Cell culture microdevices

Abstract

A cell culture microdevice for maintaining and culturing cells therein, comprising: a cell culture unit having at least one first cell carrier unit defining a cell culture chamber formed therein, the first cell carrier unit being formed from at least a chamber base shaped to support cells thereon and one or more chamber walls having one or more chamber wall surfaces surrounding the cell culture chamber around the chamber boundary, the first cell carrier unit further providing a guide surface positioned at an aperture through the chamber wall for guiding an instrument or fluid into the cell culture chamber, the cell culture microdevice being configured at a scale to substantially enclose a single cell or a cluster of cells therein. Embodiments of the cell culture microdevice may be suitable for in vitro fertilization procedures and drug efficacy testing.

Description

Technical Field

[0001] The technical field relates, in a broad sense, to the technological progress of microdevices for the culture of cells, particularly mammalian cells. The microdevices of the present invention are, in a broad sense, in the form of cell carrier devices and microdevices for cell culture. However, the carrier devices are equally applicable to cell culture, stem cell differentiation, cell array assays, infertility treatment, and specifically, procedures by in vitro fertilization (IVF). Specific embodiments relate to microdevices comprising a perfusion device with a cell culture unit, a cartridge, an array, and a cell carrier unit and a cell cover unit.

Background Art

[0002] Since tissue culture microdevices are widely available, microscale tissue culture techniques are highly sophisticated and their fields of use and application fields are becoming more diverse. Microdevices enable the manipulation of cells and their culture environment, giving rise to new therapies, products, and processes, many of which are still in their initial stages. Improvements in new microdevices and existing microdevices such as microfluidic devices, "culture on a chip" technology, micro scaffolds, and micro manipulation devices have led to new approaches to 3D tissue engineering, stem cell differentiation, and reproductive medicine, and have also brought about an epochal improvement in the success rate of these technologies.

[0003] For example, while complex in vitro tissue cultures were previously only practically applicable through limited gas exchange via diffusion, the development of microcapillary perfusion devices has enabled the culture and proliferation of larger and more complex tissues. Regarding stem cell culture technology (and its applications), organ culture systems using various embryonic tissues now allow for the culture of embryonic brain, retina, limb buds, lungs, kidneys, salivary glands, hair follicles, and dental cell lines. The application of novel tissue engineering techniques, and the microdevices that enable these techniques, will facilitate the provision of novel and improved approaches to many different therapies.

[0004] Improvements in microdevices, leading to enhanced cell culture and proliferation, have the potential to bring significant benefits to the field of reproductive medicine. A key characteristic of this field is that, due to the need to collect, grow, and reintroduce the patient's own cells, there are virtually no alternatives to in vitro culture and manipulation of the patient's own cells.

[0005] Assisted reproductive technology (IVF) has become more accessible and has recently improved, leading to an increase in the number of patients who can access it. Data published by the Human Fertilisation and Embryology Authority indicates that, overall, women who initiate IVF treatment are more likely to have children than before. 1 However, there is significant variation in the success rates of IVF treatment among individual clinics, with some achieving success rates as high as 46%, while others achieve rates as low as 10%. 1 .

[0006] The likelihood of success can vary at several stages of the IVF process, including during egg retrieval, fertilization, early embryo development, embryo vitrification, and embryo transfer. The success of egg selection, fertilization, embryo development, embryo cryopreservation, and preparation for embryo transfer currently depend heavily on the skill of the embryologist.

[0007] The success of IVF pregnancy during embryonic development is largely determined by the physical and environmental conditions present during its proliferation and development. Any kind of abrupt environmental change, physical shock, or physical stress that can occur to sperm, eggs, or developing embryos can reduce the viability of the embryo and, consequently, decrease the success rate of pregnancy after transfer.

[0008] Certain physical and environmental conditions in a cell culture system are likely to affect the viability and proliferation of cells in vitro. For example, the choice of scraping (manual / physical), trypsinization (chemical / enzymatic), and sonication (physical / gentle) for detaching adherent cells in preparation for in vivo culture significantly impacts cell viability and is highly dependent on the cell type used as well as the buffers and other culture medium components.

[0009] In tissue engineering, the construction of functional three-dimensional tissue structures between morphogenesis and organogenesis depends on intercellular interactions between tissue monolayers. These intercellular interactions are triggered and maintained by physical and environmental cues within the culture system, including the application of mechanical forces, cell shape, extracellular matrix shape or other properties, as well as physical intercellular contact and other morphogenetic factors. Similarly, stem cell differentiation is also influenced by many factors within the cell culture matrix, including the depletion or accumulation of cell-specific growth factors, enzymes, and other proteins, or their components or byproducts.

[0010] In embryogenesis, the most significant shock or stress to developing embryos occurs through handling and physical manipulation, and is influenced by the embryologist's experience, training, and fatigue. Several events during embryo proliferation and preparation for IVF procedures involve physical intervention by the embryologist and therefore pose a risk to the success of embryogenesis and its potential for implantation. The process of collecting sperm and oocytes and introducing them into the ex vivo environment poses a risk of stress or shock to sperm and oocytes from both physical damage and harmful environmental impacts during in silico placement, as well as biochemical stress from placement into different fluid environments.

[0011] The fertilization process carries the risk of damage due to the physical manipulation required to introduce sperm (whether this is intracellular or intracellular). Optimizing the environment for embryo culture and removing developed embryos for cryopreservation or transfer increases the risk of physical damage. Physical transfer of embryos creates an opportunity for physical damage or injury to the embryo. The risk of injury or shock resulting from manipulation and handling, and therefore the success or failure of the IVF procedure, is greatly influenced by the skill and attentiveness of the embryologist, as well as the precision of the tools and equipment available to the embryologist, and the laboratory environmental conditions established under the influence of the embryologist (e.g., sterility, temperature, control of any form of contaminants such as volatile organic compounds, and parental identification control systems to prevent incorrect or mixed parentage).

[0012] Precision tools and instruments reduce the likelihood of embryologists accidentally damaging or shocking sperm, eggs, or embryos, or causing incorrect parentage. Furthermore, tools or instruments that reduce or eliminate physical intervention or manipulation by embryologists reduce the likelihood of events occurring that could cause physical shock to sperm, eggs, or embryos. In addition, tools or instruments that reduce variability in an optimized ex vivo environment also reduce biochemical stress on eggs or embryos.

[0013] While there have been technological advancements in microinjection and visualization devices used in IVF therapy, there has been little progress in the development of microdevices for processing the cells involved, or in technologies for manipulating them or reducing overhandling or manipulation.

[0014] Typically, developing embryos are cultured in large volumes of liquid culture medium, minimizing the physical effects resulting from the depletion of essential culture medium components utilized by the cells, as well as the physical effects resulting from increased concentrations of waste products in the culture medium. However, even a slight increase in waste products or a slight decrease in nutrients can have a considerable impact on developing cells. These physical effects from changes in the composition of the culture medium during growth are exacerbated by changes in the metabolic needs of the developing embryo.

[0015] These challenges are similar to those experienced in the development of organoids or complex tissues in vitro. As cells develop into more complex tissues, or rather tissue structures, their metabolic needs change, imposing more complex requirements on in vitro tissue culture systems.

[0016] It is not easy to account for the physical effects of developmental changes within a given volume of culture medium. In embryo culture, the volume of culture medium must remain small enough to allow the embryo to be repositioned by the embryologist, making it difficult to dilute sufficiently to minimize the effects of changes in the medium. Furthermore, as the volume of medium increases, the known substances produced by the embryo to aid its own growth become diluted, or the embryo becomes more difficult to position, increasing the likelihood of excessive handling or manipulation.

[0017] A common approach adopted by embryologists to address this problem is to prepare a series of culture media and circulate developing embryos through each medium throughout the in vitro developmental period. The advantage of this approach is that each culture medium, fertilization medium, or cryopreservation medium can be specifically adapted to the needs of cells at a particular stage of development or manipulation. However, even if the medium components are optimized to meet the requirements of the embryo's growth stage, the stress that cells experience when moving from one liquid medium to another still negatively impacts development.

[0018] To overcome this problem, various microfluidic and liquid or gas perfusion devices have been developed, to varying degrees of success. Microfluidic "culture on a chip" microdevices are being developed to prevent shock to cells from over-handling by using microfluidics to move or "roll" cells or cell clusters from one cell culture "bath" to the next. Other microfluidic devices are adapted to allow the continuous inflow and outflow of liquid media into traditional cell culture dish systems throughout the developmental period of fertilized eggs to the embryonic stage, for cryopreservation and preparation for embryo transfer. These developed technologies are not widely adopted, and many embryologists tend to prefer static culture techniques using petri dishes or modified petri dishes, due to the convenience of positioning and retrieving embryos in these containers.

[0019] None of these approaches can address the needs for optimal gas and fluid exchange. Proper ventilation by gas diffusion is crucial for development in all tissues, particularly during morphogenesis and stem cell differentiation.

[0020] In recent years, attempts have emerged to apply microfluidic technology to static culture of embryos. 2Researchers are finding and optimizing techniques to control the retention of minute amounts of culture medium within the tiny channels of microfluidic devices, while preventing excessive movement of oocytes and developing embryos to avoid damage or stress. However, to date, there have been no attempts to combine perfusion within microfluidic devices with clinical applications to any extent.

