. 9)). Then, the bioreactor 300 is further rotated 90 degrees (FIG. 10) to complete the rotation in the second horizontal direction. In some embodiments, step 724 may be performed by one or more motors connected to bioreactor 300. These motors may be part of the oscillating device.
In some embodiments, flowchart 700 proceeds from step 724 to step 736. In this step, during a second predetermined period of time so that the cells settle in a second part of the bioreactor, such as part 1412 of hollow fiber 1400 (FIG. 14C) and part 1512 of hollow fiber 1500 (FIG. 15C). The bioreactor 300 is maintained in the second horizontal direction (FIG. 10).
In some embodiments, the flowchart 700 may have optional steps 728, 732 before performing step 736. Similar to step 708, step 728 circulates fluid in bioreactor 300. In one embodiment, at step 728, one or more pumps are activated to circulate fluid within the bioreactor 300. As described above, an IC circulation pump (eg, 812 or 911) may be activated to circulate fluid at the IC side of the bioreactor 300 at a second circulation flow rate. In at least one embodiment, the fluid carrying the cells moves from the IC side to the EC side via the hollow fiber of the bioreactor 300. In other embodiments, the cells are loaded into the EC side of the bioreactor 300 and flow fluid that carries the cells from the EC side to the IC side. In these embodiments, an EC circulation pump (eg, 828, 974) may be activated and circulated on the EC side of the bioreactor 300 at a second circulation flow rate.
In some embodiments, the second circulation flow rate may be lower than the first circulation flow rate. In certain embodiments, the second circulation flow rate may be less than about 400 ml / min, less than about 300 ml / min, or less than about 200 ml / min. In other embodiments, the second circulation flow rate may be greater than about 25 ml / min, greater than about 500 ml / min, or greater than about 75 ml / min. In one embodiment, the second circulation flow rate is between about 50 ml / min and about 150 ml / min, such as about 100 ml / min.
In some embodiments, step 728 may perform a different direction of circulation than that performed in step 708. That is, in some embodiments, at step 708, the fluid may be circulated in a counterclockwise direction (see the IC loops of FIGS. 8 and 9). In some embodiments, the circulation in step 728 may be clockwise. That is, the circulation may be a flow in the opposite direction to the circulation in step 708. In other embodiments, the circulation in step 708 may be in the same direction as step 708, ie, clockwise or counterclockwise.
In some embodiments, the optional step 728 may be performed in a specific sequence to facilitate cell distribution in the bioreactor 300 and the CES circuit fluidly associated with the bioreactor 300. A process of rotating is involved. In other embodiments, the circulation step 728 may involve rotating the bioreactor 300 for a period of time and further allowing the bioreactor 300 to be stationary for a period of time.
After optional step 728, in step 732, the fluid circulation rate is decreased again. The circulation rate may be reduced to approximately zero (0) ml / min, and in other embodiments, zero (0) ml / min or more, but the cells are bioreactor 300 (eg, hollow fiber 316 of bioreactor 300). May be reduced to a rate that allows precipitation and adhesion on the inner surface). In some embodiments, in step 732, one or more pumps used in step 728 may be stopped to circulate fluid.
In step 736, the cells are precipitated in the portion 1412 opposite the portion 1408 (or 1512 in FIG. 15C) by maintaining the bioreactor in the second horizontal direction. For example, the portion 1408 (or 1508) will be referred to as the “bottom” and the portion 1412 (or 1512 in FIG. 15C) will be referred to as the “top”. In FIGS. 14C and 15C, the cells are sedimented in portions 1412 and 1512, but in some embodiments, the reverse. In certain embodiments, the cells are not only settled, but are allowed to settle for a second predetermined period of time selected to adhere to the portion 1412 of the hollow fiber 1400 (or the portion 1512 of the hollow fiber 1500).
In some embodiments, the second predetermined period may be long enough for cells to settle and attach to the portion 1412 (the portion 1512 of FIG. 15C). In these embodiments, the cells need only travel the distance of the inner diameter of the hollow fiber 1400 or 1500. For example, for embodiments where the inner diameter of the hollow fiber is between about 150 μm and about 300 μm, the second predetermined period may be less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes. In other embodiments, the second predetermined period may be greater than about 1 minute, greater than about 2 minutes, greater than about 3 minutes, or greater than about 4 minutes. In one embodiment, the second predetermined period may be between about 3 minutes and about 8 minutes (eg, about 5 minutes).
In some embodiments, after step 736, the flowchart 700 proceeds to step 772. In this step, the cells are grown. Step 772 may include multiple sub-steps such as circulating fluid in the bioreactor to provide nutrients to cells attached within the bioreactor. As will be appreciated, step 772 may include a sub-step of supplying oxygen to the cells for growth. By controlling several other parameters in the bioreactor, proliferation, i.e. cell growth, can be optimized. In some embodiments, in step 772, the step of circulating the fluid to nourish the cells may be performed for about 24 hours or about 36 hours or about 48 hours or about 60 hours or about 72 hours. In some embodiments, the step of nourishing the cells as part of step 772 includes a period of less than about 120 hours, a period of less than about 108 hours, a period of less than about 96 hours, or a period of less than about 84 hours or about It may be performed for a period shorter than 72 hours. FIG. 14D shows a hollow fiber 1400 in the present embodiment. Flowchart 700 then ends at step 776.
