Tissue culture vessels, related methods, and systems for preparing compressed hydrogel skin grafts

Abstract

A tissue culture vessel including a graft support tray (200) and a box having a lid (400) and a base that engages and retains the tray. The tray has 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.

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 Jun. 20, 2021, and U.S. Provisional Patent Application No. 63 / 240,360, filed Sep. 2, 2021, each of these prior applications having the same invention title and applicant as this application, and each of these prior applications is hereby incorporated by reference in its entirety.

[0002] The various embodiments described herein are in the field of tissue culture experimental apparatus.

Background Art

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

[0004] When a deep wound occurs after a burn accident or the like (a full-thickness skin wound), the epidermis and the entire dermis (and in some cases the subcutaneous tissue as well) are damaged, and surgical treatment is required.

[0005] Small full-thickness skin wounds are typically treated by autografting of full-thickness skin, in which the epidermis and entire dermis are taken from a healthy part of the body and transplanted to the wound.

[0006] Large full-thickness skin wounds are typically treated by autografting of split-thickness skin. Split-thickness skin autograft consists of the epidermis and a thin and incomplete dermis layer. Split-thickness skin autograft does not provide a satisfactory clinical solution to the problem of large full-thickness skin wounds. Large full-thickness skin wounds often do not heal well and result in unsightly scars.

[0007] The reason that autograft of full - thickness skin heals deeper wounds better than autograft of split - thickness skin is that autograft of full - thickness skin contains features such as vascular structures that split - thickness skin autograft does not have. Vascular structures (blood vessels and / or lymphatic vessels) provide oxygen and nutrients as well as immune cell transport and contribute to all functions and tissue survival. The capillaries of full - thickness skin graft only need to connect with the capillaries of the wound bed, while in the case of split - thickness skin graft, the capillaries need to grow from the wound bed to the graft to perfuse through the tissue.

[0008] A further reason that autograft of full - thickness skin heals deeper wounds better than autograft of split - thickness skin is that when a split - thickness skin autograft is transplanted onto a full - thickness wound, tissue contraction that can contribute to scarring and / or fibrosis and / or other ugly appearances can occur. Alternatively or additionally, the scar can contribute to the inability of the transplanted substitute skin to grow with the patient (e.g., a child), and the skin can deteriorate further. As a result, using split - thickness skin autograft can require several consecutive surgical procedures over several years to mitigate these problems. These additional surgical procedures impose a significant economic and psychological burden on the patient.

[0009] Recently, tissue engineering of the skin has been used to create substitutes for autograft of full - thickness skin. Tissue engineering utilizes hydrogels as scaffold materials to provide 3D cell matrices. Hydrogels are biocompatible and biomimetic, have low immunogenicity (conserved across species), and are naturally reconstructed by cells that can be easily seeded between cells within the fibrous network. However, the insufficient mechanical properties of hydrogels limit their clinical use as scaffolds for tissue engineering applications. Due to the relatively large area and the thinness characteristics of skin grafts (clinically suitable sizes for skin grafts are 50 cm 2 or more, and the thickness is 2 mm or less), mechanical stability is important for the clinical application of hydrogel - based cultured skin tissue.

[0010] Mechanical stability enables the handling and processing of hydrogel-based tissue culture skin grafts during and after culturing and during and after surgical application and / or testing. The mechanical stability of the hydrogel can be enhanced by compression.

Summary of the Invention

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

[0012] One aspect of some embodiments of the present invention relates to a sealed tissue culture container that includes a graft support frame in a box having a lid and a base. According to these embodiments, the frame has two operating states: a first operating state in which the frame is slightly higher with respect to the floor of the base in which the frame is located, and a second operating state in which the frame descends and contacts the floor of the base in which the frame is located. In some embodiments, the lid includes a movable plunger that, when pushed down, moves the frame from the first to the second operating state. In some embodiments, the lid includes one or more raised compartments that accommodate the displaced medium from the frame when the plunger is pushed down. In some embodiments, the raised compartment includes a gas permeable membrane on the upper surface. These gas permeable membranes allow air to escape from the container when the plunger is lowered and allow air to enter the container when the plunger is raised. The gas permeable membrane allows gas exchange between the surrounding environment and the interior of the container while maintaining asepsis. In some embodiments, the gas permeable membrane contributes to the ability to maintain the asepsis of the contents outside the container in a tissue culture hood. The gas permeable membrane also aids in gas exchange during cell culture, for example, while being placed in an incubator with such a controlled gas environment and enriched (5% CO2).

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

[0014] Yet another aspect of some embodiments of the present invention relates to the base of a tissue culture vessel configured to receive a graft support frame. In some embodiments, the base includes a compression structure that supports the membrane floor of the graft support frame when the graft support frame is fully lowered, yet allows for the flow of media outside when the frame is lowered. In some embodiments, the base includes depressions for receiving the first and / or second sets of springs of the frame located within the base. In some embodiments, the base includes pins or holes that mate with equivalent holes or pins provided 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 provided on the plunger of the lid. In some embodiments, these pins / holes serve to orient the plunger with respect to the frame when the frame is located within 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, this gasket is integrally formed as part of the lid or base. In some embodiments, the O-ring contributes to the formation of 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 luer connectors. Alternatively or additionally, in some embodiments, the base includes a snap-fit connector for adapting to the lid. Alternatively or additionally, in some embodiments, the base includes a snap-in retainer insertion for a transport lock. In some embodiments, the transport lock holds the plunger in a slightly lowered position.

[0015] Yet another aspect of some embodiments of the present invention relates to a lid of a tissue culture vessel. In some embodiments, the lid includes a rigid frame with a flexible bellows that holds a plunger in a fixed direction with respect to the frame. In some embodiments, the bellows is normally open so as to hold the plunger in the raised position in the absence of an external force. In some embodiments, the lid includes one or more elevated sections. In some embodiments, the elevated section mates with a gas permeable membrane on the upper surface of the elevated section. According to various exemplary embodiments of the present invention, the membrane is installed inside or outside the elevated section.

[0016] In some embodiments, the rigid frame includes a snap hook that engages a corresponding structure of the base. In some embodiments, the lid includes a series of external barbed connectors attached to an outlet port inside the appropriate location. For example, provide barbed connectors for CO2 / air inflow and / or fibroblasts (FB) and / or medium and / or keratinocytes and / or collagen hydrogel. Alternatively or additionally, in some embodiments, provide an additional barbed port for CO2 / air removal. Alternatively or additionally, in some embodiments, CO2 / air exits the tissue culture vessel through the gas permeable membrane of the elevated section.

[0017] Another aspect of some embodiments of the present invention relates to a tissue culture method of introducing a hydrogel containing fibroblasts into a sealed tissue culture vessel and incubating it so that the fibroblasts can aggregate in the gel matrix. After the hydrogel is formed, compress the hydrogel while it is in the same sealed tissue culture vessel. Then seed keratinocytes (KC) into the same sealed tissue culture vessel and perform further incubation until ready to transplant the graft. In some exemplary embodiments of the present invention, hydrogel formation includes crosslinking by raising the pH (mixing the collagen / cell mixture with a buffer). This process is sometimes called polymerization, but it is not true polymerization.

[0018] Further additional aspects of some embodiments of the present invention relate to a method for transporting a compressed hydrogel skin graft, which involves growing the graft in a sealed tissue culture container, removing the growth medium, introducing a transport medium, slightly lowering an integral plunger of the lid of the sealed tissue culture container, and fixing it in place.

