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WO2025014852A2 - Systèmes de bioréacteur modulaire pour applications de médecine régénérative - Google Patents

Systèmes de bioréacteur modulaire pour applications de médecine régénérative Download PDF

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Publication number
WO2025014852A2
WO2025014852A2 PCT/US2024/037004 US2024037004W WO2025014852A2 WO 2025014852 A2 WO2025014852 A2 WO 2025014852A2 US 2024037004 W US2024037004 W US 2024037004W WO 2025014852 A2 WO2025014852 A2 WO 2025014852A2
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Prior art keywords
bioreactor
module
tissue
housing
modular
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WO2025014852A3 (fr
Inventor
Thomas SHUPE
James Yoo
Anthony Atala
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Wake Forest University Health Sciences
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Wake Forest University Health Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/44—Multiple separable units; Modules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers

Definitions

  • tissue bioreactors have been used for expanding cells in vitro, where they provide a controlled Attorney Docket 9865.236.WO biochemical growth environment.
  • tissue bioreactors utilize cells applied to a two- dimensional (2D) or three-dimensional (3D) biomaterial scaffold, which reflects the physical geometry and architecture of native tissue.
  • the tissue bioreactor provides an anchoring bracket for the tissue, an environment conducive to cell differentiation and tissue maturation, and mechanical conditioning for the engineered tissue.
  • tissue-engineered constructs do not initially maintain a degree of durability or function required for implantation in vivo.
  • the biomaterial scaffold is produced in the shape of the desired construct.
  • Human cells are then seeded onto the scaffold either as a homogenous cell population, or in a multi-cell type pattern.
  • tissue constructs may be bioprinted into desired morphologies and cellular patterns using bioinks that contain the appropriate cell types.
  • the product Following biofabrication of the construct, the product must be matured for a period of time. During this maturation phase, cells proliferate to more densely colonize the scaffold, and reorganize to form a physiologically relevant and functional microarchitecture. All of these regenerative medicine product biomanufacturing steps occur within the specialized bioreactor that can accommodate media flow, media replenishment, and transport, within a contained sterile environment.
  • Bioreactors used for preconditioning and maturing engineered skeletal muscle may include uniaxial stretching systems that expand the muscle construct through controllable static or cyclic stretch programs. This is generally accomplished by affixing the construct between a stationary and moveable mount. The construct is stretched using electro-mechanical devices like solenoids, stepper motors, electromagnets, pneumatic pumps, or syringe pumps. Most bioreactors use a single large motor to generate mechanical forces and, hence, cannot provide multiple strain ratios for stimulating different constructs simultaneously.
  • Electrode pairs immersed in the culture chamber provide the desired stimulation pattern in commercial or improvised systems. Electrical stimulation is used to assess the functionality of engineered muscle constructs through high-frequency pulse stimulation to generate peak twitch contractile forces.
  • Bioreactors have incorporated mechanisms for measuring contractile force in response to electrical stimulation to characterize the functionality of engineered skeletal muscle. A force- Attorney Docket 9865.236.WO sensing component of the bioreactor is essential for determining the maturation of the muscle tissue over time. Previously, this was achieved by optical deflection measurement followed by calculation of contractile force output. More recently, bioreactors have incorporated direct or indirect methods to quantify contractile forces.
  • Twitch forces as measured by a force transducer, are often used to measure the contractility of engineered skeletal muscle. Since the genesis of regenerative medicine and tissue engineering, it has become clear to both researchers and the regenerative medicine industry that the physical conditions within the bioreactor during all phases of engineered tissue production influence the quality and functional characteristics of the final product. Conditions within the bioreactor play a critical role in determining matrix structure, tissue micro-architecture, tissue functionality and tissue durability in terms of both physiological and mechanical stresses. The conditions within a bioreactor, particularly during the maturation phase of engineered tissue production, must be capable of simulating the physical and electrochemical conditions that the construct will experience following implantation into a patient.
  • the engineered tissue begins to adapt, and becomes conditioned to tolerate these stresses.
  • the requirements for maturation depend on the architecture of the tissue or organ, with an increasing complexity gradient present from flat to tubular, to hollow non-tubular, and finally to solid organs.
  • the stimulus requirements vary with the increasing complexity of the tissue.
  • engineered muscles are fortified by cyclic uniaxial stretching; the pliability of certain engineered tissue (e.g., skin) is increased by cyclic biaxial stretching; engineered vessels and valves are conditioned to withstand the environment of the vasculature by applying pulsatile flow; and the elasticity of non-tubular hollow organs may be bolstered by applying cyclic pressure to the construct lumen.
  • bioreactors must also be scalable to accommodate constructs across a wide range of sizes. A bioreactor for conditioning a pediatric esophagus and an adult large bowel may have similar geometry but would require vastly different volumes. Therefore, any standardized bioreactor or modular tissue actuators must be designed to function similarly across a range of clinically relevant scales.
  • bioreactor must be redesigned and revalidated, again adding cost and time to the development of the clinical product. Since bioreactor systems are designed to be semi- or fully-closed systems to prevent contamination of the construct, regular media changes are difficult and cumbersome. Therefore, integrating a real-time media monitoring system could be beneficial to ensure proper media exchanges when needed. Summary [0012] Aspects of the present invention are directed to a modular bioreactor system for regenerative medicine applications.
  • the system includes a universal docking station; a bioreactor housing configured to be received within the universal docking station; a bracket system configured to be received within the bioreactor housing; a plurality of interchangeable tissue actuators, each tissue actuator configured to be removably secured to the bracket system, the plurality of interchangeable tissue actuators including a uniaxial tissue actuator, a biaxial tissue actuator, a pulsatile tissue actuator, a fill-void tissue actuator, and/or a perfusion tissue actuator; a media exchanger subsystem configured to be connected to the bioreactor housing received within the docking station, with the media exchanger subsystem optionally configured to be received within at least a portion of the docking station; a sensor module configured to be connected to the one of the plurality of interchangeable tissue actuators received within the bioreactor housing and the docking station; and a controller operatively associated with the docking station, the plurality of interchangeable tissue actuators, the media exchanger subsystem, and the sensor module.
  • the system includes a housing; a plurality of interchangeable bioreactor modules, each module configured to be received within the housing, the plurality of Attorney Docket 9865.236.
  • WO interchangeable bioreactor modules including a uniaxial bioreactor module, a biaxial bioreactor module, a pulsatile bioreactor module, a fill-void bioreactor module, and/or a perfusion bioreactor module; a media exchanger subsystem configured to be connected to one of the plurality of interchangeable bioreactor module received within the housing; a sensor monitoring module configured to be connected to the media exchanger subsystem and the one of the plurality of interchangeable bioreactor modules received within the housing, with the sensor monitoring module optionally configured to be received within the housing; and a controller operatively associated with the housing, the plurality of interchangeable bioreactor modules, the sensor monitoring module, and the media exchanger subsystem.
  • FIG.1A is a front view of a housing (e.g., an incubator) for a modular bioreactor system according to embodiments of the present invention.
  • a housing e.g., an incubator
  • FIG.1B illustrates bioreactor modules configured to be received within the housing of FIG.1A according to embodiments of the present invention (shown from left to right, a uniaxial or biaxial bioreactor module, a pulsatile (i.e., tubular) bioreactor module, a fill-void (i.e., hollow 3D) bioreactor module, and a perfusion (i.e., solid 3D) bioreactor module).
  • FIG. 1C illustrates each of the bioreactor modules of FIG.
  • FIG. 2A is a perspective view of a support frame for a bioreactor module according to embodiments of the present invention.
  • FIG. 2B is a perspective view of an alternative support frame for a bioreactor module according to embodiments of the present invention.
  • FIG.2C is a top view of the support frame of FIG.2B.
  • FIG.2D illustrates a reactor chamber box for one of the bioreactor modules of FIG.1B secured on the support frame of FIG.2B within the housing of FIG.1A.
  • FIG.3A is a perspective view of an adapter for the reactor chamber box of the bioreactor modules according to embodiments of the present invention.
  • FIG.3B illustrates the adapter of FIG.3A inserted within the reactor chamber box with a fully inflated porcine bladder contained therein according to embodiments of the present invention.
  • FIG. 4A illustrates a reactor chamber box for the uniaxial bioreactor module with a chamber attachment and linear actuator securing thereto.
  • FIG. 4B is a perspective view of the chamber attachment for the linear actuator of FIG.4A.
  • FIG. 4C illustrates the reactor chamber box of FIG. 4A with a cover for the chamber attachment and linear actuator.
  • FIG.4D is a perspective view of the cover in FIG.4C for the chamber attachment.
  • FIG. 4E is a top perspective view of the uniaxial bioreactor module according to embodiments of the present invention.
  • FIG.4F is a top perspective view of an alternative uniaxial bioreactor module according to embodiments of the present invention.
  • FIG.5A is a top perspective view of a force transducer holder platform for the uniaxial bioreactor module according to embodiments of the present invention.
  • FIG. 5B illustrates the force transducer holder platform of FIG. 5A installed in the reactor chamber box for the uniaxial bioreactor module according to embodiments of the present invention.
  • Attorney Docket 9865.236.WO [0032]
  • FIG.6A illustrates an exemplary actuator for the biaxial bioreactor module according to embodiments of the present invention.
  • FIG.6B illustrates the actuator of FIG.6A installed in the reactor chamber box for the biaxial bioreactor module and being used with skin tissue according to embodiments of the present invention.
  • FIG. 7 illustrates the components of the pulsatile (i.e., tubular) bioreactor module according to embodiments of the present invention.
  • FIG. 35 illustrates the components of the pulsatile (i.e., tubular) bioreactor module according to embodiments of the present invention.
  • FIG. 8A illustrates a flow equalizer for the pulsatile bioreactor module of FIG. 7 according to embodiments of the present invention.
  • FIG.8B illustrates testing of the flow equalizer of FIG.8A with the pulsatile bioreactor module (right) versus using a three-way spitter (left).
  • FIG. 9A is a schematic of a fill-void mechanism for the fill-void bioreactor module according to embodiments of the present invention.
  • FIG.9B illustrates using a garter spring for a cannulation test of the fill-void mechanism of FIG.9A according to embodiments of the present invention.
  • FIG. 9A is a schematic of a fill-void mechanism for the fill-void bioreactor module according to embodiments of the present invention.
  • FIG.9B illustrates using a garter spring for a cannulation test of the fill-void mechanism of FIG.9A according to embodiments of the present invention.
  • FIG. 9C illustrates testing of the fill-void mechanism installed in the reactor chamber box of the fill-void bioreactor module using inflated silicone sacs and a porcine bladder according to embodiments of the present invention.
  • FIG. 10 illustrates integration of pressure and flow rate sensors with the fill-void bioreactor module according to embodiments of the present invention.
  • FIG. 11A illustrates testing biocompatibility and reliability of the fill-void bioreactor module using a porcine bladder according to embodiments of the present invention.
  • FIG.11B is a graph illustrating exemplary time-lapse monitoring of inflow rate (black), draining flowrate (blue), and pressure of the porcine bladder in the fill-void bioreactor module of FIG.11A.
  • FIG.11C are morphological images of the porcine bladder before and after testing (i.e., void and fill conditioning) of the fill-void bioreactor module of FIG.11A for 48 hours according to embodiments of the present invention.
  • FIG. 12A is a graph illustrating exemplary time-lapse monitoring of flow rate and pressure data of the first and the last fill-void cycle of the porcine bladder in the fill-void bioreactor module of FIG.9A according to embodiments of the present invention.
  • FIG.12B is a graph illustrating an exemplary pressure-volume curve of the first and the last fill-void cycle of the porcine bladder in the fill-void bioreactor module of FIG.9A according to embodiments of the present invention.
  • FIG.13A is a schematic of the perfusion bioreactor module according to embodiments of the present invention.
  • FIG.13B illustrates a cushioning system within the reactor chamber box of the perfusion bioreactor module of FIG.13A according to embodiments of the present invention.
  • FIG.14A illustrates testing of the perfusion bioreactor module of FIG.13A according to embodiments of the present invention.
  • FIG.14B is a graph illustrating exemplary real-time pressure and flowrate data to create a user-defined constant pressure setting using a PID controller according to embodiments of the present invention.
  • FIGS.15A and 15B are trace graphs illustrating exemplary pressure versus infusion flow rate during pressure stability testing of a PID controller and a porcine kidney with FIG. 15A showing whole organ perfusion through the ureter and FIG.15B showing whole organ perfusion through the renal artery.
  • FIG.16A is a perspective view of a support frame and locking device for a silicone sheet used as an organ sac within the reactor chamber box for the perfusion bioreactor module according to embodiments of the present invention.
  • FIG.16B shows the silicone sheet to be secured to the support frame of FIG.16A.
  • FIG. 16C illustrates the perfusion bioreactor module according to embodiments of the present invention.
  • FIG. 17A is a flow chart for the aseptic organ handling and reactor chamber box assembly for the perfusion bioreactor module according to embodiments of the present invention.
  • FIG.17B is a graph illustrating exemplary real-time pressure and flow rate data under a constant pressure perfusion setting within the perfusion bioreactor module according to embodiments of the present invention.
  • FIG.17C is a graph illustrating exemplary real-time pressure data under a constant flow rate perfusion setting within the perfusion bioreactor module according to embodiments of the present invention.
  • FIG. 18A illustrates cannulation of a porcine kidney within the perfusion bioreactor module according to embodiments of the present invention.
  • FIG. 18B are graphs illustrating exemplary flow dynamics evaluation results of the porcine kidney within the perfusion bioreactor module of FIG.18A according to embodiments of the present invention.
  • FIG. 18C illustrates morphology of the porcine kidney of FIG. 18A before and after decellularization according to embodiments of the present invention.
  • FIG. 19 illustrates the uniaxial bioreactor module connected to a media exchanger subsystem and corresponding sensors according to embodiments of the present invention.
  • FIG.20A is a flow diagram for components of a sensor monitoring module configured to be received within the housing of FIG.1A according to embodiments of the present invention.
  • FIG.20B is a graph illustrating exemplary short term data for glucose and lactate testing according to embodiment of the present invention.
  • FIG.20C is a graph illustrating exemplary long term data for glucose and lactate testing for 72 hours according to embodiments of the present invention.
  • FIG. 21A illustrates the components of the media exchanger system of the modular bioreactor system according to embodiments of the present invention.
  • FIGS.21B and 21C show level sensors connected to the reactor chamber box of one of the bioreactor modules according to embodiments of the present invention.