In other embodiments, the flowchart 700 proceeds to step 740. In this step, the bioreactor 300 is returned to the initial first horizontal direction. FIG. 11 shows the bioreactor 300 returned in the first horizontal direction. Step 740 may be performed by one or more motors connected to bioreactor 300. These motors may be part of the oscillating device. In certain embodiments, the process proceeds from step 740 to step 772 where cell growth is performed. Flowchart 700 then ends at step 776.
In other embodiments, the flowchart 700 proceeds from step 740 to step 744. Alternatively, proceed directly from step 736 to step 744 (in this case, no additional rotation is performed). In this step, the fluid is circulated at the third circulation flow rate. Similar to steps 708, 728, fluid is circulated through bioreactor 300. In one embodiment, at step 744, one or more pumps are activated to circulate fluid within the bioreactor 300. As described above, an IC circulation pump (eg, 812 or 911) may be activated to circulate fluid at the third circulation flow rate on the IC side of the bioreactor 300. In at least one embodiment, the fluid carrying the cells moves from the IC side to the EC side via the hollow fiber of the bioreactor 300. In other embodiments, the cells are loaded into the EC side of the bioreactor 300 and flow fluid that carries the cells from the EC side to the IC side. In these embodiments, an EC circulation pump (eg, 828, 974) may be activated and circulated on the EC side of the bioreactor 300 at a third circulation flow rate.
In some embodiments, the third circulation flow rate may be lower than the second circulation flow rate. In certain embodiments, the third circulation flow rate may be less than about 200 ml / min, less than about 150 ml / min, or less than about 100 ml / min. In other embodiments, the third circulation flow rate may be greater than about 10 ml / min, greater than about 20 ml / min, or greater than about 30 ml / min. In one embodiment, the third circulation flow rate is between about 20 ml / min and about 100 ml / min, such as about 50 ml / min.
In some embodiments, step 744 may perform a different direction of circulation than that performed in step 728. That is, in some embodiments, step 728 may circulate fluid in a clockwise direction. In some embodiments, the cycling in step 744 may be similar to step 708 and may be in a counterclockwise direction (see IC loops in FIGS. 8 and 9). That is, the circulation in step 744 is a flow in the opposite direction to the circulation in step 728 and may be the same direction as the circulation direction in step 708. In other embodiments, the circulation in steps 708, 728, 744 may be in the same direction, either clockwise or counterclockwise.
In some embodiments, the optional step 744 may be performed in a specific sequence to facilitate cell distribution in the bioreactor 300 and the CES circuit fluidly associated with the bioreactor 300. A process of rotating is involved. In other embodiments, the circulation step 744 may involve rotating the bioreactor 300 for a period of time and further allowing the bioreactor 300 to be stationary for a period of time.
Proceed from step 744 to step 748. In this step, the fluid circulation rate is decreased again. The circulation rate may be reduced to approximately zero (0) ml / min, and in other embodiments, zero (0) ml / min or more, but the cells are bioreactor 300 (eg, hollow fiber 316 of bioreactor 300). May be reduced to a rate that allows precipitation and adhesion on the inner surface). In some embodiments, in step 748, one or more pumps used in step 744 may be stopped to circulate fluid.
Proceed from step 748 to step 752. In this step, the bioreactor is maintained in a horizontal direction. In an embodiment that includes step 744 (rotation in the first direction), step 752 includes maintaining in the first horizontal direction. In an embodiment that does not include the rotation of step 740, step 752 includes maintaining in a second horizontal direction. In any case, step 752 is performed to re-precipitate the cells, eg, in portion 1508 (see FIG. 15D and FIG. 15E for rotation step 740). In certain embodiments, the cells are allowed to settle for a third predetermined period of time selected to attach as well as to settle.
In some embodiments, the third predetermined period may be long enough for the cells to settle and attach to portion 1508. In these embodiments, the cells need only travel the distance of the inner diameter of the hollow fiber 1500. For example, for embodiments in which the hollow fiber 1500 has an inner diameter of about 150 μm to about 300 μm, the third predetermined period may be less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes. In other embodiments, the third predetermined period may be greater than about 1 minute, greater than about 2 minutes, greater than about 3 minutes, or greater than about 4 minutes. In one embodiment, the third predetermined period may be between about 3 minutes and about 8 minutes (eg, about 5 minutes).
In some embodiments, the flowchart 700 proceeds from step 752 to step 772. In this step, cell growth is performed. FIG. 15F shows a hollow fiber 1500 in these embodiments. Then, the flowchart ends at step 776.
In other embodiments, as described below, the flowchart 700 includes an additional rotation step (756), a circulation step (760), a circulation rate reduction step (764), before proceeding to step 772 of growing cells. A direction maintaining step (768) may be included. In these embodiments, flowchart 700 proceeds from step 752 to step 756. In step 756, if it was rotating in the first horizontal direction in step 740, the bioreactor 300 returns to the second horizontal direction. FIG. 10 shows the bioreactor 300 in the second horizontal direction. Step 756 may be performed by one or more motors connected to bioreactor 300. These motors may be part of the oscillating device. In some embodiments, this step may not be necessary if step 740 of rotating the bioreactor in the first horizontal direction has not been performed.
