Tissue maturation bioreactor system with homogenous cell distribution

Abstract

A system and method for exercising cells and / or tissues in a sealed bioreactor. The system includes a cartridge having means for stretching and means for retaining cells on a scaffold assembled inside the cartridge during seeding. The system includes a bioreactor including a sealed enclosure, the sealed enclosure housing the scaffold and the cartridge, the bioreactor allowing homogenous seeding of the scaffold with cells, the seeding being performed inside the sealed enclosure to protect against environmental contamination. The bioreactor system provides means for supplying fresh medium to the cells and / or tissues and means for removing waste products.

Description

[Technical field]

[0001] The present disclosure relates to growing tissue, and more particularly to moving tissue as it grows to better simulate the dynamic mechanical environment in which real biological tissue matures from cells. [Background technology]

[0002] Tissue engineering relies on a scaffold, cells seeded on the scaffold, and the environment surrounding the scaffold and cells to produce viable, healthy tissue. The closer the environment is to what the tissue would experience in the body, the greater the likelihood that the tissue will grow to a viable, healthy state. One aspect of the environment involves simulating the mechanical stresses that cells would experience in the body. Current methods of simulating these stresses include stretch chambers that can apply stretch at cyclic frequencies.

[0003] Current systems provide stretch chambers for stretching cells. For example, the cell stretching system produced by Strex USA mechanically stimulates cells growing in culture by stretching and compressing them. The system manufactured by Strex USA attaches cells to a stretch chamber and subjects them to mechanical strain. The chamber is made of a silicone elastomer, e.g., polydimethylsiloxane (PDMS) (see "Cell Stretching System"). Other systems mechanically stimulate tissue forming cells to mechanical stress by linear translational motion of magnetic bodies in an applied magnetic field (see JP 2008-263986 A1, El-Haj et al., Treatment of microorganisms or enzymes with electrical or wave energy, egmagnetism, sonic waves). Still other systems provide culture containers with gas-permeable sides and expansion units for horizontally expanding the culture container. Such systems are designed to prepare fertilized eggs for implantation (see WO 2007 / 052653 A1, Naruse et al., Culture container and culture apparatus). Other systems provide bioreactors with mechanical actuators positioned to apply mechanical stress to tissues grown within the bioreactor. In such systems, the actuators apply normal stress to the tissue and can even inflict damage (Tuan et al., Modular, microfluidic, mechanically active bioreactor for 3D, multi-tissue, tissue culture, U.S. Patent Publication No. 2021 / 0139859). In some systems, the printing seeding process includes a cellularized 2% hyaluronic acid (HA)-based hydrogel (Christ et al., WO 2022 / 010594, Hermetically or Aseptically Sealed Bioreactor System and Related Method Thereof, published January 13, 2020).HA hydrogels can be used to guide uniform deposition of cells across the scaffold, minimizing leakage and cell loss. The hydrogel dissolves in the medium over time, and it is currently unknown whether there is a lasting effect of the transient presence of HA on the development of tissue engineered muscle repair products. In some systems, the printing process requires multiple open processing steps, and the bioreactor can only be fully closed for exercise after both sides of the scaffold have been seeded. The open processing steps are a potential contamination risk.

[0004] What is needed is a system designed to accommodate a variety of scaffold materials and geometries, such as, but not limited to, decellularized bovine or porcine acellular matrices, decellularized collagen membranes, thin polymer membranes (e.g., PDMS), or polymer fiber scaffolds. What is needed is a system designed to remain closed to the environment after sterilization, thus greatly reducing the risk of contamination and reducing manufacturing costs by requiring operations in expensive clean rooms. What is needed is a system that provides for fluid seeding of scaffolds inside a graduated bioreactor, thus facilitating integration into a tissue foundry line, promoting process robustness and reducing manufacturing costs. What is further needed is the ability to use a variety of scaffold types. What is further needed is a system that allows for closed fluid seeding through ports integrated with the cartridge inside a sealed bioreactor. Exemplary components of a tissue foundry line are described in the following documents:U.S. Patent Application No. 16 / 904,198, Andrews et al., System and Method for Cell and Tissue Preparation, filed June 17, 2020; Andrews et al., International PCT Application U.S. Patent Application No. 20 / 38214, System and Method for Cell and Tissue Preparation, filed June 17, 2020; Andrews et al., U.S. Patent Application No. 17 / 243,324, System and Method for Centralized Fluid Management and Culture Control, filed April 28, 2021; Andrews et al., International PCT Application U.S. Patent Application No. 21 / 29706, System and Method for Centralized Fluid Management and Culture Control, filed April 28, 2021; White et al., U.S. Patent Application No. 17 / 522,003, Cable / Tube Sleeve and Snorkel, filed November 9, 2021; White et al., U.S. Patent Application No. 17 / 648,466, Modular, Configurable Bioreactor System for a Manufacturing Line, filed January 20, 2022; White et al., International PCT Application No. 22 / 70265, Modular, Configurable Bioreactor System for a Manufacturing Line, filed January 20, 2022; and White et al., China Utility Model Application No. 202220160610.8, Modular, Configurable Bioreactor System for a Manufacturing Line, filed January 20, 2022. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2008-263986 A [Patent Document 2] International Publication No. 2007 / 052653 [Patent Document 3] US Patent Application Publication No. 2021 / 0139859 [Patent Document 4] International Publication No. 2022 / 010594 [Patent Document 5] U.S. Patent Application Serial No. 16 / 904,198 [Patent Document 6] U.S. Patent Application No. 20 / 38214 [Patent Document 7] U.S. Patent Application Serial No. 17 / 243,324 [Patent Document 8] U.S. Patent Application No. 21 / 29706 [Patent Document 9] U.S. Patent Application Serial No. 17 / 522,003 [Patent Document 10] U.S. Patent Application Serial No. 17 / 648,466 [Patent Document 11] U.S. Patent Application No. 22 / 70265 [Patent Document 12] China Utility Model No. 202220160610,8 Summary of the Invention

[0006] According to some configurations, the present teachings include systems and methods for motivating tissue as it grows within a confined environment.

[0007] In this disclosure, certain terms are used. The following definitions are not intended to limit the scope of the disclosure, but rather to provide context and definition to the disclosure. Tissue Engineered Muscle Repair (TEMR) Construct refers to small skeletal muscle-like tissue. (Machingal et al., A Tissue-Engineered Muscle Repair Construct for Functional Restoration of an Irrecoverable Muscle Injury in a Murine Model, Tissue Engineering:Part A, 17:17-18, 2011; Corona et al., Implantation of In Vitro Tissue Engineering Muscle Repair Constructs and Bladder Acellular Matrices Partially Restore In Vivo Skel et al., Muscle Function in a Rat Model of Volumetric Muscle Loss Injury, Tissue Engineering:Part A, 20:3-4, 2014; Corona et al., Further Development of a Tissue Engineered Muscle Repair Construct In Vitro for Enhanced Functional Recovery Following Implantation In Vivo in a Murine Model of Volumetric Muscle Loss Injury, Tissue Engineering:Part A, 18:11-12, 2012). Fluidic seeding methods that do not rely on printing of cellularized inks are referred to herein as "print-free." An individual seeded scaffold is referred to as a "sample." A scaffold unit that is surrounded and enclosed within a silicone elastomer, more preferably PDMS, frame is referred to herein as an "encapsulated scaffold."A bioreactor designed to move multiple encapsulated scaffolds is referred to herein as a first configuration bioreactor (depicted in FIG. 5A, see below). An immortalized mouse myoblast cell line is referred to herein as "C2C12". Metrics for assessing cell seeding onto scaffolds are referred to herein as process parameters. The percent area of ​​the scaffold covered by cells, as determined by area fraction of actin stain, is referred to herein as "coverage". Cell coherency and primary direction, as determined by actin fiber orientation, is referred to herein as "alignment". Polydimethylsiloxane (silicone) is referred to herein as "PDMS".

