Tissue culture vessel for preparation of compressed hydrogel skin grafts and related methods and systems

Abstract

To improve the robustness and reproducibility of a graft manufacturing process.SOLUTION: A tissue culture vessel includes a graft support tray (200) and a box having a lid (400) and a base which engages and retains the tray. The tray has two operational states: a first operational state in which a floor of the tray is slightly raised with respect to a floor of the base and a second operational state in which the floor of the tray descends to contact the floor of the base.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This PCT application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 212,662, filed June 20, 2021, and U.S. Provisional Patent Application No. 63 / 240,360, filed September 2, 2021, each of which has the same title and assignee as the present application, and each of which is incorporated herein by reference in its entirety.

[0002] Various embodiments described herein are in the field of tissue culture labware. [Background technology]

[0003] Human skin consists of three main layers: the epidermis (outermost layer), the dermis (middle layer), and the hypodermis (deepest layer).

[0004] Deep wounds (full-thickness wounds), such as those following a burn accident, involve the epidermis and the entire dermis (and sometimes the subcutaneous tissue as well), and require surgical intervention.

[0005] Small full-thickness skin wounds are typically treated with full-thickness skin autografts, in which the epidermis and entire dermis are taken from a healthy part of the body and grafted onto the wound.

[0006] Large, full-thickness wounds are typically treated with split-thickness skin autografts. Split-thickness skin autografts consist of the epidermis and a thin, incomplete dermal layer. Split-thickness skin autografts are not a satisfactory clinical solution to the problem of large, full-thickness wounds. Large, full-thickness wounds often do not heal well and result in unsightly marks and scars.

[0007] The reason that full-thickness skin autografts heal deeper wounds better than split-thickness skin autografts is that full-thickness skin autografts contain features that split-thickness skin autografts lack, such as vasculature. Vasculature (blood and / or lymphatic vessels) provides oxygen and nutrients as well as immune cell transport, contributing to overall function and tissue survival. The capillaries in a full-thickness skin graft only need to connect with those in the wound bed, whereas in a split-thickness skin graft, capillaries must grow from the wound bed to the graft to perfuse the tissue.

[0008] Another reason why full-thickness skin autografts heal deeper wounds better than split-thickness skin autografts is that transplanting split-thickness skin autografts into full-thickness wounds can result in tissue contraction that contributes to scarring and / or fibrosis and / or other disfiguring features. Alternatively or additionally, scarring can contribute to the inability of the transplanted skin substitute to grow with the patient (e.g., a child), further deteriorating the skin. As a result, the use of split-thickness skin autografts can require several successive surgical procedures over several years to alleviate these problems. These additional surgical procedures impose a significant financial and psychological burden on the patient.

[0009] More recently, skin tissue engineering has been used to create full-thickness skin autograft substitutes. Tissue engineering utilizes hydrogels as scaffolding materials that provide a 3D cellular matrix. Hydrogels are biocompatible and biomimetic, have low immunogenicity (conserved across species), and are naturally remodeled by cells, which can easily seed between cells within the fibrous network. However, the poor mechanical properties of hydrogels limit their clinical use as scaffolds for tissue engineering applications. Due to the relatively large area and thin nature of skin grafts (clinically appropriate sizes for skin grafts are 50 cm). 2 (The thickness is less than 2 mm.) Mechanical stability is important for clinical application of hydrogel-based cultured skin tissue.

[0010] Mechanical stability allows for handling and processing of hydrogel-based tissue-engineered skin grafts during and after culture and during and after surgical application and / or testing. The mechanical stability of hydrogels can be increased by compression. Summary of the Invention

[0011] A broad aspect of the present invention relates to the manufacture and delivery of compressed hydrogel skin grafts in a single container under sterile conditions.

[0012] One aspect of some embodiments of the present invention relates to a sealed tissue culture vessel including an explant support frame within a box having a lid and a base. According to these embodiments, the frame has two operational states: a first operational state in which the frame is slightly elevated relative to the floor of the base within which it resides, and a second operational state in which the frame is lowered and in contact with the floor of the base within which it resides. In some embodiments, the lid includes a movable plunger that, when depressed, moves the frame from the first to the second operational state. In some embodiments, the lid includes one or more elevated compartments that contain medium displaced from the frame when the plunger is depressed. In some embodiments, the elevated compartments include gas-permeable membranes on their upper surfaces. These gas-permeable membranes allow air to escape from the vessel when the plunger is lowered and allow air to enter the vessel when the plunger is raised. The gas-permeable membranes allow gas exchange between the ambient environment and the interior of the vessel while maintaining sterility. In some embodiments, the gas-permeable membranes contribute to the ability of a tissue culture hood to maintain sterility of the contents outside the vessel. Gas permeable membranes also support gas exchange during cell culture, for example, while placed in an incubator with a controlled gas environment enriched (5% CO2).

[0013] Another aspect of some embodiments of the present invention relates to a graft support tray comprising a medium-permeable membrane bed and an external, first, vertically extending spring that supports the membrane bed in an elevated position in the absence of an external force. In some embodiments, a second set of oppositely oriented, vertically extending springs is attached to the first spring. In some embodiments, the first and / or second sets of springs are leaf springs. In some embodiments, the first and / or second springs are attached to two opposite sides of a frame. In some embodiments, the frame includes guide holes / pins that mate with corresponding pins / holes in the base and / or lid.

[0014] Yet another aspect of some embodiments of the present invention relates to a base of a tissue culture vessel configured to accommodate a transplant support frame. In some embodiments, the base includes a compression structure that supports the membrane bed of the transplant support frame when the transplant support frame is fully lowered, while still allowing outward flow of culture medium when the frame is lowered. In some embodiments, the base includes recesses for accommodating the first and / or second set of springs of the frame located in the base. In some embodiments, the base includes pins or holes that mate with equivalent holes or pins on the frame. In some embodiments, these pins / holes serve to orient the frame within the base. Alternatively or additionally, in some embodiments, the base includes pins or holes that engage with equivalent holes or pins on the plunger of the lid. In some embodiments, these pins / holes serve to orient the plunger relative to the frame when the frame is located in the base. In some embodiments, the lid or base includes an O-ring or other gasket. According to various exemplary embodiments of the present invention, the gasket is integrally formed as part of the lid or base. In some embodiments, the O-ring contributes to forming an airtight seal. According to various exemplary embodiments of the present invention, the base includes a drain port and / or a sample removal port. In some embodiments, these ports are provided as barbed connectors. Alternatively or additionally, in some embodiments, the base includes a snap-fit connector for accommodating a lid. Alternatively or additionally, in some embodiments, the base includes a mating clasp insert for a shipping lock. In some embodiments, the shipping lock holds the plunger in a slightly lowered position.

[0015] Yet another aspect of some embodiments of the present invention relates to a lid for a tissue culture vessel. In some embodiments, the lid includes a rigid frame with a flexible bellows that holds a plunger in a fixed orientation relative to the frame. In some embodiments, the bellows are normally open to hold the plunger in an elevated position in the absence of an external force. In some embodiments, the lid includes one or more elevated lift compartments. In some embodiments, the lift compartments are mated with gas-permeable membranes on the upper surfaces of the lift compartments. According to various exemplary embodiments of the present invention, the membranes are located on the inside or outside of the lift compartments.

[0016] In some embodiments, the rigid frame includes clasp hooks that engage with corresponding structures on the base. In some embodiments, the lid includes a series of external barbed connectors attached to appropriately located internal exit ports. For example, barbed connectors are provided for CO2 / air entry and / or fibroblast (FB) and / or medium and / or keratinocytes and / or collagen hydrogel. Alternatively or additionally, some embodiments provide additional barbed ports for CO2 / air removal. Alternatively or additionally, in some embodiments, CO2 / air exits the tissue culture vessel through a gas permeable membrane in the riser compartment.

[0017] Another aspect of some embodiments of the present invention relates to a tissue culture method in which a hydrogel containing fibroblasts is introduced into a sealed tissue culture vessel and incubated to allow the fibroblasts to assemble into a gel matrix. After the hydrogel is formed, it is compressed while still in the same sealed tissue culture vessel. Keratinocytes (KCs) are then seeded into the same sealed tissue culture vessel, and further incubation is carried out until the graft is ready for implantation. In some exemplary embodiments of the present invention, hydrogel formation involves crosslinking by raising the pH (mixing the collagen / cell mixture with a buffer). This process is sometimes referred to as polymerization, but is not true polymerization.

[0018] Yet an additional aspect of some embodiments of the present invention relates to a method for transporting compressed hydrogel skin grafts in which the grafts are grown in a sealed tissue culture vessel, the growth medium is removed, a transport medium is introduced, and an integrated plunger in the lid of the sealed tissue culture vessel is slightly depressed to lock it into place.

[0019] Yet another aspect of some embodiments of the present invention relates to a system for simultaneous management and manipulation of tissue culture containers. In some embodiments, the tissue culture container is an explant culture container. In some exemplary embodiments of the present invention, the explant culture container is as described herein above. In some exemplary embodiments of the present invention, the system relies on a computer-controlled controller for dispensing cells and / or medium and / or matrix material into the container through conduits. In some embodiments, the system controls the mixing of mesenchymal cells (i.e., fibroblasts) with extracellular matrix material (e.g., collagen) and coordinates the distribution of the resulting cell / matrix mixture into the culture vessel. In some embodiments, various compartments of the system are heated and / or cooled. In some embodiments, cooling the cell matrix contributes to reducing premature gelation. Alternatively or additionally, some embodiments control the CO2 level and / or humidity of at least one system compartment.

[0020] Yet another aspect of some embodiments of the present invention relates to a system for automatically changing medium in a plurality of tissue culture containers. In some embodiments, the tissue culture containers are explant culture containers. In some exemplary embodiments of the present invention, the explant culture containers are as described herein above. In some embodiments, the system utilizes a tilting mechanism.

[0021] Yet another aspect of some embodiments of the present invention relates to a system for automatically compressing extracellular matrix material (e.g., hydrogel) in multiple tissue culture containers. According to various exemplary embodiments of the present invention, compression is performed in parallel and / or sequentially.

[0022] Yet another additional aspect of some embodiments of the present invention relates to a system that provides a visual image of a cell culture (e.g., an explant culture) at a remote location and enables remote manipulation of one or more culture parameters through a user interface at the remote location. In some embodiments, the visual image is captured by a camera and transmitted over a network to a smart device (e.g., a phone or tablet). Alternatively or additionally, in some embodiments, the user interface is a graphical user interface (GUI) of the smart device.

[0023] For purposes of this specification and the appended claims, the term "camera" includes conventional optical cameras as well as OCT (optical coherence tomography) devices. In some exemplary embodiments of the invention, the camera does not provide magnification. In other exemplary embodiments of the invention, the camera provides magnification of the captured image by 2x, 5x, 10x, 50x, 100x, 250x, 500x, 1000x, or an intermediate or greater magnification. In some embodiments, magnification is achieved by attaching or integrating a microscope to the camera.

[0024] In some of the figures, an exemplary tissue culture vessel according to some embodiments of the present invention is shown as a "graft box" (eg, FIG. 8I or FIG. 8J) or a "processing box" (eg, FIG. 12C).

[0025] It will be appreciated that the various aspects described above relate to solutions to technical problems associated with wrinkling and / or tearing of skin grafts during transport from one location to another.

[0026] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solutions to technical problems related to reducing the need for highly sophisticated tissue culture equipment for the production of skin grafts.