[0021] Downscaling engineering systems from macroscale to microscale broadens the spectrum of novel applications, a principle established through the work of Richard Feynman. However, this presents physical challenges such as evaporation, lubrication, heating, and inertia, which behave very differently at such small scales. Engineering designs adapted to this scale have not yet been adequately implemented in in vitro cell cultures. This disclosure addresses these challenges, at least in part, through design innovation.

[0022] The use of nanoscale and microscale devices offers opportunities unavailable with macroscale devices, as established in Kim Eric Drexler's research. Macroscale cell devices tend to rely on chaotic interactions between many cells, which are carried out in the hope that one or more of these interactions will produce the intended outcome within a predefined acceptable margin. Microscale devices, on the other hand, use individual cells to interact directly, enabling the production of the intended outcome more accurately and efficiently. Subsequent interactions are based on a much closer-to-ideal target, resulting in more precise outcomes.

[0023] Maintaining the physical stability of sperm, eggs, and embryos within the culture device allows embryologists to handle or manipulate sperm, eggs, or embryos more easily and / or accurately, minimizing the risk of physical shock and / or biochemical stress, or further preventing the risk of mixed parentage during fertilization, embryo culture, or embryo transfer. Stabilizing cells during culture can improve the success of IVF procedures.

[0024] This approach not only overcomes the difficulties in successfully fertilizing and culturing developing embryos, but can also be applied to culturing other cell lines that are sensitive to changes in the culture environment during gradient processes. [Overview of the Initiative]

[0025] In one aspect of the present invention, embodiments of the present disclosure relate to a cell culture microdevice for maintaining and culturing cells internally, comprising a cell culture unit having at least one first cell carrier unit defining a cell culture chamber formed internally, wherein the first cell carrier unit is formed from at least a chamber base molded to support cells on top, and one or more chamber walls having one or more chamber wall surfaces surrounding the cell culture chamber around the chamber boundary, and the first cell carrier unit further provides an guiding surface positioned in an aperture passing through the chamber wall for guiding instruments or fluids into the cell culture chamber, and the cell culture microdevice is configured on a scale substantially enclosing a single cell or cell aggregate internally.

[0026] The term “cell” as used herein should be understood to be interchangeable with the term “cellular material,” and refers to cells, cell groups, tissues, or organoids that are the subject matter of the present invention as described herein.

[0027] As used herein, the term "cell culture" shall refer to any tool or process by which cell material is isolated and maintained under controlled conditions for testing, growth, observation, experimentation, recovery of culture medium, or other bioscience processes.

[0028] As used herein, the term "microdevice" shall refer to a manufactured article produced, for example, on the micron scale of 0.1 μm to 1000 μm. Microdevices shall include both static and mechanical devices, as well as devices in the area of microfluidics.

[0029] As used herein, the term "maintaining cells" shall refer to any process by which cell material is held in a controlled environment that generates the conditions necessary for survival. The term "culturing cells" shall refer to the process of cell culture.

[0030] As used herein, the term "cryopreservation" shall refer to vitrification or freezing in an exchangeable manner.

[0031] As used herein, the term "boundary" is used to refer to a three-dimensional boundary between the inside and the outside of a cell chamber unit, which can be defined by walls, surfaces, openings, and apertures.

[0032] In a preferred embodiment, the guiding surface defines a curved bottom trench through which the instrument passes and is introduced into the cell culture chamber through an aperture. Alternatively, fluid can be guided by and through the curved bottom trench through an aperture between the inside and the outside of the cell culture chamber.

[0033] In an alternative embodiment, the guiding surface defines a narrow aperture through which the instrument passes and is introduced into the cell culture chamber via the aperture. Alternatively, a fluid may be guided and pass through a narrow aperture between the inside and outside of the cell culture chamber.

[0034] In a preferred embodiment, the cell chamber base may be substantially concave. Alternatively, the cell chamber base may be molded to be substantially convex, stepped, recessed, or any other shape that can be used to support cellular material within the cell culture chamber.

[0035] The cell carrier unit according to an embodiment of the present invention is configured to be formed on a microscale. Therefore, it is preferably formed from a material that is nontoxic to cells and suitable for microscale manufacturing.

[0036] In a particular embodiment, one or more chamber walls may comprise one or more inner wall surfaces inclined toward the proximal point of the chamber, and as a result, they are configured to guide the placement of instruments or cells within the culture chamber.

[0037] In a preferred embodiment, one or more chamber walls of the embodiment of the present invention include a proximal wall having a curved inner wall surface configured to guide the placement of instruments within the culture chamber.

[0038] In certain embodiments, the proximal wall may be stepped, recessed, or otherwise configured to guide the placement of instruments within the culture chamber.

[0039] In a preferred embodiment, the cell culture chamber according to the present invention is open from above, and the chamber base has a curved inner surface.

[0040] In embodiments of the present invention, the boundary is preferably substantially box-shaped, having an upper opening and a curved proximal wall opposite the induction aperture, thereby providing orientation and stability to the cellular material during handling. The cell culture chamber preferably includes fluid exchange apertures on both the surface of the curved proximal wall and the surface of the culture base. Alternatively, the footprint of the cell culture chamber may be substantially triangular or V-shaped at the footprint site and inclined toward the proximal point.

[0041] In alternative embodiments, the boundary in the aspects of the present invention may be substantially cylindrical, spherical, triangular, asymmetrical, or stepped.

[0042] In a preferred embodiment, one or more chamber walls of an aspect of the present invention comprises a proximal wall having a curved inner wall surface configured to guide the placement of an instrument into the culture chamber, and a distal wall having an aperture formed to define the distal chamber boundary and pass through the chamber wall, wherein the aperture defines an opening by communicating with an elongated guiding portion having an internally formed channel that projects outward from the cell culture chamber and provides a guiding surface for guiding an instrument or fluid into the cell culture chamber.

[0043] Preferably, one or more chamber walls in embodiments of the present invention comprise a curved proximal wall and an intermediate-distal wall opposite it. The intermediate-distal wall comprises an aperture and provides an guiding surface that defines a conduit formed perpendicular to the intermediate-distal wall outside the cell culture chamber.

[0044] Preferably, the inner surface of the intermediate-distal wall is substantially concave to guide the instrument or fluid from the chamber toward the guide surface. Alternatively, the inner surface of the intermediate-distal wall may be flat.

[0045] In a preferred embodiment, one or more chamber walls of the embodiment of the present invention comprises at least a left wall and a right wall, each having a left aperture and a right aperture formed to pass through it.

[0046] In a preferred embodiment, the proximal wall of the embodiment of the present invention has a proximal aperture formed to pass through it, which is configured to be aligned horizontally with a guide surface to mitigate the flow of fluid through the cell culture chamber between the aperture and the proximal aperture.

[0047] In a preferred embodiment, the aperture of the present invention is configured for perfusion through which it passes. Preferably, the aperture is positioned to pass through the chamber base and is sized so that cells cannot pass through it. Alternatively, the aperture is positioned to pass through one chamber wall or consists of several apertures distributed across one or more surfaces of the boundary.

[0048] In a preferred embodiment, the proximal wall of the present invention comprises a perfusion inlet opening adapted to a fluid perfusion flow passing through it, and a tube fitting configured to engage a perfusion tube with the perfusion inlet opening.

[0049] Alternatively, the tube fitting may be configured to engage with a perfusion manifold or other means of supplying perfusion medium.

[0050] The fluid exchange aperture may, alternatively, be positioned on other surfaces, integrated into other apertures and openings, or, in some cases, may not be necessary for the maintenance and culture of cellular material.

[0051] In a preferred embodiment, a first cell carrier unit of an aspect of the present invention comprises a cell chamber wall having an outer wall coupling adapted to engage with a corresponding outer wall coupling on at least one second cell carrier unit, thereby forming a cell carrier array. Preferably, the cell carrier array may comprise an unlimited number of cell carrier units, each adapted to engage with another. Preferably, the cell carrier array is a linear array in a horizontal plane, but may alternatively be stacked in a vertical plane, or a mixture of both. Preferably, the cell carrier units may clip together in a horizontal plane, stack in a vertical plane, or alternatively, be slidably engaged together in either a horizontal or vertical plane.

[0052] In certain embodiments, a cell culture microdevice according to an aspect of the present invention comprises at least one second cell carrier unit integrally formed with a first cell carrier unit, thereby forming a cell carrier cartridge. Preferably, the cell carrier cartridge may comprise an unlimited number of cell carrier units, each integrally connected to another. Preferably, the cell carrier cartridge is linear in the horizontal plane, but alternatively, cartridges may be formed in the vertical plane, or a mixture of both. In preferred embodiments, cell carrier cartridges may be stacked with other cell carrier cartridges or engaged with other cell carrier cartridges to define a cell carrier cartridge array.

[0053] In alternative embodiments, the cell culture array and cell culture cartridge may be formed as a circular array or cartridge, or in another form that allows the array or cartridge to be adapted for further processing.

[0054] In a preferred embodiment, the cell culture microdevice according to an aspect of the present invention further comprises a first cell cover unit having a first cover wall configured to cover at least a portion of the opening of the cell culture chamber from above when the first cell cover unit and the first cell carrier unit are connected to form a cell culture unit base.