[0008] The present disclosure relates to multi-surface seeding of human muscle progenitor cells onto acellular matrix scaffolds. The bioreactor of the present teachings facilitates print-free seeding within a closed environment to reduce contamination risk compared to open processing steps required in other systems. Seeding is followed by uniaxial extension preconditioning within a fully enclosed bioreactor environment. In some configurations, the resulting TEMR construct is ultimately intended to be surgically implanted to promote tissue regeneration and repair in cases of volumetric muscle loss. The system of the present teachings provides a hydrogel-free and print-free seeding method that produces cell coverage comparable to print seeding. To minimize open processing steps, in the bioreactor of the present teachings, the scaffolds can be fluidically seeded and moved within a single fully enclosed environment. An individual bioreactor is composed of a closed shell with seeding ports integrated into the cartridge that holds the scaffolds or ports located on the bioreactor housing, an internal frame to hold multiple cartridges, and the mechanisms required to apply forces on the cartridges and thus the scaffolds. The bioreactor design also accommodates closed processing for any necessary media exchange or continuous media flow throughout the process. The TEMR constructs produced in the bioreactor meet or exceed preselected process parameters, thus allowing for additional automation in the TEMR production process. The bioreactor includes ports to accommodate automated seeding and continuous media flow loops. The effectiveness of the system of the present teachings is exemplified by greater than 90% cell coverage on the seeded scaffold area as determined by fluorescent staining and cell body analysis (actin) and nuclear fluorescent stains, cell alignment (coherency) of 0.4 or greater across the seeded scaffold area, and cell alignment initially oriented at 0° in the extension direction. Cells within the tissue need to be tightly distributed (coherency) in the desired direction (angle).Cellular coherency refers to the alignment distribution of the actin cytoskeleton that comprises the cells; if all actin fibers in the tissue are perfectly aligned, the coherency is 1, and if they are isotropic, the coherency is 0. (Clemons et al., Coherency image analysis to quantify collagen architecture: implications in scar assessment, RSC Adv., 2018, 8, 9661 (Clemons)). According to Clemons, "the coherency value indicates the degree of orientation of the local features, and if the local structure has one dominant orientation, the coherency is 1, and if the image is isotropic in the region being analyzed, the coherency is 0." For the system of the present teachings, the local features are the actin fibers of cells within a specific tissue surface area, i.e., a predefined pixel x pixel area. Coherency is important for engineered muscle tissue analysis, since the entire muscle tissue is only as good as its alignment. Cell lines that can be used to seed the bioreactor include, but are not limited to, C2C12 mouse myoblasts, muscle progenitor cells, hMP cells, NHCN-M2 cells, mesoangioblasts, satellite cells, H2K cells, L8 rat cells, and 3T3 mouse embryonic fibroblasts.

[0009] One feature of the systems and methods of the present teachings is that they can be used for limited cell volumes, making them economically attractive. Furthermore, the low volume allows for detection of changes in the medium and / or substances secreted by the cells / tissues as they grow. Among other things, detection of changes is important for continuous, non-invasive cell and tissue monitoring to monitor cell / tissue growth, maturation, and harvestability. Determining whether muscle tissue is harvestable requires continuous monitoring of signals that indicate how the tissue condition is changing over time.

[0010] Another feature of the system and method of the present teachings is that cells are provided to the scaffold and matured all within a single enclosure. The entire process of seeding and maturation is performed inside the bioreactor of the present teachings, so the cells are never placed outside of a controlled environment. No cell printing is required, and therefore no movement of the scaffold from the printing site to the bioreactor is required. Since the number of cells to be seeded is relatively small, a device such as a special syringe pump can be used to deliver the cells onto the scaffold.

[0011] The above-recited features of the present disclosure will be more readily understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1A is a schematic block diagram of a system of the present teachings for moving tissue. [Figure 1B] FIG. 1B is a schematic block diagram of a system of the present teachings for moving tissue. [Figure 1C] FIG. 1C is a schematic block diagram of a system of the present teachings for moving tissue. [Figure 1D] FIG. 1D is a schematic block diagram of a system of the present teachings for moving tissue. [Figure 1E] FIG. 1E is a schematic block diagram of a cartridge movement system of the present teachings. [Diagram 2] FIG. 2 is a schematic block diagram illustrating details of a possible implementation of a cartridge of the present teachings. [Diagram 3] FIG. 3 is a schematic block diagram illustrating details of a possible implementation of a cartridge of the present teachings. [Figure 4A] FIG. 4A is a schematic block diagram illustrating the extension of the cartridge of FIGS. [Figure 4B] FIG. 4B is a schematic block diagram illustrating the extension of the cartridge of FIGS. [Figure 4C] FIG. 4C is a schematic block diagram illustrating the extension of the cartridge of FIGS. [Figure 4D] FIG. 4D is a schematic block diagram illustrating the extension of the cartridge of FIGS. [Figure 4E] FIG. 4E is a schematic block diagram illustrating the extension of the cartridge of FIGS. [Figure 5A] FIG. 5A is a schematic perspective view of an implementation of the system of the present teachings. [Figure 5B] FIG. 5B illustrates a schematic perspective view of an implementation of a lockdown device of the present teachings. [Figure 5C] FIG. 5C is a schematic exploded perspective view of the implementation of FIG. 5A. [Figure 5D] FIG. 5D is a schematic perspective view illustrating the linkage between the cartridge and the motor subsystem of the implementation of FIG. 5A. [Figure 5E] FIG. 5E is a schematic exploded perspective view of the environmental contamination protection of the implementation of FIG. 5A. [Figure 5F] FIG. 5F is a schematic exploded perspective view of the magnetic cap and magnet of the implementation of FIG. 5A. [Figure 5G] FIG. 5G is a schematic exploded perspective view of a motor subsystem of the implementation of FIG. 5A. [Figure 5H] FIG. 5H is a schematic exploded perspective view of a bioreactor subsystem of the implementation of FIG. 5A. [Figure 5I] FIG. 5I is a schematic exploded perspective view of various bioreactor subsystems of the implementation of FIG. 5A. [Figure 6A] FIG. 6A is a schematic exploded perspective view of a cartridge of the implementation of FIG. 7A. [Figure 6B] FIG. 6B illustrates a schematic perspective view of the cartridge in the contracted and extended configurations of the implementation of FIG. 7A. [Figure 7A] FIG. 7A is a schematic perspective view of another implementation of the system of the present teachings. [Figure 7B]FIG. 7B is a schematic perspective view of a partially exploded assembly of the bioreactor subsystem and motor linkage of the implementation of FIG. 7A. [Figure 7D] FIG. 7D is a schematic perspective view of a partially exploded assembly of the bioreactor subsystem, motor linkage and motor subsystem of the implementation of FIG. 7A. [Figure 7E] FIG. 7E is a schematic exploded perspective view of a bioreactor subsystem of the implementation of FIG. 7A. [Figure 7F] 7F-1 and 7F-2 are top views of the bioreactor subsystem of the implementation of FIG. 2A, showing the cartridge in a contracted configuration. [Figure 7G] FIG. 7G is a top view of the implementation of FIG. 7A. [Figure 7H] FIG. 7H illustrates a view of the motor subsystem from a different perspective of the implementation of FIG. 7A. [Figure 7I] Figures 7I-A, 7I-B, and 7I-C are top and cross-sectional views of the implementation of Figure 7A, respectively. [Figure 7J] FIG. 7J is a schematic exploded perspective view of the linkage and motor subsystem of the implementation of FIG. 7A. [Figure 7K] FIG. 7K is a cross-sectional view showing fluid flow of the implementation of FIG. 7A. [Figure 7L] FIG. 7L is a cross-sectional view showing fluid flow of the implementation of FIG. 7A. [Figure 7M] FIG. 7M is a cross-sectional view showing fluid flow of the implementation of FIG. 7A. [Figure 8A] FIG. 8A is a diagram of another embodiment of a cartridge of the present teachings. [Figure 8B] FIG. 8B is a diagram of another embodiment of a cartridge of the present teachings. [Figure 8C] FIG. 8C is a diagram of another embodiment of a cartridge of the present teachings. [Figure 8D] FIG. 8D is a diagram of another embodiment of a cartridge of the present teachings. [Figure 9A] FIG. 9A is a pictorial illustration of test results using a system of the present teachings. [Figure 9B] FIG. 9B is a graphical illustration of test results using a system of the present teachings. [Figure 9C] FIG. 9C is a graphical illustration of test results using a system of the present teachings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The system of the present teachings for print-free seeding and / or mechanical movement inside a bioreactor for the production of TEMR constructs is detailed herein. In particular, the system and method of the present teachings can accommodate seeded cells on a scaffold without the need for a cell printing system, and the cells can be further matured in a closed environment after being seeded on the scaffold. The bioreactor of the present teachings can accommodate multiple scaffolds that allow for simultaneous maturation of human cells, for example but not limited to, backup scaffolds with the same human cells, and a control group of cells. Other configurations are contemplated by the present teachings, including configurations with a single scaffold and configurations with two, three, or more scaffolds. The bioreactor is fully sterilizable and fully closed prior to and during all production testing with living cells and tissues. The system and method of the present teachings can also mechanically move cells. This feature can be integrated with the cell seeding feature or can exist separately from the cell seeding and closed environment. The movement feature is made possible by a flexible scaffold housed within a specially designed extensible cartridge, a frame operatively coupled to the extensible cartridge to stretch the extensible scaffold, and a processor-controlled push / pull mechanism that allows controlled movement of the frame to stretch the tissue on the scaffold. The cartridge is configured to match a variety of scaffold materials and geometries, such as decellularized bovine or porcine bladder acellular matrix. Other scaffold types include decellularized collagen membranes, thin polymer membranes, and polymer fiber scaffolds. In some configurations, the scaffold can be enclosed within a silicone framework. In some configurations, instead of silicone, split substantially rigid sides connected by flexible and extensible rings can form the top and bottom surfaces of the frame or cartridge. The frame or cartridge can surround the scaffold, and the split rigid or semi-rigid sides can be pulled apart to allow movement of the tissue on the scaffold.The cartridge and / or bioreactor can include a seeding port and a lid. Other options for seeding include seeding ports, for example, in the frame or in a covering or lid for the scaffold. The lid can have a rigid or semi-flexible structure and can be constructed, for example, of a transparent material that allows observation of the scaffold and gas exchange. Transparent materials include, for example, silicone. Gas exchange can occur inside the sealed bioreactor, for example, at about 5% CO, a gas concentration that the bioreactor can monitor and maintain. 2 The medium can be buffered to the lid. The present teachings contemplate additional options for the lid. Another feature of the present teachings is a device for pushing and pulling the cartridge to assist in the movement of the tissue on the scaffold.