[0027] Alternatively or additionally, the various embodiments described above may be used to convert small amounts of biopsy material to large amounts of graft material (e.g., 1000 cm 2 , 1500cm 2 , 2000cm2 , 2500cm 2 , 3000cm 2 , 3500cm 2 , 4000cm 2 , 4500cm 2 , 5000cm 2 , 5500cm 2 It is understood that the present invention relates to a solution to the technical problem of producing a graft area that is 100 times, 150 times, 200 times, 250 times, or an intermediate or greater factor larger than the area of the biopsy.

[0028] Alternatively or additionally, it will be appreciated that the various aspects described above relate to solutions to technical problems relating to the remote management of tissue cultures.

[0029] Alternatively or additionally, it is understood that the various aspects described above relate to solutions to technical problems relating to reducing the workload of tissue culture technicians.

[0030] Alternatively or additionally, it will be understood that the various aspects described above relate to solutions to technical problems related to improving the robustness and reproducibility of the implant manufacturing process and / or reducing batch-to-batch variability.

[0031] Alternatively or additionally, it is understood that the various technical aspects described above contribute to a reduction in overall manufacturing costs.

[0032] Alternatively or additionally, it will be appreciated that the various aspects described above relate to a solution to the technical problem of manufacturing tissue grafts in a closed, automated box. In this way, the need for heavy manufacturing and / or clean rooms is eliminated. As a result, matching grafts can be manufactured in a variety of locations, such as hospitals where patients are admitted.

[0033] In some exemplary embodiments of the present invention, a tissue culture vessel is provided that includes: (a) a graft support tray; and (b) a box having a lid and a base, the box engaging and retaining the tray, the tray having two operational states: a first operational state in which the floor of the tray is slightly elevated relative to the floor of the base, and a second operational state in which the floor of the tray is lowered to contact the floor of the base. In some embodiments, the vessel includes a movable plunger attached to the lid. Alternatively or additionally, in some embodiments, the vessel includes one or more elevated compartments. Alternatively or additionally, in some embodiments, the vessel includes a gas-permeable membrane on an upper surface of the elevated compartment. Alternatively or additionally, in some embodiments, the vessel is sterilized and packaged to maintain sterility.

[0034] In some exemplary embodiments of the present invention, a graft support tray is provided that includes (a) a rigid frame, (b) a liquid-permeable membrane bed attached to the lower edge of the frame, and (c) a first set of vertically extending springs attached to two opposing exterior sides of the frame. In some embodiments, the tray includes a second set of vertically extending springs attached in opposite directions to the first springs. Alternatively or additionally, in some embodiments, the tray includes guide holes / pins that mate with corresponding pins / holes in a base to which the frame is attached. Alternatively or additionally, in some embodiments, the tray includes guide holes / pins that mate with corresponding pins / holes in a lid located on the support frame.

[0035] In some exemplary embodiments of the present invention, a tissue culture container base is provided, the base including a compression structure designed and configured to support a membrane bed of a graft support frame fully lowered into the base, the compression structure configured to allow outward flow of culture medium when the frame is lowered. In some embodiments, the container includes a recess sized and positioned to accommodate one set of springs of a graft support tray located in the base. Alternatively or additionally, in some embodiments, the container includes a recess sized and positioned to accommodate two sets of springs of a graft support tray located in the base. Alternatively or additionally, in some embodiments, the container includes a pin or hole sized and positioned to engage with a corresponding hole or pin on a graft support tray located in the base. Alternatively or additionally, in some embodiments, the container includes a pin or hole sized and positioned to engage with a corresponding hole or pin on a plunger of a lid fitted to the base. Alternatively or additionally, in some embodiments, the container includes an O-ring or other gasket or an overmolded elastomer. Alternatively or additionally, in some embodiments, the container includes at least one liquid removal port. Alternatively or additionally, in some embodiments, at least one of the at least one port includes a barbed connector. Alternatively or additionally, in some embodiments, the container includes a snap-fit connector for attachment to a lid. Alternatively or additionally, in some embodiments, the container includes a mating clasp insert for shipping locking.

[0036] In some exemplary embodiments of the present invention, a tissue culture vessel lid is provided that includes: (a) a rigid frame; and (b) a flexible bellows disposed within the frame, the bellows holding a plunger in a fixed orientation relative to the frame. In some embodiments, the bellows is normally open to hold the plunger in an elevated position in the absence of external forces. Alternatively or additionally, in some embodiments, the lid includes one or more elevated sections extending above the plane of the upper edge of the frame. Alternatively or additionally, in some embodiments, the lid includes a gas-permeable membrane on an upper surface of the elevated section. Alternatively or additionally, in some embodiments, the lid includes a catch hook on the rigid frame sized and positioned to engage a base covered by the lid. Alternatively or additionally, in some embodiments, the lid includes one or more external barbed connectors in fluid communication with an internal outlet port. Alternatively or additionally, in some embodiments, the lid includes a barbed port for CO2 / air removal. Alternatively or additionally, in some embodiments, the lid includes a resilient seal on the side of the lid that contacts the base when the container is assembled.

[0037] In some exemplary embodiments, the present invention provides a method for producing a skin graft, comprising: (a) introducing a hydrogel containing fibroblasts (FBs) into a sealed tissue culture vessel; (b) incubating until the FBs congregate into the gel matrix; (c) compacting the gel matrix within the same sealed tissue culture vessel; (d) seeding keratinocytes (KCs) onto the compacted matrix within the same sealed tissue culture vessel; and (e) further incubating until the graft is formed and ready for transplantation. In some embodiments, the method comprises visually inspecting the growth of the FBs and / or KCs within the tissue culture vessel using a microscope. Alternatively or additionally, in some embodiments, the method comprises exchanging the culture medium, which includes tilting the sealed tissue culture vessel in one direction to allow the medium to flow into a raised compartment and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow to a waste container.

[0038] Some exemplary embodiments of the present invention provide a method for producing a skin graft, comprising: (a) preparing a compressed hydrogel skin graft in a first position in a sealed tissue culture vessel; (b) removing growth medium and introducing transport medium into the sealed tissue culture vessel; (c) slightly depressing and securing in place an integrated plunger in a lid of the sealed tissue culture vessel; and (d) transporting to a second position. In some embodiments, the removing comprises tilting the sealed tissue culture vessel in one direction to allow medium to flow into a raised compartment, and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow to a waste container.

[0039] In some exemplary embodiments of the present invention, a transport lock is provided that includes a spanning member (a) sized to fit the dimensions of the above-mentioned tissue culture vessel and having (i) two notches on the lower edge of the transport lock configured to fit the contours of the frame of the lid, (ii) a downward extension between the notches and fasteners on the base on the outer edges of each of the notches sized to engage and retain snap-fit connectors, and (iii) a series of slots on the upper edge of the spanning member sized and positioned to engage and retain corresponding ribs on the underside of the base of the above-mentioned second tissue culture vessel.

[0040] In some exemplary embodiments of the present invention, an assembly is provided that includes a plurality of the above-described culture vessels arranged in a vertical array with the above-described transport locks interspersed among the plurality of culture vessels.

[0041] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a plurality of explant culture containers; (b) storage vessels for a cell suspension, a gel matrix material, and a medium; (c) conduits connecting each of the storage vessels to each of the culture containers; and (d) a controller configured to coordinately deliver the cell suspension, the gel matrix material, and the medium to the culture containers through the conduits to produce an explant. In some embodiments, the storage vessels for the cell suspension include at least one fibroblast (FB) storage vessel and at least one keratinocyte (KC) storage vessel. Alternatively or additionally, in some embodiments, the storage vessels for the cell suspension include at least two storage vessels for at least two different cell types selected from the group consisting of fibroblasts (FB), keratinocytes (KC), adipocytes, myocytes, neurons, pericytes, stem cells, and induced pluripotent cells (IPC). Alternatively or additionally, in some embodiments, the cell suspension reservoir includes at least two reservoirs for at least a first cell type of epithelial origin and a second cell type selected from the group consisting of mesenchymal cells, skin-derived cells, adipocytes, muscle cells, neural cells, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the system includes a valve in the conduit under the control of the controller. Alternatively or additionally, in some embodiments, the gel matrix reservoir includes a cooling element. Alternatively or additionally, in some embodiments, the controller includes a pump that moves the cell suspension, gel matrix material, and medium through the conduit. Alternatively or additionally, in some embodiments, the system includes a heater positioned to heat the medium. Alternatively or additionally, in some embodiments, the system includes a mixing module that receives cells from one reservoir and gel matrix from another reservoir and mixes the cells with the matrix to produce a gel matrix cell suspension. Alternatively or additionally, in some embodiments, the mixing module mixes cells from one reservoir with a buffer from a second reservoir to produce a buffered cell suspension, and then mixes the buffered cell suspension with a gel matrix from a third reservoir to produce a gel matrix cell suspension.Alternatively or additionally, in some embodiments, the system includes an incubation chamber designed and configured to contain the plurality of explant culture containers. Alternatively or additionally, in some embodiments, the system includes a compression mechanism operable by the controller to compress the gel matrix in one or more of the explant culture containers. Alternatively or additionally, in some embodiments, the system includes a camera and a two-way data communication link to an external input device. Alternatively or additionally, in some embodiments, the controller is adapted to periodically remove medium from the explant culture containers and add new medium to one of the storage vessels. Alternatively or additionally, in some embodiments, each of the explant culture containers comprises a tissue culture vessel as described herein.

[0042] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a plurality of cell culture containers; (b) a storage vessel for a cell suspension and medium; (c) a conduit connecting each of the storage vessels to each of the culture containers; and (d) a controller configured to coordinately deliver the cell suspension and medium to the culture containers through the conduit to produce a culture in the container. In some embodiments, the storage vessel for the cell suspension contains at least one cell type selected from the group consisting of fibroblasts (FBs), keratinocytes (KCs), adipocytes, myocytes, neurons, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the storage vessel for the cell suspension contains a first cell type of epithelial origin and a second cell type selected from the group consisting of mesenchymal cells, skin-derived cells, adipocytes, myocytes, neurons, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the storage vessel for the cell suspension and medium includes a temperature control mechanism. Alternatively or additionally, in some embodiments, the controller includes a pump (932) that moves the cell suspension and medium through the conduit. Alternatively or additionally, in some embodiments, the system includes a connector for attaching the conduit to the cell culture container. Alternatively or additionally, in some embodiments, the system includes an incubation chamber designed and configured to include the plurality of cell culture containers. Alternatively or additionally, in some embodiments, the system includes a camera and a bidirectional data communication link (982) to an external input device. Alternatively or additionally, in some embodiments, the controller is adapted to periodically remove medium from the cell culture container and add new medium to one of the reservoirs.

[0043] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a cell culture container with at least one port; (b) a detector that measures a parameter of the culture medium in the container and generates an indicator signal; and (c) a controller configured to respond to a threshold value of the indicator signal by operating a pump that removes spent culture medium through the at least one port and introduces fresh culture medium through the at least one port. In some embodiments, the parameter is selected from the group consisting of pH, CO2 concentration, glucose concentration, lactate concentration, and suspended cells (number and / or percentage). Alternatively or additionally, in some embodiments, the detector includes a pH electrode and / or a camera. Alternatively or additionally, in some embodiments, the cell culture container includes an explant culture vessel as described herein.

[0044] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a closed cell culture container with at least one gas port, (b) a CO tank connected to the at least one gas port through a regulator, and (c) a water storage vessel that passes CO from the tank between the regulator and the at least one gas port. In some embodiments, the closed cell culture container includes an explant culture vessel as described herein above.

