Cell culture microdevice

Abstract

A cell culture microdevice for maintaining and cultivating cells therein includes a cell culture unit having at least a first cell carrier unit defining a cell culture chamber formed therein, the first cell carrier unit formed of at least a chamber base shaped to support cells thereon and one or more chamber walls having one or more chamber wall surfaces surrounding a chamber perimeter enclosing the cell culture chamber, the first cell carrier unit further providing a guide surface to direct positioning of an instrument or liquid through a hole in the chamber wall into the cell culture chamber, wherein the cell culture microdevice is dimensioned to substantially enclose a single cell or cell cluster therein. Embodiments of the cell culture microdevice can be adapted for use in in vitro fertilization procedures and drug efficacy testing.

Description

TECHNICAL FIELD

[0001] The technical field generally relates to advances in microdevices for cell culture, particularly mammalian cell culture. The microdevices of the present invention are generally in the form of cell carrier devices, and in the form of microdevices for cell culture. However, the carrier devices are equally applicable to cell culture, stem cell differentiation, cell array testing, infertility treatment, particularly in vitro fertilization (IVF) treatment. Specific embodiments relate to microdevices including cell culture units, cartridges, arrays, and perfusion devices including cell carrier units and cell cap units. BACKGROUND

[0002] Since the widespread use of tissue culture microdevices, microscale tissue culture technology has advanced in complexity, and the fields of use and application have become more diverse. Microdevices enable manipulation of cells and their culture environment, resulting in new therapies, products, and processes, many of which are still in their infancy. New microdevices and improvements to existing microdevices, such as microfluidic devices, "culture on a chip" technology, microscaffolds, and micro-manipulation devices, have resulted in new methods of 3D tissue engineering, stem cell differentiation, and reproductive medicine, and have enabled breakthroughs in success rates of these technologies.

[0003] For example, complex in vitro tissue culture has previously been limited in success and application by limited gas exchange, possibly by diffusion, however, the development of microcapillary perfusion devices now enables the culture and propagation of larger, more complex tissues. With respect to stem cell culture technology (and its applications), organ culture systems of various embryonic tissues now enable the culture of embryonic brain, retina, limb bud, lung, kidney, salivary gland, hair follicle, and tooth cell lines. The application of new tissue engineering technologies, and the microdevices that enable these technologies, will facilitate new and improved methods of many different therapies.

[0004] In the field of reproductive medicine, the potential benefits of improved microdevices for improved cell culture and propagation are significant. The nature of this field is almost entirely dependent on in vitro culture and manipulation of autologous cells, as these cells must be harvested, expanded, and reintroduced into the patient.

[0005] Reproductive assistance through in vitro fertilization (IVF) is becoming more readily available and has recently improved, so more and more patients are obtaining this assistance. Data published by the Human Fertilization and Embryology Authority shows that, overall, women starting IVF treatment are more likely to have a live birth than in the past 1 . However, there is a wide variation in IVF clinic success rates observed between individual clinics, with some clinics having success rates as high as 46% and others as low as 10% 1 .

[0006] The likelihood of success varies at several stages of the IVF procedure (during oocyte retrieval, oocyte fertilization, early embryo development, embryo vitrification, and embryo transfer). Currently, success in oocyte selection, fertilization, embryo development, embryo cryopreservation, and embryo transfer preparation depends largely on the skill of the embryologist.

[0007] During embryo development, determinants of successful IVF pregnancy involve, in large part, the physical and environmental conditions present during the process of embryo growth and development. Any type of rapid environmental change, physical impact, or physical stress that can occur to sperm, oocytes, or developing embryos reduces the likelihood of embryo survival, and in turn, the likelihood of successful implantation of the embryo for successful pregnancy.

[0008] The physical and environmental conditions of any cell culture system can affect the viability and proliferation of cells in vitro. For example, the choice between scraping (manual / physical), trypsinization (chemical / enzymatic), and sonication (physical / mild) to isolate adherent cells for in vitro culture preparation greatly affects cell viability and depends largely on the cell type used, the buffer, and other culture medium components.