Flowchart 700 proceeds to step 760. In this step, the fluid is circulated again at the fourth circulation flow rate. Similar to steps 708, 728, 744, fluid is circulated through the bioreactor 300. In one embodiment, at step 744, one or more pumps are activated to circulate fluid within the bioreactor 300. As described above, an IC circulation pump (eg, 812 or 911) may be activated to circulate fluid at the IC side of the bioreactor 300 at a fourth circulation flow rate. In at least one embodiment, the fluid carrying the cells moves from the IC side to the EC side via the hollow fiber of the bioreactor 300. In other embodiments, the cells are loaded into the EC side of the bioreactor 300 and flow fluid that carries the cells from the EC side to the IC side. In these embodiments, an EC circulation pump (eg, 828, 974) may be activated and circulated on the EC side of the bioreactor 300 at a fourth circulation flow rate.
In some embodiments, the fourth circulation flow rate may be lower than the third circulation flow rate. In certain embodiments, the fourth circulation flow rate may be less than about 100 ml / min, less than about 75 ml / min, or less than about 50 ml / min. In other embodiments, the fourth circulation flow rate may be greater than about 5 ml / min, greater than about 10 ml / min, or greater than about 15 ml / min. In one embodiment, the fourth circulation flow rate is between about 15 ml / min and about 35 ml / min, such as about 25 ml / min.
In some embodiments, step 760 may perform a different direction of circulation than that performed in step 744. That is, in some embodiments, step 744 may circulate fluid in a counterclockwise direction. In some embodiments, the circulation in step 760 may be similar to step 728 and may be clockwise. That is, the circulation in step 760 is a flow in the opposite direction to the circulation in step 744 and may be the same direction as the circulation direction in step 728. In other embodiments, the circulation in steps 708, 728, 744, 760 may be in the same direction, either clockwise or counterclockwise.
In some embodiments, step 760 includes rotating bioreactor 300 in a particular sequence to facilitate cell distribution in bioreactor 300 and a CES circuit that is fluidly associated with bioreactor 300. Accompanied by. In other embodiments, the circulation step 760 may involve rotating the bioreactor 300 for a period of time and further allowing the bioreactor 300 to be stationary for a period of time.
Proceed from step 760 to step 764. In this step, the fluid circulation rate is decreased again. The circulation rate may be reduced to approximately zero (0) ml / min, and in other embodiments, zero (0) ml / min or more, but the cells are bioreactor 300 (eg, hollow fiber 316 of bioreactor 300). May be reduced to a rate that allows precipitation and adhesion on the inner surface). In some embodiments, in step 764, one or more pumps used in step 760 may be stopped to circulate fluid.
Proceed from step 764 to step 768. In this step, the bioreactor is maintained in the second horizontal orientation, and the cells are reprecipitated, for example, in portion 1512 (see FIG. 15F). In certain embodiments, the cells are allowed to settle for a fourth predetermined period selected to not only settle but also reattach.
In some embodiments, the fourth predetermined period may be long enough for the cells to settle and adhere. In these embodiments, the cells need only travel the distance of the inner diameter of the hollow fiber (eg, hollow fiber 1500). For example, for embodiments in which the hollow fiber 1500 has an inner diameter of about 150 μm to about 300 μm, the fourth predetermined period may be less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes. In other embodiments, the fourth predetermined period may be longer than about 1 minute, longer than about 2 minutes, longer than about 3 minutes, or longer than about 4 minutes. In one embodiment, the fourth predetermined period may be between about 3 minutes and about 8 minutes (eg, about 5 minutes).
After step 768, flowchart 700 proceeds to step 772. In this step, the cells that have precipitated and adhered to the bioreactor 300 (for example, the hollow fiber of the bioreactor) are propagated, ie propagated. Flowchart 700 then ends at step 776.
Without being bound by theory, it is believed that in certain embodiments, cell proliferation is improved when the steps of flowchart 700 are performed. These embodiments are believed to ensure that cells are seeded on a larger portion of the bioreactor (eg, the surface of the hollow fiber within the bioreactor) prior to cell growth. As a result, more cells can be seeded at an initial stage as compared with the conventional process, and finally the cell yield is improved and the cell doubling time can be reduced.
Although flowchart 700 includes a certain number of steps for bioreactor rotation, circulation, circulation rate reduction, and direction maintenance, other embodiments are not limited to these particular numbers of steps. In other embodiments, further after step 768, the bioreactor is rotated again, cycled again, the circulation rate is reduced, the cells are allowed to settle, and the direction is maintained for a certain period of time. Cells may be attached to the part. These steps may be performed any number of times. In one embodiment, each restart of the cycle occurs at a lower rate than the previous cycle. In other embodiments, the circulation rate may be the same each time circulation is initiated. In still another embodiment, the circulation is first performed in the first direction, the circulation is stopped, the cells are precipitated and attached, and then the direction opposite to the first direction (counterclockwise direction with respect to the clockwise direction) The circulation direction may be changed such that the cell is circulated and the cells are precipitated again to precipitate the cells.
FIG. 16 shows a cross-sectional view 1600 (perpendicular to the central axis) of a bioreactor (eg, bioreactor 300). Cross-sectional view 1600 shows a plurality of hollow fibers 1608 housed in housing 1604. Cross-sectional view 1600 is depicted from one end of the bioreactor, and in addition to hollow fiber 1608, matrix material 1628 holding hollow fiber 1608 together (referred to above as a potting material) is also shown.