  • FIG.22 is a graph illustrating experimental data of the media exchanger system based the output of a pH sensor and level sensor switch, waste media pump switch, and fresh media pump switch according to embodiments of the present invention.
  • FIG. 23 is a schematic diagram illustrating the media exchanger subsystem for the modular bioreactor system according to embodiments of the present invention.
  • FIG.24 is a schematic diagram illustrating the modular bioreactor system according to embodiments of the present invention.
  • FIG. 25 is a schematic diagram illustrating the module bioreactor system utilizing an alternative mounting system according to embodiments of the present invention.
  • FIGS. 26A-26J illustrate the bioreactor modules and respective tissue actuators according to embodiments of the present invention.
  • FIG.27 illustrates an actuator assembly for the uniaxial bioreactor module according to embodiments of the present invention.
  • FIG. 28A illustrates the mechanical stimulation system of the actuator assembly of FIG.27 including three individually configurable stretch systems according to embodiments of the present invention.
  • FIG. 28B illustrates integration of a movement encoder into the stretch systems of FIG.28A according to embodiments of the present invention.
  • FIG.28C are exemplary movement protocols for a cyclic strain and stepwise strain, as read by the movement encoder of FIG.28B according to embodiments of the present invention.
  • FIG.29A illustrates the electrical stimulation system of the actuator assembly of FIG.27 including three pairs of carbon sheet electrodes held in spacers from the lid according to embodiments of the present invention.
  • FIG.29B is a schematic diagram of the electrical stimulation system of the actuator of FIG.27 according to embodiments of the present invention.
  • FIG.30A illustrates integration of force sensing into the actuator assembly of FIG.27 for mechanical property testing and for contractile/twitch force measurement in which a miniature submersible inline sensor is used to measure force in each stretch system according to embodiments of the present invention.
  • FIG. 30B illustrates the setup for force sensing with a hydrated fibrin scaffold and corresponding 1PK-stress vs.
  • FIG. 30C illustrates fresh EDL muscle anchored at tendons and corresponding contractile force recorded at incremental frequency pulses according to embodiments of the present invention.
  • FIG.31 is a schematic diagram illustrating the sensor loop and media exchange system according to embodiments of the present invention.
  • FIGS. 32A-32C illustrate the complete assembly inside an incubator along with electronics of the modular bioreactor system and schematic block diagrams of the bioreactor system with subsystems and main components according to embodiments of the present invention.
  • FIGS. 33A-33C illustrate a mechanical stimulation system of the modular bioreactor system according to embodiments of the present invention.
  • FIG. 33A shows three individually Attorney Docket 9865.236.WO configurable stretch systems having three stepper motors with ball-screw guides.
  • FIG.33B shows the integration of a movement encoder.
  • FIG. 33C compares two movement protocols and respective strain rates over time for cyclic strain and stepwise strain, as read by the encoder of FIG.33B.
  • FIGS. 34A-34B illustrate an electrical stimulation system of the modular bioreactor system according to embodiments of the present invention.
  • FIG.34A shows three pairs of carbon sheet electrodes held in spacers from the lid. The transfer of pulses from a pulse generator to the submerged electrodes uses gold-plated min-banana connector pairs on the lid (marked as "+" and "-").
  • FIG.34B shows integration of a DMT 4-channel stimulator for which pulse characteristics be defined by software and triggered to start with an MCU. Simultaneous verification of the set characteristics may be possible using the fourth channel.
  • FIGS. 35A-35E illustrate incorporation of a force sensor in the modular bioreactor system according to embodiments of the present invention.
  • FIG. 35A shows a miniaturized submersible inline sensor used to measure force in each lane.
  • FIG.35B shows an exemplary setup with a hydrated fibrin gel scaffold.
  • FIG.35C shows the corresponding 1PK – stress versus strain graph of cellular and acellular scaffolds.
  • FIG.35D shows a fresh EDL muscle anchored at tendons inside the modular bioreactor system.
  • FIG. 35A shows a miniaturized submersible inline sensor used to measure force in each lane.
  • FIG.35B shows an exemplary setup with a hydrated fibrin gel scaffold.
  • FIG.35C shows the corresponding 1PK – stress versus
  • FIGS. 36A-36B illustrate the sensor loop and media exchange system for the modular bioreactor system according to embodiments of the present invention.
  • FIG.36A show the sensor loop which automates the fluid movement for media sensing, and where sensors are exposed to either (i) media from the culture box, (ii) a PBS buffer, and (iii) a glucose/lactate calibration solution by controlling the sensor loop pump and three three-way solenoid pinch valves (PV1, PV2, PV3).
  • the media exchange system has two pumps that can be manually turned on and off for the media fill and void functions.
  • FIG.36B illustrates an exemplary assembly of the sensor loop tray formed with 3D-printed parts.
  • FIG.37A-37B illustrate an assembled uniaxial bioreactor system and operation thereof according to embodiments of the present invention.
  • FIG. 37A illustrates an assembled system inside an incubator for the bioreactor and sensor loop system.
  • FIG.37B is a schematic diagram of the subsystem operations for the bioreactor run with microcontrollers (MCUs) and computer, with a side view of the internal components of the culture chamber.
  • MCUs microcontrollers
  • FIGS. 38A-38E illustrate stimulation function validation test data with EDL muscle tissue according to embodiments of the present invention.
  • FIG.38A show histology results of the H&E and Masson's Trichrome stains.
  • FIG. 38B show the number of muscle fibers per field of view.
  • FIG. 38A show histology results of the H&E and Masson's Trichrome stains.
  • FIG. 38B show the number of muscle fibers per field of view.
  • FIGS.39A-39C illustrate stimulation function validation test data with tissue-engineered muscle constructs according to embodiments of the present invention.
  • FIG.39A show exemplary images of the three culture groups post long-term bioreactor culture.
  • FIG. 39B show quantification of a cross-sectional area using ImageJ.
  • FIG.39C shows peak stress calculated from a 5% stretch for control, mechanical stimulation (MS), and electrical stimulation (ES) at the end of long-term culture.
  • FIGS. 40A-40E illustrate IHC image analysis for long-term culture according to embodiments of the present invention.
  • FIG. 40A shows DAPI, MHC, and Desmin staining for cell proliferation and myotube formation in three culture groups.
  • FIG.40B shows quantification of myotube length.
  • FIG. 40C shows quantification of myotube width.
  • FIG. 40D shows nuclei count per myotube per field of view.
  • FIG.40E shows quantification of the number of nuclei fused to form myotubes as a percentage of the total number of nuclei per field of view.
  • FIGS. 41A-41E illustrate sensor loop system data with C2C12 cells for two days with the cell culture plate's lid modified to act as a bioreactor in the sensor loop system according to embodiments of the present invention.
  • FIG.41A shows pH change over time.
  • FIG.41B shows dissolved oxygen over time.
  • FIG.41C shows glucose concentration over time.
  • FIG.41D shows lactate concentration over time.
  • FIG. 41E shows media temperature over time. The respective arrows mark a media change.
  • FIG.42 illustrates an aseptic media exchange system according to embodiments of the present invention including two exchange system pumps, the bioreactor, and other components inside and outside of the incubation chamber.
  • FIG. 43 illustrates rat EDL muscle under electrical stimulation showing isometric twitch/tetanic contractions observed from the bioreactor's lid during low to high frequency stimulation according to embodiments of the present invention.
  • FIGS. 44A-44C illustrate fabrication of C2C12 laden fibrin gel muscle constructs according to embodiments of the present invention.
  • FIG.44A show CAD and 3D-printed mold elements.
  • FIG. 44B show assembly of the mold elements.
  • FIG. 44C show C2C12 engineered muscle fabrication in the mold elements.
  • FIG. 45 illustrates glucose and lactate sensor monitoring data taken with 72 sampling cycles over time plotted overlapped to show repeatability in glucose and lactate measurements according to embodiments of the present invention. The data points were sampled every 3 seconds and matched by the peak at point 0 on the x-axis.
  • FIGS.46A-46E illustrate force measurements during dynamic cyclic strain according to embodiments of the present invention. A sample force measurement showing three cycles during different cyclic strain protocols of peak strain at 5% (FIG. 46A), 10% (FIG. 46B), 15% (FIG.46C), 20% (FIG.46D), and a comparison of each (FIG.46E).
  • phrases such as "between about X and Y” mean “between about X and about Y.”
  • phrases such as “from about X to Y” mean “from about X to about Y.”
  • Embodiments of the present invention are directed a standardized, self-contained, and modular bioreactor system that allows for a scalable and automated process for the clinical manufacturing of a wide range of cell-based tissue engineering products.
  • the present invention provides a bioreactor system that is configured for the fabrication and maturation of a wide variety of tissue engineered regenerative medicine clinical products.
  • the modular bioreactor system of the present invention is configured to be scalable to accommodate a wide range of tissue volumes and geometries allowing for stimulation and maturation environment for different kinds of cell- based tissue engineered products (i.e., tissues and organs), together with highly automated real- time integrated cellular and environmental components sensing.
  • the present invention provides the advantage of a highly automated system using sensors and actuators enabling feedback of culture environment, cellular growth, and system performance, along with an easy user interface to fine tune culture protocols, media exchange protocols, imaging camera, data display, and file saving, before starting the tissue culture.
  • the modular bioreactor system of the present invention may be standardized across a wide range of parameters that will allow the system to be Attorney Docket 9865.236.WO validated and approved by regulatory agencies. Embodiments of the present invention will now be described in further detail below with reference to FIGS.1A-46E.
  • the main components of the bioreactor system 100 may include a plurality of interchangeable bioreactor modules 200, a sensor monitoring module 260, and a media exchanger subsystem 400 (see, e.g., FIG.19 and FIG.23).
  • the bioreactor system 100 further includes a controller (and software interface) 270 that is operatively associated with the main components of the system 100 (see also, e.g., FIG. 24 and FIG. 25).
  • the interchangeable bioreactor modules 200 are configured for preconditioning (e.g., cell seeding) and/or maturation of cell-based tissue engineered product(s) 10, for example, tissue and organs, and allow for target-specific actuation and/or stimulation of the tissue.
  • preconditioning e.g., cell seeding
  • maturation of cell-based tissue engineered product(s) 10 for example, tissue and organs
  • the interchangeable bioreactor modules 200 allow the modular bioreactor system 100 of the present invention to be easily modified for the production of any desired tissue engineered regenerative medicine clinical product.
  • a standardized mounting system 108, 108' is configured to accept each of the respective bioreactor modules 200 within a housing 102 (see, e.g., FIGS. 1A-1C) or a docking station 102' (see, e.g., FIGS. 24-25) in such a manner that the mechanical and electrical requirements of the bioreactor modules 200 (and respective actuators) are brought into communication with input contacts (e.g., the sensor module 260).
  • a standardized feed reservoir (e.g., the media exchange subsystem 400) allows for the bioreactor system 100 to replenish factors that are consumed by the tissue constructs (cell-based tissue engineered products 10) within the bioreactor modules 200, as well as includes mechanisms for driving the fillable reservoir feed module (media exchange subsystem 400) and receiving information from the integrated sensor array (sensor module 260).
  • the bioreactor system 100 further includes a user friendly interface and programmable controller hardware/software (e.g., a controller interface 270).
  • the standardized and modular bioreactor technologies of the bioreactor system 100 of the present invention can help to simplify and reduce the cost and time associated with the development of clinical manufacturing processes for engineered Attorney Docket 9865.236.WO tissues and eliminate associated challenges in gaining regulatory approval for new clinical products.
  • the bioreactor system 100 may include five interchangeable bioreactor modules 210, 220, 230, 240, 250 each having an actuator system (see also, e.g., FIGS.26A-26J) associated with the respective module 200 and one common system for sensing the culture environment and cellular metabolites (i.e., sensor monitoring module 260) within a respective bioreactor module 200. As shown in FIG.
  • the plurality of interchangeable bioreactor modules 200 may comprise one or more of a uniaxial bioreactor module 210, a biaxial bioreactor module 220, a pulsatile (or tubular) bioreactor module 230, a fill-void (or hollow 3D) bioreactor module 240, and a perfusion (or solid 3D) bioreactor module 250.
  • the bioreactor modules 200 are able to impart uniaxial mechanical stretch/electrical stimulation (e.g., bioreactor module 210), biaxial stretch (e.g., bioreactor module 220), pulsatile flow/distension (e.g., bioreactor module 230), cyclic fill/void (e.g., bioreactor module 240), or resistance modulated perfusion (e.g., bioreactor module 250), respectively.
  • the bioreactor system 100 includes a mounting system 108 in which each bioreactor module 200 may comprise a swappable (i.e., removable) tray 106 (see, e.g., FIG.
  • each of the trays 106 may be configured to slide within the shelving system 104 (e.g., similar to a drawer).
  • a door 103 may be coupled to the housing 102 to provide easy access to interior cavity 105 and mounting system 108 therein.
  • the housing 102 is a small-size commercial incubator that is customized to receive the respective bioreactor modules 200 (see, e.g., FIG.1A and FIG.1C).
  • the bioreactor system 100 may comprise an alternative mounting system 108' configured for use with a docking station 102' (see, e.g., FIGS.24 and 25).
  • the shelving system 104 within the housing 102 may include a first (or upper) shelf 104a and a second (or lower) shelf 104b. As shown in FIG.
  • one of the shelves e.g., the first upper shelf 104a
  • the other shelf e.g., the second lower shelf 104b
  • the bioreactor system 100 may include one or more additional shelves to hold additional bioreactor modules 200.
  • the bioreactor system 100 may include a larger shelving system 104 in which a larger customized incubator (i.e., housing 102) is configured to hold all five bioreactor modules 200 together within the interior cavity 105 of the housing 102, with the culture environment maintained in each individual module area to avoid cross-contamination between the bioreactor modules 200.
  • the trays 106 are configured such that one or more components for the respective bioreactor modules 210, 220, 230, 240, 250, for example, tubing, sensors, actuators, and reactor chamber, may be mounted and secured thereon.
  • the trays 106 may be formed from polycarbonate and have a thickness in a range of between about 1 centimeter and about 5 centimeters.
  • the trays 106 have a plurality of apertures comprising snap-fit couplings 126 that are configured to receive and hold the different components of the bioreactor modules 200 (see, e.g., FIG.5B).
  • the snap- fit couplings 126 may provide the advantage and ease of (1) replacing of a broken item; (2) re- adjusting of current component location on the tray; (3) adding of a new part; and (4) integrating of future bioreactor modules.