[0055] In a preferred embodiment, the cell cover unit substantially surrounds the cell carrier unit with three surfaces, including the top surface, and is molded to completely cover the top opening when positioned on the top surface.

[0056] In an alternative embodiment, the cell cover unit may be molded to substantially cover at least one surface of the cell carrier unit.

[0057] Preferably, the cell cover unit has at least one edge configured to terminate at a portion that can engage with the cell carrier unit of an embodiment of the present invention. Preferably, the portion that can engage with the cell carrier unit is fitted to engage with the base of the cell carrier unit, thereby forming a cell culture base. In a more preferred embodiment, the cell cover unit is configured to slidably engage with the base of the cell carrier unit.

[0058] In a preferred embodiment, a first cell cover unit of an aspect of the present invention comprises an outer wall coupling adapted to engage with a corresponding outer wall coupling on at least one second cell cover unit, thereby forming a cell cover array. Preferably, the cell cover array may comprise an unlimited number of cell cover units, each cell cover unit adapted to engage with another. Preferably, the cell cover array is a linear array in a horizontal plane, but may alternatively be stacked in a vertical plane, or a mixture of both. Preferably, the cell cover units may clip together in a horizontal plane and stack in a vertical plane, or alternatively be slidably engaged together in either a horizontal or vertical plane.

[0059] In a preferred embodiment, the cell culture microdevice according to an aspect of the present invention further comprises at least one second cell cover unit integrally formed with a first cell cover unit, thereby forming a cell cover cartridge. Preferably, the cell cover cartridge may comprise an unlimited number of cell cover units, each integrally connected to another. Preferably, the cell cover cartridge is linear in the horizontal plane, but alternatively, cartridges may be formed in the vertical plane, or a mixture of both. In a preferred embodiment, cell cover cartridges may be stacked with other cell cover cartridges or engaged with other cell cover cartridges to define a cell cover cartridge array.

[0060] In a preferred embodiment, the first cell cover unit and the first cell carrier unit according to an aspect of the present invention define a cell culture unit base. In a preferred embodiment, the cell culture unit base is defined by the first cell cover unit and the first cell carrier unit terminating on a flat bottom surface, providing stable support. Alternatively, the cell culture base may be defined by either the first cell carrier unit or the first cell cover unit in one configuration, and by the other in another configuration.

[0061] In a preferred configuration, the cell culture base is adapted to connect to another cell culture cartridge, cell carrier unit, or cell cover unit. The base is preferably configured to physically stabilize the cell carrier unit when placed on a surface or when connected to another component or device.

[0062] In a preferred embodiment, the first cell cover unit of an aspect of the present invention is configured to slidably engage with the first cell carrier unit. In a preferred embodiment, the first cell cover unit is molded to slidably engage with each of the two sides of the first cell carrier unit perpendicular to the proximal-distal axis.

[0063] In a particular embodiment, the first cell cover unit includes a culture inlet configured to engage with a tube fitting on the first cell carrier unit, allowing a perfusion tube to be attached to the tube fitting thereon. Alternatively, the culture inlet may be configured to engage with a perfusion manifold or other means of supplying perfusion medium.

[0064] In a preferred embodiment, the first cell cover unit further comprises an access aperture formed to pass through it, the access aperture formed to pass through the first cell cover unit configured to allow above access to the opening at a first position and to cover at least a portion of the opening from above at a second position, and is adapted to slidably engage with the first cell carrier unit from the first position to the second position.

[0065] In a preferred embodiment, the access aperture is the same size and shape as the upper opening so that the upper opening is fully accessible through the access aperture. In an alternative embodiment, the access aperture may be larger than the upper aperture and, when configured for access, may be angled to guide instruments or cells into the cell culture chamber.

[0066] In a particular embodiment, the outer surface of the chamber base according to the embodiment of the present invention is provided with notches configured to receive lugs protruding outward from a cell carrier unit or a cell cover unit.

[0067] A method of using a cell culture microdevice according to an aspect of the present invention includes the steps of: conveniently placing a single cell in the cell culture chamber of the cell culture microdevice; and culturing the cell.

[0068] A method of using a cell culture microdevice according to an aspect of the present invention includes the steps of obtaining instructions for constructing the cell culture microdevice and carrying out the instructions in an additive manufacturing process.

[0069] In a preferred embodiment of the present invention, the cell culture unit comprises at least four walls and a base defining a cell culture chamber internally. The at least four walls preferably comprise a proximal wall, a left wall, a right wall, and an intermediate-distal wall, each having a curved inner surface defining the cell culture chamber, the curvature of which provides a reference point for positioning a cell handling device. In a preferred embodiment, the intermediate-distal wall comprises an outer surface having an opening formed to pass through it, an inner surface defining the cell culture chamber, and a channel formed substantially perpendicular thereto. The channel is preferably formed by the inner surfaces of the left wall and the right wall, extending distally beyond the intermediate-distal wall and terminating substantially perpendicularly to an outer distal wall having an opening formed to pass through it.

[0070] The openings formed by the channels within the intermediate distal wall and the outer distal wall are preferably aligned to provide a line of sight from the distal end of the cell culture carrier to the cell culture chamber. This alignment preferably provides guidance for embryologists or other users to carefully introduce instruments such as micropipettes into the cell culture chamber to access the cells with minimal interference or destruction.

[0071] For example, an embryologist attempting to remove an embryo from a cell culture chamber for transplantation can introduce a micropipette through the outer distal wall opening, running and / or swaying the tip of the micropipette along the channel until it reaches the curved inner surface of the proximal wall. The curve of the proximal wall guides the micropipette to the center of the cell culture chamber, allowing the embryologist to gently aspirate or inject embryonic cells and culture medium directly beneath the micropipette. The shape of the cell culture carrier, which provides physical support for instruments such as micropipettes, and guidance on instrument positioning or placement, reduce the impact of operator error that could damage cells and hinder optimal cell growth. Therefore, the three-dimensional structure of the cell culture carrier optimizes culture techniques, optimizes cell viability, and reduces the impact of user error.

[0072] Preferably, the left and / or right walls have overflow openings formed to pass through them. Preferably, the proximal wall has an inlet opening defined by an inlet fitting positioned on the outer surface of the proximal wall. The inlet fitting may function as a connector for tubing or other instruments. It may be connected to tubing used to transfer liquid to the cell culture unit in order to perform perfusion culture. The inlet fitting may also be connected to tubing, etc., which is used simply as a reference point for the position of the cell culture unit or holder to maintain the cell culture unit in place.

[0073] In a further preferred configuration, the inlet opening, intermediate distal wall, channel, and outer distal wall are preferably aligned to provide lines of sight from the distal wall through the cell culture carrier and through the inlet opening. The line of sight alignment through the cell culture carrier is preferably adapted to allow cells to be cultured during perfusion of culture medium through the cell culture unit. Preferably, the base of the cell culture chamber is at least partially below the line of sight through the cell culture carrier. This configuration minimizes physical disturbance to the cells in culture from turbulence or flow caused by fluid perfusion.

[0074] Perfusion techniques can optimize conditions for the growth of specific cell types, particularly cells sensitive to biochemical changes in the culture medium that may result from nutrient depletion or increased waste products in the culture medium, or cells that may have varying biochemical requirements when cultured through different growth phases. Embryo culture for IVF procedures can benefit from perfusion culture, and similarly, in the culture of complex structures such as valve structures or organoids, skin, liver, kidney, lung, or other tissue grafts, or complex cell lines such as bone marrow or stem cells, or any cell line for patients prone to tissue rejection.

[0075] Herein, a wide range of embodiments of the present invention will be described with reference to the accompanying drawings, along with preferred embodiments disclosed in the Examples and Detailed Description. The present invention can be embodied in many different forms and should not be construed as being limited to the embodiments described herein. These embodiments are provided for illustrative purposes only, but by doing so, this disclosure will be thorough and complete, and the full scope and breadth of the present invention will be conveyed. [Brief explanation of the drawing]