In FIG. 16, a plurality of zones 1612, 1616, 1620, 1624 are shown. In the hollow fibers in each of these zones, the fluid circulates at different flow rates. That is, without being bound by theory, it is considered that circulation at a relatively high flow rate mainly flows through the hollow fibers in the zone 1612, such as the rates in the circulation steps 708 and 728 (FIG. 7). Without being bound by theory, it is believed that at high flow rates, the fluid cannot be sufficiently dispersed to flow evenly within the outer zone hollow fibers. When the flow rate is reduced as in steps 744 and 760, it is considered that the hollow fibers in the outer zones such as the zones 1616, 1620, and 1624 are dispersed.
Thus, without being bound by theory, by circulating at different flow rates in steps 708, 728, 744, 752, the fluid will have more hollow fibers 1608 compared to using a single flow rate. It is thought that it will begin to flow. In one embodiment of the process according to flowchart 700, at step 708 (which is the flow rate described above), fluid flows through the hollow fibers in zone 1612. In step 728 (which is the flow rate described above), the fluid flows through the hollow fibers of zone 1612 and zone 1616 because the rate is slower and the fluid is more dispersed with it. In step 744 (which is the flow rate described above), the rate is further slowed and further dispersed with it, so the fluid flows through the hollow fibers in zones 1612, 1616, 1620. In step 752 (which is the flow rate described above), the rate is even slower and, as a result, disperses in all hollow fibers in all zones, so that fluid is in all zones 1612, 1616, 1620, 1624. Flow through hollow fiber. Thus, it is believed that by using a series of different flow rates, the fluid with cells will flow through more hollow fibers compared to circulating at a single high flow rate.
In addition, different flow rates may affect the longitudinal distribution of cells along the bioreactor (eg, along the hollow fiber). That is, at higher flow rates, the cells flow further along the hollow fiber interior. For example, at higher flow rates, cells carried by the fluid will reach a position that is more than half the length of the hollow fiber. At lower flow rates, cells carried by the fluid will reach a position that is half the length of the hollow fiber. At even lower flow rates, the cells carried by the fluid will reach a position in front of half the length of the hollow fiber. Thus, in some embodiments, using different flow rates is believed to improve the longitudinal distribution of cells along the length of the bioreactor (eg, hollow fiber).
The embodiments described for flowcharts 500, 600, 700 are not limited, but include, for example, stem cells (mesenchymal cells, hematopoietic cells, etc.), fibroblasts, keratinocytes, progenitor cells, endothelial cells, and other fully differentiated cells. It can be used in the growth of any type of cell, such as cells, and combinations thereof. By using a process that includes the steps described above for flowcharts 500, 600, and / or 700, and that includes different features, and combinations of those features, proliferation can be performed on different cells.
Although flowcharts 500 (FIG. 5), 600 (FIG. 6), and 700 (FIG. 7) have been described with a plurality of steps illustrated in a particular order, the present invention is not limited thereto. In other embodiments, the steps may be performed in a different order, in parallel, or any number of times (eg, before and after another step). As described above, the flowcharts 500, 600, 700 include several optional steps and sub-steps. However, those steps that have not been shown as options should not be considered essential to the present invention, and are performed in one embodiment and not in other embodiments.
FIG. 17 illustrates example components of a basic computer system 1700 in which embodiments of the present invention are implemented. The computer system 1700 performs several steps of the method of loading and dispensing cells. The system 1700 includes components and features of the above-described CES systems 10, 430, 800, and 900 that supply and distribute cells for growth (eg, flow control devices, pumps, valves, bioreactor rotations, motors, etc.). It is a controller for controlling.
The computer system 1700 includes an output device 1704 and / or an input device 1708. The output device 1704 may have one or more displays, such as a CRT, LCD, plasma display. The output device 1704 may include a printer, a speaker, and the like. The input device 1708 may include a keyboard, a touch input device, a mouse, a voice input device, and the like.
In an embodiment of the invention, the basic computer system 1700 may include a processing unit 1712 and / or a memory 1716. Processing unit 1712 may be a general purpose processor operable to execute instructions stored in memory 1716. The processing unit 1712 may include a single processor or multiple processors in embodiments of the invention. Furthermore, each processor can be a multi-core processor having one or more cores for reading and executing instructions separately. Processors may include general purpose processors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), and other integrated circuits.
In an embodiment of the present invention, the memory 1716 may include any tangible medium that stores data and / or processor-executable instructions in the short term or in the long term. The memory 1716 includes, for example, random access memory (RAM), read only memory (ROM), or electrically erasable programmable read only memory (EEPROM). Examples of other storage media include CD-ROM, tape, digital versatile disc (DVD), or other optical storage device, tape, magnetic disk storage device, magnetic tape, and other magnetic storage devices. In an embodiment, the system 1700 is used to control rotation of the bioreactor 300 and / or various flow controllers, pumps, valves, etc. of the CES system. The memory 1716 can store protocols 1720 and procedures 1724, such as protocols and procedures for loading and dispensing cells in the bioreactor that control operations such as circulation pumps, valves, bioreactor rotation, and the like.