  • the bioreactor system 100 of the present invention further includes a support frame 110, 110' within the housing 102.
  • the support frame 110, 110' is configured to hold a reactor chamber box 120 for a respective bioreactor module 200 (see, e.g., FIG.2D).
  • the support frame 110, 110' is configured to engage with the tray 106 which is then slid onto the shelving system 104 of the housing 102.
  • the support frame 110, 110' is configured to be mounted directly to the shelving system 104 within the housing 102.
  • the support frame 110 includes one or more side members 112 that together define a central opening 113.
  • one or more flanged edges 114 extend upwardly from one or more of the side members 112.
  • the reactor chamber box 120 for a respective bioreactor module 200 is configured to sit on top of the one or more side members 112 and held between the one or more one or more flanged edges 114 (see, e.g., FIG. 2D).
  • the support frame 110 is configured to support and hold the load weight of the reactor chamber box 120 for an extended period of time.
  • An alternative support frame 110' for the reactor chamber box 120 is illustrated in FIGS.2B and 2C.
  • the support frame 110' is similar to the support frame 110 illustrated in FIG.2A having one or more side members 112' and one or more flanged edges 114' extending upwardly from the side members 112. As shown in FIGS.
  • the support frame 110' differs from the support frame 110 in that the support frame 110' has a plate member 113' (i.e., a closed bottom) rather than a central opening 113.
  • the plate member 113' is configured to provide additional structural support to the support frame 110' (compared to the support frame 110) when the reactor chamber box 120 is placed thereon.
  • FIGS. 3A-3B an adapter 129 for the reactor chamber box 120 of the bioreactor modules 200 according to embodiments of the present invention is illustrated. As shown in FIG.3A, the adapter 129 has one or more side walls 129w that together define an interior cavity 129c.
  • the interior cavity 129c is sized and configured to contain a cell-based engineered tissue product 10 therein.
  • the adapter 129 is sized and configured to fit within the interior cavity 123 of the reactor chamber box 120 of one or more of the bioreactor modules 200 of the present invention.
  • the adapter 129 may be used to adjust the storage capacity of the reactor chamber 120 based on the size of the tissue or organ 10 being cultured within the reactor chamber box 120, i.e., utilizing the adapter 129 for smaller organs.
  • the adapter 129 is inserted into the reactor chamber box 120 and a fully inflated porcine bladder 10 is contained therein.
  • each of the bioreactor modules 200 include a tissue actuator for maturing a wide range of tissue architectures and complexities (see, e.g., FIG.4A, FIG.6B, FIG.7, FIG.11A, FIG.13A, and FIGS.26A-26J).
  • Flat structures such as small muscle constructs and skin (e.g., the uniaxial and biaxial bioreactor modules 210, 220) may be considered the least complex architecturally. Mechanical stretching and electrical stimulation will promote beneficial biological adaptations such as collagen fiber alignment, elastin organization and sarcomere distribution that will improve construct function.
  • Tubular tissues such as blood vessels or tracheas (e.g., the pulsatile (or tubular) bioreactor module 230), are more complex and require consideration of structural patency. Stimuli promoting the development of circumferential smooth muscle alignment and construct barrier function will be considered.
  • Hollow non-tubular organs Attorney Docket 9865.236.WO such as the stomach or bladder (e.g., the fill-void (or hollow 3D) bioreactor module 240), require a more complex cellular architecture, and these organs usually have more interdependent interactions with other tissues or organs.
  • the present invention may provide advancement of scale-up manufacturing, decreasing design, manufacturing, and products costs, and reducing development timelines for each indication in the regenerative medicine industry.
  • Each of the interchangeable bioreactor modules 200 will now be described in further detail below.
  • the uniaxial bioreactor module 210 is configured to provide cyclic stretching and/or electrical stimulation of cell-based tissue engineered product(s) 10, such as, muscle tissue.
  • the uniaxial bioreactor module 210 provides sensor dynamic feedback about tensile or compressive force, as well as measurement of the contractile property of the cell-based tissue engineered product 10 therein.
  • the uniaxial bioreactor module 210 is also configured to be used as a mechanical testing machine for on-site characterization of the scaffold material forming the cell-based tissue engineered product 10, which replaces the need for taking samples to a mechanical testing device, such as an Instron. This also helps to improve the dynamic protocols based on plastic deformation due to cyclic stretch.
  • the uniaxial bioreactor module 210 includes a reactor chamber box 120, 120'.
  • the reactor chamber box 120, 120' has one or more side walls 122, 122' that define an interior cavity 123, 123'.
  • a cover 124, 124' is sized and configured to enclose and seal the reactor chamber box 120, 120'.
  • the interior cavity 123, 123' of the reactor chamber box 120, 120' is configured to hold one or more scaffolds for a cell-based tissue engineered product 10 being cultured or Attorney Docket 9865.236.WO conditioned therein.
  • a support platform 213, 213' is coupled or secured to one of the side walls 122, 122' of the reactor chamber box 120, 120'.
  • the support platform 213, 213' may be configured to support or hold a general uniaxial stretch actuator 212, 212' thereon.
  • the support platform 213 is also shown in FIG. 4B.
  • the uniaxial stretch actuator 212, 212' extends into the interior cavity 123 of the reactor chamber box 120, 120' and is configured to apply programmable mechanical and electrical stimulation and force sensing of the one or more scaffolds (i.e., of tissue) cultured within the reactor chamber box 120, 120' along a single axis (see also, e.g., FIG.4B and FIGS.26A-26B).
  • the uniaxial bioreactor module 210 further includes a support platform cover 214 that is sized and configured to fit over and enclose the uniaxial stretch actuator 212 (see also, e.g., FIG. 4E).
  • the support platform cover 214 is also shown in FIG. 3D.
  • the uniaxial bioreactor module 210 may be used for any tissue having a scaffold which needs compressive or tensile preconditioning, such as muscle tissue.
  • the availability of two-directional sensing enables of the uniaxial bioreactor module 210 of the present invention capable of being used with bone and muscle tissue applications.
  • the uniaxial bioreactor module 210 further includes an in-line tensile and compressive force transducer 215.
  • the transducer 215 is configured to measure tensile stresses during cyclic stretch conditioning, and twitching force during electrical pulse stimulation.
  • FIG. 5B illustrates the transducer 215 installed within the interior cavity 123 of the reactor chamber box 120 and coupled to the actuator 212.
  • the bioreactor system 100 of the present invention has a mechanical testing program to characterize the material on-site, if enabled in the user interface. For example, before starting of the program, the user interface takes in the length of the muscle scaffold for the cell-based tissue engineered product 10, actuation amplitude in percent of the original length, and speed of actuation in percent per second.
  • the biaxial bioreactor module 220 and components thereof according to embodiments of the present invention are illustrated.
  • the biaxial bioreactor module 220 is configured to provide cyclic stretch to cell-based engineered tissue products 10 having a planar configuration, for example, skin tissue.
  • the biaxial bioreactor module 220 may be used for any type of other planar or flat tissue such as but not limited to, cartilage, a segment of an organ wall, and flat scaffolding structures.
  • Attorney Docket 9865.236.WO [00121]
  • the biaxial bioreactor module 220 is configured to provide the cyclic stretch in two, three, or four axes with programmable axial stretch and multiple anchor points 224 (see, e.g., FIG.6A and FIGS.26C-26D). As shown in FIG.6A, the biaxial bioreactor module 220 utilizes a two-dimensional Hoberman mechanism 222 which turns linear motion from, for example, actuator 212, into radial motion.
  • the biaxial bioreactor module 220 may utilize a standard two-axis actuator 222' (see, e.g., FIGS.26C-26D).
  • the Hoberman mechanism 222 may be implemented in the interior cavity 123 of the reactor chamber box 120 for the biaxial bioreactor module 220.
  • the circular structure 223 of the Hoberman mechanism 222 is configured to expand diametrically with one end fixed and the opposite end actuated by a linear actuator, for example, the actuator 212 used with the uniaxial bioreactor module 210.
  • the biaxial bioreactor module 220 of the present invention reduces the number of actuators 212 compared to other known biaxial systems (which use two separate actuators).
  • the biaxial bioreactor module 220 of the present invention also increases the number of axis of strains with a uniform multiaxial stimulus to flat tissue via equal stretching up to four directions simultaneously (i.e., via the Hoberman mechanism 222).
  • the user interface of the bioreactor system 100 allows setting the initial diameter and percent area increase or optionally, percent diameter increase, of the cell-based engineered tissue 10 being cultured within the biaxial bioreactor module 220.
  • the bioreactor system 100 i.e., the biaxial bioreactor module 220
  • the bioreactor system 100 has the capability to be used with 40 mm-diameter human skin (tissue) in full- thickness which resulted in a maximum of up to 40% axial stretch ratio or 96% area increasement.
  • the pulsatile (or tubular) bioreactor module 230 and components thereof according to embodiments of the present invention are illustrated.
  • the pulsatile bioreactor module 230 is configured to provide shear stress and flow stimulation for tubular organs such as blood vessels (e.g., arteries), but is also applicable to any tubular section or organoid grow chambers, which has ports on both ends as infusion inlet and draining outlet (see also, e.g., FIGS.26E-26F).
  • the pulsatile bioreactor module 230 includes the reactor chamber box 120 and a base module or plate 121 that is configured to hold up to three sliding tracks 232 within the interior cavity 123 of the reactor chamber box 120.
  • Each of the sliding tracks 232 are configured to contain a respective tubular organ 10.
  • the sliding tracks 232 Attorney Docket 9865.236.WO allow for tubular organs having the same or different lengths to be cultured within the reactor chamber box 120 at the same time.
  • opposing ends of the respective sliding tracks 232 include a universal cannula design which has a graduation in diameter as a spherical block, thereby helping prevent the anchored tubular organ tissue 10 from slipping off the sliding tracks 232 (for example, as shear stress and/or flow stimulations are conducted on the respective tubular organs).
  • the pulsatile bioreactor module 230 of the present invention provides easy-to- connect universal anchoring points for up to three simultaneous tubular (tissue 10) constructs, with the sliding tracks 232 within the reactor chamber box 120 configured to allow individual adjustment of the length and/or pre-tensioning of the respective tubular vessel scaffolds.
  • the reactor chamber box 120 of the pulsatile bioreactor module 230 includes a lid 124 configured to cover the interior cavity 123 of the reactor chamber box 120.
  • the lid 124 has one or more windows 124w that allow a user to see into the reactor chamber box 120 without removing the lid 124 (i.e., to see the tubular organ(s) therein).
  • the lid 124 has a window 124w for each sliding track 232 within the interior cavity 123. It is noted that reactor chamber boxes 120 for the other bioreactor modules 200 may also include lids 124 with one or more windows 124w that allow a user to see into the reactor chamber box 120 (see, e.g., FIG.4E).
  • the pulsatile bioreactor module 230 further includes flow sensors, pressure sensors, and a flow regulator 235 each coupled to the reactor chamber box 120.
  • the flow regular 235 comprises a snap-fit holder design which allows easy connection to the pulsatile bioreactor module 230.
  • the flow regulator (equalizer) 235 is illustrated in FIG.8A.
  • the flow regulator 235 includes a main chamber 235c having an input 235i and three outputs 235o.
  • the main chamber 235c is divided into three equal flow compartments 235fc.
  • Each flow compartment 235fc has an aperture 235a into a respective output 235o which are in fluid communication with a respective sliding track 232 of the pulsatile bioreactor module 230 (i.e., tubular organ 10 contained within the reactor chamber box 120 of the pulsatile bioreactor module 230).
  • the design of the flow regulator 235 helps to improve equal flow distribution for the three tubular organs (tissue) 10 within the pulsatile bioreactor module 230.
  • the flow and pressure sensors may be installed within respective infusion lines 234 extending into the reactor chamber box 120 and coupled to respective sliding tracks 232 contained therein (see, e.g., FIG.8B). Installing the flow and pressure sensors within the infusion lines 234 allows for instantaneous measurements of the flow rate into and pressure inside of the respective tubular organs (i.e., tissue 10).
  • each infusion line 234 may be configured to measure flow rate, allow fine-tuning of mean flow with flow regulator, measure flow pressure, and utilize a single pump 233 to achieve programmable pulsatile flow through all three lines 234 to the respective tubular tissue 10.
  • needle valves may be used as flow regulators 235 based on user-selectable flow rates.
  • the pulsatile bioreactor module 230 of the present invention may include a motorized needle valve to automate flow equalization. The motorized needle value of the present invention replaces currently used manual rotating needle valves.
  • the equal-multichannel-flow of the pulsatile bioreactor module 230 may also be carried out with a multichannel peristaltic pump 233, but a flow dampener for each channel may be needed.
  • the gear pump 205 does not need pulse dampening as required for typical peristaltic pumps, but is usually a single- channel and bulky.
  • the pulsatile input is provided by a block programming interface for the peristaltic pump.
  • the fill-void bioreactor module 240 is configured to provide stimulation for a hollow organ or tissue 10, such as the urinary bladder.
  • the fill-void bioreactor module 240 is configured to mimic the natural fill and void cycle of the organ, with filling delivered through a gear pump 205 which is configured to provide smooth, pulse-free perfusion into the hollow organ 10 (e.g., urinary bladder). While FIGS. 9A- 9C illustrate the fill-void bioreactor module 240 being used with a porcine bladder, the fill-void bioreactor module 240 may be used for any hollow organ or tissue structure with physiological fill and void motion cycles.
  • the bioreactor system 100 (via the fill-void bioreactor module 240) is capable of measuring flow rate in and out of a hollow organ 10 utilizing at least two flow sensors 246a, 246b (see, e.g., FIG.10).
  • the fill-void bioreactor module 240 includes a flow Attorney Docket 9865.236.WO line 244 to the hollow organ 10 which comprises a pressure sensor outlet 245b and a bubble trap to help avoid filling air in the organ 10.
  • this flow line 244 forks out (e.g., using a Y-connector) into inflow 244i and outflow 244o lines.
  • the flow sensors 246a, 246b are installed in both the inflow and outflow lines 244i, 244o to measure either fill or void flow rate within the respective lines 244i, 244o.
  • the bioreactor system 100 (via the fill-void bioreactor module 240) may also be capable of measuring pressure inside the hollow organ 10 utilizing one or more pressure sensors 245a, 245b (see, e.g., FIG.10).
  • both the flow sensors 246a, 246b and pressure sensors 245a, 245b may be connected in-line at the ureter.
  • the bioreactor system 100 includes a programmable switching mechanism 242 between the void and fill cycle for the fill-void bioreactor module 240 which may be achievable based on timing, flow rate thresholds, bladder pressure, or a combination of any or all these three.