[0076] [Figure 1] The image shows a top perspective view of a culture array or cartridge according to an embodiment of the present invention. [Figure 2] This shows a front view of a cell culture array or cartridge assembled according to an embodiment of the present invention. [Figure 3] This shows a rear view of a cell culture array or cartridge assembled according to an embodiment of the present invention. [Figure 4] This shows a bottom perspective view of a cell culture array or cartridge assembled according to an embodiment of the present invention. [Figure 5] This shows a bottom view of a cell culture array or cartridge assembled according to an embodiment of the present invention. [Figure 6] This shows a top perspective view of a cell culture array or cartridge before assembly according to an embodiment of the present invention. [Figure 7] This shows a rear view of a cell culture array or cartridge before assembly according to an embodiment of the present invention. [Figure 8] This shows a bottom perspective view of a cell culture array or cartridge before assembly according to an embodiment of the present invention. [Figure 9a] Figure 9a shows a top perspective view of the cell cradle portion of a cell culture unit carrier according to an embodiment of the present invention. [Figure 9b] Figure 9b shows the cell cradle portion of a cell culture unit carrier according to an embodiment of the present invention. [Figure 9c]Figure 9c shows the cell unit cover and cell culture array before assembly. Figure 9c shows a rear perspective view of the expandable cell unit cover. [Figure 9d] Figure 9d shows the cell unit cover and cell culture array before assembly. Figure 9d shows a rear perspective view of the expandable cell culture array. [Figure 9e] Figure 9e shows the cell unit cover and cell culture array before assembly. Figure 9e shows a bottom-back perspective view of the expandable cell culture array. [Figure 10a] Figure 10a shows a cell culture unit carrier according to an embodiment of the present invention. [Figure 10b] Figure 10b shows a cell culture unit carrier according to an embodiment of the present invention. [Figure 10c] Figure 10 shows a cell culture unit carrier according to an embodiment of the present invention. Figure 10c shows a top front perspective view of a cell culture unit carrier according to an alternative embodiment. [Figure 10d] A top front perspective view of a cell culture unit carrier is provided, showing three positions for engagement with a cell culture array. [Figure 10e] A top front perspective view of a cell culture unit carrier is provided, showing three positions for engagement with a cell culture array. [Figure 10f] A top front perspective view of a cell culture unit carrier is provided, showing three positions for engagement with a cell culture array. [Figure 10g] This shows a top perspective view of the cell culture unit carrier, illustrating the two engagement points for introducing cells into the unit. [Figure 10h] This shows a top perspective view of the cell culture unit carrier, illustrating the two engagement points for introducing cells into the unit. [Figure 11a] An embodiment of the present invention shows a cell culture array or cartridge in which a unit carrier is positioned inside. Figure 11a shows a rear view. [Figure 11b]An embodiment of the present invention shows a cell culture array or cartridge in which a unit carrier is positioned. Figure 11b shows a front angle view. [Figure 11c] An embodiment of the present invention shows a cell culture array or cartridge in which a unit carrier is positioned inside. Figure 11c shows a bottom perspective view. [Figure 12] A side view of a cell culture array or cartridge according to an embodiment of the present invention is shown. [Figure 13a] This shows a side view of cells in a cell culture array or cartridge according to an embodiment of the present invention, receiving a microinjection pipette. [Figure 13b] This shows a side view of cells in a cell culture array or cartridge according to an embodiment of the present invention, receiving a microinjection pipette. [Figure 13c] This shows a side view of cells in a cell culture array or cartridge according to an embodiment of the present invention, receiving a microinjection pipette. [Figure 14] This shows an atomic force microscope (AFM) mounting assembly for a cell culture microdevice according to an embodiment of the present invention. [Figure 15a] This document provides the results of toxicity tests on polymers used in 3D printing. Figure 15a shows the percentage of embryonic development in the presence of microdevices according to the present invention and their culture media. [Figure 15b] This document provides the results of toxicity tests on polymers used in 3D printing. Figure 15b shows the percentage of embryonic development in the presence of the microdevice according to the present invention. [Figure 15c] This document provides the results of toxicity tests on polymers used in 3D printing. Figure 15c shows the percentage of DNA repair. [Figure 16] A schematic diagram is provided illustrating tissue angiogenesis through the use of a cell culture unit according to the embodiment. [Figure 17a] The results of the cell culture optimization test are provided. Figures 17a-17c show embryonic development under static culture conditions at each developmental day up to day 5, depending on the type of culture medium and intervention. [Figure 17b] The results of the cell culture optimization test are provided. Figures 17a-17c show embryonic development under static culture conditions at each developmental day up to day 5, depending on the type of culture medium and intervention. [Figure 17c] The results of the cell culture optimization test are provided. Figures 17a-17c show embryonic development under static culture conditions at each developmental day up to day 5, depending on the type of culture medium and intervention. [Figure 17d] This report provides the results of a cell culture optimization test. It shows embryonic development under static culture conditions at each developmental day up to day 5, depending on the type of culture medium and intervention. [Figure 17e] The results of the cell culture optimization test are provided. Figure 17e shows the percentage of DNA repair within the same group. [Figure 17f] The results of the cell culture optimization test are provided. Figure 17f shows the internal cell mass of the same treatment group. [Figure 18a] The results of the oxygen optimization test are provided. Figures 18a-18d show the changes in embryonic development over time when exposed to various oxygen concentrations. [Figure 18b] The results of the oxygen optimization test are provided. Figures 18a-18d show the changes in embryonic development over time when exposed to various oxygen concentrations. [Figure 18c] The results of the oxygen optimization test are provided. Figures 18a-18d show the changes in embryonic development over time when exposed to various oxygen concentrations. [Figure 18d] The results of the oxygen optimization test are provided. Figures 18a-18d show the changes in embryonic development over time when exposed to various oxygen concentrations. [Figure 18e] This shows the percentage of DNA repair within the same group.

[0077] Some embodiments of the present invention are described in the following examples. [Modes for carrying out the invention]

[0078] The cell culture microdevices described in the following embodiments are broadly constructed from a single cell culture unit having a unit carrier and a unit cover, an array of repeating units engaged to form a cell culture array having an array carrier and an array cover, or a cartridge of repeating units integrated to form a cell culture cartridge having a cartridge carrier and a cartridge cover. When these take the form of a single unit, an array of repeating units of any shape or number, or a cartridge of repeating units of any shape or number, they are broadly referred to as "carrier" and "cover" below, respectively.

[0079] Those skilled in the art will understand the advantages of manufacturing the "carrier" and "cover" embodiments in a unit format, a format that can engage in an array, and a format that integrally includes multiple "carriers" or "covers." One of these formats may be referenced in each of the following embodiments, but it should be understood that other formats may be substituted under specific circumstances.

[0080] Example 1 - Cell Culture Array Figure 1 shows an assembled cell culture array 100 having an array cover 110 placed on an array carrier 120. Figure 1 shows a linear cell culture array having five repeating cell culture units 130a, 130b, 130c, 130d, and 130e arranged side by side. The linear array cover 110 is configured such that each unit cover forms a flat surface over the upper outer surface of the array cover 110 and is adjacent to the next unit cover with a slight groove between each unit cover. The cover is formed of four walls, including a rectangular planar top wall 140, which terminates at either end of a planar left end wall 150 (not shown) and a planar right end wall 160, which are formed to extend 90 degrees downward from the top wall 140. A series of annular openings 170 are formed to pass through the top wall 140 along the length of the top wall, and one opening is formed to pass through the top wall of each unit cover. The left end wall 150 (not shown) and the right end wall 160 terminate at their bottom edges, together with the left base flange 180 (not shown) and the right base flange 190.

[0081] The left and right base flanges may be configured to engage with other components such as robotic devices, culture dishes, additional cell culture units, or other experimental equipment, or they may simply be configured to provide stability when placed on the cell culture array 100, as shown in Figure 1.

[0082] In addition to the top wall, left end wall, and right end wall, the array cover 110 further comprises a front wall. Figure 2 illustrates the front wall 200 of the array cover 120. The rectangular, planar front wall 200 of the array cover 120 extends downward at approximately 90 degrees from the top wall 140 and extends between the front edge of the left end wall 150 and the front edge of the right end wall 160. The lower edge of the front wall 200 is horizontal with the lower edges of the left end wall 150 and the right end wall 160. A vertical groove 210 within the front wall 200 defines one unit cover from the next and extends through the array cover, forming a groove that is continuous within the top wall. A series of annular openings 220a, 220b, 220c, 220d, and 220e are formed to pass through the front wall 200 across the width of the top wall, with one opening passing through the front wall of each unit cover. Each annular opening is defined by an annular connector 230 protruding from the surface of the front wall. The annular connector can be configured to connect to any number of different devices, but most commonly it is a simple silicone tube connector that can create a fluid seal with a silicone tube.

[0083] Figure 3 shows the open rear surface of the array cover 100 with the array carriers 250 positioned inside. Vertical grooves 210 between each unit cover within the array cover extend through the array cover between the inner walls 260, completing the shape of each unit cover within the array cover. As shown in Figure 3, the inner walls 260 forming each unit cover are molded or formed to fill the space between adjacent unit carriers in order to minimize the gap between the unit carrier and the unit cover. As a result, the shape of the unit cover can guide the sliding position of the unit carrier and minimize the gap between each unit cover and the outer wall surface. By providing a repetition of this configuration when formed within an array, each unit carrier of the array carrier is ensured to slide easily into the desired position relative to the array cover and be securely positioned inside it.

[0084] The sliding mechanism 270 is provided at the lower edge of each unit cover wall, thereby reversibly and securely connecting one unit cover to the next unit cover to form an array cover. The sliding mechanism not only allows individual cell culture units to be assembled in array form, but also allows for easy separation from one another, enabling different cell cultures to be handled differently from other cells, while minimizing damage or interference (resulting from unnecessary handling) of the cells in culture.

[0085] Figures 4 and 5 show the base of the cell culture array, with Figure 4 being a perspective view and Figure 5 being a direct view. The figures show the configuration of the bottom surface of the array carrier, and the shapes of the inner walls and base flanges of the individual unit covers, namely the left base flange 180 and the right base flange 190. The sliding mechanism 270 extends through only a portion of the inner wall of the individual unit cover.