Storage device 1728 is any long-term data storage device or component. In embodiments of the present invention, the storage device 1220 may include one or more of the systems described in connection with the memory 1716. The storage device 1728 may be permanently installed or removable. System 1700 is part of a CES system, and storage device 1728 can store various protocols for utilizing the CES system to load, dispense, attach, grow, and harvest various types of cells.
Examples In the following, several examples according to embodiments of the present invention will be described. However, in the following embodiments, for example, parameters, features and / or values are described below for programming CES (ie, QUANTUM® cell proliferation system), but these are only examples. Therefore, the present invention is not limited to those specific matters described below.
The purpose here is to illustrate the growth of human bone marrow derived mesenchymal stem cells (hMSCs) using two characteristic cell seeding methods in the QUANTUM® cell proliferation system.
For pre-selected hMSCs, the cell loading procedure currently used in the QUANTUM cell proliferation system distributes cells into the bioreactor via a uniform cell suspension. Cells are loaded into the IC circulation loop of the QUANTUM cell growth system and circulated for 2 minutes at a relatively high flow rate (200 mL / min). In this circulation method, a uniform cell suspension can be obtained by simultaneously performing the operation of the set bioreactor. When the cells are uniformly suspended, circulation and bioreactor movement are stopped and the cells settle on the bioreactor surface.
One of the limitations of this cell loading procedure is that cells can only be seeded in the bioreactor hollow fiber trough. hMSCs are often seeded at some specified cell density (eg, 500 cells / cm 2 ). In order to achieve the specified seeding density, only approximately 50% of the bioreactor surface can be considered when determining the appropriate number of cells to load. In the case of 500 cells / cm 2 , the QUANTUM cell proliferation system bioreactor is seeded with 10.5E + 06 cells (500 cells / cm 2 × 21000 cm 2 ). However, only 50% of the bioreactor surface area can be considered “seeded” by the above-described mechanism of the current cell input protocol. In addition, because of the use of “non-seeded” surfaces, it is necessary to overcome gravity in order for cells to grow and propagate to the “non-seeded” surface of the bioreactor. Migrating cells are thought to take the least resistance pathway and, as a result, confluence is expected to occur rapidly within the cell population as compared to similar growth in flasks.
Prepare a total of 7 sterile Quantum CES disposable sets with bioreactors and apply fibronectin coat (5 mg) overnight. All Quantum systems are seeded with precultured hMSCs. For one Quantum cell proliferation system, the current circulation / input task (Load with Circulation Task) is used as a control experiment. For three Quantum cell proliferation systems, circulation / input task: Modification 1 (Modification 1) is used, and for another three Quantum cell proliferation systems, circulation / input task: Modification 2 ( Modification 2) is used.
Disposable set: All bioreactors are integrated into the QUANTUM cell proliferation system (CES) disposable set and sterilized with ethylene oxide.
Cell source and density: The inner (IC) surface area of the bioreactor used is 2.1 m 2 . Therefore, adjustment of the seeding density for the control flask should be based on the bioreactor volume fraction of the IC loop. All bioreactors are uniformly loaded with up to 20E + 6 preselected MSCs by direct re-filling of the same cell source (existing pathway 1-3). Cells from a single donor are preferred. For comparison purposes, 3 T25 control flasks are seeded with hMSCs at the same density per cm 2 as a bioreactor.
CES medium IC input Q control and harvest: If the glucose level is below 70 mg / dL, double the medium supply rate (IC input Q). If the glucose level continues below 70 mg / dL, the IC input Q is doubled twice during the day. All disposable sets are harvested at the same time and will not delay later than day 8 to limit possible cell aggregation. Cell harvest time is determined by the metabolic characteristics exhibited by the cell culture. The target harvest time should be after the logarithmic growth phase of the cells.
Post-harvest assessment: Evaluate each harvest. These assessments include cell number and viability.
Quantum CES cell input modification 1
The current cell loading procedure is performed with the following changes shown in bold. After allowing the cells to attach for 5 minutes, all bioreactors are rotated 180 degrees and the non-attached cells are allowed to settle towards the top of the hollow fiber membrane for an additional 5 minutes. Then, the bioreactor is returned to the horizontal position, which is the home position, and a proliferation protocol is performed. The reason for making the change is to distribute the cells over the entire surface area of the bioreactor hollow fiber.
Day 0 (Day: 0) 1 turn to attach cells
Objective: To flow adherent cells to the bioreactor membrane and simultaneously run the EC circulation loop. The pump flow rate for the IC loop is set to zero.
Table 1 shows the solution bag attached to each line when cell attachment is performed. These solutions and volumes are based on the default settings for this task.
Table 1: Solution for cell attachment of Modification 1
Cell pathway: Task> Input and attachment> Cell attachment
Input each set value of cell adhesion shown in protocol table 2a-c.
Quantum CES cell input modification 2
The current cell loading procedure and preselected MSC proliferation protocol are performed with the following changes shown in bold. During the cell attachment phase (18-24 hours), the bioreactor is rotated to a 180 degree position to attach the cells to the top of the hollow fiber. The bioreactor is then returned to the home position and a growth protocol is performed. The reason for making the change is to encourage the cells to move to an open growth surface by gravity during cell proliferation.