  • FIG.9A illustrates a schematic layout of the switching mechanism 242 according to embodiments of the present invention.
  • the switching mechanism 242 between the fill and void cycle can be set to trigger based on a minimum and maximum bladder pressure (see also, e.g., FIGS.26G-26H).
  • the switching mechanism 242 between the fill and void cycle may set to trigger on more complex settings, for example, filling until a maximum pressure threshold is reached and voiding until a low threshold of outflow rate is reached.
  • the infusion flow rate into the hollow organ 10 may be directly changed using the gear pump 205.
  • a fixed flow rate of 10 ml/min may be used during the fill cycles of the organ, for example, based one maximum physiological urine infusion flow rate into the porcine bladder (see, e.g., FIG.11B and FIGS.12A-12B).
  • FIG.11B illustrates an exemplary time-lapse monitoring of inflow rate, draining flow rate, and pressure of a porcine bladder within the fill-void bioreactor module 240.
  • FIG.12A illustrates an exemplary time-lapse monitoring of flow rate and pressure data of the first and last fill-void cycle.
  • FIG.12B illustrates an exemplary pressure-volume curve of the first and last fill-void cycle.
  • FIG.11C is a morphological image of a porcine bladder before and after fill and void conditioning for a 48 hour period.
  • the reactor chamber box 120 for the fill-void bioreactor module 240 may be designed with an extension piece 120e.
  • the extension piece 120e may be quickly mounted over the reactor chamber box 120 to accommodate whole organs, such Attorney Docket 9865.236.WO as the bladder, used in the fill-void bioreactor module 240 (see, e.g., FIG. 11A).
  • the support frame 110' to hold the reactor chamber box 120 as described herein may also be used to safeguard against deformation (see, e.g., FIG.2A-2C).
  • FIGS. 13A-18C the perfusion (or solid 3D) bioreactor module 250 and components thereof according to embodiments of the present invention are illustrated.
  • the perfusion bioreactor module 250 is configured to perform stimulation of solid vascularized organs by perfusion flow. Physiologically, the flow rate is regulated by the organ's natural resistance to flow and is provided by the walls of the organ's internal vascular tree.
  • the perfusion bioreactor module 250 of the present invention implements an inlet, or arterial perfusion pressure feedback system 250e, configured to regulate the stimulus to the vessel walls in a whole organ (see, e.g., FIG.13A; see also, e.g., FIGS.26I-26J).
  • the arterial perfusion pressure feedback system 250e is capable of reading perfusion flow rate(s) and pressure at the inlet(s) of the organ 10.
  • the system 250e further allows a user to choose between constant pressure perfusion or a constant flow rate perfusion or provides the option to merge the two.
  • the perfusion bioreactor module 250 may be easily used for other solid organs such as the heart or liver.
  • the perfusion bioreactor module 250 may utilize the same reactor chamber box 120 with wall extension element 120e as the fill-void bioreactor module 240 having the infusion and draining ports for the organ 10 described herein (see, e.g., FIG.11A).
  • the reactor chamber box 120 for the perfusion bioreactor module 250 may have a redesigned lid 124 for providing multiple infusion and draining ports connections on the top of the lid and the number of ports for infusion or draining depends on different organs.
  • the perfusion bioreactor module 250 includes a pressure sensor outlet 256 next to the top inlet 251.
  • a flow sensor (not seen) may be included in the same line.
  • the arterial perfusion pressure feedback system 250e includes a proportional–integral–derivative (PID) controller 300 configured to provide constant pressure perfusion over time.
  • PID proportional–integral–derivative
  • FIG.14A illustrates an exemplary setup for the perfusion testing of a solid organ 10 (e.g., a kidney) that may be implemented within the perfusion bioreactor module 250 of the present invention.
  • a solid organ 10 e.g., a kidney
  • the user interface (i.e., controller/software interface 270) of the bioreactor system 100 of the present invention allows the choice between a constant pressure perfusion or a constant flow rate perfusion since both systems make use of a pump 253 to vary the pressure or flow rate, respectively, but uses feedback from a pressure sensor 256 and flow rate sensor 255, respectively (see, e.g., FIG.14A).
  • FIG.14B illustrates an exemplary real time pressure and flow rate data as the system 100 increases to a user-defined constant pressure setting (e.g., 12 mmHg) using the PID controller 300.
  • FIGS.15A and 15B are exemplary trace graphs illustrating pressure versus infusion flow rate during pressure stability testing of the PID controller 300 and a porcine kidney (i.e., organ tissue 10).
  • FIG.15A showing whole organ perfusion through the ureter
  • FIG.15B showing whole organ perfusion through the renal artery.
  • the perfusion bioreactor module 250 further includes an organ enclosure or cushioning system 500 configured to fit within the reactor chamber box 120.
  • the cushioning system 500 is sized and configured to provide a partial or full enclosure support of the solid 3D organ 10 to easily orient the organ 10 within the reactor chamber box 120 as desired, thereby helping to eliminate any pressure fluctuations or movements due to perfusion stream and/or user disturbances.
  • the cushioning system 500 also provides a soft support structure as compared to a hard/flat organ bed.
  • the cushioning system 500 comprises support frame 510 and an organ enclosure sac 505.
  • the support frame 510 forms a cuboid structure that is sized and configured to fit within the reactor chamber box 120 of the perfusion bioreactor module 250.
  • the organ enclosure sac 505 comprises a thin silicone sheet, for example, having a thickness of about 0.005 inches.
  • the silicone sheet 505 may be perforated using a punching machine to form a plurality of apertures 505a, 505b therein.
  • the apertures 505a residing along an upper segment of the organ enclosure sac 505 are configured to receive one or more of the protrusions 512 of the support frame 510.
  • the remaining apertures or perforations 505b of the organ enclosure sac 505 allow for weaving sutures 506 through a few of these perforations 505b configured to a desired organ shape to provide a full or partial enclosure to support the whole organ (see, e.g., FIG.16B, FIG.17A, and FIG.18A). Therefore, using different orientations while packing the organ in the organ enclosure sac 505, allows fixing the orientation for the entire duration of the culture.
  • the silicone sheet is naturally compliant providing a cushion for the organ, thereby helping to avoid local stress concentration.
  • the protrusions 512 of the support frame 510 are inserted through respective apertures 505a of the organ enclosure sac 505.
  • One of the locking devices 515 is then positioned on the support frame 510 such that the protrusions 512 extending through the apertures 505a of the organ enclosure sac 505 are received within respective recesses 515a of the locking device 515, thereby securing the organ enclosure sac 505 between the locking device 515 and the support frame 510.
  • the support frame 510 with organ enclosure sac 505 (and organ 10) may then be placed into the reactor chamber box 120 and an infusion tube 507 may be connected to the organ (e.g., the renal artery (RA) of the kidney as shown in FIG.16A; see also, e.g., FIG.18A illustrating the organ enclosure sac 505 holding a kidney 10 with infusion tubes 507 connected to the renal artery, the renal vein (RV) and the ureter).
  • the lid 124 may then be placed on the reactor chamber box 120 to enclose the organ enclosure sac 505 (and organ tissue 10) therein (FIG.17A).
  • the organ enclosure sac 505 may be used in flat-bed platform (not shown) within the reactor chamber box 120.
  • the cushioning system 500 and organ enclosure sac 505 helps to hold the solid organ 10 (and cannulation) in place within the perfusion bioreactor module 250 (see, e.g., FIG.17A) and during decellularization (see, e.g., FIG.18C).
  • the cushioning system 500 can also be used for more than one organ type inside the same organ enclosure sac 505 by changing fixation based on geometry of the organ.
  • the perfusion bioreactor module 250 of the present invention is easily scalable and can provide safe, constant, and growth-inducing stimuli to the vasculature over a long period of time, with consistent feedback and control.
  • FIG.17B illustrates an exemplary real time pressure and flow rate data under a constant pressure perfusion setting within the perfusion bioreactor module 250 according to embodiments of the present invention.
  • FIG. 17C illustrates an exemplary real time pressure data under a constant flow rate perfusion setting within the perfusion bioreactor module 250 according to embodiments of the present invention.
  • FIG.18B illustrates exemplary flow dynamics evaluation results of the renal artery, renal vein, and ureter for a kidney 10 within the perfusion bioreactor module 250 of the present invention.
  • FIG.18C shows exemplary morphology of the kidney 10 before and after decellularization within the perfusion bioreactor module 250 of the present invention.
  • the remaining decellularized extracellular matrix of the kidney may be used as a tissue scaffold within the bioreactor system 100 of the present invention.
  • Each of the bioreactor modules 200 and components thereof described herein may be designed and fabricated to be compatible with a standardized mounting system 108, 108'.
  • Each of the modules 200 are configured to apply the respective tissue stimulations, described above, to a maturing tissue construct 10.
  • FIGS.19-24 the media exchanger subsystem 400 and components thereof according to embodiments of the present invention is illustrated.
  • FIG.19 is a schematic layout of the flow loop for the individual sensors and pumps (left) and the media exchange (right) of the media exchanger subsystem 400 to perfuse media in and out of the reactor chamber box 120 for a respective bioreactor module 200 according to embodiments of the present invention.
  • FIG.20A Attorney Docket 9865.236.WO further illustrates the flow loop of the sensors and pumps of the media exchanger subsystem 400 according to embodiments of the present invention.
  • the bioreactor system 100 of the present invention includes a pump panel having a plurality of pumps.
  • the pump panel comprises five (5) pumps including two small peristaltic pumps 421 for sensor monitoring module 260 (see, e.g., FIGS.
  • the peristaltic pumps 412, 416 for the media exchanger subsystem 400 include a fresh media fill pump 416 and a waste media aspiration pump 412.
  • the peristaltic pump 421 for the sensor monitoring module 260 may include a sensor loop pump 421. It is also noted that the bioreactor system 100 may include additional components not described in detail herein, for example, impellers, paddles and agitators to produce beneficial currents within the bioreactor module 200. [00147] At the start of an experiment or culture, the media exchanger subsystem 400 controls the initial fill-up of the reactor chamber box 120 for a respective bioreactor module 200 and pumps fresh media (i.e., from fresh media bag 418) into the reactor chamber box 120 via the fresh media pump 416 until the reactor chamber box 120 is filled to a predetermined height.
  • fresh media i.e., from fresh media bag 41
  • the filling of the reactor chamber box 120 with fresh media may be aided by using non-contact level sensors 429, 430 placed outside the reactor chamber box 120 at two different heights, an upper maximum and an upper minimum (see, e.g., FIGS. 21A-21C).
  • the level sensors 429, 430 are configured to alert a controller 450 to the trigger for the fill or re-fill of fresh media into the reactor chamber box 120, replenishing of media depleted by the sensing system (i.e., sensor monitoring module 260) or through evaporation, for example, when the level of fresh media within the reactor chamber box 120 drops below the minimum threshold.
  • the exchange of media from the reactor chamber box 120 may be done manually by turning on and off the waste media pump 412, followed by turning on the fresh media pump 416 until the media is filled to the desired level in the reactor chamber box 120.
  • the integration of sensors in the Attorney Docket 9865.236.WO sensor monitoring module 260 enables a user to program an automatic exchange of media from the respective bioreactor modules 200 based on thresholds monitored by different sensors (e.g., glucose/lactate sensor 423, dissolved oxygen (DO) sensor 425, pH sensor 426, and temperature sensor 427).
  • each of these sensors are programmed with threshold values of, for example, pH, dO2, glucose and lactate, which allow the bioreactor system 100 to assure that conditions within the reactor chamber box 120 for the respective bioreactor module 200 always remain within the user defined boundaries in units of each specific sensor.
  • the sensors of the sensor monitoring module 260 are integrated together directly into a single microcontroller 450 to provide feedback to users.
  • the percent volume of media to be exchanged and flow rates during the exchange with respect to a bioreactor module 200 may be defined using the software interface of the bioreactor system 100 beforehand.
  • the bioreactor system 100 i.e., the software interface
  • the bioreactor system 100 will initiate another round of measurements from each of the sensors (e.g., glucose/lactate sensor 423, DO sensor 425, pH sensor 426, and temperature sensor 427) to ensure if that an analyte is outside of a user defined threshold for the specific bioreactor module 200.
  • the exchange of media via the media exchanger subsystem 400 is commenced.
  • the media exchanger subsystem 400 first takes the product of user-defined exchange flow rates and the volume through percent volume to obtain an "ON" time for the waste media pump 412 to remove liquid from the reactor chamber box 120 to the waste media bag 416.
  • the fresh media pump 416 pumps fresh media from the fresh media bag 418 into the reactor chamber box 120 until an upper maximum of media level has been reached within the reactor chamber box (e.g., as determined by using non-contact level sensors 429, 430 placed outside the reactor chamber box 120).
  • the media exchanger subsystem 400 if the media exchanger subsystem 400 is unable to change the sensor value after media exchange or if the next media change cycle is triggered earlier than expected, for example, in the case of contamination which would rapidly carry on to replenished media, the media exchanger subsystem 400 will alert the user.
  • FIG.22 illustrates an exemplary media exchange based on the threshold boundaries of the pH sensor 426.
  • the bioreactor system 100 includes a sensor monitoring module 260 that is configured to provide real-time monitoring of environment and cellular metabolites for a respective bioreactor module 200 This may be accomplished by continuous or semi-continuous sampling the media from within the reactor chamber box 120 for any of the bioreactor modules 200. As shown in FIGS.
  • the sensor monitoring module 260 has integrated sensors for monitoring pH (sensor 426), dissolved oxygen (sensor 425), temperature (sensor 427), and glucose/lactate (sensor 423) within the reactor chamber box 120 for a respective bioreactor module 200 with the media exchanger subsystem 400 configured to automate the tasks of sampling media, sensor calibration solutions, and neutral buffers to flush out the tubing lines 411 therebetween.
  • the sensor monitoring module 260 includes three-way pinch valves 415 to switch between buffer solutions 428, calibration solutions 424, or media from the reactor chamber box 120, and is fitted with two pumps 421, 422 together.
  • one pump 421 is configured to pull media and send it back to the reactor chamber box 120 for in-line sterilizable sensors of pH 426, dissolved oxygen 425, and temperature 427.
  • the other pump 422 is configured and responsible for pulling some part of the inline sensor line to sense it separately in the online glucose and lactate sensor 423 and send it out to the waste chamber 416.
  • the flow rate of the inline pump 421 may be kept at about 10 ml/min.