[0045] Some exemplary embodiments of the present invention provide a system including: (a) a support surface for a plurality of explant culture containers; (b) a tilting mechanism for controlling the angle of the support surface; and (c) a controller configured to operate the mechanism to provide controlled removal of culture medium from the containers through one or more ports. In some embodiments, the support surface is mounted in an incubation chamber as described hereinabove. Alternatively or additionally, in some embodiments, the controller is configured to operate the tilting mechanism to +18° and then to -30° to discharge culture medium into a waste container. Alternatively or additionally, in some embodiments, the controller is configured to operate the tilting mechanism to -5° to facilitate sample collection. Alternatively or additionally, in some embodiments, the controller is configured to operate the tilting mechanism to +18° to remove culture medium from the upper compartment to the lift compartment and then to -30° to discharge all culture medium.

[0046] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a plurality of explant culture containers, each container having a movable plunger in its lid; (b) at least one piston; and (c) a controller configured to operate a vertical displacement mechanism to lower and raise the at least one piston to depress and release each of the plungers in the lid of the container. In some embodiments, the system includes a horizontal displacement mechanism, and the controller aligns the at least one piston with the plunger. Alternatively or additionally, in some embodiments, the vertical displacement mechanism is adjustable to different force and compression patterns (e.g., linear, stepwise). Alternatively or additionally, in some embodiments, the controller is programmable. Alternatively or additionally, in some embodiments, the system includes a pressure sensor. Alternatively or additionally, in some embodiments, the system includes a camera on the piston.

[0047] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a tissue culture container including at least one port; (b) a camera that provides an image output of a culture in the container; and (c) a controller that controls a medium exchange mechanism. In some embodiments, the system includes a remote device that receives the image output and has a user interface for operating the controller. Alternatively or additionally, in some embodiments, the medium exchange mechanism includes at least one pump that removes spent medium through the at least one port and introduces fresh medium through the at least one port. Alternatively or additionally, in some embodiments, the medium exchange mechanism includes a tilt mechanism that controls the angle of a support surface that holds the tissue culture container.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can be used in practicing the present invention. In case of conflict, the present specification, including definitions, will control. All materials, methods, and examples are illustrative only and are not intended to be limiting.

[0049] As used herein, the terms "comprising" and "including," or grammatical variations thereof, shall be deemed to specify the inclusion of the recited features, integers, operations, or components without excluding the addition of one or more additional features, integers, operations, components, or groups thereof. This term is broader than and inclusive of the terms "consisting of" and "consisting essentially of," as defined by the United States Patent and Trademark Office Manual of Patent Examination Procedure. Thus, any statement that an embodiment "includes" or "comprises" a feature is a specific statement that a subembodiment "consists essentially of" and / or "consists of" the recited feature.

[0050] As used herein, the phrase "consisting essentially of" or grammatical variations thereof shall be deemed to define the stated features, integers, steps, or components, but shall not preclude the addition of one or more additional features, integers, steps, components, or groups thereof, so long as such additional features, integers, steps, components, or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, apparatus, or method.

[0051] As used in this specification and the appended claims, the phrase "adapted to" imposes additional structural limitations on previously described components.

[0052] The term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including, but not limited to, any methods, means, techniques and procedures known to a practitioner of structural and / or computer science, or those readily developed by a practitioner from known methods, means, techniques and procedures.

[0053] In order to understand the invention and how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which identical and overlapping structures, elements or portions thereof that appear in more than one drawing are generally indicated by the same or similar references in the drawings in which they appear. Dimensions of components and features in the drawings have been chosen primarily for convenience and clarity of presentation and are not necessarily drawn to scale. The accompanying drawings are as follows: [Brief explanation of the drawings]

[0054] [Figure 1A] FIG. 1A is an exploded view of a tissue culture vessel according to some exemplary embodiments of the present invention; [Figure 1B] FIG. 1B is a cross-sectional view of an assembled tissue culture vessel as in FIG. 1A; [Figure 2A] FIG. 2A is a top perspective view of an implant support tray, according to some exemplary embodiments of the present invention; [Figure 2B] FIG. 2B is a bottom perspective view of the implant support tray of FIG. 2A; [Figure 3A] FIG. 3A is a top perspective view of a base of a tissue culture vessel according to some exemplary embodiments of the present invention; [Figure 3B] 3B is a bottom perspective view of the base of the tissue culture vessel of FIG. 3A; [Figure 3C] FIG. 3C is a diagrammatic detail view of an exemplary compression structure according to some exemplary embodiments of the present invention; [Figure 3D] FIG. 3D is a diagrammatic detail of an exemplary compression structure according to some exemplary embodiments of the present invention from a top (bottom right), cross-section along line AA (top right), and top perspective (left) view; [Figure 4A] FIG. 4A is a top perspective view of a lid of a tissue culture vessel according to some exemplary embodiments of the present invention; [Figure 4B] 4B is a bottom perspective view of the lid of the tissue culture vessel of FIG. 4A; [Figure 5] FIG. 5 is a simplified flow diagram of a method for preparing a graft, according to some exemplary embodiments of the present invention; [Figure 6] FIG. 6 is a simplified flow diagram of a method for manufacturing an implant according to an additional exemplary embodiment of the present invention; [Figure 7A] FIG. 7A is a perspective view of a tissue culture vessel mated with two transport locks according to an exemplary embodiment of the invention, including an inset "L" detailing the locking mechanism; [Figure 7B] FIG. 7B is a cross-sectional view of a connection between a lid and base of a tissue culture vessel and a transport lock according to an exemplary embodiment of the invention; [Figure 7C] FIG. 7C is a side view of two tissue culture vessels assembled with two transport locks according to an exemplary embodiment of the invention; [Figure 8A] FIG. 8A is a simplified schematic diagram of an explant culture system in accordance with an exemplary embodiment of the present invention; [Figure 8B] FIG. 8B is a front view of an explant culture system in accordance with an exemplary embodiment of the present invention; [Figure 8C] FIG. 8C is a front view of a mixing module according to some exemplary embodiments of the present invention; [Figure 8D] FIG. 8D is a front view of a dispense module according to some exemplary embodiments of the present invention; [Figure 8E] FIG. 8E is a front view of a media storage vessel configured as a preheat station for media or other cell culture reagents in accordance with an exemplary embodiment of the present invention; [Figure 8F] FIG. 8F is a front view of an incubation chamber according to an exemplary embodiment of the present invention; [Figure 8G] FIG. 8G is a front view of an incubation chamber as in FIG. 8F assembled with a compression module and tilt-based medium exchange mechanism according to an exemplary embodiment of the invention; [Figure 8H] FIG. 8H is a front view of a tilt-based medium exchange mechanism in accordance with an exemplary embodiment of the present invention; [Figure 8I] FIG. 8I is a simplified schematic flow diagram of media injection in accordance with an exemplary embodiment of the present invention; [Figure 8J]FIG. 8J is a simplified schematic flow diagram of keratinocyte seeding according to an exemplary embodiment of the present invention; [Figure 8J2] FIG. 8J2 is a simplified schematic flow diagram of hydrogel formation and fibroblast seeding according to an exemplary embodiment of the present invention; [Figure 8J3] FIG. 8J3 is a simplified schematic flow diagram of hydrogel incubation and compression in accordance with an exemplary embodiment of the present invention; [Figure 8K] FIG. 8K is a simplified schematic diagram of a static mixer utilized in some exemplary embodiments of the present invention; [Figure 9A] FIG. 9A is a simplified schematic diagram of a cell culture system in accordance with an exemplary embodiment of the present invention; [Figure 9B] FIG. 9B is a schematic illustration of the adaptation of a standard tissue culture container for use in a cell culture system according to an exemplary embodiment of the present invention; [Figure 10] FIG. 10 is a simplified schematic diagram of a medium exchange system for cell culture in accordance with an exemplary embodiment of the present invention; [Figure 11] FIG. 11 is a simplified schematic diagram of a humidified CO2 supply system for cell culture according to an exemplary embodiment of the present invention; [Figure 12A] FIG. 12A is a simplified schematic diagram of a gradient-based medium exchange system for cell culture in accordance with an exemplary embodiment of the present invention; [Figure 12B] FIG. 12B is a simplified schematic flow diagram of a gradient-based medium exchange system for cell culture in accordance with an exemplary embodiment of the present invention; [Figure 12C] FIG. 12C is a simplified schematic flow diagram of a cell culture system in accordance with an exemplary embodiment of the invention (icons are the same as in FIGS. 8J, 8J2, and 8J3); [Figure 13A] FIG. 13A is a simplified schematic diagram of a compression module of an explant culture system in accordance with an exemplary embodiment of the present invention; [Figure 13B] FIG. 13B is a front view of the compression module of the explant culture system according to an exemplary embodiment of the invention, with the inset showing the piston assembly separately; [Figure 14] FIG. 14 is a schematic illustration of an explant culture system adapted for remote manipulation in accordance with an exemplary embodiment of the present invention; [Figure 15] FIG. 15 is a simplified schematic flow diagram of an environmental control system for cell culture in accordance with an exemplary embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0055] SUMMARY OF THE INVENTION Embodiments of the present invention relate to tissue culture vessels and related methods configured for producing skin grafts.

[0056] Specifically, some embodiments of the present invention can be used to manufacture skin grafts in closed containers. In some embodiments, the containers are aseptically closed and intended for single use (disposable) to limit contamination and support operations in lower GMP cleanroom classifications, such as C or D according to EU GMP regulations.

[0057] The principles and operation of tissue culture vessels and / or methods according to exemplary embodiments of the invention may be better understood with reference to the drawings and accompanying descriptions.

[0058] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or illustrated by way of example. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0059] Overview of an exemplary tissue culture vessel FIG. 1A is an exploded view of a tissue culture vessel, generally designated 100, according to some exemplary embodiments of the present invention.

[0060] The illustrated exemplary tissue culture vessel 100 includes a box having an explant support tray 200, a lid 400, and a base 300. In the illustrated embodiment, the box engages and retains a frame 210 (see FIG. 2A ) of the tray 200, which has two operational states. In the first operational state, the tray 200 is slightly elevated relative to the floor of the base 300. In the second operational state, the tray 200 is lowered to contact the floor of the base 300. In some embodiments, the tray 200 is normally in the first operational state and moves to the second operational state in response to an external force. In the illustrated embodiment, the vessel 100 includes a movable plunger 431 attached to the lid 400. In some embodiments, depressing the plunger 431 with an external force switches the tray 200 to the second operational state. For purposes of this specification and the appended claims, the term “slightly elevated” refers to sufficient space between the tray 200 and the floor of the base 300 to allow liquid medium to enter the space.

[0061] The illustrated exemplary vessel 100 includes one or more lift compartments 441. In some embodiments, the lift compartments 441 contain medium removed from the tray 200 when the plunger 431 is lowered and / or the entire vessel 100 is tilted so that the lift compartments 441 are below the rest of the vessel.

[0062] The illustrated exemplary container 100 includes a gas-permeable membrane 110 on the upper surface of the riser compartment 441. According to various exemplary embodiments of the present invention, the gas-permeable membrane 110 is attached to the inside or outside of the riser compartment. The gas-permeable membrane 110 allows air to escape from the container 100 when the plunger 431 is lowered, allows air to enter the container when the plunger 431 is raised, and / or allows gas exchange between the ambient environment and the interior of the container 100 while maintaining sterility.