[0014] 1A-1D, a system of the present teachings for exercising tissue includes, but is not limited to, a scaffold 1005 / 1005A, a cartridge 1007, a bioreactor 1009, an exerciser 1013 (FIG. 1B), and a rotating means 1012 (FIG. 1C). The scaffold 1005 provides at least one surface onto which cells can be deposited and attached. The scaffold 1005 can be of any shape, and cells can be attached on any surface of the scaffold 1005. For example, the scaffold 1005 can include a first surface and a second surface, each of which can be colonized with the same or different types of cells. The present teachings contemplate multi-surface scaffolds, or three-dimensional shapes. The shape and size of the scaffold 1005 dictates the shape and size of the cartridge 1007, which forms a retaining frame for the scaffold 1005. The cartridge 1007 is constructed to grip portions of the scaffold 1005 and hold them in place while the cartridge 1007 expands and contracts, effectively stretching and contracting the cartridge 1005. The cartridge 1005 can be configured with the cartridge 1007 to extend / contract along any axis appropriate for the tissue being moved and along any direction that may be relevant, non-limiting examples of which are shown in FIG. 1A. In response to commands from the controller 1011, a rotating means 1012 (FIG. 1C) can rotate the bioreactor 1009. The rotation can allow for cell attachment with the aid of gravity and auxiliary forces. The system of the present teachings can be part of a manufacturing line and therefore under the control of a manufacturing line processor 1014 (FIG. 1D).

[0015] 1E, an exemplary scaffold 1031 in a cartridge 1032 is stretched in a first direction 1033 to the shape of a scaffold 1037. The stretching is reversed in a second direction 1035 and the scaffold 1031 returns to its original shape and size. In the second orientation, an exemplary scaffold 1041 in a cartridge 1039 is stretched in a third direction 1045 to the shape of a cartridge 1043. As the stretching is reversed in a fourth direction 1047, the scaffold 1041 returns to its original shape and size. In the third orientation, a scaffold 1049 in a cartridge 1052 is stretched in a fifth direction 1051 to the shape of a cartridge 1053. As the stretching is reversed in a sixth direction 1055, the scaffold 1049 returns to its original shape. The present teachings contemplate other shapes, orientations and dimensions, all of which can be accommodated by the systems described herein.

[0016] 1A-1D, the scaffold 1005 can be manually seeded inside the bioreactor 1009 and / or automatically seeded with cells from a cell supply system 1001. The cell supply system 1001 can contain a single type of cell or multiple types of cells that are separated and dispensed separately from one another. The cell supply system 1001 includes a means for moving cells from a cell chamber inside the cell supply system 1001, such as, but not limited to, a pumping mechanism. Another cell movement option is a linear actuator that moves a syringe needle that deposits a preselected amount of cells at a preselected location on the scaffold 1005. In a configuration in which the scaffold 1005 is automatically seeded, the controller 1011 communicates the cell type and seed amount to the cell supply system controller, which in turn controls the cell movement option.

[0017] Continuing with reference to FIGS. 1A-1D, media can be provided to the bioreactor 1009 by a media supply system 1003. The media supply system 1003 includes at least one media type that is formulated based on the cells being seeded. Multiple types of media may be required when multiple cell types are seeded on various surfaces of the scaffold 1005. Similarly, different types of media may be required at various times during the stretching process. The media supply system 1003 includes a means for moving media from at least one media tank to the bioreactor 1009. The media can include, for example, a complement of amino acids as a source of growth factors, vitamins, inorganic salts, glucose, and serum, hormones, attachment factors, and dissolved gases. The media can circulate around the bioreactor 1009 and cartridge 1007 to maintain the viability of the cells seeded on the scaffold 1005 as the cells are stretched. A controller 1011 communicates the amount and type of medium to the medium transfer means, and the type of medium is selected and the medium is transferred to the bioreactor 1009. The system can include a drain to discard the spent medium or to recirculate it or condition it for reintroduction into the bioreactor 1009.

[0018] 1A-1D, the system of the present teachings includes a motion system 1013. The motion system 1013 is physically configured to extend the cartridge 1007 which extends the scaffold 1005 while remaining within the closed system of the present teachings. In some configurations, the motion system 1013 includes a magnetic coupling responsive to pressure from a motorized linear actuator that pulls and extends the cartridge 1007. In some configurations, the motion system 1013 includes a damping device that holds the cartridge 1007 in an extended position to complete the motion plan, if necessary. The damping device is automatically or manually activated. In other configurations, a force sensor is utilized during tissue extension / exercise to measure tissue resistance / stiffness. In some configurations, the extent to which the tissue is expanded is limited based on measurements received from the force sensor.

[0019] 2 and 3, an exemplary three-dimensional perspective block diagram of a system of the present teachings illustrates an exemplary cell supply connector 1017 and a medium supply connector 1019. The exemplary cell supply connector 1015 is configured to seed cells on both sides of the scaffold 1005. Connectors such as luer lock connectors can maintain the closure of the system of the present teachings. In some exemplary uses, the scaffold 1005 can be assembled into a cartridge 1007, and this combination is further assembled into a bioreactor 1009. The controller 1011 directs the cell supply system 1003 to seed cells on the scaffold 1005, or on both sides. The controller 1011 directs the medium supply system 1001 to provide medium to the bioreactor 1009. Once the cells reach a desired level of confluence, the controller 1011 is configured to be sealed from the environment when coupled with the cartridge 1007, as shown in FIG. 3. The motion means 1013 is directed to extend the cartridge 1007 and thus the scaffold 1005A. In one embodiment, the cartridge 1007 is configured with at least one flexible ring such that as sections of the cartridge 1007 extend, a space 1019 exposes at least one flexible ring that maintains alignment of the cartridge parts while extending, allowing the cartridge 1007 to return to its original configuration and complete the motion cycle.

[0020] 4A-4E, an exemplary configuration of a cartridge system of the present teachings is shown. Shown in FIGS. 4A and 4D is the initial geometry of the cartridge 107 and pull bar connector 102. As seen in FIGS. 4B and 4D, when the motion means 109 pulls on a push / pull mechanism (not shown) attached to the cartridge 101 by the connector 102, the cartridge 101 splits at seam 118 to form sides 117, 121 and the scaffold 107 and flexible ring 104 extend. The scaffold 107 can be held in the extended position shown in FIGS. 4B and 4E by a braking device (not shown) or, if the motion means 109 releases the tension on the push / pull mechanism, the cartridge 101 and scaffold 107 return to their original position shown in FIG. 4C.

[0021] 5A-5I, a first implementation of the bioreactor, motion means, and lockout brake means of the present teachings is shown. In this implementation, the bioreactor is composed of two parts: a bioreactor top 2005 (FIG. 5A) and a bioreactor base 2007 (FIG. 5A). A connector 2012 (FIG. 5A) securely fastens the top 2005 (FIG. 5A) to the base 2007 (FIG. 5A) to protect the contents of the bioreactor from environmental contamination. Other configurations are contemplated for the bioreactor of the present teachings. For example, the bioreactor can include two side sections that meet and connect in the middle, or four corner sections that operably join together to form a sealed container after the connectors are in place. The bioreactor can be cylindrical with top and bottom sections or side sections operatively coupled, or other geometric shapes selected to accommodate the particular scaffold and cartridge shape and size as the scaffold and cartridge are housed by the bioreactor. Coupled to the bioreactor through an associated flange 2015 is a powered device case 2001 (FIG. 5A) that covers the mechanism for tensioning and releasing the cartridge inside the bioreactor to allow movement of tissue on the scaffold within the bioreactor. A seal 2013 (FIG. 5A) between the flange 2015 (FIG. 5A) and the bioreactor helps seal the contents of the bioreactor from environmental contamination.