[0086] Figures 6, 7, and 8 show the array cover and array carrier separately, providing a top perspective view, a rear perspective view, and a bottom perspective view, respectively. Referring to Figure 6, the array carrier 120 is formed from a linear cell culture array carrier with five repeating unit carriers 280a, 280b, 280c, 280d, and 280e arranged side by side. Each unit carrier comprises a culture medium inlet 290 protruding from the outer front surface of the unit carrier, a cell cradle 300 formed within the unit carrier, and an induction channel 310 formed between the cell cradle and the outside of the unit carrier 280. The culture medium inlet 290 allows perfusion of liquid culture medium (or other fluid) into or through the cellular environment within the carrier. The fluid can also flow out of the cellular environment within the carrier through the induction channel 310 and out of the unit carrier through the channel outlet 320. Figure 7 shows the alignment of the aperture formed by the base of the channel outlet 320, the bottom of the induction channel 310, and the medium inlet 290. Figure 7 shows that a clear line of sight is formed through these openings, and therefore any turbulence caused during cell perfusion through the medium inlet 290 is unlikely to affect cell growth in the cell cradle 300.

[0087] Figures 6 and 7 show the relative size of the annular connector 230 of the array cover 110 to the culture medium inlet 290 of the array carrier 120. The outer surface of the culture medium inlet 290 is configured to be slightly smaller than the inner surface of the annular connector 230. Therefore, when the array cover slides onto the array carrier, the outer surface of the culture medium inlet 290 can fit quite closely to the inner surface of the annular connector 230. The fluid can then pass through the annular connector and the culture medium inlet without any fluid leakage through these contact points.

[0088] Figures 6 and 8 show the arrangement of the annular opening 170 through the array cover 110 relative to the array carrier 120. Referring to Figure 8, the sliding mechanism 270 is further illustrated as consisting of a runner 330 positioned between the individual unit covers of the array cover 110, terminating toward the rear of the array cover 110 in front of the gap 340 between the individual unit covers of the array cover. The runner 330 and gap 340 of the array cover 110 engage with a slider 350 on the unit carrier 120. When arranged in an overlapping manner, the slider 350 slides on the runner 330 and is guided by the space that allows the slider 350 to slide between the individual unit covers. When the slider 350 reaches the gap 340, the slide stops and it may be placed in a closed position therein. In the open position, the slider 350 is in contact with the runner 330. In this position, the annular opening 170 is positioned above the cell cradle 300 and is positioned correctly on the cell cradle 300 to optimally place oocytes or other cells in the center of the cradle from above. In the closed position, the slider 350 moves and stops within the gap 340, thereby positioning the annular opening 170 completely above the induction channel 310 and covering the cell cradle 300 from above. The cradle exit 360 is also illustrated in the array carrier 120 in Figure 8. This feature is optional but, if provided, allows for further access to the cell cradle and enables removal or movement of the embryo or its surrounding culture medium.

[0089] The figure shows a linear array configuration; however, as will be readily apparent to those skilled in the art, the array configuration can be extended beyond five units and can also be easily adapted to a non-linear array configuration. For example, a linear array configuration can be easily adapted to a circular array, which can be easily adjusted by automation or robotic processing techniques. For example, a circular array can be mounted in a carousel arranged in sequence within a microscope or other visualization device. The carousel arrangement can be easily accessed by an operator, handheld device, or robotic device for pipetting, introduction of a vacuum manifold, introduction of a pump system, or implementation of a cryopreservation process.

[0090] Example 2 - Cell Culture Unit Figures 9a and 9b illustrate the configuration of a cell cradle 300 of a single cell unit carrier in isolation of induction channel 310 (shown in Figure 6), and Figures 10a and 10b illustrate the internal configuration of a complete single cell culture unit 400. The cell unit carrier 400 is formed from five walls, including a rectangular planar left wall 410 and a rectangular planar right wall 420, each wall terminating towards the rear of the unit to connect at a 90-degree angle to a planar rear exit wall 430 and also terminating towards the front of the unit to connect at a 90-degree angle to a planar front inlet wall 440. The cell cradle shown in Figures 9a and 9b has a measured size of approximately 0.23 mm at its widest point and approximately 0.23 mm at its highest point.

[0091] An annular projection from the outer front surface of the inlet wall 420 forms a culture medium inlet 290 having an outer diameter approximately equal to (but slightly smaller than) the inner diameter of the annular connector 230 (Figure 6). The inner surface of the front inlet wall 440 is horizontally curved, allowing pipettes introduced into the cell cradle region through the channel outlet 320 and induction channel 310 to position the center point of the cell cradle region on the horizontal axis. The inner surfaces of the left wall 410, the right wall 420, and the rear outlet wall 430 are normally flat. The left wall 410 and the right wall 420 each have an overflow aperture 450 to allow overflow of culture medium from the cell cradle 300, particularly when used to perform cell perfusion. As shown in Figure 9b, the lowest point of the overflow aperture 450 is at the same height as the lowest point of the induction channel 310, ensuring that excess fluid preferentially moves away from the cell cradle region from the sides of the cell culture unit rather than passing through the induction channel 310.

[0092] Example 3 - Cell Cover Figure 9c shows a cell cover unit 600 configured for linear array assembly with other cell cover units and for housing a single cell cradle. The figure shows “puzzle piece” clip and locking systems 610a and 610b that enable each cell cover unit to engage with one another in a linear array of infinite expandability. Figure 9c also shows one embodiment of the cell cover unit, in which the cell cover unit does not feature an aperture for accessing the cell culture chamber, but rather relies on a sliding engagement inside the cell carrier unit to cover or expose at least a portion of the opening from above. This embodiment of the cell cover unit shows a notch 620 used to align with the inlet channel 470 of the cell cradle.

[0093] Figures 9d and 9e show cell cover units integrally positioned on a linear cell cover cartridge 700, which has the added capability of forming arrays in three dimensions. On the left and right sides of the cell cover cartridge are “puzzle piece” clip and lock systems 710a and 710b, which maintain orientation with subsequent units and allow arrays to be formed in these directions. Also illustrated on the front and rear of this system 720a and 720b are upper deformations in these systems, which also maintain orientation with subsequent units and allow arrays to be formed in these directions. On top of the cell cover cartridge are lugs 730a configured to receive into inverted notches 730b below the cell cover cartridge in order to allow stacking in that direction.

[0094] Figures 10a and 10b (Figures 9a and 9b) show the arrangement of the inner wall 460 that separates the cell cradle 300 from the induction channel 310 (Figures 10a and 10b). The inner wall 460 maintains a consistent height with respect to the front inlet wall 440, left side wall 410, right side wall 420, and rear outlet wall 430, otherwise opening from above, so that the array cover 120 (or unit cover) is flat across the top of the unit carrier. The inner wall 460 has a longitudinal inlet channel 470 formed through it. As shown in Figures 10a and 10b, the lowest point of the inlet channel 470 is at the level of the base of the induction channel 310. The inlet channel 470 opens toward the induction channel 310 toward the rear of the unit carrier and toward the inner surface of the cell cradle 300 toward the front of the unit carrier. The inner surface of the cell cradle defines a cell culture cavity 510 for culturing and / or growing cells inside.

[0095] The size and shape of the cell culture cavity 510 are determined by the maximum size of the embryo after development to ensure the physical stability of the embryo contained within. The size of the cell cradle 300 is approximately 0.23 mm × 0.23 mm. The shape of the surface defining the cell cradle is typically rounded to conform to the general shape of the cell mass. In particular, each wall is tapered downward to give the cell cradle 300 a more rounded internal shape.

[0096] The cell culture cavity 510 is located away from the flow of the perfusion fluid. The aperture, defined by the medium inlet, rear outlet wall, induction channel, longitudinal outlet channel, and overflow aperture, is typically aligned horizontally and defines the fluid pathway. The cell culture cavity is located lower than the fluid pathway, which ensures that the cells in the cell cradle remain immersed in the liquid medium and are not physically disturbed or otherwise destroyed by the flow along the fluid pathway. The wall of the cell culture cavity terminates at the location of the cell cradle base 520, which is typically formed on a horizontal plane and is lower than the aperture defined by the medium inlet, rear outlet wall, induction channel, longitudinal outlet channel, or overflow aperture. The cell cradle base 520 also has a cell cradle outlet 530, which is closed during use but can be opened to drain fluid from within the cell cradle 300.

[0097] Figure 10c illustrates an alternative embodiment of the features of the cell carrier unit 800, which comprises a narrow induction channel 810, a rounded channel inlet 820, and a triangular footprint cell culture chamber 840. The narrow induction channel 810 and the rounded channel inlet 820 work together to allow the instrument to be guided into and inserted into the cell culture chamber 840, while still allowing the instrument to be visible as it moves through the narrow induction channel, without moving through or exiting the narrow induction channel. The triangular footprint of the cell culture chamber 840 illustrates another shape that the cell culture chamber can adopt without requiring a curved proximal wall.