[0063] In some exemplary embodiments of the invention, container 100 is sterilized and packaged to maintain sterility. For example, one or more containers 100 are assembled, packaged in a plastic sleeve, and then X-ray sterilized. In some embodiments, connections to container 100 are made by sterile tubing connections (e.g., tubing welds), so that under normal use conditions, exposure to the environment inside the container does not occur until the implant is removed for implantation or other use.

[0064] In the illustrated embodiment, an O-ring 120 is used to provide an airtight seal between the lid 400 and the base 300. In other exemplary embodiments of the invention, the O-ring 120 is replaced by a gasket or is overmolded directly onto the base 300 and / or lid 400.

[0065] Each of the implant support tray 200, base 300, and lid 400 are described in further detail herein below. The properties used to characterize these individual parts also characterize the container 100.

[0066] FIG. 1B is a cross-sectional view of an assembled tissue culture vessel as in FIG. 1A showing the interrelationships between the parts, generally designated as 101.

[0067] Exemplary Support Tray FIG. 2A is a top perspective view of an implant support tray, generally designated as 200, according to some exemplary embodiments of the present invention.

[0068] FIG. 2B is a bottom perspective view of the implant support tray of FIG. 2A, generally designated as 201.

[0069] In the illustrated embodiment, the graft support tray 200 includes a rigid frame 210, a liquid-permeable membrane bed 220 attached to a lower edge 212 (FIG. 2B) of the frame 210, and a first set of vertically extending springs 230 attached to two opposite exterior sides of the frame 210. The springs 230 function to support the membrane bed 220 in a raised position in the absence of external forces, as described herein above in the "first operating state" in the context of FIG.

[0070] In the illustrated embodiment, the implant support tray 200 includes a second set of vertically extending springs 232 attached in an opposite direction to the first springs 230. In some embodiments, the first and / or second springs are attached to two opposite sides of the frame.

[0071] In the illustrated embodiment, springs 230 and 232 are leaf springs. In other exemplary embodiments of the invention, one or both of the springs in the set are provided as coil springs.

[0072] In the illustrated embodiment, the implant support tray 200 includes guide holes / pins (240) that mate with corresponding pins / holes in the frame-mounted base.

[0073] In the illustrated embodiment, the implant support tray 200 includes guide holes / pins (242) that mate with corresponding pins / holes in the lid located on the support frame.

[0074] In some embodiments, a slope 211 is disposed on one side of the graft support tray 200. In embodiments featuring a slope 211, the slope directs the flow of infused fluid to the area having the membrane bed 220. In some embodiments, the angle of the slope is 2°, 3°, 4°, 5°, 6°, 7°, or 8°, or an intermediate or larger angle. Alternatively or additionally, in some embodiments, the length of the slope 211 is determined by the placement of the fluid inlet 460 and the size of the membrane 420.

[0075] Alternatively or additionally, in some embodiments, a structure with holes 213 is located on the other side of the implant support tray 200, which allows for efficient drainage in the event of spillage or tilting.

[0076] The graft support tray 200 is in an assembled state prestressed by four spacers 411 on the lid (two visible in FIG. 4A), which exert pressure on two ears 215 flush with the rigid frame 210.

[0077] The rigid frame 210 is designed to be flat and provide improved support for the attached membrane 220 .

[0078] The rigid frame 210 shown in the drawings has two holes and a rib in the middle designed to provide the added rigidity needed to ensure the membrane 220 is flat.

[0079] Exemplary Base FIG. 3A is a top perspective view of a base of a tissue culture vessel, generally designated as 300, according to some exemplary embodiments of the present invention.

[0080] FIG. 3B is a bottom perspective view of the base of the tissue culture vessel of FIG. 3A, generally designated as 301.

[0081] The illustrated exemplary base 300 includes a compression structure 310 designed and configured to support the membrane bed of a graft support frame fully lowered into the base. When the base 300 is assembled to the tissue culture vessel 100, the lid plunger 431 is lowered, pressing the graft support tray downward, causing medium to pass through the compression structure 310 and enter the elevated compartments (441 and / or 440, FIGS. 1 and 4, respectively). In some exemplary embodiments of the invention, the compression structure 310 is provided as a pattern molded into the base that, upon compression, forces medium to flow into the elevated compartments. According to these embodiments, the pattern includes channels that facilitate liquid flow. FIGS. 3C and 3D provide detailed views of the exemplary compression structure 310. In other exemplary embodiments of the invention, the compression structure 310 is constructed from a liquid-permeable material. In either case, the compression structure 310 allows medium to flow outward when the frame is lowered.

[0082] According to various exemplary embodiments of the present invention, compression is achieved by application of a suitable weight and / or an external linear force-controlled or displacement-controlled drive.

[0083] The illustrated exemplary base 300 includes a recess 320 sized and positioned to accommodate one set of springs of an implant support tray positioned in the base 300. In the illustrated embodiment, the base 300 includes recesses (320) sized and positioned to accommodate two sets of springs of an implant support tray positioned in the base. In the illustrated embodiment, the base 300 includes a step 322 that positions the implant support tray.

[0084] The illustrated exemplary base 300 includes pins 330 or holes sized and positioned to engage corresponding holes or pins on the implant support tray located in the base. In some embodiments, these pins / holes help to orient the implant support tray within the base. In some embodiments, the orientation of the implant support tray within the base 300 contributes to the precision of alignment of the implant support tray with the lid plunger 431.

[0085] The illustrated exemplary base 300 includes pins and / or holes 332 sized and positioned to engage corresponding holes or pins on the plunger of a lid that fits over the base 300. In some embodiments, these pins or holes 332 help to orient the plunger relative to the frame when the frame is placed in the base 300. In some embodiments, the holes 332 are simple recesses that provide space for the pins of the plunger. According to these embodiments, a pin 470 in the plunger engages with a guide hole 242 in the implant support tray 200.

[0086] In the illustrated embodiment, the base 300 includes an O-ring 340 or other gasket or overmolded elastomeric structure. In some embodiments, the O-ring 340 (or gasket) contributes to forming an airtight and / or watertight seal.

[0087] In the illustrated embodiment, base 300 includes at least one liquid removal port 344 and / or 346. In the illustrated embodiment, port 344 is a drain port and 346 is a sample removal port. Figure 3B shows that these ports mate with barbed connectors 345 and 347.

[0088] In the illustrated embodiment, the base 300 includes a snap-fit connector 350 for attachment to the lid. In some embodiments, the lid 400 snaps onto the connector 350, while the shipping lock snaps onto the exterior of the same connector structure.

[0089] In some exemplary embodiments of the invention, base 300 includes a mating catch insert 350 for a shipping lock. In some embodiments, the shipping lock holds the plunger of a lid attached to the base in a slightly lowered position.

[0090] Also visible in FIG. 3B are ribs 352 and braces 354, legs 356 and stacking spacers 358 for structural support and / or strength.

[0091] Further details of the transport lock are provided herein below in the context of Figures 7A, 7B and 7C.

[0092] Exemplary lid FIG. 4A is a top perspective view of a tissue culture vessel lid, generally designated as 400, according to some exemplary embodiments of the present invention.

[0093] FIG. 4B is a bottom perspective view of the lid of the tissue culture vessel of FIG. 4A, generally designated as 401.

[0094] The illustrated exemplary lid 400 includes a rigid frame 410 within which is disposed a flexible bellows 420. The bellows 420 holds a plunger 431 in a fixed orientation relative to the frame 410. In some embodiments, the plunger 431 is constructed from a transparent material to facilitate visual inspection of cells being cultured in an explant support tray mounted beneath the lid 400.

[0095] In some exemplary embodiments of the invention, bellows 420 is normally open to hold plunger 431 in an elevated position in the absence of external forces. According to various exemplary embodiments of the invention, the elevated position is above the plane of frame 410. Alternatively or additionally, in some embodiments, bellows 420 is manually removable by the end user of the implant (e.g., due to adhesive attachment). In some embodiments, a manually removable bellows contributes to the convenience of use in removing the implant from the container.

[0096] In the illustrated embodiment, lid 400 includes one or more raised sections 440 that extend in the plane of the upper edge of frame 410. FIG. 4B shows a structural support 442 that divides raised section 440 into two sections. In the illustrated embodiment, raised section 440 includes gas-permeable membrane 110 on its upper surface. According to various exemplary embodiments of the invention, gas-permeable membrane 110 is attached from the inside or the outside. In some embodiments, gas-permeable membrane 110 is provided as OXYPADS (Oxyphen GMBH, Switzerland). OXYPADS include a membrane center surrounded by an adhesive edge that is pressed onto a surface to seal the pad in place.

[0097] In the illustrated embodiment, the lid 400 includes a catch hook 450 on the rigid frame 410 that is sized and positioned to engage the base covered by the lid.

[0098] In the illustrated embodiment, the lid 400 includes one or more external barbed connectors 460 (five are shown, but fewer or more may be used) in fluid communication with an internal exit port 462 ( FIG. 4B ). For example, the barbed connectors 460 can be connected to supply lines for CO2 / air inlet and / or fibroblasts (FB) and / or collagen gel (with or without cells therein) and / or medium and / or keratinocytes (KC). In some embodiments, one of the barbed ports 460 is used to remove CO2 / air. Alternatively or additionally, in some embodiments, the CO2 / air exits the tissue culture vessel through the gas permeable membrane 110 in the riser compartment 440. The barbed connectors 460 can be arranged next to each other or in tandem with each other for more efficient mass production.

[0099] In some exemplary embodiments of the invention, the lid 400 is fitted with a resilient seal (see 120 in FIG. 1) on the side of the lid that contacts the base when the container is assembled. According to these embodiments, the seal forms an airtight and leak-proof connection with the base.

[0100] Exemplary Methods FIG. 5 is a simplified flow diagram of a method for preparing an implant, generally designated 500, according to some exemplary embodiments of the present invention.

[0101] In the illustrated embodiment, the method 500 includes introducing a hydrogel containing fibroblasts (FBs) into a sealed tissue culture vessel 510. After introducing the hydrogel-containing fibroblasts, the gel is "polymerized" (crosslinked by adding a buffer to the cell culture medium).

[0102] In the illustrated embodiment, the FBs are then incubated (typically after cross-linking) until they assemble into a gel matrix 520. Once the FBs have assembled into a gel matrix, the matrix is compressed 530 within the same sealed tissue culture vessel.

[0103] In some exemplary embodiments of the invention, after compaction, the gel is allowed to incubate for several days (typically 5) to allow the cells to attach, aggregate, and remodel the gel, hi some embodiments, the cell culture medium is changed every two days.

[0104] In the illustrated embodiment, keratinocytes (KCs) are seeded onto the compacted matrix within the same sealed tissue culture vessel 540 and further incubated 550 until the graft is ready. In some embodiments, the graft is considered ready for transplantation when a fibroblast-rich dermal layer and a continuous keratinocyte / epidermal layer have formed. Alternatively or additionally, in some embodiments, medium exchange / removal during skin culture is performed during incubation 520 and / or 550 (e.g., using the elevated compartment 441). In some embodiments, the exchange involves tilting the sealed tissue culture vessel in one direction to allow the medium to flow into the elevated compartment and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow to a waste container. As with all medium exchanges, the result is the introduction of fresh nutrients for the cells and / or the removal of metabolic waste products.