[0019] Yet another aspect of some embodiments of the present invention relates to a system for the simultaneous management and operation of tissue culture containers. In some embodiments, the tissue culture container is a graft culture container. In some exemplary embodiments of the present invention, the graft culture container is as described above herein. In some exemplary embodiments of the present invention, the system relies on a computer-controlled controller for distributing cells and / or media and / or matrix materials to the containers through conduits. In some embodiments, the system controls the mixing of mesenchymal cells (i.e., fibroblasts) with an extracellular matrix material (e.g., collagen) and cooperatively distributes the resulting cell / matrix mixture to the culture containers. In some embodiments, various compartments of the system are heated and / or cooled. In some embodiments, the cooling of the cell matrix contributes to the reduction of early gelation. Alternatively or additionally, in some embodiments, the CO2 level and / or humidity of at least one system compartment is controlled.

[0020] Yet another aspect of some embodiments of the present invention relates to a system for automatically changing the media of a plurality of tissue culture containers. In some embodiments, the tissue culture container is a graft culture container. In some exemplary embodiments of the present invention, the graft culture container is as described above herein. In some embodiments, the system utilizes an inclination mechanism.

[0021] Yet another aspect of some embodiments of the present invention relates to a system for automatically compressing an extracellular matrix material (e.g., hydrogel) of a plurality of tissue culture containers. According to various exemplary embodiments of the present invention, the 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 visual images of cell cultures (e.g., graft cultures) at a remote location and enables remote manipulation of one or more culture parameters via a user interface at the remote location. In some embodiments, the visual images are captured by a camera and transmitted through 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 the 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 present invention, the camera does not magnify. In other exemplary embodiments of the present invention, the camera magnifies the acquired image by 2 times, 5 times, 10 times, 50 times, 100 times, 250 times, 500 times, 1000 times or an intermediate magnification or a greater magnification. In some embodiments, the magnification is achieved by attaching a microscope to the camera or integrating the camera with a microscope.

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

[0025] The various aspects described above are understood to relate to solutions to technical problems associated with the generation and / or rupture of wrinkles in a skin graft during transportation from one location to another.

[0026] Alternatively or additionally, the various aspects described above are understood to relate to solutions to technical problems associated with reducing the need for very sophisticated tissue culture facilities for the production of skin grafts.

[0027] Alternatively or additionally, the various aspects described above relate to obtaining a large amount of graft material (e.g., 1000 cm 2 , 1500 cm 2 , 2000 cm2 、 2500 cm 2 、 3000 cm 2 、 3500 cm 2 、 4000 cm 2 、 4500 cm 2 、 5000 cm 2 、 5500 cm 2 or alternatively, it is understood to relate to a solution to the technical problem of manufacturing (a graft of an intermediate or wider area). According to various exemplary embodiments of the present invention, the graft area is 100 times, 150 times, 200 times, 250 times, or an intermediate or larger multiple larger than the biopsy area.

[0028] Or alternatively, the various aspects described above are understood to relate to a solution to the technical problem of remote management of tissue cultures.

[0029] Or alternatively, the various aspects described above are understood to relate to a solution to the technical problem of reducing the workload of tissue culture technicians.

[0030] Or alternatively, the various aspects described above are understood to relate to a solution to the technical problem of improving the robustness and reproducibility of the graft manufacturing process and / or reducing the variation between batches.

[0031] Or alternatively, the various technical aspects described above are understood to contribute to reducing the total manufacturing cost.

[0032] Or alternatively, the various aspects described above are understood to relate to a solution to the technical problem of manufacturing tissue grafts within a closed automated box. In this way, the need for a heavy manufacturing room and / or cleanroom is eliminated. As a result, matching grafts can be manufactured in various locations such as hospitals where patients are housed.

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

[0034] In some exemplary embodiments of the present invention, there is provided a graft support tray including: (a) a rigid frame; (b) a liquid permeable membrane floor attached to the lower edge of the frame; and (c) a first set of vertically extending springs attached to the outside of two opposite sides of the frame. In some embodiments, the tray includes a second set of vertically extending springs attached in a direction opposite to the first springs. Alternatively or additionally, in some embodiments, the tray includes guide holes / pins that engage corresponding pins / holes of a base to which the frame is attached. Alternatively or additionally, in some embodiments, the tray includes guide holes / pins that engage corresponding pins / holes of a lid located on the support frame.

[0035] In some exemplary embodiments of the present invention, a compression structure is designed and configured to support the membrane floor of a graft support frame that is fully recessed within this base, the compression structure being configured to allow for the flow of media to the exterior when the frame is recessed, and providing a base of a tissue culture vessel that includes this compression structure. In some embodiments, the container is dimensioned and positioned to contain a set of springs of a graft support tray located within this base and includes depressions. Alternatively or additionally, in some embodiments, the container is dimensioned and positioned to contain two sets of springs of a graft support tray located within this base and includes depressions. Alternatively or additionally, in some embodiments, the container includes pins or holes dimensioned and positioned to engage corresponding holes or pins provided on a graft support tray located within this base. Alternatively or additionally, in some embodiments, the container includes pins or holes dimensioned and positioned to engage corresponding holes or pins provided on a plunger of a lid fitted to this base. Alternatively or additionally, in some embodiments, the container includes an O-ring or other gasket or 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 luer 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 snap-in insert for a transport lock.

[0036] In some exemplary embodiments of the present invention, there is provided a lid for a tissue culture vessel comprising: (a) a rigid frame; and (b) a flexible bellows disposed within the frame, the bellows holding a plunger in a fixed direction with respect to the frame. In some embodiments, the bellows is normally open such that it holds the plunger in an elevated position in the absence of an external force. Alternatively or additionally, in some embodiments, the lid includes one or more raised sections extending on a plane of an upper edge of the frame. Alternatively or additionally, in some embodiments, the lid includes a gas permeable membrane on an upper surface of the raised section. Alternatively or additionally, in some embodiments, the lid includes a snap hook sized and positioned on the rigid frame to engage a base covered by the lid. Alternatively or additionally, in some embodiments, the lid includes one or more external luer connectors in fluid communication with an internal outlet port. Alternatively or additionally, in some embodiments, the lid includes a luer port for removing CO2 / air. Alternatively or additionally, in some embodiments, the lid includes an elastic seal on a side of the lid that contacts the base when the container is assembled.

[0037] In some exemplary embodiments of the present invention, a method for manufacturing a skin graft is provided that includes: (a) introducing a hydrogel containing fibroblasts (FB) into a sealed tissue culture vessel; (b) incubating until the FB aggregates in the gel matrix; (c) compressing the gel matrix within the same sealed tissue culture vessel; (d) seeding keratinocytes (KC) onto the compressed 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 includes visually inspecting the growth of the FB and / or the KC within the tissue culture vessel using a microscope. Alternatively or additionally, in some embodiments, the method includes changing the medium, which includes tilting the sealed tissue culture vessel in one direction to flow the medium into a raised compartment and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow into a waste container.

[0038] In some exemplary embodiments of the present invention, a method for manufacturing a skin graft is provided that includes: (a) preparing a compressed hydrogel skin graft at a first position in a sealed tissue culture vessel; (b) removing the growth medium and introducing a transport medium into the sealed tissue culture vessel; (c) slightly lowering and fixing in place an integral plunger of the lid of the sealed tissue culture vessel; and (d) transporting to a second position. In some embodiments, the removing includes tilting the sealed tissue culture vessel in one direction to flow the medium into a raised compartment and tilting the sealed tissue culture vessel in a second direction to remove the medium by gravity flow into a waste container.

[0039] In some exemplary embodiments of the present invention, (a) a transport lock is provided that is sized to fit the dimensions of the tissue culture vessel described above and includes a spanning member having: (i) two notches configured to fit the contour of the frame of the lid at the lower edge of the transport lock; (ii) a retainer sized to engage and hold a snap-fit connector on the base at the downward extension between the notches and on the outer edge of each of the notches; and (iii) a series of slots at the upper edge of the spanning member, the slots being sized to engage and hold corresponding ribs on the lower surface of the base of the second tissue culture vessel located above.