[0022] Referring now primarily to Figures 5A and 5B, the bioreactor is operably coupled to a lockout brake 2003 (Figures 5A and 5B) that can hold the cartridge in a desired position. In one embodiment, the lockout brake 2003 (Figures 5A and 5B) is comprised of two parts, such as, but not limited to, a lockout brake top 2023 (Figure 5B) and a lockout brake bottom 2021 (Figure 5B). A thumb screw 2029 (Figure 5B) operably couples the lockout brake 2023 (Figure 5B) to the lockout brake bottom 2021 (Figure 5B). The lockout brake 2003 (Figures 5A and 5B) is configured to surround the powered device case 2001 (Figure 5A) to allow for an operable coupling between the lockout device 2003 (Figures 5A and 5B) and a device enclosed within the powered device case 2001 (Figure 5A). In one embodiment, the coupling is secured by a connector, such as, for example and without limitation, a clevis pin 2025 (FIG. 5B) that fits into a matching cavity 2006 (FIG. 5C) in a mechanism partially enclosed by the electromotive device case 2001 (FIG. 5A). In one embodiment, the lockout device 2003 (FIGS. 5A and 5B) is operably coupled to the bioreactor 2005 (FIG. 5A) by a thumb screw 2027 (FIG. 5B). The present teachings contemplate other configurations, such as, for example, a remotely controlled internal lockout mechanism. This lockout mechanism can include sensors that detect various levels of tissue stretch and provide these data to a controller, which can temporarily or permanently disable further movement of the device partially enclosed by the electromotive device case 2001 (FIG. 5A). In one embodiment, the lockout mechanism can engage a cartridge. In one aspect, no lockout mechanism is required since the controller determines the cartridge position and inputs that information into a controller associated with the motorized device, assuming the tissue motion plan starts or stops at predetermined or dynamically determined times or a combination thereof.

[0023] Referring now to FIG. 5C, the bioreactor top 2005 has been removed to reveal the bioreactor contents. The geometry of the bioreactor bottom 2007 is configured to provide secure mounting of the push / pull mechanism 2033 and cartridge 2031. In one embodiment, a bioreactor with a larger footprint than the depicted bioreactor can provide mounting for more cartridges 2031 and larger push / pull mechanisms 2033. The depicted bioreactor providing mounting for three cartridges 2031 (of which two cartridges 2031 are depicted) is merely exemplary. Furthermore, the size and shape of the motion area depends on the desired type, extent, and direction of tissue motion. Cell seeding is performed automatically or manually into the closed bioreactor through luer lock connectors 2009, one on each side of each scaffold 2032 (FIG. 5D) assembled on the cartridge 2031. Other configurations are contemplated by the present teachings. In one embodiment, multiple seeding ports can be configured per scaffold side, and these ports can provide different cell types to different portions of the scaffold. In one embodiment, the scaffold 2032 (FIG. 5D) can include more than two sides, e.g., can be in a three-dimensional form, and one seeding port can be required for each surface of the scaffold 2032 (FIG. 5D). In one embodiment, multiple scaffold surfaces can be accommodated with a single seeding port, by delivering the same cell type to each surface and / or sequentially delivering different cell types to different scaffold surfaces. In some configurations, the push / pull mechanism 2033 is operably coupled, e.g., to the powered device using connector 2008, and to the cartridge 2031 by connector 2041 (FIG. 5D). The cartridge 2031 can further be operably coupled to the bioreactor base 2007 by connector 2004.In this manner, when the push / pull mechanism 2033 moves, a first edge 2043 of the cartridge moves while a second edge 2045 of the cartridge remains stationary, forcing the sides of the cartridge 2031 apart and forcing the scaffold 2032 (FIG. 5D) to extend. Any form of coupling can be used and / or the cartridge 2031 can be formed as a monoblock with the push mechanism 2033 at one end. In other words, the cartridge 2031, or a part of the cartridge 2031, can be formed as a monoblock with the bioreactor base 2007, so long as one edge of the cartridge 2031 is free to move with the push mechanism 2033.

[0024] 5E-5G, components of an exemplary electromotive device are shown. The electromotive device moves a push / pull mechanism 2033 (FIG. 5D) as discussed herein. Specifically, an electromotive device case 2001 (FIG. 5A), positioned relative to the bioreactor by standoffs 2062 (FIG. 5E), partially encases a linear actuator 2065, which couples with a motor / magnet coupling 2051 (held in place by pin 2063), a magnet / motor coupling 2053, and a magnet 2061. A flange 2013 and a diaphragm 2015 couple the electromotive device to the bioreactor 2007 (FIG. 5A). In the depicted configuration, the linear actuator 2065, when actuated by the motor, urges the magnet 2061 (FIG. 5E) through couplings 2051 and 2053 in the direction of an opposing magnet 2059 (FIG. 5F), which is held in place by a connector 2058 (FIG. 5F). A magnet 2059 (FIG. 5F) is assembled into a magnetic coupling 2055 (FIG. 5F) that is coupled to a push / pull mechanism 2033 (FIG. 5D). The cartridge 2031 (FIG. 5D) is pulled toward the motorized device when the magnet is engaged, removing any tension in the scaffold and positioning it in a relaxed state. The magnet transfers the positive displacement of the motor to the cartridge, exerting a pushing force on the cartridge 2031 (FIG. 5D), causing the edges of the cartridge 2031 (FIG. 5D) to disengage from each other, stretching the flexible rings connecting the parts of the cartridge 2031 (FIG. 5D), and stretching the scaffold 2032 (FIG. 5D). When the linear actuator 2065 pulls the associated magnets in the opposite direction and the cartridge 2031 (FIG. 5D) is moved in the opposite direction, the edges of the cartridge 2031 (FIG. 5D) re-engage, the flexible ring returns to a resting, unstretched configuration, and the scaffold 2032 (FIG. 5D) relaxes. The flexible ring can be a band, and an O-ring or any other retainer that allows the cartridge to stretch and return to its original position. The present teachings contemplate other configurations for the movement mechanism.For example, servo motors, stepper motors, pneumatic or vacuum based actuators can replace the linear actuators and magnets can be replaced with weights for example. The motor enclosure can be decoupled from the bioreactor base to allow the bioreactor to be transported around. One method for decoupling the motor enclosure from the bioreactor base includes a clevis pin 2052 that can release the magnet motor coupling from the motor magnet coupling to allow attachment of a lockout plate (shown in FIG. 5B) that can prevent further movement of tissue. The bioreactor magnetic coupling can be coupled to the motor enclosure with a diaphragm flange and a bioreactor diaphragm that together seal the bioreactor contents from environmental contamination while allowing energy transfer from the motor subsystem to the cartridge.

[0025] 5H and 5I, a bioreactor base implementation includes the currently described features. The bioreactor base 2007 includes a cell seeding output (to the interior of the bioreactor) port 2073 (FIG. 5H) and a cell seeding input (from an external cell supply (of the bioreactor)) port 2081 (FIG. 5I) that are used to seed the bottom side of the scaffold. In one embodiment, there are as many cell seeding ports 2073 as there are cartridges 2031, but matching is not required. More or fewer cell seeding ports can be configured in the bioreactor base 2007 and lid 2005. A push / pull mechanism 2033 can be present on the bar 2077 on the bioreactor base 2007 and can be operatively coupled to a magnet mount 2055 (FIG. 5D) present in the cavity 2084 (FIG. 5I) as described herein. The bioreactor base 2007 can include a well 2083 into which media is delivered from a luer lock 2010 (FIG. 5C). In some configurations, rotation allows seeding from the "top," the side facing away from the gravitational force of the Earth, thus using gravity to aid in seeding the scaffold. The ports on the opposite side of the cartridge 2009 (FIG. C) are seeded after rotating the bioreactor, so they are at the top. In some configurations, one side is seeded, and then after a preselected period of time, the bioreactor is inverted so that the unseeded side of the cartridge faces up and the scaffold is seeded. In some configurations, the scaffold is framed with silicone or another flexible material using a mold. The frame results in silicone walls around the scaffold that hold the seeded cells on the top surface of the scaffold after the second cells are deposited. After the cells are exercised and the process is completed, the silicone walls can be cut away.

[0026] 8A-8D, a fixture for creating a second configuration of the cartridge is shown. An upper magnet 2501 used with a lower magnet 2511 creates compression on the decellularized bovine collagen-based matrix (BAM) 2505 (FIG. 8A) to inhibit the silicone from invading the surface of the BAM 2505 (FIG. 8A) when added to the mold. Although magnets are shown to create compression of the mold form on the BAM, any known technique for creating compression such as between mold forms can be utilized and falls within the scope of this disclosure. A scaffold pinch 2503 includes an upper component in contact with the BAM 2505 (FIG. 8A) that inhibits the silicone from invading the surface of the BAM 2505 (FIG. 8A). A tray 2507 is used to remove the framed BAM cartridge surrounded by a silicone frame from the mold after the silicone has fully cured. The silicone mold 2509 is where the silicone is injected to create the shape of the framed BAM cartridge. Silicone mold 2509 includes a table top 2512 (FIG. 8A) which is the lower surface of the silicone mold 2509 in contact with the lower side of the BAM 2505 (FIG. 8A) to inhibit silicone from penetrating the lower surface. Outer edge 2510 (FIG. 8B) is a clarification of the line in the drawing between the tray 2507 and the silicone mold 2509.