[0105] Alternatively or additionally, in some embodiments, the sample port is used to remove a sample of the cell culture medium. In some embodiments, measuring lactate and / or glucose and / or other metabolites allows for assessment of cell growth and determination of transplant readiness. Alternatively or additionally, sampling the cell culture medium via the sample port allows for measurement of the microbiological load of the medium (e.g., sterility, mycoplasma, endotoxin, etc.).

[0106] In some embodiments, visual inspection of the growth of FBs and / or KCs in the tissue culture vessel is performed using a microscope. According to various exemplary embodiments of the invention, visual inspection is performed, for example, at 520 and / or 550 and / or during 510 and / or 540.

[0107] In some exemplary embodiments of the invention, method 500 includes providing hydrogel, FB, and KC to multiple sealed tissue culture vessels using a common set of reagent reservoirs (e.g., 810, 812, 814, and 816) using controller 830 (FIG. 8) and pump 832.

[0108] Additional Exemplary Methods FIG. 6 is a simplified flow diagram of a method for manufacturing an implant according to an additional exemplary embodiment of the present invention, generally designated 600.

[0109] The illustrated exemplary method 600 includes preparing 610 a compressed hydrogel skin graft in a first position (as described in the context of FIG. 5) in a sealed tissue culture vessel. The illustrated exemplary method 600 includes removing growth medium and introducing transport medium into the sealed tissue culture vessel 620, and partially lowering 630 an integral plunger in the lid of the sealed tissue culture vessel and locking the plunger in place 640, and then moving to a second position 650. In some embodiments, locking 640 contributes to the ability to securely hold the graft in place.

[0110] In the illustrated embodiment, removing 620 includes tilting the sealed tissue culture vessel in one direction to allow the medium to flow into a raised compartment 622 and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow to a waste container 624.

[0111] Exemplary Transport Lock 7A is a perspective view of a tissue culture vessel mated with two transport locks 710 according to an exemplary embodiment of the invention, generally designated as 700. Inset L provides a close-up view of connector 716.

[0112] FIG. 7B is a cross-sectional view of the connection between lid 400 and base 300 of a tissue culture vessel, generally designated as 701, and a shipping lock 710 according to an exemplary embodiment of the invention.

[0113] In the illustrated embodiment, the transport lock includes a spanning member 710 sized to fit the dimensions of the tissue culture vessel 100 (FIG. 1A). The transport lock has two notches 720 on its lower edge configured to fit the contours of the frame 410 of the lid 400, as well as downward extensions 712 between the notches 720 and catches 716 on the outer edges of each of the notches sized to engage and retain snap-fit connectors 350 on the base 300. During use of the transport lock, the downward extensions 712 and catches 716 work in concert to hold the tray 200 in the second operational state described herein above. Alternatively or additionally, in some embodiments, the catches 716 are of the "snap-fit" type and are constructed of flexible plastic for manual removal.

[0114] In the illustrated embodiment, the transport lock includes a series of slots (714) on the upper edge of the spanning member 710. In some embodiments, the slots 714 are sized and positioned to engage and retain corresponding ribs 352 (see FIG. 3B) on the underside of the base 300 of a second tissue culture vessel 100 located above the transport lock.

[0115] 7C is a side view of two tissue culture vessels 100 assembled with two transport locks 710 according to an exemplary embodiment of the invention, generally designated as 703. The exemplary assembly shown includes multiple culture vessels arranged in a vertical array with the aforementioned transport locks interspersed between the multiple culture vessels 100 (two are shown for clarity, but in practice there will typically be more).

[0116] Exemplary Measurements and Materials In some embodiments, tissue culture vessel 100 is sized to hold 160-170 ml of medium.

[0117] In some embodiments, the graft support tray 200 has a height of 5-15 mm (e.g., 10 mm), a width of 110-120 mm (e.g., 115 mm), and a length of 170 mm-180 mm (e.g., 175.4 mm). A graft support tray of this size weighs 30-35 grams (e.g., 32.1 g) when constructed from polystyrene, such as Ineos Styrolution PS 158N / L. In some embodiments, the membrane bed of the tray is Oxyphen Unique-Mem track-etched membrane corona treated, 51.9069.101.111, polyester (PET), 12 μm thick, 3.0 μm pore size, and 0.6 e pore density. 6 / cm 2 In some embodiments, the tray 200 is manufactured by injection molding and subsequent heat bonding of the film.

[0118] In some embodiments, lid 400 has a height of 35-45 mm (e.g., 40.5 mm), a width of 155-165 mm (e.g., 158 mm), and a length of 255-265 mm (e.g., 259.3 mm). In some embodiments, the rigid portion is constructed from polycarbonate, such as Trinseo Caliber Megarad 2081-15 polycarbonate, and flexible bellows 420 is constructed from a thermoplastic elastomer, such as Kraiburg TPE Thermolast M TM5ADT (50Sh). In some embodiments, lid 400 is manufactured by injection molding followed by overmolding and drilling of ports.

[0119] In some embodiments, base 300 has a height of 20-30 mm (e.g., 24 mm), a width of 160-165 mm (e.g., 162.3 mm), and a length of 250-255 mm (e.g., 253.6 mm). A box of this size, when constructed from polycarbonate such as Trinseo Caliber Megarad 2081-15 polycarbonate, would weigh 200-204 grams (e.g., 202 grams). In some embodiments, base 300 is manufactured by injection molding and then drilling the ports.

[0120] Example Operational Considerations To remove medium from container 100, the container is first tilted backward (away) along its horizontal axis from waste outlet port 344 to remove medium from the upper compartment into riser compartment 440. Once medium has accumulated in riser compartment 440, container 100 is tilted at an angle of <20° toward waste outlet port 344. Port 344 is connected to a waste container and is open. At a 20° angle, less than 10% of the medium remains in container 100. Returning container 100 to a neutral position raises port 344 above the liquid level, allowing fresh medium to be introduced into one of ports 460 in lid 400.

[0121] For seeding with KCs (keratinocytes), the vessel is first tilted backward (away) from waste outlet port 344 along the horizontal axis at a 26° angle to remove liquid from the upper compartment into the elevated compartment 440. At a 26° angle, only 4 ml of medium is held in the tray 200. After returning the vessel 100 to the neutral position, KCs can be seeded through one of the ports 460 in the lid 400.

[0122] A container 100 of the dimensions described above holds 165 ml ± 10% of medium, enough to ensure 4 mm of immersion in the explant growth in the support tray 200. Approximately 40 ml of medium is present in the explant support tray 200. The remainder is within the base 300 but is external and / or underneath the tray 200.

[0123] During the introduction of the hydrogel into the graft support tray 200, the membrane bed 220 must be elevated above the support surface 310 of the base 300. A first spring 230 on the support tray 200 ensures this. After a first incubation (which crosslinks the hydrogel), the gel is compressed. To facilitate compression, the membrane must be in contact with the support surface 310 of the base 300 so that it is wet and / or provides counter-pressure support (such as a tent cloth that begins to leak when touched). After compression, the springs 230 and / or 232 return the support tray 200 to its initial position. The space below the support tray 200 is sufficient for the amount of medium needed for cell growth on the graft. In some embodiments, the liquid-permeable membrane bed 220 allows nutrients from the medium in the space below the tray 200 to nourish the cells growing in and / or on the hydrogel in the tray.

[0124] Exemplary Explant Culture System FIG. 8A is a simplified schematic diagram of an explant culture system, generally designated 800, according to an exemplary embodiment of the present invention.

[0125] FIG. 8B is a front view of an explant culture system, generally designated 801, according to an exemplary embodiment of the invention.

[0126] In the illustrated embodiment, the system (800) includes multiple explant culture containers (820A, 820B, 820C) and reservoirs (810, 812) of cell suspension, gel matrix material (814), and culture medium (816). In the illustrated embodiment, conduits (818, 822) connect each of the reservoirs (810, 812, 814, and 816) to each of the culture containers (820A, 820B, 820C). While three culture containers are shown for clarity, in many embodiments of the invention, many more are actually present. The illustrated exemplary system 800 includes a controller (830) configured to coordinately deliver the cell suspension, gel matrix material, and culture medium to the culture containers through the conduits to produce the explant. In some exemplary embodiments of the invention, the cell suspension reservoirs include at least one fibroblast (FB) reservoir (810) and at least one keratinocyte (KC) reservoir (812).

[0127] According to various exemplary embodiments of the present invention, the cell suspension reservoirs include at least two reservoirs for at least two different cell types selected from the group consisting of fibroblasts (FBs), keratinocytes (KCs), adipocytes, myocytes, neurons, pericytes, stem cells, and induced pluripotent cells (IPCs). According to various exemplary embodiments of the present invention, the stem cells include epithelial stem cells and / or mesenchymal stem cells. In some exemplary embodiments of the present invention, the use of multiple cell types contributes to the ability to generate tissues (soft and / or hard tissues) and / or organs.

[0128] In some embodiments, the cell suspension reservoirs include at least two reservoirs for at least a first cell type of epithelial origin and a second cell type selected from the group consisting of cells of mesenchymal origin, cells of skin origin, adipocytes, muscle cells, neurons, pericytes, and stem cells.

[0129] In some exemplary embodiments of the invention, system 800 includes valves (see, e.g., 2910 in Figure 8D) in the conduits under the control of controller 830. In Figure 8D, 2930 is a support structure for unused conduits.

[0130] Alternatively or additionally, in some embodiments, the storage container for gel matrix 814 includes a cooling element 840. In some embodiments, cooling the gel matrix inhibits premature gelation and / or contributes to fluidity in conduits 818 and / or 822. In some exemplary embodiments of the invention, cooling element 840 includes a Peltier cooler. In the illustrated embodiment, controller 830 includes a pump 832 that moves the cell suspension, gel matrix material, and medium through conduits 818 and / or 822. In some embodiments, pump 832 is external to controller 830. In the illustrated embodiment, system 800 includes a heater 850 positioned to heat the medium in storage container 816. In some exemplary embodiments of the invention, a common Peltier unit cools the matrix material in storage container 814 and heats the medium in storage container 816.

[0131] In the illustrated embodiment, system 800 includes a mixing module 835 that receives cells from one storage container (e.g., 810) and a gel matrix from another storage container (e.g., 814) and mixes the cells with the matrix to produce a gel matrix-cell suspension of cells.

[0132] Referring to Figure 8K, in some embodiments, a mixing module (835) mixes cells from one reservoir (e.g., 810) with a buffer solution from a second reservoir (e.g., 817) to produce a buffered cell suspension. In the illustrated embodiment, this is accomplished using a static mixer 837. In the illustrated embodiment, module 835 then mixes the buffered cell suspension with a gel matrix from a third reservoir (e.g., 814) to produce a gel matrix cell suspension. In the illustrated embodiment, this is accomplished using a static mixer 837.

[0133] In the illustrated embodiment, system 800 includes an incubation chamber 860 designed and configured to hold a plurality of explant culture containers. In some embodiments, the temperature and / or humidity and / or CO2 concentration within chamber 860 is controlled by controller 830.

[0134] In the illustrated embodiment, the system 800 includes a compression mechanism 870 operable by the controller 830 to compress the gel matrix in one or more explant culture containers 100 .

[0135] In the illustrated embodiment, system 800 includes a camera 880 and a two-way data communication link 882 to an external user input device. In some embodiments, the external user input device includes a user interface (e.g., a graphical user interface (GUI)) for displaying output images from camera 880 and inputting commands to controller 830. In some exemplary embodiments of the invention, images output by camera 880 are used to assess the color of the medium (an indicator of changes in pH and associated nutritional value of the medium) and / or assess turbidity (indicative of the presence of contaminants). Alternatively or additionally, in some embodiments, output by camera 880 is used to assess the smoothness of the gel surface and / or detect the presence of air bubbles as part of a quality assessment of the tissue formation process.