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

[0041] In some exemplary embodiments of the present invention, there is provided a system comprising: (a) a plurality of graft culture containers; (b) storage containers for a cell suspension, a gel matrix material, and a culture medium; (c) conduits connecting each of the storage containers to each of the culture containers; and (d) a control device configured to cooperatively deliver the cell suspension, the gel matrix material, and the culture medium to the culture containers through the conduits to produce grafts. In some embodiments, the storage container for the cell suspension includes at least one fibroblast (FB) storage container and at least one keratinocyte (KC) storage container. Alternatively or additionally, in some embodiments, the storage container for the cell suspension includes at least two storage containers for at least two different cell types selected from the group consisting of fibroblast (FB), keratinocyte (KC), adipocyte, myocyte, neuron, pericyte, stem cell, and induced pluripotent cell (IPC). Alternatively or additionally, in some embodiments, the storage container for the cell suspension includes at least two storage containers for at least a first cell type derived from epithelium and a second cell type selected from the group consisting of cells of mesenchymal origin, skin-derived cells, adipocytes, myocytes, neurons, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the system includes a valve in the conduit under the control of the control device. Alternatively or additionally, in some embodiments, the storage container for the gel matrix includes a cooling element. Alternatively or additionally, in some embodiments, the control device includes a pump for moving the cell suspension, the gel matrix material, and the culture medium through the conduit. Alternatively or additionally, in some embodiments, the system includes a heater positioned to heat the culture medium. Alternatively or additionally, in some embodiments, the system includes a mixing module that receives cells from one storage container and a gel matrix from another storage container and mixes the cells with the matrix to produce a gel matrix cell suspension. Alternatively or additionally, in some embodiments, the mixing module mixes the cells of one storage container with a buffer of a second storage container to produce a buffered cell suspension, and then mixes the buffered cell suspension with the gel matrix of a third storage container to produce a gel matrix cell suspension.Alternatively or additionally, in some embodiments, the system includes an incubation chamber designed and configured to receive the plurality of graft culture containers. Alternatively or additionally, in some embodiments, the system includes a compression mechanism operable by the control device to compress the gel matrix in one or more of the graft culture containers. Alternatively or additionally, in some embodiments, the system includes a bidirectional data communication link to a camera and an external input device. Alternatively or additionally, in some embodiments, the control device is adapted to periodically remove media from the graft culture containers and add fresh media from one of the storage containers. Alternatively or additionally, in some embodiments, each of the graft culture containers includes the tissue culture container described hereinabove.

[0042] In some exemplary embodiments of the present invention, there is provided a system including (a) a plurality of cell culture containers, (b) storage containers for cell suspensions and media, (c) conduits connecting each of the storage containers to each of the culture containers, and (d) a control device configured to cooperatively deliver the cell suspension and the media through the conduits to the culture containers to produce a culture in the containers. In some embodiments, the storage container for the cell suspension contains at least one cell type selected from the group consisting of fibroblasts (FB), keratinocytes (KC), adipocytes, myocytes, neurons, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the storage container for the cell suspension contains a first cell type derived from epithelium and a second cell type selected from the group consisting of cells of mesenchymal origin, skin-derived cells, adipocytes, myocytes, neurons, pericytes, and stem cells. Alternatively or additionally, in some embodiments, the storage containers for the cell suspension and the media include a temperature control mechanism. Alternatively or additionally, in some embodiments, the control device includes a pump (932) for moving the cell suspension and the media through the conduits. Alternatively or additionally, in some embodiments, the system includes connectors for attaching the conduits to the cell culture containers. 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 control device is adapted to periodically remove media from the cell culture containers and add one fresh media of the storage containers.

[0043] In some exemplary embodiments of the present invention, a system is provided that includes a medium exchange mechanism including: (a) a cell culture container having at least one port; (b) a detector that measures parameters of the medium in the container and generates an indicator signal; and (c) a control device configured to respond to a threshold value of the indicator signal by operating a pump that removes spent medium through the at least one port and introduces fresh 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 floating cells (number and / or ratio). 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 the graft culture container described above herein.

[0044] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a closed cell culture container having at least one gas port; (b) a CO2 tank connected to the at least one gas port via a regulator; and (c) a water storage container that passes CO2 from the tank between the regulator and the at least one gas port. In some embodiments, the closed cell culture container includes the graft culture container described above herein.

[0045] In some exemplary embodiments of the present invention, a system is provided that includes: (a) a support surface for a plurality of graft culture containers; (b) an inclination mechanism that controls the angle of the support surface; and (c) a control device configured to operate the mechanism to provide controlled removal of medium from the container through one or more ports. In some embodiments, the support surface As described above in this specificationIt is installed in the incubation chamber. Alternatively or additionally, in some embodiments, this control device is configured to operate this tilting mechanism to +18° and then -30° to discharge the culture medium into the waste container. Alternatively or additionally, in some embodiments, this control device is configured to operate this tilting mechanism to -5° to facilitate sample collection. Alternatively or additionally, in some embodiments, this control device is configured to operate this tilting mechanism to +18° to remove the culture medium from this upper section to the ascending section and then to -30° to discharge all the culture medium.

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

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

[0048] Unless defined otherwise, 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. Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used when practicing the present invention. In case of conflict, the present specification, including definitions, will control. All materials, methods, and examples are illustrative only and not intended to be limiting.

[0049] As used herein, the terms "comprising" and "including" or grammatical variations thereof are considered to define the inclusion of the recited features, integers, operations, components, or groups thereof without precluding the addition of one or more additional features, integers, operations, components, or groups thereof. This term is broader than and includes the terms "consisting of" and "consisting essentially of" as defined by the United States Patent and Trademark Office's Manual of Patent Examination Procedure. Thus, any recitation that an embodiment "includes" or "comprises" a certain feature encompasses specific recitations that the sub - embodiment "consists essentially of" and / or "consists of" the recited feature.

[0050] As used herein, the phrase "consisting essentially of" or grammatical variations thereof is considered to define the recited features, integers, steps, or components, but does 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 change the basic and novel features of the claimed composition, apparatus, or method.

[0051] The phrase "configured to" as used in this specification and the appended claims imposes additional structural limitations on previously recited components.

[0052] The term "method" refers to a method, means, technique, and procedure for accomplishing a given task, including, but not limited to, any of those known to, or readily developed from those known to, a person having ordinary skill in the art of structure and / or computer science.

[0053] To understand the present invention and how it may be actually implemented, embodiments will be described with reference to the accompanying drawings, only as non-limiting examples. In the drawings, the same and disclosed structures, elements, or portions thereof that appear in more than one drawing are generally denoted by the same or similar references in the drawings in which they appear. The dimensions of the components and features of the drawings are selected primarily for convenience of presentation and clarity and are not necessarily drawn to scale. The accompanying drawings are as follows.

Brief Description of the Drawings

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[0055] Embodiments of the present invention relate to a tissue culture vessel configured to produce a skin graft and related methods.

[0056] Specifically, some embodiments of the present invention can be used to produce a skin graft in a closed container. In some embodiments, the container is aseptically closed and is intended for single use (disposable) to limit contamination and assist in operations in a low-grade GMP cleanroom classification such as C or D according to EU GMP rules.

[0057] The principles and operation of the tissue culture vessel and / or method according to an exemplary embodiment of the present invention may be better understood with reference to the drawings and the accompanying description.

[0058] Before detailing at least one embodiment of the present invention, it should be understood that the present invention is not limited to the details of the present invention described in the following description or exemplified by the examples. The present invention can take other embodiments and can be carried out and performed in various ways. Also, the expressions and technical terms used herein are for the purpose of explanation and should not be regarded as limiting.