[0027] Returning to FIG. 6A, an exploded view of an exemplary scaffold frame, cartridge 2031, is shown. Cartridge 2031 is an example of a frame that allows mechanical movement of scaffold 2207. In some configurations, cartridge 2031 is configured to grip scaffold 2207, whose edges are captured between frame parts 2201 / 2211 and 2203 / 2213. In such configurations, the scaffold is not surrounded by a silicone frame. Instead, scaffold frame and O-rings 2255 / 2209 seal the perimeter of the scaffold. In some configurations, frame parts 2201 and 2211 are operably coupled by fasteners 2225. Frame parts 2203 and 2213 are operably coupled in a similar manner. In some configurations, as described herein, when the cartridge 2031 stretches, the frame parts 2201 / 2211 and 2203 / 2213 separate 2221 / 2223 at seam 2222 / 2224 (FIG. 6B). At the same time, the scaffold 2207, which is gripped between the frame parts, is stretched. Once the seam 2221 / 2223 is closed, the O-ring 2255 / 2209 and frame create a wall around the scaffold, resulting in a well into which seeding medium can be injected and the cells are forced to settle on the scaffold contained within the desired scaffold seeding area. After the cells attach to the scaffold, movement begins and the cartridge is completely surrounded by medium. Now the cells are fixed to the scaffold, so opening the seam of the cartridge does not provide an opening for the cells to flow out of the scaffold.

[0028] 6B, the motion of the scaffold 2207 can include movement in at least two directions, with elongation followed by contraction of the frame parts 2201 / 2211 and 2203 / 2213 back to a resting position where the space at the seam 2221 / 2223 is closed. The cartridge 2031 and scaffold 2207 can undergo further elongation and contraction as required for tissue maturation. The present teachings contemplate various shapes and sizes of the scaffold and its associated cartridge. In one embodiment, the scaffold can take on virtually any two-dimensional geometry for which a complementary cartridge can be constructed. For example, a circular scaffold can be surrounded by a circular cartridge that can stretch in various directions, not limited to two, depending on the location of the seam between the sections of the cartridge. Alternative configurations in which silicone walls are molded around the scaffold can allow stretching in various directions as well. In one embodiment, three-dimensional geometries can be accommodated. The bioreactor can take on a general shape that accommodates both two- and three-dimensional scaffolds, or it can take on specific shapes depending on the geometry of the scaffold.

[0029] Referring again now to FIG. 6A, in one embodiment, cartridge 2031 includes a flexible material that couples frame part 2201 to frame part 2203 and frame part 2211 to frame part 2213. In one embodiment, flexible ring 2255 can be in the first channel and flexible ring 2209 can be in channel 2217. When the frame parts are clamped together, the flexible ring pressed between the frame clamps can stretch when the frame parts are stretched and can simultaneously couple the frame parts back to their original configuration when the stretch is complete. Flexible rings that can provide sealing as well as stretching can be constructed of materials selected based on operating temperature, operating pressure, finish and stretch. Such materials can include, but are not limited to, nitrile, fluoroelastomer, silicone, highly saturated nitrile and PTFE, among others. Friction due to intermittent movement (breakout friction) and friction due to continuous movement (running friction) can cause high pressure and heat buildup, respectively. For optimal performance, the type of motion as well as size, squeeze, stretch, chemical compatibility, temperature, pressure and friction of the application are considered in the selection of the flexible ring material. The system of the present teachings can accommodate a variety of designs, materials and configurations of the cartridge and flexible ring combination.

[0030] 7A, a second implementation of the system of the present teachings is shown. The second implementation includes a motor subsystem that drives the movement of the cartridge inside the bioreactor and a mechanism for rotating the bioreactor. In the second implementation, the motor subsystem can be decoupled from the bioreactor and cell seeding is accomplished through a seeding port integrated with the cartridge. An example of the second implementation is a system 2300 that includes, but is not limited to, a rotating yoke 2303 for rotating the bioreactor and a removable motor subsystem. The motor subsystem can drive the movement of a cartridge 2363 enclosed within the bioreactor subsystem. As in the first implementation described herein, the cartridge 2363 includes frame parts connected by a flexible ring. The frame parts hold the scaffold between them and split to allow movement of the scaffold. As in the first implementation, the motor subsystem includes a magnetic coupling to allow movement of the frame and scaffold.

[0031] 7B and 7C, a magnet tube 2357 surrounds a first magnet 2355. A cooperating magnet 2351 is positioned across a diaphragm 2353 from the first magnet 2355, and an O-ring 2399 (FIG. 7C) and diaphragm 2353 can seal the bioreactor from environmental contamination. The O-ring 2399 (FIG. 7B) can be constructed of a material that can prevent gas and fluid leakage and meet environmental requirements. For example, leakage protection can be provided by materials such as polytetrafluoroethylene, nitrile, neoprene, ethylene propylene diene monomer rubber, and fluorocarbons. If strong chemical resistance is required, the O-ring 2399 can be constructed of a copolymer of ethylene, tetrafluoroethylene, and perfluoromethyl vinyl ether, for example. Cells can be provided directly to the scaffold in the cartridge 2363 through a fluidic connector 2373 (FIG. 7B), through which the cells enter the cartridge wells. The scaffold is positioned between two ports that connect to the fluid connectors (FIG. 7M), so that one seeding fluid connector 2373 can seed a first surface of the scaffold and the other seeding fluid connector 2373 can seed a second surface of the scaffold. Fluid connector 2375 can simultaneously bring media and nutrients into the bioreactor chamber and vent the chamber to remove air from the chamber.

[0032] Referring now to FIG. 7D, the magnet cup 2314 is coupled to the motor subsystem by a connector 2316 that couples the linkage 2311 and the magnet cup 2314 in a prismatic joint. The motor subsystem can be decoupled from the bioreactor subsystem by removing the connector 2316. The position of the pull mechanism 2369 (FIG. 7A) is maintained as described herein, and the bioreactor subsystem can be transported to a location where the tissue to be moved on the cartridge 2363 (FIG. 7A) is required. In a similar manner, the motor subsystem can be recoupled with the bioreactor subsystem to further move the tissue. Other forms of coupling between the bioreactor subsystem and the motor subsystem are also contemplated by the present teachings. For example, the linkage 2311 and the magnet cup 2314 can be constructed as a click-together coupling. Additionally, the connector 2316 can include a pin or a screw. The magnet cup 2314 and the linkage 2311 can be two parts of a ball and socket joint or a knuckle joint.

[0033] 7E, the components of the bioreactor subsystem are shown. The various components of the bioreactor subsystem are held together by frame clamps 2368T and 2368. Resting on one side of each frame clamp is a film 2364 that surrounds the cartridge 2363 and the scaffolds they move. In some configurations, the film 2364 is gas permeable, obviating the need to provide a sterile filter to add gas to the environment of the cartridge 2363. The cartridge 2363 is assembled to a pull mechanism 2369. Coupled to the pull mechanism 2369 is a rail 2366 that slides into a guide rail mount within a guide rail 2378 cut into the base 2361. When the pull mechanism 2369 is forced forward or backward by a motor subsystem coupled to the pull mechanism 2369 through a cavity 2380 in the base 2361, the rail 2366 slides back and forth within the guide rail 2378, moving the cartridge 2363 and the scaffold held within the cartridge 2363.

[0034] 7F-1 and 7F-2, to move the scaffold, the motor linkage 2405 is activated to move the pull mechanism 2403 from its rest position shown in FIG. 7F-1 to its extended position shown in FIG. 7-2 by sliding the rail within the guard rail 2378 (FIG. 7E). The cartridge seam 2409 forms a gap 2411 when the pull mechanism 2403 is moved to its extended position, pulling the first cartridge part 2407 away from the second cartridge part 2415 and moving the scaffold held within the cartridge. The cartridge part 2415 is fixed to the base 2413 while the cartridge part 2407 is free to slide within the guard rail 2378.

[0035] 7G-7J, an implementation of the movement means of the present teachings is depicted. Protruding from the side of the bioreactor system is a first part of the movement means. The first part directly interfaces with a pull mechanism 2369 (FIG. 7G) at the end of the bioreactor and with a motor subsystem at the opposite end. The interface with the pull mechanism 2369 (FIG. 7G) includes a magnet tube 2357 held in place by a slotted spring pin 2367 (FIG. 7H). The present teachings contemplate other coupling means. Once coupled, the magnets 2355 and 2351 (FIG. 7J) provide pressure to the pull mechanism 2369 (FIG. 7G) to pull the cartridge apart at its seam 2409 (FIG. 7G) and move the scaffold. The motor motion directly couples the magnet 2351 and moves the magnet 2351. Magnet 2351 is coupled to pull mechanism 2369 directly by magnet 2355. The magnetic force between magnets 2351 and 2355 results in the movement of magnet 2355 when magnet 2351 moves. Magnet cup 2314 houses magnet 2351 and provides one half of the linkage between the bioreactor interface components and the motor subsystem. Magnet cup 2314 and linkage structure 2311 form a rotatable connection at tongue 2492 that slides into the gap of magnet cup 2314. The geometry of the gap allows linkage structure 2311 to rotate and move the motor subsystem to the side of the bioreactor. When the motor structure is needed to move the scaffold, it can be rotated to face the bioreactor so that the linear actuator can exert pressure on the interface structure and thus the pull mechanism 2369 (FIG. 7G). The motor pressure moves the cartridge to a retracted position completing the motion cycle initiated by the magnetic force.