[0098] Example 4 - Cell cover and cell carrier Figures 10d, 10e, 10f, 10g, and 10h illustrate how the cell cover cartridge 900 according to the embodiment engages with the cell carrier unit 950 according to the embodiment. Figure 10d shows a rear perspective view of the cell cover cartridge 900 and cell carrier unit 950 in their pre-assembled configuration, allowing the cell carrier unit to be inserted into the central cover unit. Figure 10d further shows a cell culture chamber 960 that opens from above, and an annular opening 920 configured to allow access through it. Each side of the cell culture chamber 960 in this embodiment features an overflow aperture 965 that allows potential overflow from the cell culture chamber through a similarly positioned cover overflow aperture 925.

[0099] Figures 10e and 10g show a cell cover cartridge 900 and cell carrier unit 950 partially engaged in a first position, where the cell culture chamber 960 is accessible from above through an annular opening 920 for cell deposition, handling, and retrieval, and the induction channel 980 is accessible both from above and through the channel inlet 970.

[0100] Figures 10f and 10g show the cell cover cartridge 900 and cell carrier unit 950 fully engaged in a second position, where the cell culture chamber 960 is covered by the cell cover cartridge 900 and the medium inlet 990 is exposed through the front of the cell cover cartridge. In this position, the overflow aperture 965 is continuous with the cover overflow aperture 925, allowing the overflow to pass through it. Lines of sight are maintained through the channel inlet into the cell culture chamber 960 and through the medium inlet 990.

[0101] Regarding the external shape of the carrier unit 400 shown in Figures 10a and 10b, the left wall 410 and the right wall 420 are connected to the base wall 480 at approximately 100 degrees outward, while the rear exit wall 430 and the front inlet wall 440 are connected to the base wall 480 at 90 degrees. Figures 9a, 9b, 10a, and 10b show the left sliding flange 490 and the right sliding flange 500 protruding from the base wall 480. The left flange 490 and the right flange 500 are molded or configured to fix the base of the unit carrier inside or on another object, or to slide on or through another object. Figures 11a, 11b, and 11c show the arrangement of the unit carrier 400 within the array cover 110. Figure 11a provides a rear view of the unit carrier 400 and shows the positioning of the channel exit 320. Figure 11b provides a front view of the unit carrier 400, showing the positioning of the culture medium inlet 290 within the annular connector 230 of the array cover 110. Figure 11c shows a bottom perspective view of the unit carrier 400, which has a left flange 490 and a right flange 500, respectively, positioned for sliding engagement on the runner 330.

[0102] In a particular embodiment, the slider 350 shown in Figure 8 may be weakened to allow the individual unit carriers of the array carrier 120 to be snapped or disassembled into individual unit carriers (with or without a dedicated tool). When the array carrier 120 is configured to be disassembled into individual unit carriers in this manner, the array is configured to provide a left-side flange and a right-side flange when disassembled.

[0103] The ability to separate individual cell culture carriers or unit carriers from an array can offer advantages in processing cells for cryopreservation. For example, individual units can be separated from the array without further physical manipulation of the cells, then prepared on the carrier for cryopreservation and stored as aliquots.

[0104] The small batches of the above cell culture arrays can be manufactured using 3D printing technology with biocompatible polymer materials. Certain polymers have been shown to be printable biocompatible materials; for example, nanoscribe polymers or crystalline polystyrene have been shown to be shatter-resistant when prepared for cryopreservation.

[0105] Example 5 - Use of Cell Culture Array The embodiments described herein can be used for any type of cell culture, but may find particular use in the culture of mammalian cell lines. The embodiments described herein are particularly useful for cell cultures involving embryonic development, and thus particularly useful for their subsequent use in IVF procedures.

[0106] The embodiments may also be used for general cell culture, static perfusion, or dynamic perfusion of cells in culture.

[0107] As shown in Figure 12, a single cell culture unit can be used individually outside of an array configuration. A single silicone tube can be attached to the unit via an annular connector and can be used to position or locate the unit as needed. The tube can be used to fix the unit in place for observing cells during microscopy or for manipulating them using a micropipette or the like. The tube can also be used to fix the unit in place in a liquid medium, which may be static or fluid (for example, in a medium in a larger container, the unit can be held and continuously perfused or replenished). The tube can also be connected to a pump or vacuum manifold to force fluid through the cell culture unit and perfuse the cell culture fluid maintained within it.

[0108] As shown in Figure 13, in a cell culture array having five cell culture units, 100 μm inner diameter silicone tubes were attached to three of the five cell culture units. Perfusion of the culture medium through each unit carrier was performed by passing the cell culture medium through each of the three units, but the other two units were not perfused.

[0109] Figure 14 provides an assembly for holding a cell culture array within an atomic force microscope (AFM) mounting assembly. Variations of this assembly may be employed to adapt the mounting assembly for users of other devices that can reduce or eliminate manual intervention by embryologists during cell culture, such as lasers, piezo injectors, micropumps, visually "trained" robotic devices, or other devices for mounting cell culture arrays within automated or other instruments.

[0110] The assembly shown in Figure 14 holds a petri dish base with a 35 mm diameter × 1 mm thick glass disk for mounting the cell culture array or cell culture unit shown in Figures 12 and 13. The petri dish can be specifically fitted to hold the silicone tubes shown in Figures 12 and 13 on it, or they can simply be clamped in place. The petri dish is fitted with a special mount that can accommodate 12 mm or 25 mm circular coverslips, allowing the use of a high NA inversion optical microscope. The assembly allows cells maintained in the array or unit to be cultured and imaged in the petri dish.

[0111] The closed assembly shown in Figure 14 provides a fitting for a Petri dish on a closed fluid cell clamp. The clamp is sealed against a membrane threaded clamp and secured onto the closed fluid cell dish. Inlet and outlet access to the dish is provided via port plugs and inlet / outlet tubes delivered through the closed fluid cell dish. The assembly is sealed against a threaded bottom clamp attached to the AFM by installing an O-ring between the closed fluid cell dish and the glass disc. Those skilled in the art will know that the replacement or use of additional O-rings, or the use of assembly tools, tweezers, and cleaning brushes, is necessary for proper mounting.

[0112] Cell culture arrays are designed for use in addition to petri dishes, microscope slides, or other culture plates. While they do not replace these devices, they are used alongside these existing culture devices to position cells or cell aggregates within such devices, to handle cells or cell aggregates without disturbing them, to manipulate cells more easily, and to store cells. The assemblies described above are illustrative and can be readily adapted to specific applications by those skilled in the art. For example, a particular user might prefer to place microdevices on slides that can be easily accommodated within the assemblies described above, instead of petri dishes.

[0113] The assembly shown in Figure 14 was implemented in a perfusion assembly for perfusing the enclosed and sealed array. Using the access port, array perfusion was performed via syringe injection, gravity supply, and a micropump system, allowing for fluid and gas exchange. The AFM mount can also be used for fertilization with or without intracytoplasmic sperm injection (ICSI), as well as for time-lapse microscopy, embryo culture (with or without perfusion), and vitrification. Polymers, materials used, and properties (devices were microfabricated using a Nanoscribe GT Professional machine (Nanoscribe GmbH, Germany)).

[0114] Example 6 - In vitro test design All experiments were approved by the University of Adelaide Animal Ethics Committee (M-2019-008) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Pre-pubescent CBAxC57Bl / 6F1 hybrid and Swiss albino female mice (3-4 weeks old), weighing 9-11g, were housed in Laboratory Animal Services (University of Adelaide, Australia) and kept under controlled temperature, a 12-hour daylight saving time cycle (12 hours light:12 hours dark), and with free access to water and feed.

[0115] Pre-pubescent female mice were intraperitoneally administered 5 IU of equine chorionic gonadotropin (eCG; Follignon, Intervet, Boxmeer, The Netherlands), and 47 hours later, human chorionic gonadotropin (hCG; Humanon, Orgenon) was administered intraperitoneally to induce ovulation in the mice.

[0116] Next, male mice of the same strain were mated with female mice (1 male:1 female), and the mating plug was checked the following morning. 22 hours after hCG administration, the mice were culled by neck dislocation, and the presumed zygotes were collected from the ampoules and randomly assigned to each treatment group.

[0117] The culture media used in the experiment (including embryo washing and cleavage medium) were sourced from ART Lab Solutions (Adelaide, Australia).

[0118] The microfabrication design was developed using CAD, and microfabrication was performed using Nanoscribe GT Professional (Nanoscribe GmbH, Germany) with the polymers, materials, and settings recommended by the manufacturer.

[0119] All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, California). Statistical analyses were performed to compare embryonic development in standard embryo cultures with embryonic development in standard cultures in docked Pods within the Garage, in the presence of other variables, as described below. A normality test was performed first to determine whether to use a parametric or nonparametric test. The statistical significance of differences in mean values ​​between groups was assessed using an unpaired t-test for normally distributed data and the Kruskal-Wallis test for non-normally distributed data. A p-value of <0.05 was considered significant, and a 10% difference was considered biologically significant.

[0120] Example 7 - Preliminary Safety Analysis We investigated the toxicity of 3D-printable polymers provided by Nanoscribe, used to construct cell culture arrays and units, and conducted preliminary experiments to determine their potential effects on embryonic development.