Gravity is used to affect cell migration during growth. This is accomplished by seeding the cells as described in current cell loading procedures and rotating the bioreactor 180 degrees during growth. In this case, the unoccupied growth surface of the bioreactor is below the seeded cells. The cells grow in the direction with the least resistance (for example, the downward direction promoted by gravity).
Objective: To flow adherent cells to the bioreactor membrane and simultaneously run the EC circulation loop. The pump flow for the IC loop is set to zero.
Table 5 shows the solution bags that are attached to each line when performing cell attachment. These solutions and volumes are based on the default settings for this task.
The core cell loading procedure in the present invention is to enable a more uniform cell distribution within the bioreactor of the QUANTUM® cell growth system and to reduce the number of cells lost in the seeding process, A series of steps designed to increase cell yield.
The cell input technology at the heart of the present invention for the QUANTUM cell growth system is commonly used for seeding bioreactors (Quantum Cell Growth System Manual in Software Version 2.0) “Cells with Uniform Suspension” A series of steps including and added to the “Load Cells with Uniform Suspension” protocol is provided. In cell loading with uniform suspension (LCWUS), suspended cells enter the inner surface of one hollow fiber with a bioreactor after the cell suspension circulates through the IC loop at 200 mL / min. There is only one opportunity to do that. In the cell loading technique of the present invention, cells that do not adhere after the initial suspension and cells that remain in the IC loop rather than in the bioreactor are resuspended and are separated from another in the bioreactor for subsequent attachment. Carried to hollow fiber.
The input method of the present invention is based on the principle that the cell suspension introduced into the bioreactor through the circulation of the IC loop passes through different hollow fiber sets depending on the circulation rate of the cell suspension in the IC loop. .
In the cell injection (LCWUS) with a uniform suspension, the suspension is initially circulated at 200 mL / min. After such a state, the cell suspension in the IC loop is performed by alternately changing the circulation direction between the forward direction and the reverse direction, and at the same time, the circulation rate is decreased sequentially (ie, −100 mL / min, 50 mL / minute). Min, as −25 mL / min). By gradually slowing the circulation of the IC loop, the cells remaining in the suspension can be given another opportunity to enter and adhere to the inner surface of the bioreactor hollow fiber.
After each cycle of fluid in the IC loop, a cell attachment period is allowed for 7 minutes to bring the IC circulation rate to zero. In bioreactors used in the QUANTUM cell proliferation system, MSC cells have been demonstrated to attach to the inner surface of the hollow fiber within 5 minutes. Therefore, in the adhesion period of 7 minutes, it is possible to attach cells in 5 minutes, and it is possible to cope with cells that adhere more slowly in 2 minutes. After a total of 4 cycles of cell suspension and cell attachment in the IC loop, a 24 hour attachment period may be maintained, and then an appropriate cell feeding schedule may be applied as needed.
1 day before (Day: -1) Bioreactor coating
Purpose: Coating bioreactor membrane with reagents
Step 1: Put reagents into the IC loop until the bag is empty.
Step 2: Drive reagent from ARC into IC loop.
Step 3: Recirculate reagents in the IC loop.
Satisfy the following conditions before this task begins:
Include at least 40 mL of air in the cell input bag.
Table 10 shows the solution bags used attached to each line when performing bioreactor coating. These solutions and volumes are based on the default settings for this task.
Bioreactor coating route: Task> System management> Bioreactor coating
Enter the values for each setting in Step 1 shown in Table 11.
Enter values for each setting in step 2 shown in Table 12.
Enter the values for each setting in step 3 shown in Table 13.
Day 0 (Day: 0) IC / EC cleaning
Purpose: Exchange fluid in both IC circulation loop and EC circulation loop. The exchange volume is specified by the number of IC volumes and EC volumes to be exchanged. Table 14 shows a solution bag attached to each line when performing IC / EC cleaning. These solutions and volumes are based on the default settings for this task.
IC / EC cleaning route: Task> Cleaning> IC / EC cleaning
Check the values of each setting for IC / EC cleaning shown in Table 15.
Day 0 (Day: 0) Medium adjustment
According to the instructions for this task, the medium is allowed to equilibrate with a defined gas supply before loading the cells. This task has two steps.
Step 1: Rapid contact between media and gas supply by using high EC circulation rate.
Step 2: Maintain the system in an appropriate state until the operator is ready to load cells.
Table 16 shows the solution bag attached to each line when adjusting the culture medium. These solutions and volumes are based on the default settings for this task.
Medium adjustment route: Task> System management> Medium adjustment
Enter the values for each setting in Step 1 shown in Table 17.
Enter the values for each setting in step 2 shown in Table 18.
Day 0 (Day: 0) Cell loading with uniform suspension
Objective: Load cells into the bioreactor from the cell loading bag until the bag is empty. In this task, only IC circulation is used to distribute cells and not drive cells from the line into the bioreactor. This task has three steps.
Step 1: Cells are loaded from the cell loading bag into the bioreactor.
Step 2: Drive cells from ARC into bioreactor. The larger the chase volumes, the more the cells are dispersed and moved to the IC exit.
Step 3: Promote cell distribution to the membrane by IC circulation without IC inlet and thus ultrafiltration.
Before starting this task, satisfy the following conditions:
Table 19 shows the solution bag attached to each line when performing cell injection with a uniform suspension. These solutions and volumes are based on the default settings for this task.