[0136] Alternatively or additionally, in some embodiments, controller 830 is adapted to periodically remove medium from the explant culture containers (e.g., 820A, 820B, and 820C) and add new medium from one of the reservoirs (e.g., 816). In some exemplary embodiments of the invention, controller 830 is programmed with a schedule. In some embodiments, controller 830 reverses the flow direction of pump 832 to remove medium from the culture containers via conduit 822. In some exemplary embodiments of the invention, different conduits are used for the removal of spent medium and the introduction of fresh medium.

[0137] In some exemplary embodiments of the invention, each of the explant culture containers (e.g., 820A, 820B, and 820C) is a tissue culture vessel 100 as described herein above. In some embodiments, the controller 830 tilts the culture container 100 at a predetermined angle to remove the medium, as described in further detail herein below.

[0138] FIG. 8C is a front view of a mixing module, generally designated as 835, according to some exemplary embodiments of the present invention.

[0139] 8C shows a more detailed exemplary embodiment of the mixing module 835. In the illustrated embodiment, the mixing module 835 includes a static mixer using a branched tubing strategy. In the illustrated embodiment, the mixing module 835 includes multiple peristaltic pumps 2832 (three shown as an example) mounted at an angle (45°) on a support plate 2851. In the illustrated embodiment, fluid sensors 2840 are located below and above the peristaltic pumps to allow for control of fluid dispensing. The illustrated exemplary mixing module utilizes standard, single-use tubing kits in a closed system configuration. The illustrated exemplary configuration includes a retention structure 2850 for a sterile connection point. In the illustrated embodiment, additional retention structure for single or branched tubing sets is included in the design 2860.

[0140] Figure 8K is a simplified schematic diagram of a static mixer utilized in mixing module 835 of Figure 8C, according to some exemplary embodiments of the invention. In the illustrated embodiment, mixer 837 receives liquid / suspension from reservoirs 810, 814, 817 via conduit 818 and outputs a mixture via conduit 822. In the illustrated embodiment, mixer 837 is a connector with two inputs and one output. The two input streams mix due to the turbulence created when they meet in mixer 837. In some embodiments, this configuration eliminates the need for moving parts in the mixer.

[0141] FIG. 8D is a front view of a dispense module, generally designated 2900, according to some exemplary embodiments of the invention. In some exemplary embodiments of the invention, the dispense module utilizes single-use tubing trees for dispensing multiple liquids simultaneously. In the illustrated exemplary embodiment of FIG. 8D, dispense module 2900 includes a holder 2920 for multiple branching tubing trees. In some exemplary embodiments of the invention, holder 2920 contributes to the ability of liquids to flow evenly through the tubing and / or helps prevent kinking of the tubing, which could cause flow blockages. In the illustrated embodiment, 2930 secures unused tubing.

[0142] In the illustrated embodiment, module 2900 has four solenoid valves 2910. This arrangement contributes to the ability to control liquid distribution. For example, if solenoid valves 2910 are operated sequentially every second, even liquid distribution is achieved. In other exemplary embodiments of the invention, more valves are used to distribute liquid to more containers simultaneously.

[0143] FIG. 8E is a front view of a media storage vessel, generally designated as 851, configured as a preheat station for media or other cell culture reagents according to an exemplary embodiment of the present invention.

[0144] FIG. 8E illustrates an exemplary media storage vessel 816 (FIG. 8A) configured as a preheating station 851 for media or other cell culture reagents prior to dispensing into the single-use containers 100 described herein. The illustrated exemplary station 851 is used in connection with a disposable plastic tubing and bag system. For example, a tubing system that connects aseptically (e.g., ASEPTIQUICK) to a plastic bag (media storage vessel 816 (FIG. 8A)) on one end was stored at room temperature (external storage hook on the left side of the device). The storage vessel may also be stored in a refrigerated compartment. In the illustrated embodiment, the tubing that connects to the media storage vessel (816, FIG. 8A) on one side connects to a small media bag 2816 on the other side. The bag 2816 is filled with a volume of media when preheating is required. The bag 2816 has a second tubing outlet for dispensing preheated media into the container 100.

[0145] In the illustrated embodiment, chamber 2940 is filled with preheated air (to a desired temperature, such as 37°C) from the outside via connection point 2942. In some exemplary embodiments of the invention, connection point 2942 connects to an environmental control system, such as a LIFE IMAGING SYSTEM environmental control system "CUBE" or an incubator, via large-bore tubing. In the illustrated embodiment, chamber 2940 contains a storage unit 2944 in which medium bag 2816 is placed. The unit is shaped to support the bag with a hook on top for holding the bag. Storage unit 2944 has a lid 2946. In some embodiments, storage unit 2944 and lid 2946 contain Peltier elements that heat medium bag 2816 to the required temperature. In some exemplary embodiments of the invention, the use of Peltier elements partially or completely eliminates the need to connect chamber 2940 to an environmental control system. In either case (with or without connection to an environmental control system), the chamber 2940 is fitted with guides and exit ports 2953 for the incoming and outgoing tubing.

[0146] FIG. 8F is a front view of an incubation chamber, generally designated 860, according to an exemplary embodiment of the present invention. In the illustrated embodiment, a single-use explant culture container 100, as described herein above, is incubated in the incubation chamber 860. In some exemplary embodiments, the chamber 860 is adapted for compaction of the three-dimensional matrix (hydrogel) and / or culturing of the explant for days or weeks. In some exemplary embodiments of the present invention, some of the construction of the 3D explant occurs outside of the chamber 860. In the illustrated embodiment, the chamber 860 has two sliding doors 2862 on the front for assembly of the container 100. In the illustrated embodiment, a smaller door 2864 is provided for interaction during processes where temperature fluctuations within the chamber are undesirable. In the illustrated embodiment, four smaller doors 2864 are suitable, although the number of smaller doors 2864 may be increased depending on the number of containers 100. The sliding doors are fitted with rubber-covered slits 2863 to allow tubing to connect from the outside to the interior of the environmental chamber. A similar rubber covered slit 2866 is fitted to the bottom of the device to allow waste disposal tubing to exit the environmental chamber and connect to a trash bag.

[0147] In some embodiments, the incubation chamber 860 is filled with preheated air to create an environment at a desired temperature (e.g., 37°C). The air is preheated by an environmental control system, such as a LIFE IMAGING SYSTEM environmental control system "CUBE" or an incubator. In some embodiments, the heater inlet port 2868 is connected to the environmental control system via large diameter tubing. In the illustrated embodiment, the return air port 2865 returns air to the environmental control system via tubing.

[0148] In the illustrated embodiment, opening 2869 provides an attachment interface for compression module 1301 (see FIGS. 8G and 13B and corresponding text), described herein below. In the illustrated embodiment, connection point 2861 facilitates attachment to tubing connected to a reservoir of CO2-enriched humidified air on one side and a culture container (e.g., 820A in FIG. 8A) on the other side.

[0149] A suitable environment for cell growth in the culture container 100 inside chamber 860 requires that the air be humidified and contain 5% CO2. In some exemplary embodiments of the invention, an air mixture containing 5% CO2 is pumped into humidity chamber 2870. In some embodiments, the air mixture is provided by a system such as a LIFE IMAGING SYSTEMS "BRICK" or an incubator. In the illustrated embodiment, water column 2872 ensures that the air is humidified before being distributed to container 100. In the illustrated embodiment, 2861 serves as a connector port for humidity chamber 2870. In some embodiments, culture container 100 connects to connector 2861 via tubing to receive the air.

[0150] FIG. 8G is a front view of an incubation chamber such as that of FIG. 8F assembled with a compression module 1301 and a tilt-based medium exchange mechanism 3000, according to an exemplary embodiment of the invention. In the illustrated embodiment, the incubation chamber 860 is assembled with the compression module 1301, described below in connection with FIG. 13B. In the illustrated embodiment, a valve 2910 can be seen on the top (outside) front of the chamber 860 (see FIG. 8D and corresponding text). In the illustrated embodiment, the compression system 1301 is inserted into the incubation chamber 860 from above. In the illustrated embodiment, a metal tilt table 3000 is placed within the chamber 860. The tilt table 3000 is tilted so that the medium can be ejected from the container 100, as described below in connection with FIG. 8H, 12A, and 12B. In the illustrated embodiment, the tilt table 3000 is actuated by an actuator 3010 (see FIG. 8H). In some embodiments, the tilt table 3000 is designed with a secondary function of heat storage in mind. According to these embodiments, the use of thick metal plates in the construction of table 3000 creates a thermal mass. In some exemplary embodiments of the invention, the thermal mass contributes to temperature uniformity in chamber 860.

[0151] In the illustrated embodiment, a piston head 1354 can be seen (see FIG. 13B and accompanying text for the function of the piston head).

[0152] 8H is a front view of a tilt-based medium exchange mechanism 3000 according to an exemplary embodiment of the invention removed from the chamber 860. To improve handling, a structure 3020 was designed that includes a metal plate with two handles. The metal plate is shaped to provide space for the bioreactor tubing. In this particular design, four culture containers 100 (possibly extended further) are assembled onto the metal plate. A frame 3030 is used to hold the culture containers 100 in place on the structure. In some embodiments, the structure 3020 is assembled with one or more containers 100, lifted into the incubation chamber 860, and placed on top of the tilt table 3000.

[0153] FIG. 8I is a simplified schematic flow diagram of media injection according to an exemplary embodiment of the present invention.

[0154] FIG. 8J is a simplified schematic flow diagram of keratinocyte seeding (such as 540 in FIG. 5) according to an exemplary embodiment of the present invention.

[0155] FIG. 8J2 is a simplified schematic flow diagram of hydrogel formation and fibroblast seeding (such as 510 in FIG. 5) according to an exemplary embodiment of the invention.

[0156] FIG. 8J3 is a simplified schematic flow diagram of hydrogel incubation and compression (such as 520 and 530 in FIG. 5) according to an exemplary embodiment of the invention.

[0157] Exemplary Cell Culture Systems 9A is a simplified schematic diagram of a cell culture system according to an exemplary embodiment of the invention, generally designated as 900. The illustrated exemplary system 900 includes multiple cell culture containers (e.g., 920A, 920B, and 920C), a reservoir 910 for a cell suspension and a medium 916, and conduits (e.g., 918 and 922) connecting each of the reservoirs to each of the culture containers. In the illustrated embodiment, a controller (930) is configured to coordinately deliver the cell suspension 910 and the medium 916 to the culture containers 920A, 920B, and 920C through the conduits 918 and 922 to produce a culture therein. For clarity, only three culture containers are illustrated, although typically many more are present.

[0158] In some exemplary embodiments of the invention, the cell suspension reservoirs (910 and / or 912) contain at least one cell type selected from the group consisting of fibroblasts (FB), keratinocytes (KC), adipocytes, myocytes, neurons, and stem cells. According to various exemplary embodiments of the invention, the cell suspension reservoirs include at least two reservoirs for at least two different cell types selected from the group consisting of fibroblasts (FB), keratinocytes (KC), adipocytes, myocytes, neurons, pericytes, and stem cells. According to various exemplary embodiments of the invention, the stem cells include epithelial stem cells and / or mesenchymal stem cells and / or induced pluripotent cells (IPC).