  • the online sensing pump 422 may be kept below 1.7 ml/min based on manufacturer thresholds for the glucose/lactate sensor 423.
  • FIG. 20B is a graph illustrating exemplary short term data for glucose and lactate testing according to embodiment of the present invention.
  • FIG.20C is a graph illustrating exemplary long term data for glucose and lactate testing for 72 hours according to embodiments of the present invention.
  • the sensor monitoring module 260 of the present invention may be configured to sample less than 3 ml of media from the reactor chamber box 120 and perform a one-shot measurement across the all sensors and dispose it off to the waste media bag 416.
  • the sensors are placed close to each other within the sensor monitoring module 260, and the media pulled from the reactor chamber box 120 is halted when it is inside the Attorney Docket 9865.236.WO flow-through volume of the respective sensor.
  • This halt timing accuracy is achieved by a tubing bubble sensor to automatically detect the presence of liquid (media). It is then used to control the stationing of media at desired location by stopping the pump as soon a liquid is present at the desired location. The restarting of the pump is based on the maximum time of all sensor response times in the loop. [00154] To maintain cell, tissue, or organ viability, within the respective bioreactor modules 200, it is crucial to remove accumulated cellular waste over time and replenish the reactor chamber box 120 with fresh cell culture media.
  • the present invention provides the automated media exchanger subsystem 400 that may be used for all tissue types (i.e., each of the bioreactor modules 200) based on user-defined (or pre-defined) or programmable threshold values for each of the sensors integrated into the sensor monitoring module 260 for media exchange, to initiate and accomplish the exchange of media within the reactor chamber box 120.
  • the percentage of media volume inside the reactor chamber box 120 for replenishing with fresh media is also user- programmable in each trigger event (e.g., reaching a sensor threshold value).
  • the bioreactor system 100 of the present invention further includes common hardware and software controls 270 allowing operation, sensor data reading, and programming of the system by the end user (see, e.g., FIG. 24).
  • the software control program is developed in Labview, and a common hardware microcontroller 450 as described herein may be used to run the entire bioreactor system 100 platform.
  • the bioreactor system 100 may use, for example, a NI MyRio as the main control/processing unit (see, e.g., FIG.24).
  • Each of the bioreactor modules 200 may include a common multi-wire junction with a common mating system to the part permanently inside the housing 102 (e.g., incubator) and connected to the controller system.
  • the connection of each bioreactor module 200 has a unique identity saved digitally and activates on connection of the respective bioreactor module 200 within the system 100. This provides the advantage of eliminating the need to change the entire wiring, thus making it the system 100 of the present invention modular.
  • many of the components on for each bioreactor module 200 such as sensors and valves may be designed Attorney Docket 9865.236.WO to either be part of a 4-wire (two data and two power) system or a 2-wire system by systematic use of digital communication protocols such as a two-wire I2C.
  • all analog sensors or analog controllers in the bioreactor system 100 may be designed to have a dedicated I2C ADC/DAC on-site eliminating need for tedious processing in conversion for the main controller 450 and enabling a common two-wire bus line for extraction of media with the synced system clock.
  • FIGS. 24 and 25 are schematic diagrams illustrating the components of the modular bioreactor system 100 according to embodiments of the present invention, and as described herein, but utilizing an alternative mounting system 108'. As shown in FIG. 24 and FIG. 25, in some embodiments, each of the bioreactor modules 200, as described herein, may be configured to be affixed to a standardized bracket system 104' which can be inserted into a bioreactor housing 106'.
  • the bracket system 104' comprises a three-dimensional bracket configured to slide into the bioreactor housing 106'.
  • the bioreactor housing 106' is a disposable module that mates internally with the bracket 104' and externally with a universal docking station 102'.
  • the bioreactor housing 106' may comprise a rail system integral with the internal wall of the housing 106' that is configured to receive the bracket 104' (and bioreactor module 200).
  • the bracket 104' may comprise an array of graduated attachment points to secure a respective bioreactor module 200 thereto.
  • the bracket 104' (and rail system) is configured to bring the bioreactor module 200 into alignment with input/output contact points on the internal wall of the bioreactor housing 106' to allow for precise communication with the respective bioreactor module 200 across the housing 106'.
  • the universal docking station 102' is configured to provide standardized contact points for mechanical and electrical input and output to the bioreactor module 200 placed therein.
  • the docking station 102' may be configured to be used as either a stand-alone unit or within a series of parallel stations that are controlled from a single interface (e.g., controller 270).
  • the docking station 102' is configured such that when the bioreactor housing 106' is inserted into the docking station 102', all of the input/output points on the bioreactor housing 106' (and bioreactor module 200 inserted therein) are brought into alignment with the corresponding contact points Attorney Docket 9865.236.WO within the docking station 102', i.e., similar to the interaction between the bracket 104' and bioreactor housing 106'.
  • the bioreactor housing 106' may be designed with a molded internal structure that will allow for the standardized bracket 104' to be inserted into the housing 106' such that it will bring the bioreactor module 200 into a position that will allow electrical and mechanical communication with the docking station 102'.
  • the docking station 102' is also configured provide thermal regulation and receive sensor telemetry from within the respective bioreactor module 200 (e.g., via the sensor module 260) to maintain a controllable, uniform, and constant temperature throughout the bioreactor module 200 held therein.
  • the docking station 102' comprises a heating unit to maintain the media within the bioreactor module 200 at a controlled and constant temperature.
  • the docking station 102' is also connected with the sensor suite (i.e., sensor module 260) either through a physical contact point or using radio frequency.
  • the sensor suite i.e., sensor module 260
  • at least a portion of the media exchange subsystem 400 associated with the bioreactor housing 106' may extend into the docking station 102'.
  • the docking station 102' may include an insulated refrigeration unit intended to preserve heat-labile factors.
  • the docking station 102' may further include a mechanism for injecting factors into the bioreactor media (i.e., bioreactor module 200), as the factors become depleted.
  • the standardized bracket 104' may be configured to enable the affixation of a wide variety of modular bioreactor hardware and tissue actuators including spinners, agitators, tissue anchors, mechanical actuators, cannula, and electrodes, as described herein.
  • the bracket 104' is sized and configured to fit within the disposable bioreactor housing 106' to provide mechanical and electrical input to the bioreactor module 200 placed therein, as well as permitting output from system sensors (i.e., sensor module 260), all without the need for breaking containment of the bioreactor system 100.
  • the bioreactor system 100 comprises a reliable gasket closure that results in a reliable hermetically sealed environment.
  • the internal geometry of the bioreactor housing 106' is configured to provide a consistent current within the bioreactor housing 106' (and corresponding bioreactor Attorney Docket 9865.236.WO module 200), for example, driven by an impeller positioned at the bottom of the bioreactor housing 106'.
  • the bioreactor system 100 may provide computer modeling and colored dye studies to indicate and minimize dead zones within the bioreactor module 200.
  • the bioreactor system 100 includes an integrated reservoir (i.e., the media exchange subsystem 400) for replenishment of nutrients and oxygen. All electrical and mechanical components of the system 100 may be regulated by a peripheral interface card that will provide output for the system operator.
  • the system includes an operating system (i.e., controller interface 270) which includes user-friendly software that allows the end-user to easily create programs to operate the bioreactor system 100 in any desired manner.
  • the sealants, tubing, connectors and all hardware components of the bioreactor system 100 that come into contact with biomaterials, cells or fluid are fabricated from materials that are considered safe and appropriate by regulatory agencies.
  • glass or poly(methyl methacrylate) have been used for bioreactor housings due to minimal chemical leeching/adsorption.
  • materials such as glass, polymers, ceramics, metal alloys and a variety of sealants may be used in the bioreactor system 100 of the present invention.
  • a major concern is shedding of particles that may become incorporated into the tissue construct.
  • Biocompatible metals such as titanium may be primary used for moving parts within the bioreactor system 100 of the present invention. However, new polymers and alloys that have sufficient strength and limited reactivity may also be used. Stainless steel offers an inexpensive option for internal hardware that does not move or contact another moving part. When internal components, such as batteries, that are comprised of unsatisfactory materials are required, they will be portioned within a hermitically sealed shell.
  • the core functional units of the bioreactor system 100 of the present invention are the bioreactor modules 200 and tissue actuators therein. These units will provide all of the stimuli that will condition the tissue engineered constructs 10 for implantation.
  • each of the tissue actuators for the respective bioreactor modules 200 may also be modular and interchangeable (see, e.g., FIGS. 26A-26J).
  • the actuators are compatible with the mounting systems 108, 108' described herein such that they are able to receive mechanical and electrical input from the sensor module 260 and transmit these forces to a maturing tissue engineered construct within the respective bioreactor module 200 in a controllable manner.
  • adjustable push/pull rods are aligned with tissue anchors and configured to affect uniaxial or biaxial tissue exercise in the uniaxial bioreactor module 210 (see, e.g., FIGS.4A- 5B and FIGS.26A-26B) and the biaxial bioreactor module 220 (see, e.g., FIGS. 6A-6B and FIGS.26C-26D).
  • the uniaxial bioreactor module 210 may also contain integrated electrodes for providing electrical stimulation to conductive tissues.
  • a peristaltic pump in the pulsatile (or tubular) bioreactor module 230 is configured to provide pulsatile flow through a tubular construct in either direction (see, e.g., FIGS.7-8B and FIGS.26E-26F).
  • a volumetric tissue anchor system with associated pumps in the fill-void bioreactor module 240 is configured to induce cyclic fill/void stimulation (see, e.g., FIGS.9A-12B and FIGS.26G-26H).
  • a perfusion system with resistance feedback capabilities the perfusion bioreactor module 250 is configured for conditioning the vasculature within three dimensional solid constructs (see, e.g., FIGS.13A-18C and FIGS.26I-26J).
  • An integrated sensor suite (e.g., sensor module 260) comprising a sensor array including oxygen, pH, temperature, glucose, lactate, pressure and flow sensors is configured to interface with the bioreactor housing 106'. Temperature and flow sensors are included to maintain consistent environmental conditions within the bioreactor module 200. Glucose, lactate and oxygen sensors are configured to provide feedback for replenishment of required factors through an integrated and hermetically sealed interface with the docking station 102'.
  • the sensor module 260 may be configured to be amenable to accepting additional sensors as needed for future applications.
  • a conditioning module e.g., media exchange subsystem 400 including a disposable and fillable feed reservoir containing nutrients, waste scavengers (carbon dioxide and lactic acid) and oxygen generation is configured to interface with the bioreactor housing 106'.
  • the media exchange subsystem 400 includes mechanisms for input/output within the bioreactor module 200, as well as strategies for operation through the bioreactor housing 106' without the need to break containment.
  • the media exchange subsystem 400 may be designed with reservoirs that extend into the docking station 102' to isolate these reservoirs from the higher temperatures within the bioreactor module 200.
  • the docking station 102' is configured to provide both input and output to the bioreactor system 100 without the need for breaking containment.
  • the mechanical operation of the bioreactor system 100 may be included in the docking station 102', and thus, will be hermetically isolated from the reservoir contents (i.e., the respective bioreactor module 200). In other words, mechanical input into the bioreactor system 100 (i.e., into the bioreactor module 200) may be driven external to the bioreactor module 200.
  • mechanical input of the bioreactor system 100 may be designed around magnetic transmission of force.
  • a magnetically-controlled drive shaft may be configured through various gears to apply mechanical force in any required axis or geometry to facilitate fluid circulation or tissue exercise.
  • these magnetically-controlled drive shafts are configured to mate with worm gears, helical gears, universal joints, screws and other mechanical force transmission devices to convert force to any desired vector for powering mechanical components of the modular bioreactor system 100 internal hardware structures.
  • electrical input into the bioreactor system 100 either to provide electrical stimulation to conductive tissue or to charge batteries associated with internal components, may be provided either through external induction coil technology or trans-housing integrated electrical contact points.
  • sensor telemetry output may be either through radio frequency or hermetically sealed trans-housing contact points.
  • a battery compartment may be required within the bioreactor system 100 to buffer against transient periods of high electrical demand.
  • a peripheral interface card e.g., controller interface 270
  • the controller interface 270 is configured to automate and regulate the activities of all components associated with the bioreactor system 100.
  • the controller interface 270 is configured operate and regulate all input and output from the bioreactor modules 200 through the docking station 102' and may be mated with a user-friendly display and interface device (see, e.g., FIG.25).
  • the controller 270 is configured to interface with a standard PC, and has sufficient capabilities to handle multiple simultaneous platforms, in parallel. Attorney Docket 9865.236.WO [00170]
  • a customized software may be developed for programing system operations and provide an easily interpreted display output for sensor data.
  • the controller interface 270 and associated software may be amenable to production scale-up by supporting multiple-parallel systems.
  • the controller interface 270 may allow for simultaneously operating multiple parallel bioreactor systems 100.
  • the bioreactor system 100 may be configured to interface with a centralized integrated system that is able to monitor and control multiple bioreactor systems 100 for scaled- up mass production, thereby providing a standardized and modularly configurable bioreactor platform that could be applied for the fabrication and maturation of a wide variety of regenerative medicine clinical products and provide the capability to monitor and control the internal bioreactor environment that could condition tissue engineered constructs for implantation.
  • interface software of the bioreactor system 100 is engineered using a commercially available package.
  • the software is designed to be very user friendly and to run in a Windows environment.
  • the software has the capability to address each electrical component of the bioreactor system 100.
  • the graphical user interface is configured to display the current system configurations and allow the user to select individual bioreactor modules in order to access further control options.
  • the speeds of respective pumps and motors of the bioreactor system are adjustable in through the interface, with options for changing speed over time at either a linear or logarithmic rate.
  • the ability to program operation cycles at any frequency is also available.
  • the software is further configured to interface with the environmental control module (i.e., sensor module 260 and the media exchange subsystem 400) to provide easy adjustment of environmental conditions within the bioreactor system 100.
  • the bioreactor system 100 may comprise a display for reporting current and historical information regarding environmental conditions as well as system hardware performance provides data for identifying and troubleshooting problems.
  • the modular bioreactor system 100 of the present invention described herein provides a standardized, self-contained, and modular bioreactor platform that allows scale-up and automation for the clinical manufacturing of a wide range of regenerative medicine products over a range of sizes and geometries.
  • the interchangeable modular bioreactor system 100 of the present invention provides the advantage for maturation and preconditioning of multiple tissues/organs in a single unified plug and play setup.