[0009] In tissue engineering, the assembly of functional three-dimensional tissue structures during morphogenesis and organogenesis relies on cell-cell interactions between tissue monolayers. These cell-cell interactions are triggered and maintained by physical and environmental cues within the culture system, including the application of mechanical forces, cell shape, extracellular matrix geometry or other properties, and physical cell-cell contact, and other morphogenic factors. Likewise, differentiation of stem cells is influenced by many factors in the cell culture matrix, including cell-specific growth factors, enzymes, and other proteins, or depletion or accumulation of these components or their byproducts.

[0010] For embryogenesis, the most important impact or stress on developing embryos occurs through handling and physical manipulation, which is influenced by the experience, training, and fatigue of the embryologist. For IVF procedures, several events during embryo growth and preparation involve physical intervention by the embryologist, and thus pose a risk to the embryo for success and its likelihood of implantation. During the process of collecting sperm, oocytes, and introducing them into an in vitro environment, there is a risk of stress or impact on sperm and oocytes, including physical injury and deleterious environmental effects during placement in silicon chips, and biochemical stress from placement in different liquid environments.

[0011] The process of fertilization presents a risk of injury from the physical manipulation required to introduce sperm, whether or not it is intracytoplasmic manipulation. Optimization of the embryo culture environment and the removal of the embryo once it has developed for freezing or implantation increases the risk of physical injury. Physical implantation of the embryo also creates the opportunity for physical damage or harm to the embryo. The risk of injury or shock from manipulation and handling, and the success of the IVF procedure, is greatly influenced by the skill and care of the embryologist, and the precision of the tools and instruments available to the embryologist, and the laboratory environmental conditions influenced by the embryologist (e.g. sterility, temperature, prevention of any form of contamination, including volatile organic compounds, and management of parentage identification to prevent errors or mixed parentage).

[0012] Precise tools and equipment reduce the likelihood of errors by the embryologist that result in injury or shock to sperm, oocytes, or embryos, or errors in parentage. Furthermore, tools or equipment that reduce or eliminate physical intervention or manipulation by the embryologist can reduce the likelihood of events that can cause physical shock to sperm, oocytes, or embryos. Furthermore, tools or equipment that reduce optimization of changes in the in vitro environment can also reduce biochemical stress to oocytes or embryos.

[0013] While there has been progress in the microinjection devices used in IVF treatment and in visualization devices, there has been little progress in developing microdevices for handling the relevant cells that must be manipulated, or in ways to house these cells that reduce excessive handling or manipulation.

[0014] Typically, developing embryos are cultured in large volumes of liquid media, which can minimize the physical impact of the depletion of important media components as they are used by the cells, and can minimize any physical impact of the increasing concentration of waste products in the media. However, even small increases in waste products and small reductions in nutrients can have a significant impact on developing cells. The changing metabolic demands during embryo development exacerbate the physical impact of changes in media composition during growth.

[0015] These challenges are similar to those encountered in the process of culturing organoids or complex tissues in vitro. The changing metabolic demands of cells during development into more complex tissues and tissue structures place more complex demands on in vitro tissue culture systems.

[0016] It is not simple to include the physical impact of developmental changes in the volume of media contained. For embryo culture, it is difficult to provide sufficient dilution to minimize the impact of media changes, because the volume of media must remain small enough for the embryologist to be able to locate the embryo, and larger volumes of media increase the likelihood that the embryo will be diluted of known substances that the embryo produces to help it grow itself, or become difficult to locate, resulting in excessive handling or manipulation.

[0017] One common approach taken by embryologists to address this problem is to prepare a series of media through which the developing embryo is cycled throughout its in vitro development. The benefit of this approach is that each medium, fertilization medium or cryopreservation medium, can be specifically tailored to the needs of the cells at a particular stage of development or manipulation, however, even with optimization of medium composition to meet the requirements of the stage of embryo growth, the stress experienced by the cells as they move from one liquid medium to another still has a detrimental effect on development.

[0018] To overcome this problem, various microfluidic and liquid or gas perfusion devices have been developed with varying degrees of success. Microfluidic "on-chip culture" microdevices have been developed that utilize microfluidics to move or "roll" cells or cell clusters from one cell culture "bath" to the next to prevent cell shock from excessive manipulation. Other microfluidic devices have been developed that are suitable for use in more traditional cell culture dish systems that allow for continuous flow of liquid media in and out of the system throughout the development of the zygote to embryo stage, as well as for preparation for cryopreservation and embryo transfer. These developments have been little adopted and many embryologists still prefer to use static culture techniques in culture dishes or variations of culture dishes because embryos are easier to locate and retrieve in these vessels.