[0159] In some embodiments, the cell suspension reservoirs (910, 912) contain a first cell type of epithelial origin and a second cell type selected from the group consisting of cells of mesenchymal origin, cells of skin origin, adipocytes, muscle cells, neurons, pericytes, and stem cells.

[0160] In some exemplary embodiments of the invention, the use of multiple cell types contributes to the ability to generate tissues (soft and / or hard tissues) and / or organs. Alternatively or additionally, according to various exemplary embodiments of the invention, additional reservoirs for culture medium 916 and / or buffers and / or cell matrices are provided. According to various exemplary embodiments of the invention, the contents of the various reservoirs are mixed (e.g., by mixer 935) in all possible combinations in controlled ratios. In some exemplary embodiments of the invention, program instructions of controller 930 control the proportions and / or components of the mixture being prepared.

[0161] In the illustrated embodiment, cell suspension reservoirs 910 and / or 912 and / or culture medium reservoir 916 each include a temperature control mechanism (950 and / or 952). In some exemplary embodiments of the invention, the temperature control mechanism comprises a Peltier element capable of heating and / or cooling. In the illustrated embodiment, controller 930 includes a pump 932 that moves the cell suspension and culture medium through the conduits. In some exemplary embodiments of the invention, the pump is external to the controller.

[0162] In some exemplary embodiments of the invention, system 900 includes connectors for attaching conduit 922 to cell culture containers (920A, 920B, and 920C) as shown in FIG. 9B.

[0163] The illustrated exemplary system 900 includes an incubation chamber 960 designed and configured to contain a plurality of tissue culture containers (920A, 920B, and 920C). In some exemplary embodiments of the invention, the incubation chamber 960 includes temperature and / or humidity and / or CO2 control mechanisms as discussed herein above and herein below.

[0164] In the illustrated embodiment, the system 900 includes a camera 980 and a two-way data communication link 982 to an external input device. In some embodiments, the external user input device includes a user interface (e.g., a graphical user interface (GUI)) for displaying output images or video from the camera 980 and for inputting commands to the controller 930.

[0165] In some exemplary embodiments of the invention, controller 930 is adapted to periodically remove medium from the culture containers (920A, 920B, and 920C) and add new medium from storage vessel 916. In some exemplary embodiments of the invention, controller 930 is programmed with a schedule to reverse the flow direction of pump 932 to remove medium from containers 920A, 920B, and 920C via conduits 922. In some embodiments, the removed medium is pumped to a waste container. Although only one set of conduits 922 is shown for simplicity, some embodiments include separate conduits 922 for waste removal and introduction of fresh medium.

[0166] In the illustrated embodiment, the system 900 includes a compression mechanism 970 operable by the controller 930 to compress the gel matrix in one or more of the containers 920A, 920B, and 920C.

[0167] FIG. 9B is a schematic diagram of the adaptation of a standard tissue culture container for use in a cell culture system according to an exemplary embodiment of the invention. FIG. 9B illustrates a tissue culture flask 920 fitted with a cover 921 through which tubing (conduit) 923 passes. In the illustrated embodiment, the distal end of tubing 923 fits with a sterile connector 925. In some embodiments, an ASEPTQUICK connector is used as 925. According to various exemplary embodiments of the invention, tubing 923 is used to introduce / remove culture medium to / from flask 920 and / or introduce a humidified mixture of air and CO2. Alternatively or additionally, in some embodiments, tubing 923 is connected to a pump 932 (FIG. 9A). In other exemplary embodiments of the invention, a roller bottle is used in place of flask 920.

[0168] Exemplary Pump-Based Medium Exchange System FIG. 10 is a simplified schematic diagram of a medium exchange system for cell culture, generally designated as 1000, according to an exemplary embodiment of the present invention.

[0169] The illustrated exemplary system 1000 includes a cell culture container 1010 with at least one port 1012, a detector 1020 that measures a parameter of the medium 999 in the container 1010 and generates an indicator signal 1022. In the illustrated embodiment, the system 1000 includes a medium exchange mechanism that includes a controller 1030 configured to respond to a threshold value of the indicator signal 1022 by operating a pump 1040 that removes spent medium through the at least one port 1012 and introduces fresh medium 1042 through the at least one port 1012. According to various exemplary embodiments of the invention, the parameter is selected from the group consisting of pH, CO2 concentration, glucose concentration, lactate concentration, and suspended cells (number and / or percentage).

[0170] Alternatively or additionally, according to various exemplary embodiments of the present invention, detector 1020 includes a pH electrode and / or a camera. In some exemplary embodiments of the present invention, a change in the pH of medium 999 causes a color change in a medium pH indicator that generates a machine-readable change in a digital output signal from a camera acting as detector 1020.

[0171] Alternatively or additionally, in some embodiments, detector 1020 measures dissolved solutes (e.g., glucose and lactate) in the medium. In some embodiments, measurement of dissolved solutes in the medium is achieved by a biosensor that utilizes an enzyme-based amperometric mechanism. In some exemplary embodiments of the invention, cell culture container 1010 is explant culture vessel 100 described herein above. In other exemplary embodiments of the invention, cell culture container 1010 is a tissue culture flask 920 (FIG. 9B) or a roller bottle.

[0172] Exemplary Humidified CO2 Supply System 11 is a simplified schematic diagram of a humidified CO2 supply system for cell culture according to an exemplary embodiment of the invention, generally designated 1100. The illustrated exemplary system 1100 includes a closed cell culture container 1110 with at least one gas port 1112. In the illustrated embodiment, the container 1110 is partially filled with culture medium 999. In the illustrated embodiment, the system 1100 includes a CO2 tank 1120 connected to the at least one gas port 1112 via a regulator 1122, and a water storage vessel 1130 partially filled with water 1132 through which CO2 from the tank 1120 passes between the regulator 1122 and the at least one gas port 1112. According to the illustrated embodiment, as gas from the regulator 1122 bubbles through the water 1132 in the storage vessel 1130, the gas is humidified. In some exemplary embodiments of the invention, the closed cell culture container 1110 is the explant culture vessel 100 described hereinabove.

[0173] Exemplary Gradient-Based Medium Exchange System FIG. 12A is a simplified schematic diagram of a gradient-based medium exchange system for explant culture, generally designated as 1200, according to an exemplary embodiment of the present invention. FIG. 12B is a simplified schematic flow diagram of a gradient-based medium exchange system for explant culture, generally designated as 1201, according to an exemplary embodiment of the present invention. The illustrated exemplary system 1200 includes a support surface 1210 for multiple tissue culture vessels 100 and a tilting mechanism 1220 that controls the angle of the support surface 1210, and a controller 1230 configured to operate the mechanism 1220 to provide controlled removal of culture medium from the container 100 through one or more ports.

[0174] In some embodiments, the support surface 1210 is mounted in the incubation chamber 860 described herein above.

[0175] In some exemplary embodiments of the invention, controller 1230 is configured to operate tilt mechanism 1220 to +18° and then to -30° to discharge medium into a waste container, as shown in FIG. 12B. In FIG. 12B, the angle (+) or (-) is defined by the orientation of container 100. If ascending section 441 ascends, the angle is (-). If ascending section 441 descends, the angle is (+). Alternatively or additionally, in some embodiments, controller 1230 is configured to operate tilt mechanism 1220 to -5° to facilitate sample collection, as shown in FIG. 12B.

[0176] Alternatively or additionally, in some embodiments, the controller 1230 is configured to operate the tilt mechanism 1220 to +18° to remove medium from the upper compartment of the container 100 to the lift compartment 441. The tilt mechanism then returns to its initial position of 0° to an angle of -30°. Given the design of the container 100, medium above the normal medium level at 0° automatically flows into a waste bag. Therefore, medium does not re-enter the upper compartment. At a tilt angle of 30°, all medium exits the container 100. In certain configurations of the container 100, medium from the upper compartment is always removed via the lower compartment.

[0177] Figure 12C is a simplified schematic flow diagram of a cell culture system according to an exemplary embodiment of the invention (icons are the same as in Figures 8J, 8J2, and 8J3). The process illustrated in Figure 12C is similar to that described in connection with Figure 8J3.

[0178] Exemplary Compression Module FIG. 13A is a simplified schematic diagram of a compression module, generally designated as 1300, of an explant culture system according to an exemplary embodiment of the present invention.

[0179] FIG. 13B is a front view of a compression module of an explant culture system according to an exemplary embodiment of the invention, generally designated as 1301, with the inset separately showing the piston assembly, generally designated as 1311.

[0180] The illustrated exemplary system 1300 includes a plurality of explant culture containers 100, each having a movable plunger 431 in its lid 400 (see FIGS. 4A and 4B and the accompanying description above). In the illustrated embodiment, the system 1300 includes a controller 1320 configured to operate at least one piston 1310 and a vertical displacement mechanism 1330 to lower and raise the piston 1310 to depress and release each of the plungers 431 in the lid 400 of the containers 100.

[0181] In some embodiments, the system 1300 includes a horizontal displacement mechanism (1332). According to these embodiments, the controller 1320 aligns the at least one piston 1310 with the at least one plunger 431.

[0182] In some exemplary embodiments of the invention, all containers 100 are lined up in a row, with an equal number of pistons 1310 in corresponding rows. In this configuration, no horizontal displacement is required; otherwise, only one horizontal displacement aligns the row of pistons 1310 with the row of containers 100. In either case, when the vertical displacement mechanism 1330 lowers the pistons 1310, all plungers 431 are depressed simultaneously. When the vertical displacement mechanism 1330 raises the pistons 1310, all plungers 431 are released simultaneously. This is a single-cycle parallel operation.

[0183] In some exemplary embodiments of the invention, all containers 100 are lined up in a row or in parallel, and a small number of pistons 1310 are provided. In the simplest case, only one piston 1310 is provided. In this configuration, horizontal displacement is required to align the piston 1310 with the plungers of the sequential containers 100. Each time the vertical displacement mechanism 1330 lowers the piston 1310, it depresses an additional plunger 431. When the vertical displacement mechanism 1330 raises the piston 1310, all of the depressed plungers 431 are released simultaneously. This is a sequential operation.

[0184] In some exemplary embodiments of the invention, the containers 100 are in parallel, and a corresponding number of pistons 1310 are provided. In this configuration, horizontal displacement is required to align the pistons 1310 with the next row after each series of motion. Each time the vertical displacement mechanism 1330 lowers a piston 1310, it depresses a row of plungers 431. When the vertical displacement mechanism 1330 raises the pistons 1310, all of the depressed plungers 431 in that row are simultaneously released. This is a multi-cycle parallel motion.

[0185] Regardless of the mode of operation, depression of plunger 431 by piston 1310 compresses the hydrogel matrix in container 100. In some exemplary embodiments of the invention, vertical displacement mechanism 1330 is adjustable for different forces and compression patterns (e.g., linear, stepwise).

[0186] For example, in some embodiments, the force is first increased to a first stop, maintained for a period of time, and then increased to a second stop, e.g., Stop 1: 268 Pa and maintained for 5 minutes, Stop 2: increased to 625 Pa and maintained for 5 minutes, Stop 3: increased to 1517 Pa and maintained for 5 minutes.

[0187] In other exemplary embodiments of the invention, piston 1310 continuously increases the force until a final force is reached and maintained. For example, in some embodiments, piston 1310 applies a continuously increasing pressure at a rate of 4 Pa per second until a final force of 1517 Pa is reached after 15 minutes and maintained at 1517 Pa for 5 minutes.