[0121] The table below shows the results of the first iteration of the analysis of embryonic development from day 1 (zygote) and day 2 (2-cell) to day 5 (blastocyst) after treatment. [Table 1]

[0122] The table below shows the results of the second iteration of the embryonic development analysis. [Table 2]

[0123] The table below shows the results of the third iteration of the embryonic development analysis. [Table 3]

[0124] Furthermore, we conducted an experiment in which four carrier units were inserted into two array carriers. Embryo culture was performed in 20 μL of fission medium. Mouse embryos, which were hyperstimulated and mated with 4-week-old female F1 CBAxC57Bl6 mice, were cultured for 24 hours from the zygote stage to the 2-cell stage. The zygotes were cultured for 4 days in four repeats until they became blastocysts, with each group undergoing 40 replications. As a result, no statistically significant difference in viability was observed between cells cultured in carrier units and cells cultured in petri dishes.

[0125] Figure 15a shows the results of toxicity tests on 3D-printed polymers, specifically the percentage of development from cleavage embryos to CBAF1 mouse embryos. Briefly, 10 embryos were cultured in 20 μL of cleavage medium. For the treatment group, embryos were cultured in a device manufactured using 3D printing technology, which consisted of two array covers and 10 unit carriers configured as 2 × 5 unit arrays. The 10 embryos were placed in drops of 20 μL of cleavage medium. The culture medium was covered with paraffin oil.

[0126] Embryos assigned to Group 1 were cultured in fresh fission medium (control). Embryos in Group 2 were cultured in fission medium that had been pre-exposed to 10 Pods and 2 Garages. Embryos in Group 3 were cultured in fresh fission medium and co-cultured with 10 Pods and 2 Garages per drop of culture medium. Embryos in Group 4 were cultured in fresh fission medium and co-cultured with 10 Pods and 2 Garages per drop.

[0127] Figure 15b shows the rate of CBAF1 mouse embryo development from cleavage embryos. Embryo culture was performed in 10 μL of cleavage medium. Embryos assigned to the control group were cultured under standard culture conditions, while embryos assigned to the test treatment group were cultured under standard culture conditions in pods docked to a Garage. The test treatment culture group had 5 pods and 1 Garage per drop (mean ± SEM).

[0128] Figure 15c shows the rate of DNA repair in CBAF1 mouse embryo development after γH2A.X DNA repair staining of blastocysts in the control and test treatment groups. Embryos assigned to the control group were cultured under standard culture conditions, and embryos assigned to the test treatment group were cultured under standard culture conditions in Pods docked in the Garage (mean ± SD).

[0129] No significant differences were observed between the treatment groups in any of the tests. The materials used in manufacturing did not show toxicity, suggesting the potential safety of the microdevice.

[0130] Example 8 - Optimization of embryo culture conditions in a cell culture unit Figure 16 provides a schematic diagram illustrating tissue angiogenesis achievable through the use of a cell culture unit according to an embodiment. Under optimal conditions, organoids are expected to develop normally within the cell culture chamber. The chamber, coated with Matrigel, provides a scaffolding environment for promoting the proliferation of adherent cells. Directional, linear growth of the cell aggregate is provided through a further aperture between the cell culture chamber and the external environment. The arrangement and size of the aperture through the unit carrier and unit cover are selected in terms of promoting angiogenesis and supporting in-situ growth. These depend on the cell type and organoid type and can be determined by those skilled in the art.

[0131] Cell culture conditions for embryonic development were optimized. Optimal culture media and mixed air were determined using a method that can be adapted for the optimization of other conditions. The optimized cell culture conditions are expected to be applicable to the proliferation of other cell types. Presumptive zygotes were collected and randomly assigned to five treatment groups. Embryonic development was observed and recorded daily.

[0132] The embryos were divided into five groups, each treated with a different culture medium. Embryos in Group 1, if they developed on time, were transferred to a drop of fresh fission medium in the same dish. Embryos in Group 2 were placed in fission medium and then transferred to a fresh dish using a drop of fresh fission medium on day 3. Embryos in Group 3 were placed in G1+ medium, and viable embryos were transferred to fresh G1+ medium in the same dish. Embryos in Group 4 were placed in G1+ medium and then transferred to fresh G1+ medium on day 3. Embryos in Group 5 were cultured in G1+ medium and then transferred to a fresh dish using a drop of G2+ medium. Embryo culture was carried out in a humidified oven-style conventional incubator at 6% CO2, 5% O2, and a temperature of 37°C.

[0133] Figure 17 shows the percentage breakdown of embryonic development results for embryos cultured in a standard 10 μL culture medium with oil layered in a Petri dish. Embryos in Group 1 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 5. Embryos in Group 2 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 3, then transferred to fresh ART Lab Solutions embryonic division medium and cultured until day 5. Embryos in Group 3 were cultured in Vitrolife G1+ medium from day 1 to day 5. Embryos in Group 4 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G1+ medium and cultured until day 5. Embryos in Group 5 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G2+ medium and cultured until day 5.

[0134] Embryonic development was recorded daily, and the percentage of embryonic development for each treatment group is shown in Figure 17. Figure 17a shows the percentage of embryonic development from day 1 to day 2, Figure 17b shows the percentage of embryonic development from day 1 to day 4, and Figure 17c shows the percentage of embryonic development from day 1 to day 5 (mean ± SD). Embryos cultured in Vitrolife G1+ medium by day 5, followed by Vitrolife G2+ medium from day 3, showed a significant improvement in development.

[0135] Figure 17d provides the results of further tests in which cells were cultured in a cell unit carrier and covered with a cell unit cover. Embryos in Group 1 were cultured in Vitrolife G1+ medium from day 1 to day 5. Embryos in Group 2 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G1+ medium and cultured until day 5. Embryos in Group 3 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G2+ medium and cultured until day 5. Figure 17d shows the percentage of embryonic development from day 1 to day 5 for each treatment group when cells were cultured in the device according to the present invention.

[0136] Figure 17e shows the percentage of γH2a.x staining intensity indicating DNA repair in embryos cultured in standard 10 μL culture medium with oil layered in a Petri dish (in vivo blastocysts were used as a control). Embryos in Group 1 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 5. Embryos in Group 2 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 3, then transferred to fresh ART Lab Solutions embryonic division medium and cultured until day 5. Embryos in Group 3 were cultured in Vitrolife G1+ medium from day 1 to day 5. Embryos in Group 4 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G1+ medium and cultured until day 5. Embryos in Group 5 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G2+ medium and cultured until day 5 (mean ± SD).

[0137] Figure 17f shows the percentage of inner cell mass (ICM) to total cell number (TCN) in embryos cultured in standard 10 μL culture droplets with oil layered in a Petri dish (in vivo blastocysts were used as a control). Embryos in Group 1 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 5. Embryos in Group 2 were cultured in ART Lab Solutions embryonic division medium from day 1 to day 3, then transferred to fresh ART Lab Solutions embryonic division medium and cultured until day 5. Embryos in Group 3 were cultured in Vitrolife G1+ medium from day 1 to day 5. Embryos in Group 4 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G1+ medium and cultured until day 5. Embryos in Group 5 were cultured in Vitrolife G1+ medium from day 1 to day 3, then transferred to fresh Vitrolife G2+ medium and cultured until day 5 (mean ± SD).

[0138] Compared to embryos that were not moved within the same medium, all treatment groups in which embryos were moved showed improvements in DNA repair and the percentage of inner cell mass. For both DNA repair and the percentage of inner cell mass, embryos cultured in Vitrolife G1+ medium, followed by Vitrolife G2+ medium, showed improvement compared to embryos moved to Vitrolife G1+ medium on day 3.

[0139] The inadequate development observed in embryos cultured in Vitrolife G1+ medium, followed by Vitrolife G2+ medium, was resolved by further testing with the same intervention applied to embryos cultured in cell unit carriers and cell unit covers. While Vitrolife G1+ medium, followed by Vitrolife G2+ medium, still demonstrated optimal growth medium conditions, cells cultured in cell unit carriers and cell unit covers showed improvement with the medium change compared to no change. The results indicate that cell unit carriers and cell unit covers improved inadequate growth by maintaining the same medium over a 5-day growth period.

[0140] Figures 18a-18d show the percentage breakdown of embryonic development outcomes for embryos cultured in standard 10 μL culture medium with oil overlay in a Petri dish, under either a mixed air of 6% CO2, 5% O2, 89% N2, or 6% CO2, 20% O2, 74% N2, and under humidified conditions of 37°C. Figure 18a shows the percentage of embryonic development from day 1 to day 2, Figure 18b shows the percentage of embryonic development from day 1 to day 3, Figure 18c shows the percentage of embryonic development from day 1 to day 4, and Figure 18d shows the percentage of embryonic development from day 1 to day 5 (mean ± SD). Figure 18e shows the percentage of γH2a.x staining intensity, indicating DNA repair with and without increased oxygen in the mixed air, in embryos cultured in standard 10 μL culture medium droplets with oil overlay in a Petri dish (control: in vivo blastocyst).

[0141] The percentage of embryonic development showed a slight decrease by day 5 when embryos were cultured in 20% O2 instead of 5% O2, although this result was not consistent throughout all culture days. However, the rate of DNA repair showed a significant improvement by day 5 when cells were cultured in the presence of 20% O2.