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

[0060] The illustrated exemplary tissue culture container 100 includes a box having a graft support tray 200, a lid 400, and a base 300. In the illustrated embodiment, the box engages and holds with a frame 210 (see FIG. 2A) of the tray 200 having two operating states. In the first operating state, the tray 200 is slightly higher with respect to the floor of the base 300. In the second operating state, the tray 200 drops and contacts the floor of the base 300. In some embodiments, the tray 200 is normally in the first operating state and moves to the second operating state in response to an external force. In the illustrated embodiment, the container 100 includes a movable plunger 431 attached to the lid 400. In some embodiments, depressing the plunger 431 by an external force switches the tray 200 to the second operating state. For the purposes of this specification and the appended claims, the term "slightly higher" means that there is sufficient space for a liquid medium to enter the space between the tray 200 and the floor of the base 300.

[0061] The illustrated exemplary container 100 includes one or more raised compartments 441. In some embodiments, the raised compartments 441 receive the medium transferred from the tray 200 when the plunger 431 is depressed and / or when the entire container 100 is tilted such that the raised compartments 441 are below the rest of the container.

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

[0063] In some exemplary embodiments of the present invention, the 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, the connection to the container 100 is made by a sterile tube connection (e.g., tube welding), such that under normal use conditions, exposure to the environment inside the container does not occur until the implant is removed for transplantation or other use.

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

[0065] Each of the implant support tray 200, the base 300, and the lid 400 will be described in further detail below herein. 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 container like that of FIG. 1A showing the interrelationships between parts, generally designated 101.

[0067] Exemplary support tray FIG. 2A is a top perspective view of an implant support tray, generally designated 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 201.

[0069] In the illustrated embodiment, the implant support tray 200 includes a rigid frame 210, a liquid-permeable membrane floor 220 attached to the lower edge 212 (FIG. 2B) of the frame 210, and a first set of vertically extending springs 230 attached to two opposite outer sides of the frame 210. The springs 230 function to support the membrane floor 220 in an elevated position in the absence of an external force, as described in the context of the "first operating state" of FIG. 1 above herein.

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

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

[0072] In the illustrated embodiment, the graft support tray 200 includes guide holes / pins (240) that engage corresponding pins / holes of the base to which the frame is attached.

[0073] In the illustrated embodiment, the graft support tray 200 includes guide holes / pins (242) that engage corresponding pins / holes of the lid located on the support frame.

[0074] In some embodiments, an incline 211 is disposed on one side of the graft support tray 200. In embodiments characterized by the incline 211, the incline guides the injected liquid to flow into the region having the membrane floor 220. In some embodiments, the angle of the incline 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 incline 211 is determined by the placement of the liquid inlet 460 and the size of the membrane 420.

[0075] Alternatively or additionally, in some embodiments, a structure having holes 213 is located on the other side of the graft support tray 200, which structure allows for efficient drainage during overflow or inclination.

[0076] The graft support tray 200 is in an assembled state with initial stress applied by four spacers 411 of the lid (two are visible in FIG. 4A), and the spacers apply pressure on two ears 215 at the same height as the rigid frame 210.

[0077] The rigid frame 210 is designed to be flat and improve the support of the bonded film 220.

[0078] The rigid frame 210 shown in the drawings has two holes and ribs in the middle and is designed to increase the rigidity required to make the film 220 flat.

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

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

[0081] The illustrated exemplary base 300 includes a compression structure 310 designed and configured to support the membrane floor of the graft support frame that is fully recessed in the base. When the base 300 is assembled into the tissue culture vessel 100, the plunger 431 of the lid descends and pushes the graft support tray downward, so that the culture medium passes through the compression structure 310 and enters the upper compartments (441 and / or 440, FIGS. 1 and 4 respectively). In some exemplary embodiments of the present invention, the compression structure 310 is provided as a pattern formed in the base that channels the culture medium into the upper compartments during compression. According to these embodiments, the pattern includes channels that facilitate the flow of liquid. FIGS. 3C and 3D provide detailed views of the exemplary compression structure 310. In other exemplary embodiments of the present invention, the compression structure 310 is composed of a liquid-permeable material. In any case, the compression structure 310 allows the flow of the culture medium to the outside when the frame descends.

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

[0083] The illustrated exemplary base 300 is dimensioned and positioned to accommodate a set of springs of a graft support tray located within the base 300 and includes recesses 320. In the illustrated embodiment, the base 300 is dimensioned and positioned to accommodate two sets of springs of a graft support tray located within the base and includes recesses (320). In the illustrated embodiment, the base 300 includes a step 322 for positioning the graft support tray.

[0084] The illustrated exemplary base 300 is dimensioned and positioned to engage corresponding holes or pins provided on a graft support tray located within the base and includes pins 330 or holes. In some embodiments, these pins / holes serve to orient the graft support tray within the base. In some embodiments, the orientation of the graft support tray within the base 300 contributes to the accuracy of alignment between the graft support tray and the plunger 431 of the lid.

[0085] The illustrated exemplary base 300 is dimensioned and positioned to engage corresponding holes or pins provided on a plunger of a lid fitted to the base 300 and includes pins and / or holes 332. In some embodiments, these pins or holes 332 serve to orient the plunger with respect to the frame when the frame is located within the base 300. In some embodiments, the holes 332 are simple recesses that provide space for pins of the plunger. According to these embodiments, the pins 470 within the plunger engage with the guide holes 242 of the graft 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 the formation of an airtight and / or watertight seal.

[0087] In the illustrated embodiment, the 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 these ports mating with luer connectors 345 and 347.

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

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

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

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

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

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

[0094] The illustrated exemplary lid 400 includes a rigid frame 410 with a flexible bellows 420 disposed therein. The bellows 420 holds the plunger 431 in a fixed direction with respect to the frame 410. In some embodiments, the plunger 431 is composed of a transparent material to facilitate visual inspection of cells cultured in a graft support tray attached under the lid 400.

[0095] In some exemplary embodiments of the present invention, the bellows 420 is normally open so as to hold the plunger 431 in the raised position in the absence of an external force. According to various exemplary embodiments of the present invention, the raised position is at a height above the plane of the frame 410. Alternatively or additionally, in some embodiments the bellows 420 is manually removable by the end user of the graft (e.g., for adhesive attachment). In some embodiments, the manually removable bellows contributes to the convenience of removing the graft from the container.

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

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

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

[0099] In some exemplary embodiments of the present invention, the lid 400 mates with an elastic 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 method FIG. 5 is a simplified flow diagram of a graft conditioning method according to some exemplary embodiments of the present invention, generally shown as 500.

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

[0102] In the illustrated embodiment, then incubate 520 the FB until it aggregates in the gel matrix (typically after crosslinking). Once the FB has aggregated in the gel matrix, compress 530 the matrix within the same sealed tissue culture vessel.

[0103] In some exemplary embodiments of the present invention, after compression, the gel is incubated for several days (typically 5) so that the cells can adhere to, aggregate, and reconstruct the gel. In some embodiments, the cell culture medium is changed every two days.

[0104] In the illustrated embodiment, keratinocytes (KC) are seeded into the compressed matrix in the same sealed tissue culture vessel 540 and further incubated 550 until the graft is ready. In some embodiments, when a fibroblast-rich dermis and a continuous keratinocyte / epidermal layer are formed, the graft is considered ready for transplantation. Alternatively or additionally, in some embodiments, medium exchange / removal during skin culture is performed during incubations 520 and / or 550 (e.g., using the raised compartment 441). In some embodiments, the exchange includes tilting the sealed tissue culture vessel in one direction to flow the medium into the raised 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, a 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 enables the evaluation of cell growth and the determination of transplant readiness. Alternatively or additionally, sampling the cell culture medium via the sample port enables the measurement of the microbiological load of the medium (e.g., sterility, mycoplasma, endotoxin, etc.).