[0019] Neither of these approaches addresses the need for optimal gas exchange and liquid exchange. In all tissues, and especially in morphogenesis and stem cell differentiation, adequate aeration by means of gas diffusion is important for development.

[0020] Attempts to apply microfluidic technology to static culture of embryos have only recently emerged 2 Researchers are still exploring and optimizing techniques to ensure that the oocyte and developing embryo are not moved excessively, thereby avoiding damage or stress, while managing very small volumes of media that can be contained in the very small channels of microfluidic devices. However, to date, no perfusion combination within a microfluidic device has achieved any degree of clinical application.

[0021] Engineering principles established in the writings of Richard Feynman have well proven that the scaling down of engineering systems from the macroscopic scale to the microscopic scale presents a wider range of new applications, however, it brings with it physical challenges such as evaporation, lubrication, heating and inertia that work very differently at such small scales. For in vitro cell culture, engineering design to accommodate this scale has not been adequately addressed. The present invention addresses these problems, at least in part, through design innovation.

[0022] The writings of Kim Eric Drexler also demonstrate that the use of nanoscopic and microscale devices offers opportunities that are not available to macroscopic devices. Large scale cellular devices often rely on random interactions between multiple cells, with the hope that one or more of the interactions will produce the intended result within a predetermined acceptable range. Microscale devices allow for direct interaction with a single cell to produce the intended result more accurately and more efficiently. Subsequent interactions are based on a more idealized object to produce a more accurate result.

[0023] By maintaining the physical stability of sperm, oocytes, and embryos within their culture devices, embryologists can more easily and / or accurately handle or manipulate sperm, oocytes, or embryos and minimize or further prevent the risk of physical shock and / or biochemical stress, or the risk of mixing parental contributions during fertilization, embryo culture, or embryo transfer. The stability of cells during culture can improve the success rate of IVF procedures.

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

[0025] In one aspect of the invention, embodiments of the invention relate to a cell culture microdevice for maintaining and culturing cells therein, the cell culture microdevice 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 formed of at least one chamber base shaped to support cells thereon and one or more chamber walls having one or more chamber wall surfaces surrounding a chamber perimeter enclosing the cell culture chamber, the first cell carrier unit further providing a guide surface to direct an instrument or fluid positioned through a hole in the chamber wall into the cell culture chamber, wherein the cell culture microdevice is dimensioned to substantially enclose a single cell or cell mass therein.

[0026] As used herein, the term "cell" shall be understood to be interchangeable with the term "cellular material" and shall refer to a cell, cell population, tissue, or organoid as the subject of the inventive subject matter described herein.

[0027] As used herein, the term "cell culture" shall be described as any tool or process for isolating and maintaining cellular material under controlled conditions for testing, growth, observation, experimentation, harvesting of media, or other biological science processes.

[0028] As used herein, the term "microdevice" shall be described as fabrication produced on a micrometer scale (e.g., between 0.1 pm and 1000 pm). Microdevices shall include static and mechanical devices as well as devices in the field of microfluidics.

[0029] As used herein, the term "maintaining cells" refers to any process that stores cell material in a controlled environment to create conditions necessary for viability. The term "culturing cells" refers to the process of cell culture.

[0030] As used herein, the term "cryopreservation" refers to vitrification or freezing interchangeably.

[0031] As used herein, the term "boundary" refers to the three-dimensional demarcation between the interior of a cell chamber unit and the exterior of a cell lumen unit and can be defined by walls, surfaces, openings and pores.

[0032] In preferred embodiments, the guide surface will define a curved bottom trench through which the instrument will be passed and introduced into the cell culture chamber through the pore. Alternatively, fluid can be directed and passed through the curved bottom trench and through the pore between the interior and exterior of the cell culture chamber.

[0033] In alternative embodiments, the guide surface will define a narrow pore through the chamber wall through which the instrument will be passed and introduced into the cell culture chamber through the pore. Alternatively, fluid can be directed and passed through the narrow pore between the interior and exterior of the cell culture chamber.