  • the system 100 is capable of producing optimal conditions for both Attorney Docket 9865.236.WO the tissue biofabrication and tissue maturation phases of engineered tissue clinical manufacturing. All of the materials used in the construction of system 100 components that come into contact with biomaterials, cells or fluids will meet regulatory standards for clinical manufacturing equipment.
  • the physical arrangement of components for each of the modules 200 described herein may be configured to include a quick disconnect fitting system for each component mounted on respective module 200, thus making the bioreactor system 100 of the present invention a user friendly, easy to modify, and easy to upgrade system.
  • bioreactor systems are often seen with customized industrial solutions for just one function in the assembly. These solutions can be costly because of the product quantity. Therefore, the current state of device development such as bioreactors are often done as individual projects and hence there exists a huge potential for a standardized platform allowed for by embodiments of the present invention.
  • the modularity of the system 100 provides the ability to repurpose the system for multiple applications at greatly reduced cost.
  • modular bioreactor system 100 of the present invention is tailored for use in the biofabrication and tissue maturation phases of engineered tissue production
  • components of the system such as the standardized bioreactor housing and associated hardware/software could be adapted for use with any cell-based therapy.
  • the sensor array that will interface with the bioreactor system, along with associated display/interface software could have applications in a wide variety of clinical manufacturing applications.
  • EXAMPLES [00176] The following EXAMPLES provide illustrative embodiments. Certain aspects of the following EXAMPLES are disclosed in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the embodiments. In light of the present Attorney Docket 9865.236.WO disclosure and the general level of skill in the art, those of skill will appreciate that the following EXAMPLES are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently claimed subject matter.
  • Example 1 Multifunctional Uniaxial Bioreactor
  • Bioreactors have been traditionally used for growing in vitro cell products such as fermentation bioreactors, and provided a strict biochemical growth environment where a particular cell type can multiply in a controlled manner.
  • Tissue bioreactors are different as the culture involves cells that are usually loaded on or adhered to 2D or 3D porous growth sites to allow cells to form colonies in a certain physical geometry, and the bioreactor provides anchoring site, maturation environment, and conditioning to this combined structure.
  • These tissue products do not have the strength to function like native tissue and need further differentiation and conditioning in a tissue bioreactor.
  • uniaxial bioreactors Since a skeletal muscle has two anchoring points via tendons and generates uniaxial contraction force along that line, the devices made to mature these cultures are often called uniaxial bioreactors.
  • biophysical and biochemical environments should match the in vivo conditions, which means loading and unloading cycles, shears due to blood flow, and response to electrical pulses through neural activation. Therefore, to achieve a system with multiple conditioning components combined for better tissue growth, uniaxial bioreactors must overcome challenges in physical manipulation of tissue, stimulus delivery component integration, biochemical environment sensing and regulation, material property, biocompatibility, and sterility.
  • these bioreactors have specifically evolved into a uniaxial stretching system that strains the live muscle tissue under assignable static or cyclic, stretch protocols. This is usually done by making one end of this muscle stationary and the other end connected to a movable end. This is the first subcomponent that has been added for providing a tensile force on the tissue as means of exercise, which is seen to improve cell alignment, increase cell proliferation, facilitate myogenesis and myotube formation, increase force production and hypertrophy and also seen to maintain the viability of native tissue.
  • the second subcomponent in these bioreactors is electrical stimulation delivery to the growing tissue.
  • bioreactors need a system for sensing media in real-time so that the media exchange step can be done based on change in these parameters and not arbitrarily.
  • a highly multifunctional uniaxial bioreactor has been developed that can be configured for dynamic culture protocols, and mechanical and electrical stimulation parameters independently for three 3D muscle cultures, while using off the shelf components and 3D printing to enable this, along with giving feedback about actuator movement, electrical stimulation pulses, force generated, macro imaging, and media sensing.
  • the uniaxial bioreactor includes (i) a mechanical subsystem with three stepper linear actuators, which facilitates programming separate strain ratios, (ii) an electrical stimulation subsystem by integrating carbon electrode pairs to each culture lane that can take pulse input from any commercial stimulator, (iii) force sensor integration to each culture lane to read mechanical stretch forces and isometric contraction forces, (iv) and off the shelf media sensors such as pH, dissolved oxygen, temperature, glucose, and lactate integrated to the system.
  • the designed system also has a space on lid for monitoring via macro Attorney Docket 9865.236.WO imaging.
  • a fixed volume media exchange system has been integrated which can be triggered manually through on/off buttons or timed via a system clock and relays.
  • CAD components were developed using Solidworks and exported them in stereolithography format.
  • the wetted incubation components were printed using a commercial biocompatible photopolymer resin (Formlabs Biomed Clear) printed using SLA desktop 3D printer (Formlabs 3B) (see, e.g., FIG.27).
  • Formlabs 3B SLA desktop 3D printer
  • the system is configured to separate stimulation patterns to be individually defined in three lanes of the bioreactor (see, e.g., FIG.28A).
  • FIG. 29A Parallel carbon electrode plates were integrated for stimulating the tissue during culture and force response measurement from contractile activity (see, e.g., FIG. 29A). This provides a way to generate constant unidirectional electric field on the tissue volume.
  • Three pair of positive and negative male mini-banana connectors are designed to individually receive the stimulus pulses up to the box, with platinum wires used on the inside to connect to the carbon plates.
  • a 4-channel electrical stimulator (DMT-CS4) is integrated, and digital oscilloscope was used for verification.
  • Futek uniaxial force sensor
  • a third three-way valve is used which pulls incubator air and creates an air gap between two consecutive fluids going to the sensor.
  • the perfusion loop also has an added ultrasonic no-contact liquid sensor (AE-301, Panasonic Industrial Automation) which automates the liquid travel up to the sensor point by sensing the presence and absence of liquid in the tubing non- invasively. All tubing used are C-Flex Process tubing (Masterflex) of 1/16” or 1/8” inner diameter, and pump tubing is platinum cured silicon. Tubing and flow through sensor ports are coupled to tubing using appropriate polypropylene hose barb or luer connectors.
  • Bioreactor assembly There are three parts to the physical assembly of the whole device: (1) assembly of the bioreactor box, (2) sensor loop, and (3) media exchanger. Assembly of the bioreactor involves preparing three lanes for culture experiment. The physical system has two parts, the linear actuator box, and the culture chamber (see, e.g., FIG.27). The actuator box holds three stepper motors, loaded with encoders, and fixed to the base with resin (see, e.g., FIG.28A).
  • the moving base on top of the linear ball screw guide of each of these motors connect to a tissue holder shaft in each lane passing through culture chamber wall, making a movable end for the muscle tissue.
  • a 3D printed base piece is fitted inside the culture chamber, which holds the force sensor mounts on the other static end for three lanes.
  • the tissue holder shaft loads to the stepper guide using a push-rotate locking mechanism for easy loading.
  • the box is ready to hold the tissue (see, e.g., FIG.30A).
  • the last step in physical assembly is sealing the chamber by taking the lid with electrodes assembly and carefully placing it around the three culture lanes.
  • the lid also has three imaging ports of rectangular shape to fit a glass slide on top to see through. These two boxes after joining via shafts, are loaded onto a frame fitted on top of a 9.5 mm thick polycarbonate Attorney Docket 9865.236.WO sheet with UV/Scratch resistant properties, using snap-fit connectors with 12 mm diameter feet for locking.
  • the perfusion system for the bioreactor is shown in FIG.32A, and shows the flow line, normally open and normally close (NO & NC) lines of the three-way pinch valves which switch between the sampling solution for glucose and lactate sensor.
  • media is pulled from culture box at defined intervals to measure the analytes and sent out to a waste bag.
  • the sensing system is assembled on another polycarbonate tray with all components mounted on dedicated 3D printed holders.
  • One end of the system has tubing with a quick disconnect connector to quickly hook it up to bioreactor for perfusion as shown in FIGS. 32B and 32C for all the components in the perfusion system.
  • the tray holds the PBS solution and glucose/lactate calibration solution (2747 Standard, YSI) in two different bags (250 ml 2D labtainer, Thermofisher Scientific) for intermittent wash and calibration in the glucose and lactate sensor.
  • the other end is connected to a waste plastic container (2000 ml 2D labtainer, Thermofisher Scientific).
  • the last part is the media exchange system (FIG.31), which has two peristaltic pumps (KDM- OCM, Kamoer, CN) for media aspiration and replacement.
  • the first one connects the box to a waste media bag (2000 ml 2D labtainer, Thermofisher Scientific) for removing old media, and the other one for taking media from the fresh media bag (250 ml 2D labtainer, Thermofisher Scientific) to the culture chamber.
  • the waste media bag, and the media exchanger pumps are placed outside the incubator with tubing passing through the rear access hole of the incubator.
  • Both pumps are controlled using physical on/off buttons and are calibrated for flow rates and set to 100 ml/min for withdrawal and 60 ml/min for refill, which was kept fast enough to finish the replacement quickly while not disturbing the construct.
  • the USB Camera(s) held inside a 3D printed assembly gets placed on top of the culture chamber lid and can image through the glass slide window. An adjustable light source is kept below the upper tray to light up the scene for capturing images and videos.
  • both polycarbonate trays for the bioreactor and sensor perfusion loop are cut and designed to be easily loaded on to a CO2 Incubator (MCO-50AIC-PE, Panasonic Healthcare) in a tray format.
  • MCO-50AIC-PE CO2 Incubator
  • the encoder can provide information about current position, speed, and direction of rotation of the stepper motor and based on resolution of stepper driver, the resolution, or pulses per revolution of the encoder can be set.
  • the motor Attorney Docket 9865.236.WO software uses parameters such as (i) length of cultured tissue, and (ii) percent maximum strain to generate amplitude of strain. For this study, we have generated two protocols that we use: a stepwise strain protocol with further choosing (iii) step size as percentage of original length and (iv) wait time between steps (FIG. 30B).
  • the second protocol of cyclic stretch protocol with further user inputs of (v) frequency in hertz to define speed, (vi) train time to internally calculate number of stretch cycles in nearest whole number by truncating the decimal part, and (vii) wait time between two trains (FIG.30C).
  • Electrical Stimulation Since electrical stimulation is important for testing the contractile activity, and stimulus during culture, it is important that tissue receives a uniform electric field where it is placed inside the culture media. Since this is a 3D tissue, we try to make use of a plate rather than a rod or point electrode to ensure unidirectional field. The carbon plate electrodes placed inside the culture chamber get immersed in media inside the chamber box.
  • the force sensor utilized in the system is an inline compression and tension sensor and has M3 male thread mounts on both ends (FIG. 30A).
  • the sensor body is made of stainless steel and wiring point is covered with a silicon elastomer to prevent contact with polymer coating from the manufacturer.
  • the body is further locked inside a 3D printed shell to minimize fluid contact and three of these assemblies get fixed on the shells facing the culture lane on force sensor holders designed as part of the base plate.
  • the base plate can be temporarily held vertically with a custom vise to hang the weights and use small calibration points by droplets of water through calibrated pipettes.
  • the force sensor amplifier can save four calibration curves in the EEPROM and can be accessed through the software.
  • the main objectives of using a force sensor are to (i) measure contractile force, (ii) quantify mechanical property non-destructively, and (iii) measure stretch forces during mechanical stimulation. To qualify it for use with engineered muscle, we performed experiments using a small size fast twitch muscle and selected rat EDL to read twitch forces. Force output data for different frequency were recorded for contraction force measurement (FIG.30C). Mechanical property of the tissue can be quantified using the force output during stretch, and with knowledge of 3D morphology of the cross-section area over time, applied strain, and force output for each time point (FIG. 30B).
  • the pH and dissolved oxygen sensor have longer lifetime sufficient for long term culture, without re-calibration.
  • PBS was sampled and stopped.
  • calibration solution is sampled and stopped at the sensor area in the perfusion loop. The system can alter the sample source from the three solutions automatically overtime by defining (i) the interval of re-measurement for sampling media again from bioreactor, (ii) duration of data sampling from sensors to write to file, and (iii) number of measurements before recalibration.
  • the liquid sensor ON or OFF is utilized to make triggers to start and stop pump, rather than time, so that the perfusion system can define functions such as "wait till ON", or "wait till OFF” to automate movements of sampled fluids.
  • the appropriate stoppage of each fluid at the required location and their measurement recording thereof in the perfusion loop is programmed based on the maximum of the time individual sensors take to reach 95% of the sensor's final response value through reaction time studied by exposure to different analyte solutions for each sensor.
  • the hardware to Attorney Docket 9865.236.WO read glucose and lactate sensor is an OEM transmitter connected to controller via UART and has physical connection to the sensor with perfusion chamber.
  • the hardware to read pH and DO sensor connects to sensors via two optical fiber cable lines and calibrates and saves data via USB software.
  • the temperature sensor connects to a simple voltage divider circuit to read change in resistance in the thermistor-based flow through temperature sensor and saves data via analog input to microcontroller.
  • a USB camera sits on top of the lid facing the culture lane. Through the imaging ports, the 120-degree wide angle lens captures entire culture lane, capable of collecting up to 16MP at 30 fps, and minimal focus adjustments by slightly adjusting height using the camera holder fixture. Focus can also be adjusted by distance taken of the lens from the tissue surface distance from CAD models and adjusting the M12 threaded lens.
  • the two sets of trays were designed for bioreactor and media sensing for full functionality of the system as shown in FIG.32A. All wet parts can be sterilized using low temperature EO treatment.
  • the 3D printed parts are made of biocompatible resin with minimal toxicity for culture period and are washed/cured using manufacturer guidelines.
  • the first tray has the bioreactor culture chamber with a mechanical stimulation system, force sensors and electrical stimulation electrodes.
  • the second tray has pinch valves, five media sensors, glucose lactate transmitter, liquid sensor and bags with PBS and calibration solution.
  • a mechanical stimulation subsystem was designed with real-time feedback capable movements that can be defined via a protocol. The system is capable of micro stepping up to 1800 steps per revolution to provide high resolution in stepping and an absolute rotary encoder up to 4096 pulses per revolution to read correct data about movement.
  • the system can provide stimulation of at least 10% to tissue of length up to 5 cm, due to limitation in size of the box and length of the stepper guide.
  • Culture protocol and stepwise strain Attorney Docket 9865.236.WO protocol recorded through encoder are shown in FIG.28C which showed no error in the stepper's movement as compared to the desired or output profile.
  • Multiple protocols were designed to stimulate the tissue including cyclic, static, stepwise static and intermittent cyclic and more can be easily defined using parametric protocol editing.