[0106] In some embodiments, a visual inspection of the growth of FB and / or KC in the tissue culture vessel using a microscope is performed. According to various exemplary embodiments of the present invention, the 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 present invention, method 500 includes providing hydrogel, FB, and KC to a plurality of sealed tissue culture vessels using a common set of reagent storage containers (e.g., 810, 812, 814, and 816) and using a control device 830 (FIG. 8) and a pump 832.

[0108] Additional Exemplary Methods FIG. 6 is a simplified flowchart of a graft manufacturing method according to an additional exemplary embodiment of the present invention, generally shown as 600.

[0109] The illustrated exemplary method 600 includes preparing a compressed hydrogel skin graft at a first position of a sealed tissue culture vessel (as described in the context of FIG. 5) 610. The illustrated exemplary method 600 includes removing the growth medium and introducing a transport medium into the sealed tissue culture vessel 620 and partially lowering an integral plunger of the lid of the sealed tissue culture vessel 630 and fixing the plunger in place 640, and then moving it to a second position 650. In some embodiments, fixing 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 flow the medium into the upper 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 FIG. 7A is a perspective view of a tissue culture vessel mated with two transport locks 710 according to an exemplary embodiment of the present invention, generally shown as 700. Insert L provides a close-up view of connector 716.

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

[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 configured to fit the contour of the frame 410 of the lid 400 at the lower edge of the transport lock, as well as a lower extension 712 between the notches 720 and fasteners 716 sized to engage and hold a snap-fit connector 350 on the base 300 on the outer edge of each of the notches. During use of the transport lock, the lower extension 712 and the fasteners 716 cooperate to hold the tray 200 in the second operating state described above herein. Alternatively or additionally, in some embodiments, the fasteners 716 are of the "snap-fit" type and are composed of flexible plastic so as to be removable by hand.

[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 hold corresponding ribs 352 (see FIG. 3B) on the lower surface of the base 300 of a second tissue culture vessel 100 located on top of the transport lock.

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

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

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

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

[0119] In some embodiments, the base 300 has a height of 20 to 30 mm (e.g., 24 mm), a width of 160 to 165 mm (e.g., 162.3 mm), and a length of 250 to 255 mm (e.g., 253.6 mm). When the box of this size is composed of polycarbonate such as Trinseo Calibre Megarad 2081-15 polycarbonate, it is 200 to 204 grams (e.g., 202 grams). In some embodiments, the base 300 is manufactured by injection molding and subsequent port drilling.

[0120] Exemplary Operational Considerations To remove the culture medium from the container 100, first tilt the container backward (away) along the horizontal axis from the waste discharge port 344 to remove the culture medium from the upper compartment to the rising compartment 440. Once the culture medium has accumulated in the rising compartment 440, tilt the container 100 at an angle of <20° to the waste discharge port 344. The port 344 is connected to a waste container and is open. At an angle of 20°, less than 10% of the culture medium remains in the container 100. By returning the container 100 to the neutral position, the port 344 rises above the liquid level and fresh culture medium can be introduced through one of the ports 460 of the lid 400.

[0121] For the seeding of KC (keratinocytes), first tilt the container backward (away) along the horizontal axis from the waste discharge port 344 at an angle of 26° to remove the liquid from the upper compartment to the rising compartment 440. At an angle of 26°, only 4 ml of the culture medium is retained in the tray 200. After returning the container 100 to the neutral position, KC can be seeded through one of the ports 460 of the lid 400.

[0122] The container 100 of the dimensions described above holds 165 ml ± 10% of the culture medium, which is sufficient to ensure a 4 mm immersion in the graft growth in the support tray 200. Approximately 40 ml of the culture medium is present in the graft support tray 200. The remaining amount is inside the base 300 but outside and / or below the tray 200.

[0123] While introducing the hydrogel into the graft support tray 200, the membrane floor 220 needs to be raised above the support surface 310 of the base 300. The first spring 230 on the support tray 200 ensures this situation. After the first incubation (to crosslink the hydrogel), the gel is compressed. To facilitate compression, the membrane needs to contact the support surface 310 of the base 300 so that it can be wetted and / or support counterpressure (such as a tent cloth that starts to leak when touched). After compression, the spring 230 and / or 232 returns the support tray 200 to its initial position. The space under the support tray 200 is sufficient for the amount of medium required for cell growth of the graft. In some embodiments, the liquid-permeable membrane floor 220 allows nutrients from the medium in the space under the tray 200 to provide nutrients for cell growth in and / or on the hydrogel in the tray.

[0124] Exemplary graft culture system FIG. 8A is a simplified schematic view of a graft culture system according to an exemplary embodiment of the present invention, generally shown as 800.

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

[0126] In the illustrated embodiment, the system (800) includes a plurality of graft culture containers (820A, 820B, 820C) and storage containers (810, 812) for cell suspensions, a gel matrix material (814), and a culture medium (816). In the illustrated embodiment, conduits (818, 822) connect each of the storage containers (810, 812, 814 and 816) to each of the culture containers (820A, 820B, 820C). For clarity, three culture containers are illustrated, but in many embodiments of the invention, there are actually many more. The illustrated exemplary system 800 includes a control device (830) configured to cooperatively deliver cell suspensions, gel matrix material, and culture medium to the culture containers through the conduits to produce grafts. In some exemplary embodiments of the invention, the storage containers for cell suspensions include at least one fibroblast (FB) storage container (810) and at least one keratinocyte (KC) storage container (812).

[0127] According to various exemplary embodiments of the invention, the storage containers for cell suspensions include at least two storage containers for at least two different cell types selected from the group consisting of fibroblast (FB), keratinocyte (KC), adipocyte, myocyte, neuron, pericyte, stem cell, and induced pluripotent cell (IPC). According to various exemplary embodiments of the invention, the stem cells include epithelial stem cells and / or mesenchymal stem cells. In some exemplary embodiments of the invention, the use of multiple cell types contributes to the ability to generate tissue (soft tissue and / or hard tissue) and / or organs.

[0128] In some embodiments, the storage containers for cell suspensions include at least two storage containers for at least a first cell type derived from epithelium and a second cell type selected from the group consisting of cells of mesenchymal origin, skin-derived cells, adipocytes, myocytes, neurons, pericytes, and stem cells.

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

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

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

[0132] Referring to FIG. 8K, in some embodiments, the mixing module (835) mixes cells from one storage container (e.g., 810) with buffer from a second storage container (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, the module 835 then mixes the buffered cell suspension with the gel matrix from a third storage container (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 that is designed and configured to receive a plurality of graft culture containers. In some embodiments, the temperature and / or humidity and / or CO2 concentration within chamber 860 is controlled by a control device 830.

[0134] In the illustrated embodiment, system 800 includes a compression mechanism 870 that is operable by control device 830 to compress the gel matrix within one or more graft culture containers 100.

[0135] In the illustrated embodiment, system 800 includes a camera 880 and a bi-directional 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 control device 830. In some exemplary embodiments of the present invention, the images output by camera 880 are used for the evaluation of the color of the culture medium (an indicator of pH change and related nutrient value of the culture medium) and / or turbidity (indicating the presence of contaminants). Alternatively or additionally, in some embodiments, the output by camera 880 is used for the evaluation of the smoothness of the surface of the gel and / or the detection of the presence of air bubbles as part of the quality assessment of the tissue formation process.

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

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

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

[0139] FIG. 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 tube strategy. In the illustrated embodiment, the mixing module 835 includes a plurality of peristaltic pumps 2832 (illustrated as three by way of example) fixed at an angle (45°) on a support plate 2851. In the illustrated embodiment, fluid sensors 2840 are positioned below and above the peristaltic pumps so that the liquid distribution can be controlled. The illustrated exemplary mixing module utilizes a standard, single-use tube kit in a closed system arrangement. The illustrated exemplary configuration includes a holding structure 2850 for aseptic connection points. In the illustrated embodiment, the design 2860 includes additional holding structures for single or branched tube sets.