[0036] Continuing with reference to FIGS. 7G-7J, the linear actuator 2307 provides pressure through a plunger 2310. The plunger 2310 relies on a washer 2354 / stacked wave disc spring 2352 mounted on a linkage 2311 through a shoulder screw 2398 to provide pressure on a pull mechanism 2369 (FIG. 7G) that is damped by a spring 2352. A shaft mount 2303 rigidly couples the linear actuator 2307 to the linkage 2311. A position sensor mounting brick 2499 provides one mounting option for a position sensor to track the location of the plunger 2310 from which data usable to control the scaffold motion plan originates. Power and environmental protection for the linear actuator 2307 may be provided by a junction box 2305 capped by a junction box cap 2395.

[0037] The rotating shaft mount 2304 (FIG. 7G) provides a means for the rotary activation means 2360 to fully rotate the bioreactor for the purpose of seeding cells on both sides of the scaffold.

[0038] With particular reference to Figures 7I-A, 7I-B and 7I-C, cell seeding port 2413 interfaces directly with cartridge 2301. Media to fill the bioreactor cavity is provided through media port 2411. Cells are seeded on one side of the scaffold and allowed to settle and attach onto the scaffold. A rotational actuator flips the bioreactor so that the opposite side of the scaffold can be seeded with cells and allowed to settle and attach. The movement / stretching routine begins after both sides have been seeded and cells have attached.

[0039] 7K-7M, components of a configuration that allows for multi-surface cell seeding are shown. In particular, referring to FIG. 7K, seeding of a first surface begins at fluid inlet 2373. The interior of the bioreactor is sealed from the environment beginning at seal 2601. The direction of travel of cells introduced through inlet 2373 is indicated by the arrow beginning at inlet 2373. As shown, cells are directed to exit ports 2612 and 2614 (FIG. 7M) and deposited above where a scaffold would be (scaffold not depicted in FIG. 7K). Seals 2609 and 2607 similarly provide a barrier from environmental contamination that could be introduced by inlet 2373. Also shown is media / aeration inlet / outlet 2375. Media and / or nutrients introduced into the wells of the bioreactor enter the bioreactor through cavity 2375 and exit into interior region 2375A that surrounds the bioreactor (FIG. 7K). The seeded cells are trapped on the scaffold 2605 by a flexible ring present in the cavity 2603, described elsewhere herein. Detail B (FIG. 7M) of section AA (FIG. 7L) is a cross-sectional view of a detail of FIG. 7K. Seeding of the second side of the scaffold 2605 is shown in detail D (FIG. 7M) of section CC (FIG. 7L). The cells entering the inlet 2373A travel on the path indicated by the arrows in section CC, with seals 2602 and 2611 preventing environmental contamination from the inlet 2373A. In this case the cells do not cross the scaffold 2605. Instead, the cells are deposited on the scaffold 2605 at the outlet 2612 (FIG. 7M) on the same side of the scaffold 2605 that the cells entered. Section CC and detail D show a component that may be rotated 180° before the cells are deposited in order to utilize gravity to encourage cell attachment.

[0040] In the first test, one of the systems of the present teachings was incubated at 37° C., 5% CO 2In this test, C2C12 cells were fluidically seeded inside a sealed bioreactor on both sides of the scaffold through designated seeding ports in the sealed bioreactor or directly into the cartridge. The first scaffold side was seeded, followed by the second side after a preselected time. Throughout the seeding process, the bioreactor was completely closed to the environment, allowing seeding to be performed without any contamination issues in a non-cleanroom environment but using aseptic technique and a specific sample port. After the second seeding, again a preselected time elapsed and samples were stained and imaged to assess cell viability and coverage. Samples were stained with NucBlue and Calcein. In this test, cells were present on both sides of each scaffold, indicating correct delivery of the seeding input through the seeding port to the cartridge.

[0041] In the second test, the cell harvest solution was quenched with an equal volume of growth medium, and the cells were counted, centrifuged, and resuspended at the desired concentration for seeding. A volume of the cell solution was added to a sterile syringe to seed each scaffold with a preselected number of cells. The syringe was connected to the seeding port of side B above each cartridge position, and the seeding input was manually delivered. A syringe filled with sterile air was then attached to each seeding port to expel the remaining seeding input volume. Side B was seeded first, such that side A was already in the upright position when the motion was initiated. After 24 hours, the bioreactor was inverted so that side A was facing up, ensuring that the top space voids and isolated air bubbles were completely displaced from side B. The cell harvest and seeding procedure was repeated to seed side A.

[0042] For motility studies, 24 hours after the second seeding, the bioreactor was drained. The reactor was refilled with differentiation medium and the motility protocol was initiated.

[0043] In all tests where samples were seeded inside the assembled bioreactor, high coverage was observed, suggesting that the print-free seeding mechanism of the bioreactor is effective in delivering and maintaining cells within the seeding window. Based on the results presented here, it appears that the tension state of the scaffold may be important for homogeneous seeding of the scaffold. The bioreactor configuration used for the tests is easy to assemble, incorporates a leak-proof cartridge and a sealed tank, and has a robust motion mechanism capable of applying strains up to 25%. The bioreactor demonstrates an effective method for the fully enclosed hydrogel-free delivery of cells to the BAM scaffold resulting in uniform cell distribution and high cell coverage. The tank also allows for a reduction of approximately 60% in the required medium volume compared to other similar bioreactors. The cartridge and bioreactor concept is suitable for cost-effective production and scale-up.

[0044] One method of using the configuration of the system of the present teachings was tested using the following steps: (1) wash all components of the bioreactor with soap and water, rinse with DI and dry; (2) autoclave all components (except the BAM scaffold) at 121° C. for 30 minutes; (3) rehydrate the sterilized BAM scaffold by covering it in sterile PBS for at least 10 minutes; (4) drape the BAM over the bottom cartridge piece such that the BAM covers all the O-rings but not the seeding ports, cover with the top cartridge and seal the top and bottom pieces together; (5) trim excess BAM from the edges of the cartridge and store the assembled cartridge in sterile PBS to prevent dehydration during subsequent cartridge assembly; (6) load the cartridge into the bioreactor; (7) autoclave the bioreactor at 121° C. for 30 minutes. (8) fill the bioreactor with 60 ml of growth medium through the fill / drain port system and remove any air from the bioreactor by tipping the port side up and sealing the fill port while drawing the air out through the drain; (9) rotate the reactor until the cartridge is top side up and the reactor is flat; (10) seed on side A of the BAM, mature for 24 hours, rotate the bioreactor, seed on side B, mature for 24 hours; (11) remove the growth medium; (12) add 100 ml of differentiation medium, rotate the bioreactor 90° so that the port is on the top side, draw excess air out of the bioreactor and rotate to the original orientation; and (13) exercise the tissue for 5 days. The test results indicate that the bioreactor is ready for immediate integration into a manufacturing line. With regard to closed, fluid seeding, growth and maturation of TEMR products, the performance of the bioreactor is superior to existing bioreactors.

[0045] Referring now to Figures 9A-9C, shown are results from human muscle progenitor cells using three cartridges of two sides each, using the bioreactor system depicted in Figures 6 and 7. Figure 9A is a pictorial illustration of the results of static seeding followed by five days of exercise. In Figure 9A, actin is shown in red and nuclei in blue. Figures 9B and 9C are graphical illustrations of the results. In Figure 9B, it can be seen that the coverage of Side A and Side B was measured to be approximately 96% on average, with Side A and Side B capping out at over 97% in the first and second positions, respectively. In Figure 9C, it can be seen that the alignment angle of the primary actin was within 5° for all cartridges / sides, and the coherency of actin alignment was consistently high (combined sides approximately 0.70).

[0046] Those skilled in the art can devise various variations and modifications without departing from the present disclosure. Therefore, the present disclosure is intended to cover all such variations, modifications and differences. Moreover, although some exemplary configurations of the present disclosure have been shown in the drawings and / or discussed herein, the present disclosure is not intended to be limited thereto, and the present disclosure has as broad a scope as the art will permit, and the specification is intended to be read accordingly. Thus, the above description should not be considered limiting, but merely illustrative of a particular configuration. Moreover, those skilled in the art will envision other modifications that fall within the scope and spirit of the claims appended hereto. Other elements, steps, methods and techniques that are insubstantial different from those described above and / or in the appended claims are also intended to fall within the scope of the present disclosure.