  • Electrical Stimulation Results Continuity tests of the electrode circuit showed a closed circuit when placed in media, and the current output read through voltage drop across a known value resistor has proportional increase on increasing voltage.
  • An automated sampling perfusion sensing system has been designed to collect more information about the cellular metabolites and environment of culture (FIG.31).
  • the time taken to read a new analyte was 45 seconds for glucose and lactate sensor, 20 seconds for the temperature sensor and 10 minutes for both pH and DO sensors. Since maximum settlement time was seen in the pH and DO sensors, the time spent in measuring different analytes was set to trigger after 10 minutes and set to collect data for a minute from all sensors. Because of this, the sensor loop can re-measure only after 11 minutes period on exposure. Through calibration solution in the system, the auto sampling can automatically calculate the gain for the output even when sensor gets old.
  • the designed sensing system therefore is capable of reading temperature between 20- 40 degrees Celsius, owing to the resistor configuration used to increase sensitivity in the range of interest.
  • the Glucose and lactate concentrations measurement ranges are 0-25 mM, and 0-12.5 mM, respectively.
  • the pH sensor range is limited to 6–8 as cell cultures are usually neutrophilic.
  • the dissolved oxygen saturation range that can be monitored is 0-21%.
  • the error % of these sensor hardware from their manufacturers are within the scope of our experiments.
  • Attorney Docket 9865.236.WO Example 2: Multifunctional Uniaxial Bioreactor [00203] Bioreactors are used to dynamically condition engineered tissues to achieve a required degree of maturation prior to implantation in vivo.
  • Integrating sensors and imaging capabilities into these devices could advance understanding of how the culture environment influences tissue maturation and growth. Additionally, this allows for monitoring tissue constructs within the bioreactor and provides critical information for quality control.
  • the present example aimed to develop a standardized, self-contained, uniaxial bioreactor module for the clinical manufacturing of tissue constructs that would benefit from unidirectional mechanical and/or electrical stimulation. Toward this goal, integrated stimulation and sensing components may provide optimal culture environment and monitoring capabilities to improve tissue manufacturing outcomes.
  • the uniaxial bioreactor module according to embodiments of the present invention includes integrated, user-friendly mechanical and electrical stimulation with force measurement to enhance the preconditioning of engineered tissues.
  • a sensor loop and media exchange system are integrated to monitor the culture environment and cellular metabolites over time, and a camera system placed above the tissue construct allows for macroscopically visualizing the tissue maturation process.
  • an onboard media exchange system may be programmed into the module to maintain aseptic culture conditions in the long term. The performance of the uniaxial bioreactor module was validated using native skeletal muscle tissue and tissue-engineered skeletal muscle constructs to confirm the utility of the multifunctional bioreactor to precondition and enhance tissue maturation.
  • the module was designed to fit inside a compact cell culture incubator (MCO- 50AIC, PHCbi, Wood Dale, IL), which maintains consistent temperature (37° C) and CO2 (5%). Attorney Docket 9865.236.WO [00206] Due to the availability of digital communication ports, the AVR-based ATMega2560 microcontrollers were used as the main microcontroller system during development. All wiring was PVC-insulated copper conductors. [00207] Integration of mechanical stimulator.
  • the encoder's internal pulse per revolution resolution can be increased or decreased.
  • the motor software uses parameters such as (i) length of tissue construct and (ii) percent maximum strain to generate the strain amplitude.
  • Two protocols were generated for complete system validation studies (FIG.33C).
  • Protocol 1 A cyclic strain with operational inputs, such as frequency (Hz), to define speed and train time to calculate the number of stretch cycles internally rounded to the nearest integer and wait time between the two training cycles.
  • Protocol 2 A stepwise strain with the tunable step size as a percentage of the original length and adjustable rest time between steps to generate a stress-relaxation testing system.
  • a program was developed to increase or decrease the distance between anchors for correct pre-tensioning.
  • MyoPulse software (DMT, Ann Arbor, MI) was used to control the stimulator functions, including pulse width, frequency, amplitude, and train parameter protocols, and could be triggered to start channels individually or in combination using the microcontroller of the bioreactor.
  • the digital output from the microcontroller was used as a trigger signal received through the rear panel of the stimulator.
  • the fourth channel of the stimulator was connected to a digital oscilloscope (Picoscope, Pico Technology, UK) (FIG.34B).
  • FIG.34B Digital oscilloscope
  • Integration of force sensor Three stainless-steel uniaxial force sensors (Customized LCM100, FUTEK, USA) with waterproof and corrosion resistance properties were integrated into the system (FIGS. 35A-35E).
  • the inline compression and tension sensors have M3 male thread mounts on both ends.
  • the body was locked inside a 3D-printed shell to minimize fluid contact with the cable, and one of the M3 threads was mounted to the removable base plate inside the culture chamber.
  • the tissue gripper was screw mounted to the other M3 thread at the end of the sensor.
  • the output was fed to a digital gain amplifier with a high signal-to-noise ratio (USB520, FUTEK, USA) and then to calibration and measurement software (SENSIT, FUTEK, USA).
  • USB520 digital gain amplifier with a high signal-to-noise ratio
  • SENSIT FUTEK, USA
  • the base plate mounted with a force sensor was temporarily held vertically within a custom vise to hang standard weights of 1-5 grams for stepwise calibration up to 100 grams.
  • small calibration steps were measured by the addition of small volumes of water with a micropipette (PIPETMAN G, Gilson Incorporated, Middleton, WI). A 1 g/ml for the density of the water was assumed to calculate force.
  • the force sensor amplifier can save four separate calibration curves in the EEPROM that can be easily accessed and selected through the software. After the calibration of each sensor, the base plate was fixed on the bottom of the bioreactor chamber. [00212] Integration of sensor loop system.
  • a sensor loop was developed that contained Attorney Docket 9865.236.WO several single-use flow-through sensors connected to the bioreactor through a perfusion loop system using a peristaltic pump (P625, Instech Labs) (FIG. 36A). All tubing used was flexible process-tubing (C-FLEX Clear Process tubing, MASTERFLEX) with a 1/16” inner diameter, and the pump tubing used was Pharmed BPT tubing (Pharmed Pump Tubing/P625, Instech Labs). The flow-through sensor ports were coupled to the tubing using polypropylene hose barbs, lure-locks, and valved quick disconnect body and insert pairs (Colder Products Company).
  • the pH and DO sensors are pre-calibrated and come with a calibration code that can be manually entered through the software.
  • Temperature sensor A single-use temperature sensor (Pendotech, Princeton, NJ) was placed in line with the pH and DO sensors. The temperature sensor connects to a simple voltage divider circuit to read the change in resistance in the thermistor-based flow. Data was saved via analog input to the microcontroller.
  • the sensor loop system was set to withdraw a fixed volume sample ( ⁇ 200uL) from a desired source.
  • a small peristaltic pump placed at the end of the one-way sensor loop can be switched to collect a sample from either a PBS buffer solution bag, a glucose/lactate calibration solution bag, a bioreactor media bag, or an incubator air supply via three three-way solenoid pinch valves, labeled PV1 to 3, which were activated by a microcontroller using a standard relay circuit (FIG. 36A).
  • a hit-and-hold circuit CoolCube Cold-Parmer, Vernon Hills, IL
  • PV1 was used at the beginning of the sensor loop to pull incubator air to separate different fluids going to the sensor.
  • the flow rate was set to 1.6 ml/min without exceeding Attorney Docket 9865.236.WO the limits of the glucose and lactate sensors, which have the smallest flow rate limit among all the sensors in the loop. [00217]
  • the no-contact liquid-bubble sensor (AE-301, Panasonic Industrial Automation, Newark, NJ) was installed to automate the movement of fluid in the sensor loop by non-invasively detecting the presence and absence of liquid in the latched tubing. Altogether, the system was designed for semi-continuous monitoring to minimize the exposure of the glucose and lactate sensors in highly concentrated glucose media, which could degrade sensor performance during long-term operation.
  • a buffer wash (PBS) system was added to rinse residual media in the sensor loop tubing.
  • a calibration solution containing fresh, complete media with known glucose and lactate concentrations was sampled to calibrate the sensors after a user-defined number of rounds.
  • the initial glucose and lactate content in complete media was measured by the CEDEX Bioanalyzer (Roche Diagnostics, Switzerland).
  • the system autonomously selects sample sources (bioreactor media, PBS washing buffer, or calibration solution) with air gaps to prevent liquid collapse.
  • the system records glucose and lactate sensed values by pre-defining variables like measurement interval, count before re-calibration, known concentrations in the calibration solution, time to plateau, and data averaging duration before file recording.
  • the liquid-bubble sensor's ON and OFF outputs function as triggers to start and stop the pump and switch relays running the solenoid valves.
  • the perfusion system can define functions such as "wait till ON” or "wait till OFF” to automate the movement of sampled fluids.
  • the appropriate stoppage of each fluid at the required location and the measurement and recording thereof within the perfusion loop were programmed based on the maximum time for individual sensors to reach 95% of the sensor’s final response value. This time was examined through reaction time studied by exposure to different analyte solutions for each sensor.
  • the last component to be assembled was the media exchange system, which has two peristaltic pumps (KDM-OCM, Kamoer, CN) for media aspiration and replacement (FIG. 42).
  • the first one connects the bioreactor housing to a media waste bag (2000 mL 2D Labtainer, Thermofisher Scientific) for the removal of old media, and the second for transferring media from the fresh media bag (250ml 2D Labtainer, Thermofisher Scientific) to the culture chamber.
  • the waste media bag and the media exchange pumps were placed outside the incubator with tubing passed through the rear access hole of the incubator.
  • Both pumps were controlled using physical on/off buttons, calibrated for flow rates, and set to 100 ml/min for withdrawal and 40 ml/min for refill. This refill rate was selected for a fast media exchange that did not disturb the tissue- engineered construct.
  • Integration of imaging system The lid of the bioreactor system was designed with three rectangular ports, into which a glass microscope slide is secured for imaging the construct in real time.
  • the USB-16MP (Arducam, China) low light camera assembly consisted of a 1/2.8” 16MP IMX298 image sensor secured within a 3D printed case. The entire assembly was placed on the top of the culture chamber lid facing the culture lanes, allowing for imaging through the glass slide window.
  • the tendons at each end of the EDL muscle were aseptically secured to tissue grippers in the culture chamber, with one end affixed to the force sensor and the other attached to the linear actuator.
  • DMEM Modified Eagle Medium
  • rat EDL muscles were loaded into two separate bioreactors for electrical and mechanical stimulation, respectively.
  • the mechanical stimulation protocol for the muscles consisted of a 10% cyclic stretch every 20 seconds for 5 minutes, followed by a 55-minute rest period over 3 days.
  • the electrical stimulation protocol consisted of 10 V electrical pulses with a pulse width of 0.3 ms, a frequency of 70 Hz, and a pulse train period of 400 ms, which was repeated every 1.6 min for 3 days.
  • Static control muscles were placed into the bioreactor culture chamber without anchoring the tissue grips and left free floating. After 3-days of culture, all muscles were collected and fixed with 10% neutral buffered formalin for processing and histological assay. After fixation, muscles were paraffin-embedded, sectioned, and stained with H&E and Masson’s Trichrome. All sections were imaged using an optical upright microscope (BX-63, Olympus Life Science Solutions, Japan) and quantified using ImageJ software (NIH, Bethesda, MD). [00224] Tissue-engineered muscle construct model.
  • C2C12 myoblast cells (American Type Culture Collection, Manassas, VA) were used at passage 6 or lower, cultured in growth medium (low-glucose DMEM and 10% fetal bovine serum), and used to generate tissue-engineered skeletal muscle.
  • the engineered muscle mold was designed using SolidWorks software (Hawk Ridge Systems, Mountainview, CA) and manufactured using the Form 3B+ 3D printer (Formlabs, Somerville, MA).
  • the 3D-printed mold consisted of 3 parts: the base piece with anchoring pillars, the middle well element, and the top lid element (FIGS.44A-44C).
  • the engineered muscle was fabricated in the mold using a gel-based casting method.
  • the base and middle well pieces were assembled, and two 4x8 mm polyethylene cutouts with pore sizes of 45–90 microns (SP Bel- Art Fritware, Warminster, PA) were inserted through pillars of the base elements, followed by the addition of a fibrin-cell mixture with a cell density of 5x106 cells/ml in DMEM supplemented with 10 mg/ml fibrinogen, 10 units/ml thrombin, 0.08 mg/ml aprotinin and 50 ng/ml insulin growth factor.
  • the engineered muscle constructs were polymerized in molds inside the culture hood for 10 minutes prior to equilibration inside the incubator for another 50 minutes.
  • Tissue-engineered muscle morphology was evaluated after a 14-day differentiation period. To observe tissue transparency, all engineered muscles were placed on an LCD panel (LightPad-Go, Cricut) with the same backlit light density.
  • Engineered muscles were then loaded into the base element of the mold with the two sets of pillars to observe differences in length after stimulations compared with the original length.
  • All engineered muscles were processed for histology and immunostaining. Briefly, samples were fixed with 10% neutral buffered formalin at room temperature for one hour, followed by washing with PBS. The central region of each fixed sample, approximately 5 mm in length, was excised for immunostaining. Samples were treated with 0.2% Triton-X in PBS for 5 min, followed by rinse in PBS, and blocked using 1X protein block (AB126587, Abcam, UK) for 20 min.
  • the samples were incubated with primary antibodies against myosin (MF20, Invitrogen Antibodies) and desmin (ab32362, Abcam) overnight at 4°C. The next day, the samples were washed with PBS and then incubated with the secondary goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (A-11001, Invitrogen) or goat anti-rabbit IgG antibody conjugated with Alexa Fluor 568 (A-11036, Invitrogen) at room temperature for 30 min. After a final wash in PBS, the samples were mounted in an antifade mounting medium with DAPI (Vector Laboratories, Newark, CA) and imaged by confocal microscopy.
  • DAPI Vector Laboratories, Newark, CA
  • Image J software was Attorney Docket 9865.236.WO used to quantify myotube length, width, and nuclei per myotube. The fusion index is defined as the percentage of nuclei fused into myotubes per total number of nuclei in a field of view.
  • Statistical Analysis T-tests were used to establish the significance of quantification results for the EDL and engineered muscle experiments. A p-value ⁇ 0.05 was considered significant.
  • Results Bioreactor system assembly. Two sets of trays were designed for the bioreactor and sensing modules of the full system, as shown in FIGS. 37A-37B.