[0142] These results indicate that, despite trauma sustained when cells are disturbed during development, supplementing the culture medium results in a significant improvement in the proliferation and viability of cultured cells. This improvement is expected to be further enhanced by employing continuous perfusion to introduce fresh culture medium, and even further when cells are not damaged during perfusion. The temporal results regarding developmental and viability discrepancies reflect the diverse requirements of developing embryos at each growth stage. Static perfusion in the presence of optimized growth medium and different culture conditions for each growth stage are expected to further improve growth outcomes.

[0143] Throughout this specification, the conjugations "comprise," "comprises," or "comprising" mean to include the element, integer, or step, or group of elements, integers, or steps, that is described, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

[0144] Various devices and components of the apparatus described herein may be supplied in various sizes and / or dimensions as needed. The appropriate size and / or dimensions will vary depending on the specifications or application field of the connecting components, which can be selected by those skilled in the art.

[0145] It should be understood that the properties, elements, and / or features described in relation to one embodiment of this disclosure may be used in conjunction with other embodiments of the invention as needed.

[0146] Preferred embodiments of this disclosure are disclosed for illustrative purposes only, but those skilled in the art will understand that various modifications, additions, and substitutions are possible without departing from the scope and spirit of this disclosure and the appended claims.

[0147] When an element or layer is said to be on or within another element or layer, it will be understood that the element or layer can exist directly on or within the other element or layer, or an intervening element or layer. In contrast, when an element is said to be "directly" on or "directly within" another element or layer, there is no intervening element or layer.

[0148] As used herein, the term "and / or" includes combinations of one or more and all of the related enumerated items.

[0149] The terms First, Second, Third, etc., may be used herein to describe various elements, components, regions, layers, and / or sections, but it will be understood that these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used solely to distinguish one element, component, region, layer, or section from another region, layer, or section. Accordingly, a First element, component, region, layer, or section may also be referred to as a Second element, component, region, layer, or section without departing from the teachings of this disclosure.

[0150] Spatially relative terms such as “bottom,” “top,” “left,” and “right” may be used herein to facilitate explanation in describing the relationship between one element or feature and another element or feature(s), as shown in the figures. It is understood that spatially relative terms are intended to encompass different orientations of the structure in use or operation, in addition to the orientation shown in the drawings. For example, if the illustrated device is turned over, an element described as “lower” relative to another element or feature will thereby be oriented “top” relative to the other element or feature. Thus, the exemplary term “lower” may encompass both upward and downward orientations. The device may be oriented in other ways (it may be rotated 90 degrees or rotated in other orientations), and spatially relative terms used herein should be interpreted accordingly.

[0151] The technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit this disclosure. Where used herein, the singular forms “a,” “an,” and “the” are also intended to include plural forms unless the context clearly indicates otherwise. Where used herein, the terms “including,” “comprises,” and / or “comprising” identify the presence of a specified feature, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0152] Embodiments in the detailed description of the invention are described herein by reference, for example, to diagrams and / or cross-sectional views that are schematic representations of preferred embodiments (and intermediate structures) of the description. Therefore, deformations that differ from the illustrated shapes are expected, for example, as a result of manufacturing techniques and / or tolerances. Accordingly, the embodiments described herein should not be construed as being limited to specific shapes of the components illustrated herein, but rather should encompass, for example, deviations in shape that occur during manufacturing.

[0153] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art to which this specification belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with those in the context of the relevant technical field, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0154] Any reference herein to “one embodiment,” “a particular embodiment,” or “an exemplary embodiment” means that any specific feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the detailed description of the invention. The occurrence of such phrases in various places in this specification does not necessarily all refer to the same embodiment. Furthermore, when a particular property, structure, or feature is described in relation to any embodiment, it is within the scope of the skill of those skilled in the art to utilize and / or use such feature, structure, or characteristic in relation to other embodiments.

[0155] The embodiments are intended to include, or encompass, methods of using and manufacturing any or all of the elements disclosed above.

[0156] Although the present invention has been described above with respect to specific embodiments, it should be understood that the present invention is not limited to these disclosed embodiments. By reading the teachings of this disclosure, it will be understood by those skilled in the art that many modifications and other embodiments of the present invention are relevant to the present invention and are intended and covered by both this disclosure and the appended claims.

[0157] All publications referenced herein are incorporated herein by reference. Any discussion of literature, acts, materials, devices, articles, etc. contained herein is intended solely to provide context to the present invention. The mere fact that any or all of these matters existed in Australia or elsewhere prior to the priority date of each claim of this application does not constitute part of the foundation of the prior art or general knowledge in the art relating to the present invention.

[0158] The scope of the present invention is actually intended to be determined by a proper interpretation and construction of the appended claims and their legal equivalents, as can be understood by those skilled in the art based on the disclosures herein and in the appended drawings.

[0159] References 1.Data on IVF clinics show wide variation in success rate,BMJ 2002;325 doi:https: / / doi.org / 10.1136 / bmj.325.7362.460 / e(Published 31 August 2002).

[0160] 2.JMST Advances, June 2019, Volume 1, Issue 1-2, pp 1-11| Cite as Microfluidic technology for in vitro fertilization(IVF).

Claims

1. A cell culture microdevice for maintaining and culturing cells internally, The cell culture unit comprises at least one first cell carrier unit that defines a cell culture chamber formed inside, The first cell carrier unit comprises at least, A chamber base molded to support the cells at the top, It is formed from one or more chamber walls having one or more chamber wall surfaces surrounding the cell culture chamber around the chamber boundary, The first cell carrier unit further provides a guide surface positioned in an aperture that passes through the chamber wall for guiding an instrument or fluid into the cell culture chamber, The dimensions of the cell culture microdevice are 0.1 μm to 1000 μm in width, height, and length. The cell culture microdevice is maintained in a liquid culture medium within a larger container and is configured to allow passive exchange of liquid fluid with the larger container. A cell culture microdevice wherein the cell culture microdevice is configured on a scale that substantially encloses a single cell or cell aggregate within it.

2. The cell culture microdevice according to claim 1, wherein one or more chamber walls include one or more inner wall surfaces inclined toward the proximal point of the chamber, and are configured to guide the arrangement of instruments or cells within the culture chamber.

3. The cell culture microdevice according to claim 1 or 2, wherein the cell culture chamber is open from above and the chamber base has a curved inner surface.

4. The one or more chamber walls comprise a proximal wall and a distal wall, The proximal wall has a curved inner wall surface configured to guide the placement of instruments into the culture chamber. The cell culture microdevice according to claim 1 or 3, wherein the distal wall defines the distal chamber boundary and has an aperture formed to pass through the chamber wall, the aperture defining an opening by communicating with an elongated guiding portion having an internally formed channel that protrudes outward from the cell culture chamber and provides a guiding surface for guiding instruments or fluids into the cell culture chamber.

5. The cell culture microdevice according to claim 4, wherein one or more chamber walls comprise at least a left wall and a right wall, each having a left aperture and a right aperture formed to pass through it.

6. The cell culture microdevice according to claim 4, wherein the proximal wall has a proximal aperture formed to pass through it, configured to be aligned horizontally with the guiding surface, and mitigating the flow of the fluid through the cell culture chamber between the aperture and the proximal aperture.

7. The cell culture microdevice according to claim 6, wherein the proximal wall comprises a perfusion inlet opening adapted to a fluid perfusion flow passing through it, and a tube fitting configured to engage a perfusion tube with the perfusion inlet opening.

8. The cell culture microdevice according to any one of claims 1 to 7, wherein the first cell carrier unit comprises a cell chamber wall having an outer wall coupling adapted to engage with a corresponding outer wall coupling on at least one second cell carrier unit, thereby forming a cell carrier array.

9. The cell culture microdevice according to any one of claims 1 to 7, further comprising at least one second cell carrier unit formed integrally with the first cell carrier unit, thereby forming a cell carrier cartridge.

10. The cell culture microdevice according to claim 3, further comprising a first cell cover unit having a first cover wall configured to cover at least a portion of the opening of the cell culture chamber from above when the first cell cover unit and the first cell carrier unit are connected to form a cell culture unit base.

11. The cell culture microdevice according to claim 10, wherein the first cell cover unit comprises an outer wall coupling adapted to engage with a corresponding outer wall coupling on at least one second cell cover unit, thereby forming a cell cover array.

12. The cell culture microdevice according to claim 11, further comprising at least one second cell cover unit integrally formed with the first cell cover unit, thereby forming a cell cover cartridge.

13. The cell culture microdevice according to claim 12, wherein the first cell cover unit further comprises an access aperture formed to pass through it, the access aperture formed to pass through the first cell cover unit is configured to allow access from above to the opening at a first position and to cover at least a portion of the opening from above at a second position and is adapted to slidably engage with the first cell carrier unit from the first position to the second position.

14. The cell culture microdevice according to any one of claims 1 to 13, wherein the outer surface of the chamber base is provided with a notch configured to receive a lug protruding outward from a cell carrier unit or a cell cover unit.

15. A method for using a cell culture microdevice according to any one of claims 1 to 14, comprising the steps of: conveniently placing one cell in the cell culture chamber of the cell culture microdevice; and culturing the cell.

16. A method for using a cell culture microdevice according to any one of claims 1 to 14, comprising the steps of obtaining instructions for constructing the cell culture microdevice and carrying out the instructions in an additive manufacturing process.

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