[0047] The drawings are presented only to demonstrate some embodiments of the present disclosure, and the drawings described are merely exemplary and non-limiting. In the drawings, for illustrative purposes, the size of some elements may be exaggerated and not drawn to a particular scale. Moreover, elements shown in the drawings having the same number may be identical or similar elements, depending on the context.

[0048] When the term "comprising" is used in the present specification and claims, it does not exclude other elements or steps. When an indefinite or definite article, such as "a", "an" or "the", is used when referring to a singular noun, this includes a plurality of that noun, unless something is specifically stated otherwise. Thus, the term "comprising" should not be interpreted as being limited to the items listed thereafter; it does not exclude other elements or steps, and therefore the scope of the expression "a device comprising items A and B" should not be limited to a device consisting of only components A and B.

[0049] Moreover, terms such as "first," "second," "third," and the like, whether used herein or in the claims, are provided to distinguish between similar elements and not necessarily to describe an order or chronology. It should be understood that terms so used are interchangeable under appropriate circumstances (unless otherwise expressly disclosed) and that the disclosure described herein is capable of operating in orders and / or arrangements other than those described or illustrated herein.

Claims

1. 1. A system for mobilizing cells and tissues produced from said cells, comprising: at least one cartridge having a cavity therein and being expandable in at least one dimension; at least one scaffold configured to retain the cells on the at least one surface and configured to reside within the cavity in the at least one cartridge; a flexible ring contained within the at least one cartridge, holding the at least one cartridge in an unextended position and holding the cells within the cavity; an extension means operably coupled to the at least one cartridge; a bioreactor including a sealed enclosure housing the at least one scaffold and the at least one cartridge, the bioreactor configured to allow seeding of the at least one scaffold with the cells, the seeding occurring within the sealed enclosure when the cartridge is in an unextended position; A system including:

2. The system of claim 1, wherein the cells are selected from the group consisting of immortalized mouse myoblasts (C2C12), muscle progenitor cells, hMP cells, NHCN-M2 cells, mesoangioblasts, satellite cells, H2K cells, L8 rat cells, 3T3 mouse embryonic fibroblasts, and combinations thereof.

3. The at least one cartridge comprises: a seeding inlet configured to distribute the cells onto the at least one surface of the at least one scaffold within the sealed enclosure; The system of claim 1 , comprising:

4. The at least one cartridge comprises: a channel configured to allow fluid flow to and from the at least one scaffold; The system of claim 1 , comprising:

5. The at least one cartridge prepared by a method comprising: a compression means configured to provide compression to the at least one scaffold, the compression inhibiting silicone penetration into the at least one surface of the at least one scaffold; a scaffold pinch configured to inhibit silicone penetration into said at least one surface; a silicone mold configured to provide a reservoir of silicone to create the shape of at least one scaffold, the silicone mold including a table top configured to inhibit infiltration of silicone into the at least one surface, the silicone mold configured to surround the at least one scaffold with the silicone embedded around the at least one scaffold to form a sidewall of a well with the at least one scaffold being a base of the well, the well retaining the cells; and a tray configured to remove the at least one scaffold after the silicone has cured; The system of claim 1 further comprising:

6. The stretching means comprises: a frame configured to elongate the at least one cartridge in the at least one dimension when a force is applied thereto; The system of claim 1 , comprising:

7. The flexible ring comprises: a band surrounding the at least one cartridge, the flexible ring configured to seal a cavity of the at least one cartridge to prevent loss of cells while the cells are seeded, the flexible ring bridging a separation distance when the cartridge is altered in the at least one dimension and urging the cartridge to return to its original dimension; The system of claim 1 .

8. The at least one scaffold comprises: Acellular matrix, The system of claim 1 , comprising:

9. The at least one scaffold comprises: extensible membrane, The system of claim 1 , comprising:

10. 10. The system of claim 1, wherein the at least one scaffold is selected from the group consisting of decellularized bovine bladder, decellularized porcine bladder, an acellular matrix, a decellularized collagen membrane, a thin polymer membrane, and a polymer fiber membrane.

11. 10. The system of claim 1, wherein the at least one scaffold is surrounded by a silicone enclosure.

12. 10. The system of claim 1, wherein the at least one surface is configured to receive the cells seeded on the at least one scaffold within the sealed enclosure.

13. a gas-permeable lid covering said bioreactor, said lid including at least one cell seeding port; The system of claim 1 further comprising:

14. 14. The system of claim 13, wherein the lid is formed from a transparent or translucent material configured to allow observation of gas exchange between a bioreactor environment inside the bioreactor and the at least one scaffold and observation of the scaffold and cells and tissues attached thereto.

15. The system of claim 13 , wherein the lid is a solid or semi-solid structure.

16. The bioreactor comprises: an inlet configured to introduce culture medium into the sealed enclosure; The system of claim 1 further comprising:

17. 17. The system of claim 16, wherein the medium is selected from the group consisting of: amino acids, vitamins, inorganic salts, glucose, growth factors, serum, hormones, attachment factors, dissolved gases, and combinations thereof.

18. The bioreactor comprises: a drain configured to drain waste products from the sealed enclosure; The system of claim 1 further comprising:

19. a rotating means configured to rotate the bioreactor, the rotating means being responsive to commands from a controller; The system of claim 1 further comprising:

20. a controller operably coupled to the system, the controller executing instructions for controlling medium supply, controlling cell seeding, controlling rotation, controlling motion, and controlling interface with a manufacturing line; The system of claim 1 further comprising:

21. an electronic coupling between said production line and said controller; 21. The system of claim 20, further comprising:

22. a lockout brake configured to maintain the at least one cartridge in place after the at least one dimensional change; The system of claim 1 further comprising:

23. 2. The lockout brake: two sections operably coupled to the at least one cartridge by removable pins; 23. The system of claim 22, further comprising:

24. The bioreactor comprises: at least one environmental barrier to protect said sealed enclosure from environmental contamination; The system of claim 1 further comprising:

25. a push / pull mechanism configured to exert a force on the at least one cartridge across the at least one environmental barrier to cause the at least one dimensional change within the at least one cartridge and the at least one scaffold supported therein, thereby moving the cells on the scaffold; 25. The system of claim 24, further comprising:

26. 26. The system of claim 25, wherein the force is a magnetic coupling force engaged by a linear actuator, the magnetic coupling providing force transmission across the at least one environmental barrier between the at least one scaffold and an exterior of the bioreactor.

27. The push / pull mechanism comprises: a decoupling means configured to decouple the push / pull mechanism from the bioreactor; 26. The system of claim 25, further comprising:

28. 26. The system of claim 25, wherein a force sensor measures the resistance / stiffness of the tissue and initiates interruption of the movement when the resistance / stiffness reaches a predetermined limit.

29. a push / pull mechanism removably coupled to said bioreactor; The system of claim 1 further comprising:

30. Durable components that are sterilizable and / or reusable; The system of claim 1 further comprising:

31. Disposable components that are sterilizable and / or single-use; The system of claim 1 further comprising:

32. 1. A system for mobilizing cells and tissues produced from said cells, comprising: at least one cartridge comprising an internal cavity, a flexible ring that retains the cells within the cavity, and a spreading means; at least one scaffold configured to retain the cells on at least one surface and configured to reside within the cavity in at least one cartridge; a bioreactor housing having a sealed enclosure that houses the at least one cartridge and at least one scaffold; Including, The bioreactor housing and at least one cartridge are configured to seed the at least one scaffold with the cells, the seeding occurring within the sealed enclosure. system.

33. The system of claim 32, wherein the cells are selected from the group consisting of immortalized mouse myoblasts (C2C12), muscle progenitor cells, hMP cells, NHCN-M2 cells, mesoangioblasts, satellite cells, H2K cells, L8 rat cells, 3T3 mouse embryonic fibroblasts, and combinations thereof.

34. The at least one cartridge comprises: a seeding inlet configured to distribute the cells onto the at least one scaffold within the sealed enclosure; 33. The system of claim 32, comprising:

35. The at least one cartridge comprises: a channel configured to allow fluid flow to and from the at least one scaffold within the cavity; 33. The system of claim 32, comprising:

36. The at least one cartridge comprises: a compression means configured to provide compression to the at least one scaffold, the compression inhibiting silicone penetration into the at least one preselected surface of the at least one scaffold; a scaffold pinch configured to inhibit silicone penetration into said at least one surface; a silicone mold configured to provide a reservoir of the silicone to create a shape of a cartridge containing at least one scaffold, the silicone mold including a table top configured to inhibit penetration of the silicone into preselected ones of the at least one surface, the silicone mold configured to surround the at least one scaffold with the silicone; and a tray configured to remove the at least one scaffold after the silicone has cured; 33. The system of claim 32, further comprising:

37. The stretching means comprises: a frame configured to elongate the at least one cartridge in the at least one dimension when a force is applied thereto; 33. The system of claim 32, comprising:

38. The flexible ring comprises: a band surrounding the at least one cartridge, the flexible ring configured to seal the cavity of the at least one cartridge to prevent cell loss while the cells are seeded, the flexible ring bridging a separation distance when the cartridge is altered in at least one dimension and urging the cartridge to return to its original dimension; 33. The system of claim 32.