  • the bioreactor tray accommodating the bioreactor culture chamber with a capacity of up to 600 ml, was successfully integrated with the mechanical stimulation system, force sensors, and electrical stimulation electrodes.
  • the sensor module tray included the pinch valves, media sensors, biosensor transmitter, liquid-bubble sensor, and fluid bags containing PBS and calibration solution. Materials used in all system parts were selected to be microbe-resistant and non-leaching. The two trays are simple to assemble in a sterile hood and easy to handle for assembly within the tissue culture incubator. [00231] Mechanical and Electrical Stimulation.
  • a mechanical stimulation subsystem was designed with real-time feedback-capable movement that can be defined via customized protocols.
  • the system capable of micro-stepping up to 1800 steps per revolution, provided high-resolution steps and an encoder with up to 4096 pulses per mm to acquire accurate movement data.
  • the system also provided a mechanical stretch of at least 10% to a tissue of up to 4 cm in length with a maximum actuator speed locked at 20 mm per second in the software.
  • the motor driver allowed real-time access to change the micro-step function, sleep, and step profiles without changing the physical setup.
  • the motors were rated at 600 mA, which is safely paired with a driver chip of maximum 750 mA of current. through the motor. Further, due to the availability of the encoder, a high-resolution 1/4096 mm movement control was possible with the recording of data to an SD Card.
  • the electrodes were placed parallel to the long axis on both sides of the muscle tissue, creating an electrical field perpendicular to the construct.
  • the parallel plate electrode configuration allowed for generating a straight field line in a 3D tissue and performs better than rod-shaped electrodes. This capacitive field aligns the cells perpendicular to the field in the direction of the long axis.
  • Voltage and current were tested using the system placed in the media. Continuity testing of the electrode circuit showed a closed circuit when placed in media, and the current output was read through voltage drop measured across a 0.01% accuracy resistor, which varied linearly with stimulator voltage, similar to previously published data using equivalent circuits. Due to the limitations of the stimulator, the constant voltage output was a maximum of 30 V.
  • the pulse protocol used for the culture and excitation of EDL muscle for contractile activity is shown in FIG.34C.
  • the availability of more than one stimulation channel provides the ability to study multiple tissues in the bioreactor simultaneously.
  • the ability of the microcontroller to start the defined pulse protocol enables studies that have in-phase and out-of-phase co-stimulation for electrical stimulators, such as those used in this study.
  • Calibration of the force sensor in the vertical orientation was performed inside an incubator at 37°C.
  • the force sensing resolution was defined by a small range calibration at 100 ⁇ N steps.
  • the calibration result was saved as a profile in the internal memory of the digital amplifier, which resulted in accurate readings when tested against known standard weights and pipetted water droplets.
  • the theoretical resolution of the system is calculated from the maximum capacity, or range of force sensor divided by the Analog to Digital Converter’s (ADC) bit resolution, yielding 0.53 ⁇ N for 24-bit ADC.
  • the maximum force measurement of the sensor is 1kg-force, or around 9.8 N.
  • the low-force calibration profile ensured our resolution of 50 ⁇ N, owing to noise.
  • the force sensor integrated into the bioreactor can withstand multiple cycles of sterilization and long-term use submersed in tissue culture media.
  • Automated sampling, calibration, and sensor loop system An automated sampling and sensor calibration system was developed to monitor metabolic activity in cultured tissue and the general culture environment within the bioreactor. The sensing system was assembled on a polycarbonate tray with all components mounted on dedicated 3D-printed holders.
  • FIG. 36A A diagram of the bioreactor perfusion system (FIG. 36A) shows the flow line and the normally open and Attorney Docket 9865.236.WO normally closed (NO & NC) lines of the three-way pinch valves, which can switch between the sampling solutions for glucose and lactate sensors.
  • One end of the system has tubing with a quick disconnect connector linking it to the bioreactor, as shown in FIG.36B.
  • the tray holds the PBS buffer solution (DPBS, Cytiva) and a glucose/lactate calibration solution, which may be either a commercial standard (2747 Standard, YSI) or serum-supplemented media used in the experiment.
  • the initial glucose and lactate contents were 23.42 mM/L and 1.89 mM/L, respectively, attributed to added serum.
  • FIG. 45 details the sequence of exposure events for each sensor per measurement.
  • the first sensor exposure is to air, which shows no reading (yellow), followed by a PBS wash (blue), air (yellow), and the calibration solution (green).
  • This cycle is then repeated to test bioreactor media.
  • the data described above was plotted by matching the peaks in the first 8 minutes of hourly exposures using a custom Python code. The peaks matched at the zero-point are shown overlapped in FIG.45.
  • the system automatically re-calculates the calibration gains for the output by automatically sampling Attorney Docket 9865.236.WO the calibration solution before each measurement, thereby providing accurate readings even as the sensor performance degrades over time.
  • the sensor system can read temperatures between 20°C - 45°C due to the resistor configuration used to increase sensitivity in the range of interest.
  • the measurement of glucose and lactate concentrations range from 0 - 25 mM and 0 - 12.5 mM, respectively.
  • the pH sensor range is limited to 6 – 8, as cell cultures are almost exclusively maintained within this range.
  • the dissolved oxygen range that can be monitored is 0-21%.
  • the % error range of the sensor hardware, as listed by their manufacturers, is within the scope of the experiments described herein.
  • the cross-sectional area (CSA) of the myofibers in the static group was also significantly larger than in freshly excised controls and the mechanical and electrical stimulation groups (FIG.38C).
  • the nuclei count per field of view (FIG.38D) was significantly lower for all the experimental groups compared to the fresh control, with the static group having the lowest nuclei count.
  • the circumference of the static group was significantly larger than the fresh control and mechanical and electrical stimulation groups (FIG.38E).
  • the circularity of myofibers can be determined by (4 ⁇ x CSA)/(perimeter)2. Myofibers from healthy skeletal muscle tissue are angular, with necrotic myofibers becoming more rounded.
  • Muscle constructs from both the electrical and mechanical stimulated groups had longer myotubes than the control group (FIG. 40B), but the width of the myotubes in the electrically stimulated group was thinner than the other groups (FIG.40C). Based on this data, the myotubes in the muscle constructs exposed to electrical stimulation had a thinner and more elongated morphology than the control or mechanical stimulation groups. With DAPI and MHC staining, nuclei number per myotube may be determined (fusion index). Quantification of DAPI and MHC staining showed that both the electrical and mechanical stimulated groups had significantly higher nuclei count per myotube than the control, with the electrical stimulation group showing the highest nuclei count (FIG.40D).
  • the electrically stimulated group had the highest fusion index, as compared to the other groups (FIG. 40E), suggesting that electrical stimulation may provide a better differentiation stimulus than mechanical stimulation, although both stimulation protocols resulted in a higher fusion index compared to the static control.
  • the development of the uniaxial bioreactor of the present invention was guided by the objective of creating a user-friendly and multifunctional system with high throughput feedback capabilities for long-term tissue culture, maturation, and support of a functional cell phenotype. To achieve this, a medical-grade photopolymer resin was used to design a leakproof bioreactor module chamber box assembly into which components and actuation elements may be snap-fit.
  • the bioreactor system features independent cycle programming, parametric strain protocol definition, software handling of micro-stepping, and encoder feedback, enabling precise control and customization of mechanical stimulation. It was Attorney Docket 9865.236.WO opted not to use sine waves for strain cycles to keep the strain rates constant, as previously used by others, although the disclosed system is compatible with this approach. Commercially available systems are very costly and limited in the types of tissue geometries and scaffold biomaterials that can be accommodated. [00247] The integration of a force sensor into the disclosed system enabled the measurement of contractile force (FIG. 35E), the non-destructive quantification of mechanical properties (FIG.35C), and force feedback during dynamic strain conditioning (FIGS. 46A-46E).
  • Two validation experiments of force sensor functionality were performed.
  • the rat EDL muscle tissue was used to determine isometric twitch and tetanic contractile force at different frequencies of electrical stimulation. Force output during the stretch, 3D morphology of the cross-section areas over time, and the applied strain were determined at each time point for more comprehensive mechanical property characterization.
  • the material property of cellular and acellular engineered muscle scaffolds was characterized, and peak forces were measured from force feedback of cyclic strain cycles of different maximum strain percentages. Including a force measurement system further expanded the capabilities of our bioreactor to enable the measurement of forces exerted by tissue during mechanical or electrical stimulation, providing valuable insights into tissue differentiation and maturation.
  • force sensing can be utilized for studies involving in situ scaffold testing by combining it with a stepper motor program to generate force-displacement curves. This data, along with knowledge of tissue geometry, can be used to calculate stress-strain/stiffness data for the wet testing of mechanical properties.
  • Force sensing has also been shown to help resolve the issue of uniform pre-tension loading of the scaffolds by checking force generation curves with a small incremental strain, starting from a zero-force sagging position and moving the tissue holder away while measuring force. Having a readout of the force of stretching in real time can provide information about changes in the material property of the construct following many stretch cycles.
  • the availability Attorney Docket 9865.236.WO of force sensors within the bioreactor can also assist in comparative studies between an engineered construct and native tissue, such as freshly excised muscle.
  • an essential aspect of the pre-tensioning system has been shown to be relevant for both dynamic and isometric force testing of muscle tissues.
  • a more sensitive micro-newton force sensor could be easily incorporated to gather cell-level force data.
  • factors such as electrode material and geometry were considered to develop a non-cytotoxic, non-corrosive, sterilizable system that would produce a straight field stimulus generated over the desired fluid volume.
  • the detachable electrode holders designed for the device can be easily reconfigured to adjust the field shape.
  • integrated plate electrodes are used instead of rod or point electrodes, ensuring uniform unidirectional field distribution. This supports a more uniform and consistent electrical stimulation across the tissue. Previous studies have also highlighted the significance of maintaining a uniform electric field for testing contractile activity and stimulating tissue development.
  • in-phase or out-of-phase electrical and mechanical co-stimulation studies can be easily performed in this system, as the initiation of the electrical pulse can triggered prior to, during, or following the corresponding mechanical cycle. It would be trivial to generate a variety of strain protocols combining these two stimuli.
  • Monitoring physiological conditions within the tissue culture is critical, as previously discussed. Dissolved oxygen, pH, glucose, lactate, and temperature sensors provide real-time data on essential parameters that influence the structure and functionality of a maturing tissue construct. To demonstrate the reliability of repeated measurements in this system, peak matching was performed. This avoids the slight timestamp drift when using a low-frequency microcontroller for long-term continuous testing. The results established that the sampling system reliably automates correct fluid routing to the sensors.
  • Laboratory and commercial media exchange systems typically use two pumps for small or large culture volumes (AMX, Agilent Biotek, Winooski, VT).
  • the present system was designed to maintain a constant culture media volume by utilizing a commercially available single-channel stepper motor-based peristaltic pump with a multifunctional driver board. This provides easy control of pump speed, frequent flow rate calibrations, and easy integration with the larger bioreactor system. Additionally, threshold sensor outputs can be programmed to trigger media exchanges automatically.
  • Commercial media exchange systems designed for research laboratory use are generally much more costly than the developed system.
  • the pump driving the sensor loop and media exchanger in the present system are panel-mounted, reducing the device footprint. [00255] Rat EDL muscles were used to validate both mechanical and electrical conditioning within the bioreactor.
  • C2C12 cells cast in molds were cultured in our bioreactor system and differentiated under electrical or mechanical stimulation for 14 days. Stimulated constructs demonstrated increased myotube formation compared to the control group (FIGS. 40A-40E), which is similar to the results of other groups.
  • engineered muscle was cultured for 14 days, followed by 15% mechanical stimulation, and reported that mechanical stimulation induced hypertrophy and improved force production of engineered muscle with evidence of wider myotubes, more nuclei per myotube and higher fusion index.
  • the uniaxial bioreactor according to embodiments of the present invention has shown tremendous potential for conditioning engineered muscle tissues. Integrating mechanical, electrical, and sensor capabilities offers researchers a versatile tool for investigating cellular responses, tissue development, and contractile activity under controlled stimulations. Overall, this system has undergone rigorous qualification processes, ensuring its robustness and reliability.
  • the utilization of 3D printing technology, leakproof assembly, customizable mechanical and electrical stimulation, force sensing capabilities, sensor loops, and advanced features in the measurement systems make the disclosed uniaxial bioreactor an asset for tissue engineering and biomedical research.
  • This system has the ability to support long-term biological experiments and offers hands- free operation that can be managed without disturbing tissue samples. Stimulation programs can be updated in real time either manually or through sensor feedback.
  • This design can serve as the foundation for other types of complex tissue engineering bioreactors with improved feedback and a higher degree of automation, which can be rapidly developed and prototyped.
  • the development of a multifunctional bioreactor with two types of integrated stimulation systems and feedback from physical and biological sensing enables the execution of conditioning protocols to enhance the consistency, functionality, and durability of engineered skeletal muscle.
  • the components and production methods used to develop this bioreactor can be easily adapted for other tissue applications that benefit from unidirectional stimulation to mature multiple constructs or large volumetric tissues fully.
  • the feedback obtained from this device on tissue maturation status, along with the ability to take real-time in-situ measurements that indicate tissue differentiation, maturation, and growth, provides new manufacturing strategies to advance the field of tissue engineering to clinical applications.

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Abstract

La présente divulgation concerne un système de bioréacteur modulaire. Le système comprend un boîtier ; une pluralité de modules de bioréacteur interchangeables, chaque module étant conçu pour être reçu à l'intérieur du boîtier, la pluralité de modules de bioréacteur interchangeables comprenant un module de bioréacteur uniaxial, un module de bioréacteur biaxial, un module de bioréacteur pulsatile, un module de bioréacteur de remplissage de vide et/ou un module de bioréacteur de perfusion ; un sous-système d'échangeur de milieux conçu pour être connecté à l'un de la pluralité de modules de bioréacteur interchangeables reçus à l'intérieur du boîtier ; un module de surveillance de capteur conçu pour être connecté au sous-système d'échangeur de milieux et à l'un de la pluralité de modules de bioréacteur interchangeables reçus à l'intérieur du boîtier, le module de surveillance de capteur étant éventuellement conçu pour être reçu à l'intérieur du boîtier ; et un dispositif de commande associé de manière fonctionnelle au boîtier, à la pluralité de modules de bioréacteur interchangeables, au module de surveillance de capteur et au sous-système d'échangeur de milieux.
PCT/US2024/037004 2023-07-11 2024-07-08 Systèmes de bioréacteur modulaire pour applications de médecine régénérative Pending WO2025014852A2 (fr)

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