[0140] FIG. 8K is a simplified schematic view of the static mixer utilized in the mixing module 835 of FIG. 8C according to some exemplary embodiments of the present invention. In the illustrated embodiment, the mixer 837 receives liquid / suspension from the storage containers 810, 814, 817 via a conduit 818 and produces a mixture via a conduit 822. In the illustrated embodiment, the mixer 837 is a connector having two inputs and one output. These two input streams are mixed by the turbulence generated when they merge in the mixer 837. In some embodiments, in this configuration, the need for moving parts in the mixer is eliminated.

[0141] FIG. 8D is a front view of a dispensing module according to some exemplary embodiments of the present invention, generally shown as 2900. In some exemplary embodiments of the present invention, the dispensing module utilizes a single-use tube tree for simultaneously dispensing a plurality of liquids. In the illustrated exemplary embodiment of FIG. 8D, the dispensing module 2900 includes a holder 2920 for a plurality of branched tube trees. In some exemplary embodiments of the present invention, the holder 2920 contributes to the ability of the liquid to flow evenly through the tubing and / or contributes to preventing the formation of twists in the tubing that could cause flow blockages. In the illustrated embodiment, 2930 secures the unused tubes.

[0142] In the illustrated embodiment, the module 2900 has four solenoid valves 2910. This arrangement contributes to the ability to control liquid dispensing. For example, when the solenoid valves 2910 operate continuously every second, even liquid dispensing is achieved. In other exemplary embodiments of the present invention, more valves are used to dispense liquid into more containers simultaneously.

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

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

[0145] In the illustrated embodiment, the chamber 2940 is filled with air preheated from the outside (to a desired temperature such as 37° C.) via the connection point 2942. In some exemplary embodiments of the present invention, the connection point 2942 is connected to an environmental control system such as the environmental control system "CUBE" of LIFE IMAGING SYSTEM or an incubator via a large-diameter tubing. In the illustrated embodiment, the chamber 2940 contains a storage unit 2944 in which the media bag 2816 is installed. The unit is formed to support the bag with an upper hook for holding the bag. The storage unit 2944 has a lid 2946. In some embodiments, the storage unit 2944 and the lid 2946 include a Peltier element for heating the media bag 2816 to the required temperature. In some exemplary embodiments of the present invention, the use of the Peltier element partially or completely eliminates the need to connect the chamber 2940 to an environmental control system. In any case (regardless of whether there is a connection to an environmental control system), the chamber 2940 mates with guides and outlet ports 2953 for incoming and outgoing tubing.

[0146] Figure 8F is a front view of an incubation chamber according to an exemplary embodiment of the present invention, generally designated 860. In the illustrated embodiment, the single-use graft culture container 100 described above herein is incubated in the incubation chamber 860. In some exemplary embodiments, the chamber 860 is adapted for the compression of a three-dimensional matrix (hydrogel) and / or the culture of grafts over several days or weeks. In some exemplary embodiments of the present invention, part of the construction of the 3D graft occurs outside the chamber 860. In the illustrated embodiment, the chamber 860 has two sliding doors 2862 on the front for the assembly of the container 100. In the illustrated embodiment, the small door 2864 is suitable for interactions during processes where temperature fluctuations within the chamber are undesirable. In the illustrated embodiment, four small doors 2864 are suitable, but the number of small doors 2864 may increase depending on the number of containers 100. The sliding doors fit into slits 2863 covered with rubber so that tubing can connect from the outside to the inside of the environmental chamber. Similar rubber-covered slits 2866 fit into the bottom of the device so that tubing for waste disposal can exit the environmental chamber and connect to a garbage bag.

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

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

[0149] For an environment suitable for cell growth in the culture container 100 inside the chamber 860, it is required to humidify the air and contain 5% CO2. In some exemplary embodiments of the present invention, an air mixture containing 5% CO2 is injected into the humidity chamber 2870. In some embodiments, the air mixture is provided by a system such as the "BRICK" of LIFE IMAGING SYSTEM or an incubator. In the illustrated embodiment, the water column 2872 ensures humidification of the air before distributing it to the container 100. In the illustrated embodiment, 2861 functions as a connector port of the humidity chamber 2870. In some embodiments, the culture container 100 is connected to the connector 2861 via tubing to receive air.

[0150] FIG. 8G is a front view of an incubation chamber such as FIG. 8F assembled with a compression module 1301 and an inclination-based medium exchange mechanism 3000 according to an exemplary embodiment of the present invention. In the illustrated embodiment, the incubation chamber 860 is assembled with a compression module 1301 described below in connection with FIG. 13B. In the illustrated embodiment, a valve 2910 can be seen at the upper front (outside) of the chamber 860 (see FIG. 8D and the 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 disposed within the chamber 860. The tilt table 3000 is tilted so that the medium can be discharged from the container 100 as described below in connection with FIGS. 8H, 12A, and 12B. In the illustrated embodiment, the tilt table 3000 is driven by an actuator 3010 (see FIG. 8H). In some embodiments, the tilt table 3000 is designed considering a second function of heat storage. According to these embodiments, using a thick metal plate in the configuration of the table 3000 creates a heat storage body. In some exemplary embodiments of the present invention, the heat storage body contributes to the temperature uniformity in the chamber 860.

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

[0152] FIG. 8H is a front view of an inclination-based medium exchange mechanism 3000 according to an exemplary embodiment of the present invention removed from the chamber 860. A structure 3020 including a metal plate having two handles is designed to improve handling. The metal plate is formed to secure a space for the pipes of the bioreactor. In this particular design, four culture containers 100 (which may be further expanded) are assembled on the metal plate. A frame 3030 is used to hold the culture containers 100 in predetermined positions on the structure. In some embodiments, the structure 3020 is assembled with one or more containers 100, lifted into the incubation chamber 860, and installed on top of the tilt table 3000.

[0153] Figure 8I is a simplified illustrative flow diagram of medium injection according to an exemplary embodiment of the present invention.

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

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

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

[0157] Exemplary cell culture system Figure 9A is a simplified schematic diagram of a cell culture system according to an exemplary embodiment of the present invention, generally shown as 900. The illustrated exemplary system 900 includes a plurality of cell culture containers (such as 920A, 920B, and 920C), a storage container 910 for cell suspension, a medium 916, and conduits (such as 918 and 922) connecting each of the storage containers to each of the culture containers. In the illustrated embodiment, the control device (930) is configured to coordinately deliver the cell suspension 910 and the medium 916 through the conduits 918 and 922 to the culture containers 920A, 920B, and 920C to produce a culture within the containers. For clarity, only three culture containers are illustrated, but typically there are many more.

[0158] In some exemplary embodiments of the present invention, the storage containers (910 and / or 912) of the cell suspension 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 present invention, the storage containers of the cell suspension include at least two storage containers 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 present invention, the stem cells include epithelial stem cells and / or mesenchymal stem cells and / or induced pluripotent cells (IPCs).

[0159] In some embodiments, the storage containers (910, 912) of the cell suspension contain a first cell type derived from epithelium and a second cell type selected from the group consisting of cells of mesenchymal origin, skin-derived cells, adipocytes, myocytes, neurons, pericytes, and stem cells.

[0160] In some exemplary embodiments of the present invention, the use of multiple cell types contributes to the ability to generate tissue (soft tissue and / or hard tissue) and / or organs. Alternatively or additionally, according to various exemplary embodiments of the present invention, additional storage containers for medium 916 and / or buffer and / or cell matrix are provided. According to various exemplary embodiments of the present invention, the contents of the various storage containers are mixed in all possible combinations at a controlled ratio (e.g., by mixer 935). In some exemplary embodiments of the present invention, the program instructions of the control device 930 control the ratio and / or components of the mixture to be prepared.