Cell input path with uniform suspension: Task> Input and attachment> Cell input with uniform suspension
Check the values for each setting in Step 1 shown in Table 20.
Check the values for each setting in step 2 shown in Table 21.
Check the values for each setting in step 3 shown in Table 22.
Day 0 (Day: 0) Cell attachment (Bull's Eye)
Objective: Adherent cells are attached to the bioreactor while flowing through the EC circulation loop. The pump flow for the IC loop is set to zero.
Step 1: Cells are allowed to attach for 7 minutes to the inner surface of the bioreactor at 180 degrees.
Step 2: Circulate the IC fluid and residual suspended cells at a high rate in the direction opposite to the initial input direction.
Step 3: This step is a second 7 minute attachment period to further attach the cells. Cells that have migrated from the IC loop or from other areas of the bioreactor are given the opportunity to settle and attach to the bioreactor.
Step 4: Recirculate cells remaining in the IC loop and cells not yet attached to the surface. Circulation is carried out in the positive direction, and the circulation rate is lowered to avoid leaving attached cells away and to preferentially seed the areas of the bioreactor that have not been seeded in the previous steps.
Step 5: This step is a third 7 minute attachment period to further attach the cells. Cells that have migrated from the IC loop or from other areas of the bioreactor are given the opportunity to settle and attach to the bioreactor.
Step 6: Recirculate cells remaining in the IC loop and cells not yet attached to the surface. Circulation takes place in the negative direction, and the circulation rate is lowered to avoid leaving already attached cells away.
Step 7: Cell attachment period of 24 hours. The cells are firmly attached to the bioreactor for 24 hours before feeding begins.
Table 23 shows the solution bag attached to each line when performing the cell attachment which is the center in the present invention. These solutions and volumes are based on the default settings for this task.
Central cell attachment pathway: Task> Custom> Custom
Enter a value for each setting shown in Table 24.
Enter a value for each setting shown in Table 25.
Enter the values for each setting shown in Table 26.
Enter values for each setting shown in Table 27.
Enter values for each setting shown in Table 29.
Day 1 (Day: 1) Cell feed
Objective: To provide a continuously low flow rate for the IC circulation loop and / or EC circulation loop. There are several outlet settings that can be used to remove fluid added to the system during this task.
Table 31 shows the solution bag attached to each line when performing cell feed. These solutions and volumes are based on the default settings for this task.
Cell Feed Path: Task> Feed and Add> Cell Feed
Check the values for each setting in Step 1 shown in Table 32.
If necessary, increase the IC input rate.
Detachment and harvest of adherent cells
Objective: Detach cells from the membrane, release to IC loop, and transfer suspended cells (including cells in bioreactor) from IC circulation loop to harvest bag.
Step 1: Perform IC / EC cleaning task in preparation for reagent addition. For example, in the system, the IC • EC medium is replaced with PBS to remove proteins, Ca ++, and Mg ++ in preparation for trypsin addition.
Step 2: Fill the system with reagents until the bag is empty.
Step 3: Drive reagent into the IC loop.
Step 4: Mix reagents in the IC loop.
Step 5: Transfer suspended cells (including cells in bioreactor) from IC circulation loop to harvest bag.
Fill the cell input bag with at least 40 mL of air.
Table 33 shows the solution bags attached to each line when peeling and harvesting adherent cells. These solutions and volumes are based on the default settings for this task.
Adherent cell detachment path: Task> Detachment and harvesting> Adherent cell detachment and harvesting
Check the value of each setting for step 1 shown in Table 34.
Check the values for each setting in step 2 shown in Table 35.
Check the values for each setting in step 3 shown in Table 36.
Check the values for each setting in step 4 shown in Table 37.
Check the values for each setting in step 5 shown in Table 38.
The input technology of the present invention is evaluated using MSCs from four different donors. The yield based on the input harvest number according to the present invention is consistently higher than the yield when using LCWUS and culturing under the same conditions. The average cell yield increase when using the present invention (n = 6) over LCWUS (n = 4) is 25%.
The survival rate of the MSC sample taken from the IC loop immediately after the input according to the present invention is 100%. The survival rate of MSC based on the harvest number of the present invention is over 98% in all samples. MSCs by harvest using the present invention show typical morphology in culture and all MSC biomarkers measured by flow cytometry are compatible with ISCT standards.
Using the same protocol described in Example 2, a variation of the deposition protocol at the heart of the present invention was studied. A modification to the above form and the above protocol (Embodiment 2) is the omission of the deposition period after the circulation rate (100 ml / min; -50 ml / min; 25 ml / min). That is, instead of the 7-minute stop condition described above, the stop condition is omitted and the next circulation rate immediately follows the previous circulation rate. A control experiment and an experiment of the present invention of Example 2 (Embodiment 1) were also performed for comparison.
The various components may be referred to herein as “operably associated”. As used herein, “operably associated” refers to components being operatively coupled to each other in an embodiment in which the components are directly coupled and the two Also encompassed are embodiments in which additional components are placed between the combined components.