39. 33. The system of claim 32, wherein the at least one scaffold is an acellular matrix.

40. 33. The system of claim 32, wherein the at least one scaffold is a compliant membrane.

41. 33. The system of claim 32, wherein the at least one scaffold is selected from the group consisting of decellularized bovine bladder, decellularized porcine bladder, an acellular matrix, a decellularized collagen membrane, a thin polymer membrane, and a polymer fiber membrane.

42. 33. The system of claim 32, wherein the at least one scaffold is surrounded by a silicone enclosure.

43. 33. The system of claim 32, wherein the at least one surface is configured to receive the cells seeded on the at least one scaffold within the sealed enclosure.

44. a gas-permeable lid covering said bioreactor, said lid including at least one cell seeding port; 33. The system of claim 32, further comprising:

45. 45. The system of claim 44, wherein the lid is formed from a transparent or translucent material configured to allow observation of the at least one scaffold.

46. 45. The system of claim 44, wherein the lid is a solid or semi-solid structure.

47. The bioreactor comprises: an inlet configured to introduce culture medium into the sealed enclosure; 33. The system of claim 32, further comprising:

48. 48. The system of claim 47, wherein the medium is selected from the group consisting of: amino acids, vitamins, inorganic salts, glucose, growth factors, serum, hormones, attachment factors, dissolved gases, and combinations thereof.

49. The bioreactor comprises: a drain configured to drain waste products from the sealed enclosure; 33. The system of claim 32, further comprising:

50. a rotating means configured to rotate the bioreactor, the rotating means being responsive to commands from a controller; 33. The system of claim 32, further comprising:

51. a controller operably coupled to the system, the controller executing instructions for controlling medium supply, controlling cell seeding, controlling rotation, controlling motion, and controlling interface with a manufacturing line; 33. The system of claim 32, further comprising:

52. an electronic coupling between said production line and said controller; 52. The system of claim 51, further comprising:

53. a lockout brake configured to maintain the at least one cartridge in a predetermined position after at least one dimensional change has been made; 33. The system of claim 32, further comprising:

54. 2. The lockout brake: two sections operably coupled to the at least one cartridge by removable pins; 54. The system of claim 53, further comprising:

55. The bioreactor comprises: at least one environmental barrier to protect said sealed enclosure from environmental contamination; 33. The system of claim 32, further comprising:

56. a push / pull mechanism configured to exert a force on the at least one cartridge across the at least one environmental barrier to cause the at least one dimensional change within the at least one cartridge and the at least one scaffold supported therein, thereby moving the cells on the scaffold; 56. The system of claim 55, further comprising:

57. 57. The system of claim 56, wherein the force is a magnetic coupling force engaged by a linear actuator, the magnetic coupling providing force transmission across the at least one environmental barrier between the at least one scaffold and an exterior of the bioreactor.

58. The push / pull mechanism comprises: a decoupling means configured to decouple the push / pull mechanism from the bioreactor; 57. The system of claim 56, further comprising:

59. The push / pull mechanism comprises: a force sensor for measuring the resistance / stiffness of the tissue and for initiating the interruption of the movement when the resistance / stiffness reaches a predetermined limit.

57. The system of claim 56.

60. a push / pull mechanism removably coupled to said bioreactor; 33. The system of claim 32, further comprising:

61. Durable components that are sterilizable and / or reusable; 33. The system of claim 32, further comprising:

62. Disposable components that are sterilizable and / or single-use; 33. The system of claim 32, further comprising:

63. 1. A method for moving cells in a sealed bioreactor by a force device, the sealed bioreactor and the force device being components of a manufacturing line, the method comprising: sending, by a controller, a command to a seeding device to seed cells onto a first surface within the sealed bioreactor, the first surface including a cell barrier configured to retain the seeded cells; sending, by the controller, a command to the force device to exert a pressure on a first surface configured to alternately stretch and relax the seeded cells on the first surface to move the cells; The method includes:

64. sending a command by a controller to a rotation device that rotates the sealed bioreactor; sending, by a controller, a command to a seeding device to seed cells onto a second surface within the sealed bioreactor, the second surface including a cell barrier configured to retain the seeded cells; sending, by the controller, a command to the force device to exert a pressure on a second surface configured to alternately stretch and relax the seeded cells on the second surface to move the cells; 64. The method of claim 63, further comprising:

65. 64. The method of claim 63, wherein the cells are selected from the group consisting of immortalized mouse myoblasts (C2C12), muscle progenitor cells, hMP cells, NHCN-M2 cells, mesoangioblasts, satellite cells, H2K cells, L8 rat cells, 3T3 mouse embryonic fibroblasts, and combinations thereof.

66. 65. The method of claim 64, wherein the first surface is enclosed within at least one cartridge, the at least one cartridge comprising a seeding inlet configured to distribute the cells onto the at least one scaffold within a sealed bioreactor.

67. The at least one cartridge comprises: a channel configured to allow fluid flow to and from the at least one scaffold within the cavity; 67. The method of claim 66, further comprising:

68. The at least one cartridge comprises: a compression means configured to provide compression to the at least one scaffold, the compression inhibiting silicone from penetrating a preselected one of the at least one surface of the at least one scaffold; a scaffold pinch configured to inhibit silicone penetration into said at least one surface; a silicone mold configured to provide a reservoir of silicone to create a shape of the cartridge containing at least one scaffold, the silicone mold including a table top, the table top configured to inhibit penetration of silicone into preselected ones of the at least one surface, the silicone mold configured to surround the at least one scaffold with the silicone; and a tray configured to remove the cartridge containing the at least one scaffold after the silicone has cured; 67. The method of claim 66, further comprising:

69. The stretching means comprises: a frame configured to elongate the at least one cartridge in the at least one dimension when a force is applied thereto; 67. The method of claim 66, comprising:

70. The flexible ring comprises: a band surrounding the at least one cartridge, the flexible ring configured to seal the cavity of the at least one cartridge to prevent cell loss while the cells are seeded, the flexible ring bridging a separation distance when the cartridge is altered in at least one dimension and urging the cartridge to return to its original dimension; 67. The method of claim 66.

71. 70. The method of claim 69, wherein the at least one scaffold is an acellular matrix.

72. 70. The method of claim 69, wherein the at least one scaffold is a compliant membrane.

73. 70. The method of claim 69, wherein the at least one scaffold is selected from the group consisting of decellularized bovine bladder, decellularized porcine bladder, acellular matrix, decellularized collagen membrane, thin polymer membrane.

74. 70. The method of claim 69, wherein the at least one scaffold is surrounded by a silicone frame.

75. 70. The method of claim 69, wherein the first surface and / or the second surface are configured to receive cells seeded onto the at least one scaffold within the sealed bioreactor.

76. The sealed bioreactor comprises: a lid including at least one cell seeding port; 64. The method of claim 63, further comprising:

77. 77. The method of claim 76, wherein the lid is formed from a transparent or translucent material configured to allow observation of the at least one scaffold.

78. 45. The method of claim 44, wherein the lid is a solid or semi-solid structure.

79. The bioreactor comprises: an inlet configured to introduce culture medium into the sealed enclosure; 64. The method of claim 63, further comprising:

80. 80. The method of claim 79, wherein said medium is selected from the group consisting of: amino acids, vitamins, inorganic salts, glucose, growth factors serum, hormones, attachment factors, dissolved gases and combinations thereof.

81. The bioreactor comprises: a drain configured to drain waste products from the sealed enclosure; 64. The method of claim 63, further comprising:

82. Controlling medium supply; Controlling the interface with the production line; 64. The method of claim 63, further comprising:

83. coupling said production line with said controller; 83. The method of claim 82, further comprising:

84. retaining said at least one cartridge in a preselected position with a lockout brake; 67. The method of claim 66, further comprising:

85. 2. The lockout brake: two sections operably coupled to the at least one cartridge by removable pins; 85. The method of claim 84, further comprising:

86. The bioreactor comprises: at least one environmental barrier to protect said sealed enclosure from environmental contamination; 64. The method of claim 63, further comprising:

87. exerting a pressure with the force device across the at least one environmental barrier into the sealed bioreactor; 87. The method of claim 86, further comprising:

88. The pressure: A magnetic k-coupling engaged by a linear actuator; 64. The system of claim 63, further comprising:

89. The force device comprising: a decoupling means configured to decouple the push / pull mechanism from the bioreactor; 88. The method of claim 87, further comprising:

90. 90. The method of claim 89, wherein the decoupling means is a swing-away mount.

91. decoupling the force device from the sealed bioreactor; 64. The method of claim 63, further comprising:

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