[0161] In the illustrated embodiment, each of the storage containers 910 and / or 912 of the cell suspension and / or the storage container 916 of the culture medium includes a temperature control mechanism (950 and / or 952). In some exemplary embodiments of the present invention, the temperature control mechanism comprises a Peltier element that can heat and / or cool. In the illustrated embodiment, the control device 930 includes a pump 932 that moves the cell suspension and the culture medium through a conduit. In some exemplary embodiments of the present invention, the pump is external to the control device.

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

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

[0164] In the illustrated embodiment, the system 900 includes a camera 980 and a bi-directional 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 the output image or video from the camera 980 and for inputting commands to the control device 930.

[0165] In some exemplary embodiments of the present invention, the control device 930 is adapted to periodically remove the culture medium from the culture containers (920A, 920B, and 920C) and add fresh culture medium from the storage container 916. In some exemplary embodiments of the present invention, the control device 930 is programmed using a schedule and reverses the flow direction of the pump 932 to remove the culture medium from the containers 920A, 920B, and 920C through the conduit 922. In some embodiments, the removed culture medium is pumped into a waste container. Although only one set of conduits 922 is shown for simplicity, in some embodiments, separate conduits 922 are provided for waste removal and introduction of fresh culture medium.

[0166] In the illustrated embodiment, the system 900 includes a compression mechanism 970 operable by the control device 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 present 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 the tubing 923 mates with a sterile connector 925. In some embodiments, an ASEPTQUICK connector is used as 925. According to various exemplary embodiments of the present invention, the tubing 923 is used to introduce / remove the culture medium from the flask 920 and / or introduce a humidified mixture of air and CO2. Alternatively or additionally, in some embodiments, the tubing 923 is connected to a pump 932 (FIG. 9A). In other exemplary embodiments of the present invention, a roller bottle is used instead of the flask 920.

[0168] Exemplary pump-based culture medium exchange system FIG. 10 is a simplified schematic diagram of a culture medium exchange system for cell culture according to an exemplary embodiment of the present invention, generally shown as 1000.

[0169] The illustrated exemplary system 1000 includes a cell culture container 1010 having at least one port 1012, and a detector 1020 that measures parameters of the culture medium 999 in the container 1010 and generates an indicator signal 1022. In the illustrated embodiment, the system 1000 includes a medium exchange mechanism including a control device 1030 configured to respond to a threshold of the indicator signal 1022 by operating a pump 1040 that removes the consumed medium via at least one port 1012 and introduces fresh medium 1042 via at least one port 1012. According to various exemplary embodiments of the present invention, the parameters are selected from the group consisting of pH, CO2 concentration, glucose concentration, lactate concentration, and floating cells (number and / or ratio).

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

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

[0172] Exemplary humidified CO2 supply system FIG. 11 is a simplified schematic view of a humidified CO2 supply system for cell culture according to an exemplary embodiment of the present invention, generally designated 1100. The illustrated exemplary system 1100 includes a closed cell culture container 1110 having at least one gas port 1112. In the illustrated embodiment, the container 1110 is partially filled with a medium 999. In the illustrated embodiment, the system 1100 includes a CO2 tank 1120 connected to at least one gas port 1112 via a regulator 1122 and a water storage container 1130 partially filled with water 1132 that passes the CO2 of the tank 1120 between the regulator 1122 and at least one gas port 1112. According to the illustrated embodiment, the gas is humidified when the gas from the regulator 1122 bubbles through the water 1132 in the storage container 1130. In some exemplary embodiments of the present invention, the closed cell culture container 1110 is the graft culture container 100 described above herein.

[0173] Exemplary Inclination-Based Medium Exchange System FIG. 12A is a simplified schematic view of an inclination-based medium exchange system for graft culture according to an exemplary embodiment of the present invention, generally designated 1200. FIG. 12B is a simplified illustrative flow diagram of an inclination-based medium exchange system for graft culture according to an exemplary embodiment of the present invention, generally designated 1201. The illustrated exemplary system 1200 includes a support surface 1210 for a plurality of tissue culture containers 100, an inclination mechanism 1220 that controls the angle of the support surface 1210, and a control device 1230 configured to operate the mechanism 1220 to provide a controlled removal of the 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 above herein.

[0175] In some exemplary embodiments of the present invention, the control device 1230 is configured to operate the tilting mechanism 1220 to +18° and then to -30° to discharge the culture medium into the waste container, as illustrated in FIG. 12B. In FIG. 12B, the (+) or (-) of the angle is defined by the direction of the container 100. When the ascending section 441 ascends, the angle is (-). When the ascending section 441 descends, the angle is (+). Alternatively or additionally, in some embodiments, the control device 1230 is configured to operate the tilting mechanism 1220 to -5° to facilitate sampling, as illustrated in FIG. 12B.

[0176] Alternatively or additionally, in some embodiments, the control device 1230 is configured to operate the tilting mechanism 1220 to +18° to remove the culture medium from the upper section of the container 100 to the ascending section 441. The tilting mechanism then returns to the initial position of 0° and the angle becomes -30°. Considering the design of the container 100, the culture medium exceeding the standard culture medium level of 0° automatically flows into the garbage bag. Thus, the culture medium does not re-enter the upper section. At a tilting angle of 30°, all the culture medium exits the container 100. In a specific configuration of the container 100, the culture medium from the upper section is always removed via the lower section.

[0177] FIG. 12C is a simplified illustrative flow diagram of a cell culture system according to an exemplary embodiment of the present invention (the icons are the same as those in FIGS. 8J, 8J2, and 8J3). The process illustrated in FIG. 12C is similar to that described in relation to FIG. 8J3.

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

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

[0180] The illustrated exemplary system 1300 includes a plurality of graft culture containers 100, each container having a plunger 431 movable within its lid 400 (see FIGS. 4A and 4B and the foregoing description in the present specification). In the illustrated embodiment, the system 1300 includes a control device 1320 configured to operate at least one piston 1310 and a vertical displacement mechanism 1330 to lower and raise the piston 1310 to push down and release each of the plungers 431 in the lid 400 of the container 100.

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

[0182] In some exemplary embodiments of the present invention, all of the containers 100 are arranged in a row, and the same number of pistons 1310 are provided in a matching row. In this configuration, no horizontal displacement is required, or otherwise the row of pistons 1310 is aligned with the row of containers 100 by a single horizontal displacement. In either case, when the vertical displacement mechanism 1330 lowers the piston 1310, all of the plungers 431 are pushed down simultaneously. When the vertical displacement mechanism 1330 raises the piston 1310, all of the plungers 431 are released simultaneously. This is a single-cycle parallel operation.

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

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

[0185] Regardless of the operating mode, when the pistons 1310 push down the plungers 431, they compress the hydrogel matrix in the containers 100. In some exemplary embodiments of the present invention, the vertical displacement mechanism 1330 is adjustable to different forces and compression patterns (e.g., linear, stepped).

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

[0187] In other exemplary embodiments of the present invention, the pistons 1310 continuously increase the force until the final force is reached and maintained. For example, in some embodiments, the pistons 1310 apply a pressure that continuously increases at a rate of 4 Pa per second until they reach 1517 Pa after 15 minutes and are finally maintained at 1517 Pa for 5 minutes.

[0188] Alternatively or additionally, in some embodiments, the control device 1320 is programmable (e.g., a 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, 200 in the container 100 moves by compression (see FIGS. 1A, 2A, 2B and the corresponding text). When the desired compression stage is reached, 230 and 232 are in positions visually indicated within the container 100. This position is detected by a camera (e.g., 1340 and / or 880 of FIG. 8A and / or 980 of FIG. 9), and the camera provides an output to the control device 1320. The control device 1320 translates the camera output into a stop signal. In the current embodiment, the camera is aligned with the container 100, located on the compression system for use in visual inspection, and not involved in the compression itself.