[0188] Alternatively or additionally, in some embodiments, the controller 1320 is programmable (e.g., horizontal displacement pattern, etc.). Alternatively or additionally, in some embodiments, the system 1300 includes a pressure sensor 1340. Alternatively or additionally, in some embodiments, the system 1300 includes a camera 1340 on the piston 1310. In some embodiments, compression causes 200 to move in the container 100 (see FIGS. 1A, 2A, 2B and corresponding text). When the desired compression stage is reached, 230 and 232 assume a visually indicated position within the container 100. This position is detected by a camera (e.g., 1340 and / or 880 in FIG. 8A and / or 980 in FIG. 9), which provides an output to the controller 1320. The controller 1320 translates the camera output into a stop signal. In the current embodiment, the camera is aligned with the container 100 and is located on the compression system for use in visual inspection and is not involved in the compression itself.

[0189] FIG. 13B illustrates an exemplary compression module 1301 inserted into an environmental chamber (e.g., 860 in FIG. 8B) from the top. In some embodiments of the invention, the environmental chamber is fixed there as part of the device. In the illustrated design, four pistons 1311 are arranged in line with four controllers 1321 with vertical displacement mechanisms 1331 for compressing the four hydrogels. The controllers 1321 generate a constant force by depressing the pistons 1311 using the vertical displacement mechanisms 1331. A pressure sensor 1341 monitors the buildup of pressure. This pressure is transferred via a spring 1350 to a pressure plate 1352 of a piston head 1354. In some embodiments, the piston head 1354 is first coupled to the explant culture container 100 and then to the pistons 1311. In some exemplary embodiments of the invention, the spring 1350 is a removable part and is not fixed to the pistons 1311 or the container 100.

[0190] In some embodiments, once the explant culture container 100 is filled with hydrogel and placed in the incubation chamber, a piston head 1354 is placed on the container 100. Pins 1360 fit into matching connection points on the container 100. In the illustrated embodiment, the pressure generated by 1321 / 1331 is transferred through spring 1350 to pressure plate 1352, and from there evenly via four pins 1360 to plunger 431 on cover 430 of container 100 (see FIG. 13A).

[0191] In the illustrated embodiment, camera 1343 is held in place by camera holder 1344. The camera is capable of capturing photographs (not photomicrographs) of the 3D tissue culture.

[0192] Exemplary Remote Control System FIG. 14 is a schematic diagram of an explant culture system adapted for remote operation, generally designated 1400, according to an exemplary embodiment of the invention.

[0193] The illustrated exemplary system 1400 includes a tissue culture container 1410 with at least one port 1412, a camera 1420 that provides an image output 1422 of the culture in the container 1410, and a controller 1430 that controls a medium exchange mechanism 1450.

[0194] In some exemplary embodiments of the invention, the image output 1422 is received by a remote device 1440 and displayed on a display device 1423 to a user of the device 1440. The user then utilizes a user interface 1442 for operation of the control device 1430.

[0195] In the illustrated embodiment, the medium exchange mechanism 1450 includes at least one pump 1452 that removes spent medium through at least one port 1412 and introduces fresh medium through at least one port.

[0196] Alternatively or additionally, in some embodiments, medium exchange mechanism 1450 includes a tilt mechanism 1456 that controls the angle of a support surface 1458 that holds tissue culture container 1410. The use of tilt mechanisms such as 1450 is described herein above in connection with Figures 12A and 12B.

[0197] In some exemplary embodiments of the invention, exchange mechanism 1450 is used to remove a sample of the medium for analysis, as shown in FIG. 12B. According to various exemplary embodiments of the invention, metabolic indicators such as glucose and / or lactate are analyzed. In some exemplary embodiments of the invention, pump 1452 pumps the sample to an analytical device (not shown). In other exemplary embodiments of the invention, a biosensor (not shown) is disposed within container 1410 and provides an output signal to remote device 1440.

[0198] Exemplary Environmental Control System Figure 15 is a simplified schematic flow diagram of an environmental control system for cell culture according to an exemplary embodiment of the present invention, showing how a tubing manifold is utilized to deliver air at a desired temperature / CO2 content and humidity to multiple explant boxes (tissue culture vessels 100 in other figures).

[0199] Example Usage Scenarios While most of the above description relates to dermal fibroblasts and epidermal keratinocytes, additional embodiments of the present invention utilize other cell types isolated from human or animal tissues of different origins. Fibroblast cell lines (human or animal) are typically derived from sources including, but not limited to, dermis, tendon, lung, umbilical cord, cartilage, urethra, corneal stroma, oral mucosa, and intestine. Human / animal sources include, but are not limited to, fibroblasts, smooth muscle cells, chondrocytes, and other connective tissue cells of mesenchymal origin. In some exemplary embodiments of the present invention, fibroblasts are isolated by microdissection from the dermal papilla of hair follicles. In some exemplary embodiments of the present invention, corneal constructs are fabricated using matrix-producing cells derived from corneal stroma. In some exemplary embodiments of the present invention, cell donors vary with development and age. Alternatively or additionally, in some embodiments, cells are derived from donor tissues of other individuals, including embryonic, neonatal, or adult. In some exemplary embodiments of the present invention, embryonic progenitor cells, such as mesenchymal stem cells, are used in embodiments of the present invention and induced to develop into the desired tissue using known differentiation factors and / or conditions.

[0200] Alternatively or additionally, human or animal epithelial cells are from sources including, but not limited to, human or animal epithelium, skin, lung, umbilical cord, urethra, corneal stroma, oral mucosa, intestine, urinary bladder, esophagus and cornea.

[0201] While human cells are used in many embodiments of the invention, animal cells are also used in many embodiments. Cells from other mammalian species, including, but not limited to, equine, canine, porcine, bovine, ovine sources, and rodent species (e.g., mouse and / or rat and / or rabbit), are also used. Additionally, spontaneously, chemically, or virally transfected cells, or recombinant or genetically modified cells are used in some embodiments of the invention. In those embodiments incorporating more than one cell type, chimeric mixtures of normal cells from two or more sources, mixtures of normal and genetically modified or transfected cells, or mixtures of cells from two or more species or tissue sources are used in different embodiments of the invention.

[0202] It is anticipated that many cell culture media, membrane types, and polymers will be developed during the life of this patent, and the scope of this invention proactively includes all such new technologies.

[0203] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0204] In particular, various numerical designations are used. It should be understood that these numerical designations may further vary based on various engineering principles, materials, intended uses, and designs incorporated into various embodiments of the present invention. Furthermore, components and / or operations belonging to exemplary embodiments of the present invention and illustrated as single units may be divided into sub-units. Conversely, components and / or operations belonging to exemplary embodiments of the present invention and illustrated as sub-units / separate operations may be combined into a single unit / operation with the functionality described / illustrated.

[0205] Alternatively, or in addition, features used to describe a method can be used to characterize an apparatus, and features used to describe an apparatus can be used to characterize a method.

[0206] It should further be understood that the individual features described herein above can be combined in all possible combinations and subcombinations to create additional embodiments of the present invention. The foregoing examples are exemplary in nature and are not intended to limit the scope of the present invention, which is defined solely by the claims that follow.

[0207] Each description of an embodiment of the invention that includes a particular feature, part, component, module, or process is an explicit statement that there are additional embodiments of the invention that do not include the described feature, part, component, module, or process.

[0208] Alternatively, or in addition, various exemplary embodiments of the present invention exclude any particular feature, part, component, module, process, or element not specifically disclosed herein.

[0209] Specifically, although the present invention has been described with respect to the ex vivo production of skin grafts, it may also be used to produce implants of other tissue types amenable to fabrication in compressed hydrogels and / or to produce tissue for uses other than transplantation.

[0210] All publications, references, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.

[0211] As used herein, the terms "include" and "have" and their cognates mean "including but not necessarily limited to."

Claims

1. 1. A method for producing a skin graft, comprising: (a) introducing a hydrogel containing fibroblasts (FB) into a sealed tissue culture vessel; (b) incubating until the FBs are assembled into a gel matrix; (c) compressing the gel matrix in the same sealed tissue culture vessel; (d) seeding the compacted matrix with keratinocytes (KCs) in the same sealed tissue culture vessel; (e) further incubating and changing the medium until the graft is formed and ready to be transplanted; The exchanging tilting the sealed tissue culture vessel in one direction to allow the medium to flow into the rising compartment; tilting the sealed tissue culture vessel in a second orientation to remove the medium by gravity flow into a waste container. method.

2. The method of claim 1, further comprising visually observing the growth of FBs and / or KCs in a plurality of the sealed tissue culture vessels using a microscope.

3. 10. The method of claim 1, comprising using a common set of reagent reservoirs and using a controller and pumps to deliver the hydrogel, FB and KC to a plurality of said sealed tissue culture vessels.

4. (f) removing the growth medium and introducing a transport medium into the sealed tissue culture vessel; (g) slightly depressing the integrated plunger on the sealed tissue culture vessel lid and locking it into place; (h) transporting to a second location; The method of claim 1 , comprising:

5. A transportation lock, comprising: (a) a spanning member sized to fit the dimensions of the tissue culture vessel, the spanning member comprising: (i) having two notches on the lower edge configured to fit the contours of the lid frame; (ii) a fastener having a downward extension between said notches and sized on each outer edge of said notches to engage and retain a snap-fit connector on the base; (iii) a series of slots on its upper edge sized and positioned to engage and retain corresponding ribs on the underside of the base of a second tissue culture vessel of claim 1 disposed above; Each of the tissue culture vessels comprises: a graft support tray; a box having a lid and a base, the box engaging and holding the tray; The tray is In a first operating state, the floor of the tray is slightly elevated relative to the floor of the base; In a second operating state, the floor is lowered into contact with the floor of the base. Transport lock.

6. 6. An assembly comprising the transport lock of claim 5 and a plurality of vertically arranged tissue culture vessels.

7. (a) a plurality of explant culture containers; (b) storage containers for the cell suspension, gel matrix material, and culture medium; (c) conduits connecting each of said storage vessels to each of said culture containers; (d) a controller configured to coordinately supply the cell suspension, gel matrix material, and culture medium through the conduit to the culture container to produce a graft; system.

8. 8. The system of claim 7, wherein the cell suspension reservoirs include at least one fibroblast (FB) reservoir and at least one keratinocyte (KC) reservoir.

9. The system of claim 7 including a valve in the conduit under the control of the controller.

10. The system of claim 7 , wherein the reservoir for the gel matrix material includes a cooling element.

11. The system of claim 7 , wherein the control device comprises a pump that moves the cell suspension, gel matrix material, and culture medium through the conduit.

12. The system of claim 7 , comprising a heater positioned to heat the culture medium.

13. 10. The system of claim 7, further comprising a mixing module that receives cells from one storage container and a gel matrix from another storage container and mixes them to produce a gel matrix cell suspension.

14. 14. The system of claim 13, wherein the mixing module mixes cells with a buffer to produce a buffered cell suspension, which is then mixed with a gel matrix to produce a gel matrix cell suspension.

15. The system of claim 7 , comprising an incubation chamber designed to accommodate the plurality of explant culture containers.

16. The system of claim 7 , comprising a compression mechanism operable by the controller to compress a gel matrix in one or more of the explant culture containers.

17. 8. The system of claim 7, including a camera and a bidirectional data communication link to an external input device.

18. 8. The system of claim 7, wherein the controller is adapted to periodically remove medium from the explant culture container and supply fresh medium from one of the reservoirs.

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