The above description of one or more embodiments of the invention has been made for the purpose of illustrating the invention and is not intended to limit the invention. In the foregoing detailed description, for example, various features of one or more embodiments of the invention have been grouped for ease of understanding the disclosure of the invention. However, such disclosed approaches should not be interpreted as reflecting an intention that embodiments of the invention require more features than are expressly recited in the claims. Rather, as the claims of this application describe, aspects of the present invention are less than all the features of one embodiment disclosed herein. Therefore, the scope of the claims is intended to be included in the detailed description, and each scope of the claims is independent of the embodiments of the present invention.
Further, the description of the invention includes descriptions of one or more embodiments and certain variations, modifications, although other variations and modifications are within the scope of the present invention (eg, after understanding the present invention). It will be understood by those skilled in the art because of the skill and knowledge of those skilled in the art. To the extent permitted, ie to publish patentable subject matter, whether or not other, interchangeable and / or equivalent structures, functions, ranges, steps are disclosed. It is not intended, but is intended to acquire rights to include other embodiments having such structures, functions, ranges, and processes.
A method of growing cells in a cell proliferation system comprising:
Circulating a fluid having a plurality of cells in a bioreactor of a cell growth system;
Reducing the fluid circulation rate;
Maintaining the bioreactor in a first horizontal direction for a first predetermined period of time to precipitate at least a portion of the plurality of cells and attach to the first portion of the bioreactor;
After the first predetermined period, rotating the bioreactor in a second horizontal direction rotated approximately 180 degrees from the first horizontal direction;
After the rotating step, growing at least the first portion of the plurality of cells in the bioreactor in the second horizontal direction;
2. The method of claim 1, wherein the bioreactor comprises a hollow fiber membrane.
3. The method of claim 2, wherein the hollow fiber membrane comprises a plurality of hollow fibers.
4. The method of claim 3, wherein the first portion of the bioreactor is at least the top of a hollow fiber.
5. The method of claim 4, wherein the step of proliferating at least the first portion of the plurality of cells affects the growth of the first portion of the plurality of cells by causing gravity to affect the plurality of cells. Growing a first portion toward a second portion of the bioreactor located below the first portion.
6. The method of claim 5, wherein the second portion of the bioreactor is at least the bottom of a hollow fiber.
2. The method of claim 1, wherein the first predetermined period of time is a length of time sufficient to attach and proliferate the portion of the cells.
8. The method of claim 7, wherein the first predetermined period is longer than about 10 hours.
The method of claim 1, wherein the first predetermined period is less than about 8 minutes.
10. The method of claim 9, wherein the first predetermined period is about 5 minutes.
The method of claim 1, further comprising after the first predetermined period and before rotating the bioreactor in a second horizontal direction.
Rotating the bioreactor in a first vertical direction and maintaining the bioreactor in the first vertical direction for a second predetermined period of time;
After the second predetermined period, the bioreactor is rotated in a second vertical direction rotated approximately 180 degrees from the first vertical direction, and the bioreactor is rotated in the second vertical direction for a third predetermined period. Step to maintain,
12. The method of claim 11, wherein the second predetermined period is long enough to grow the portion of the cell.
13. The method of claim 12, wherein the third predetermined period is the same length as the second predetermined period.
13. The method of claim 12, wherein the second predetermined period is at least about 10 hours.
A system for growing cells, the system comprising:
At least one motor connected to the bioreactor for rotating the bioreactor;
At least one fluid circuit fluidly coupled to the bioreactor;
At least one pump for circulating fluid in the at least one fluid circuit and the bioreactor;
A processor for executing processor-executable instructions;
Memory for storing processor executable instructions;
The processor-executable instructions when executed by the processor;
Activating the at least one pump to circulate a fluid having a plurality of cells in the bioreactor;
Reducing the flow rate of the at least one pump;
Activating the at least one motor after the first predetermined period to rotate the bioreactor in a second horizontal direction rotated approximately 180 degrees from the first horizontal direction;
After the rotation, activating the at least one pump to circulate fluid in the bioreactor and to grow the cells in the second horizontally positioned bioreactor;
A system comprising performing a method comprising:
16. The system according to claim 15, wherein the bioreactor comprises a hollow fiber membrane.
17. The system according to claim 16, wherein the hollow fiber membrane comprises a plurality of hollow fibers.
18. The system of claim 17, wherein the first portion of the bioreactor is at least the top of a hollow fiber.
19. The system of claim 18, wherein the step of expanding the first portion of the plurality of cells affects the growth of the first portion of the plurality of cells by gravity and the first portion of the plurality of cells. Growing a portion toward a second portion of the bioreactor located below the first portion.
The system of claim 18, wherein the second portion of the bioreactor is at least the bottom of a hollow fiber.
JP2016530921A 2013-11-16 2014-11-14 Cell growth in bioreactors Pending JP2016536998A (en)
US201361905182P true 2013-11-16 2013-11-16
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PCT/US2014/065829 WO2015073918A1 (en) 2013-11-16 2014-11-14 Expanding cells in a bioreactor
JP2016536998A true JP2016536998A (en) 2016-12-01
JP2016536998A5 JP2016536998A5 (en) 2017-12-21
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JP2016531019A Pending JP2016537001A (en) 2013-11-16 2014-11-14 Cell growth in bioreactors
JP2016530921A Pending JP2016536998A (en) 2013-11-16 2014-11-14 Cell growth in bioreactors
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JP (2) JP2016537001A (en)
CN (2) CN105793411B (en)
WO (2) WO2015073918A1 (en)
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