[0189] FIG. 13B illustrates an exemplary compression module 1301 inserted into the environmental chamber (e.g., 860 of FIG. 8B) from above. In some embodiments of the present invention, the environmental chamber is fixed therein as part of the device. In the illustrated design, four pistons 1311 are arranged in a row in four control devices 1321 with a vertical displacement mechanism 1331 for compressing four hydrogels. The control device 1321 generates a constant force by using the vertical displacement mechanism 1331 to lower the piston 1311. The pressure sensor 1341 monitors the increasing pressure. This pressure moves via the spring 1350 to the pressure plate 1352 of the piston head 1354. In some embodiments, the piston head 1354 first couples to the implant culture container 100 and then to the piston 1311. In some exemplary embodiments of the present invention, the spring 1350 is a removable component and is not fixed to the piston 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, the piston head 1354 is placed on the container 100. The pin 1360 fits into a corresponding connection point on the container 100. In the illustrated embodiment, the pressure generated by 1321 / 1331 moves through the spring 1350 to the pressure plate 1352, and from there it evenly moves to the plunger 431 of the cover 430 of the container 100 via four pins 1360 (see FIG. 13A).

[0191] In the illustrated embodiment, the camera 1343 is fixed in place by the camera holder 1344. The camera can capture a photograph of the 3D tissue culture (not a microscopic photograph).

[0192] Exemplary remote control system FIG. 14 is a schematic diagram of an explant culture system adapted for remote operation according to an exemplary embodiment of the present invention, generally shown as 1400.

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

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

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

[0196] Alternatively, or additionally, in some embodiments, the media exchange mechanism 1450 includes an inclination mechanism 1456 that controls the angle of the support surface 1458 that holds the tissue culture container 1410. The use of an inclination mechanism such as 1450 is described hereinabove in connection with FIGS. 12A and 12B.

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

[0198] Exemplary environmental control system FIG. 15 is a simplified schematic flow diagram of an environmental control system for cell culture according to an exemplary embodiment of the present invention. FIG. 15 shows how a tubing manifold is utilized to deliver air of a desired temperature / CO2 content and humidity to a plurality of graft boxes (tissue culture containers 100 in other drawings).

[0199] Exemplary usage scenario Most of the above description relates to dermal fibroblasts and epidermal keratinocytes, but in additional embodiments of the invention other cell types isolated from human or animal tissues of different origins are utilized. Fibroblast cell lines (human or animal) are always derived from sources including, but not limited to, dermis, tendon, lung, umbilical cord, cartilage, urethra, corneal stroma, oral mucosa, and intestine. Human / animal includes, but is not limited to, fibroblasts, smooth muscle cells, chondrocytes and other connective tissue cells of mesenchymal origin. In some exemplary embodiments of the invention, fibroblasts are isolated by microdissection from the dermal papilla of hair follicles. In some exemplary embodiments of the invention, corneal constructs are manufactured using matrix-producing cells derived from corneal stroma. In some exemplary embodiments of the invention, the cell donor varies by development and age. Alternatively or additionally, in some embodiments, the cells are derived from donor tissue of other individuals including embryos, neonates, or adults. In some exemplary embodiments of the invention, embryonic progenitor cells such as mesenchymal stem cells are used in embodiments of the invention and are induced to differentiate to develop into the desired tissue using known differentiation factors and / or differentiation conditions.

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

[0201] Human cells are used in many embodiments of the invention, but animal cells are also used in many embodiments. Cells of other mammalian species are used including, but not limited to, horse, dog, pig, cow, sheep sources and rodent species (e.g. mouse and / or rat and / or rabbit). Additionally, cells that are spontaneously, chemically or virally transfected or recombinant cells or genetically modified cells are used in some embodiments of the invention. In these embodiments incorporating more than one cell type, chimeric mixtures of normal cells from two or more sources, mixtures of normal and genetically recombinant or transfected cells, or mixtures of cells from two or more species or tissue sources are used in different embodiments of the invention.

[0202] During the term of this patent, many cell culture media, membrane types, and polymers are expected to be developed, and the scope of the present invention is intended to include all such new technologies in advance.

[0203] Although the present invention has been described with its specific embodiments, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

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

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

[0206] It should be further understood that additional embodiments of the present invention can be created by combining all the individual features described herein in all possible combinations and sub-combinations. The above-described examples are illustrative in nature and are not intended to limit the scope of the present invention, which is defined only by the following claims.

[0207] Each description of an embodiment of the present invention that includes a particular feature, part, component, module, or process is an explicit statement that there are additional embodiments of the present 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 specific features, parts, components, modules, processes, or elements 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 for the production of other tissue types of implants according to production in compressed hydrogels and / or for the production of tissues for uses other than transplantation.

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

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

Claims

1. (a) a graft support tray, (b) a box having a lid and a base, the box engaging and holding the tray, A tissue culture container comprising: The tray has a first operating state in which the floor of the tray is slightly higher than the floor of the base, and a second operating state in which the floor of the tray is lowered to contact the floor of the base, The graft support tray comprises (a) a rigid frame, (b) a liquid-permeable membrane floor attached to the lower edge of the frame, (c) a set of first vertically extending springs attached to the outside of two opposite sides of the frame. A tissue culture container.

2. Comprising a movable plunger attached to the lid, The tissue culture container according to claim 1.

3. Comprising one or more raised compartments, The tissue culture container according to claim 1.

4. Comprising a second set of vertically extending springs attached in a direction opposite to the first springs, The tissue culture container according to claim 1.

5. The base comprises a compression structure designed and configured to support the membrane floor of the graft support frame that has been fully lowered into the base, the compression structure being configured to allow the flow of the culture medium to the outside when the frame is lowered. The tissue culture container according to claim 1.

6. The base is dimensioned to accommodate a set of springs of the graft support tray located in the base and has recesses positioned therein. The tissue culture container according to claim 1. **Claim 7**: The base is dimensioned to accommodate two sets of springs of the graft support tray located in the base and has recesses positioned therein. The tissue culture vessel according to claim 1. **Claim 8**: The base is dimensioned to engage corresponding holes or pins provided on the graft support tray located in the base and has pins or holes positioned therein. The tissue culture vessel according to claim 1. **Claim 9**: The base comprises an O-ring or other gasket or overmolded elastomer. The tissue culture vessel according to claim 1. **Claim 10**: The base comprises at least one liquid removal port. The tissue culture vessel according to claim 1. **Claim 11**: The base comprises a snap-fit connector for attachment to the lid. The tissue culture vessel according to claim 1. **Claim 12** (a) A graft support tray, and (b) A box having a lid and a base, the box engaging and holding the tray, comprising The tray has two operating states: a first operating state in which the floor of the tray is slightly higher with respect to the floor of the base, and a second operating state in which the floor of the tray is lowered to contact the floor of the base. The lid (a) has a rigid frame, and (b) a flexible bellows disposed inside the frame, the bellows holding a plunger in a fixed direction with respect to the frame. The lid comprises one or more raised sections extending on the plane of the upper edge of the frame. Tissue culture vessel. **Claim 13**: (a) A graft support tray, and (b) A box having a lid and a base, the box engaging and holding with the tray, a box, comprising, The tray is, A first operating state in which the floor of the tray is slightly higher than the floor of the base, and A second operating state in which the floor of the tray descends and contacts the floor of the base, having two operating states, The lid is, (a) A rigid frame, (b) A flexible bellows disposed inside the frame, the bellows holding a plunger in a fixed direction with respect to the frame, the flexible bellows, comprising, The lid is dimensioned and positioned with a fastening hook on the rigid frame to engage with the base covered by the lid, The lid comprises one or more external spiked connectors in fluid communication with an internal outlet port, Tissue culture vessel.

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