[0034] In preferred embodiments, the cell chamber base is substantially concave. Alternatively, the cell chamber base can form a substantially convex, stepped, concave or other shape that can be used to support cell material within the cell culture chamber.

[0035] The cell carrier unit of aspects of the invention is configured to be formed on a microscale. Thus, it is also preferably formed from materials that are suitable for microscale production that are not toxic to cells.

[0036] One or more chamber walls of certain embodiments can include one or more interior wall surfaces that are angled toward the proximal point of the chamber so as to be configured to guide placement of an instrument or cells within the culture chamber.

[0037] In preferred embodiments, one or more chamber walls of aspects of the invention include a proximal wall having a curved interior wall surface that is configured to guide placement of an instrument within the culture chamber.

[0038] In certain embodiments, the proximal wall can be stepped, concave or otherwise configured to guide placement of an instrument within the culture chamber.

[0039] In preferred embodiments, the cell culture chamber of aspects of the invention is open from above and the chamber base includes a curved interior surface.

[0040] The boundary of aspects of the present invention is preferably substantially box-shaped, shaped with a top opening and a curved proximal wall opposite a guide hole that can provide direction and stability to the cell material during processing. The cell culture chamber preferably includes a fluid exchange hole located at the surface of the curved proximal wall and the surface of the culture base. Alternatively, the footprint of the cell culture chamber is substantially triangular or V-shaped and tilted towards the proximal point.

[0041] In alternative embodiments, the boundary of aspects of the present invention is substantially cylindrical, spherical, triangular, asymmetric or stepped.

[0042] In preferred embodiments, one or more chamber walls of aspects of the present invention include a proximal wall having a curved inner wall surface configured to guide placement of an instrument within the culture chamber and a distal wall defining a distal chamber boundary and having a hole through the chamber wall, the hole formed through the chamber wall defining an opening in communication with an elongated guide portion projecting outward from the cell culture chamber having a channel formed therein providing a guide surface to guide an instrument or fluid into the cell culture chamber.

[0043] Preferably, one or more chamber walls of aspects of the present invention include a medial distal wall opposite the curved proximal wall. The medial distal wall includes the hole and provides the guide surface defined outside the cell culture chamber and formed perpendicular to the medial distal wall.

[0044] Preferably, the inner surface of the medial distal wall is substantially concave to direct an instrument or fluid from the chamber towards the guide surface. Alternatively, the inner surface of the medial distal wall can be planar.

[0045] In preferred embodiments, one or more chamber walls of aspects of the present invention include at least one left side wall and at least one right side wall, each having a left side hole and a right side hole formed therethrough.

[0046] In preferred embodiments, the proximal wall of aspects of the present invention has a proximal hole formed therethrough configured to be horizontally aligned with the guide surface to mitigate flow of fluid through the cell culture chamber between the hole and the proximal hole.

[0047] In preferred embodiments, the hole of aspects of the present invention is configured for perfusion therethrough. Preferably, the hole is located through the chamber base and is sized such that cells cannot pass therethrough. Alternatively, the hole is located through the chamber wall or consists of a plurality of holes distributed on one or more surfaces of the boundary.

[0048] In a preferred embodiment, the proximal wall of various aspects of the invention includes an irrigation inlet opening adapted for fluid irrigation therethrough, and a tubing fitting configured for connecting an irrigation tube to the irrigation inlet opening.

[0049] Alternatively, the fittings can be configured for connection with irrigation manifolds or other means of supplying irrigation media.

[0050] Liquid exchange pores can also be located on other surfaces, can be integrated into other pores and openings, or may not be necessary for the maintenance and culture of cell material in some cases.

[0051] In a preferred embodiment, a first cell carrier unit of various aspects of the invention includes a cell chamber wall having an outer wall coupling member adapted to engage with a corresponding outer wall coupling member on at least a second cell carrier unit, thereby forming a cell carrier array. Preferably, the cell carrier array may include an unlimited number of cell carrier units, each adapted to engage with another unit. Preferably, the cell carrier array may be a linear array in a horizontal plane, but may also be stacked in a vertical plane or a combination of both. Preferably, the cell carrier units will be sandwiched together in a horizontal plane and stacked in a vertical plane, or may be slidably engaged together in either a horizontal or vertical plane.

[0052] In some embodiments, the cell culture microdevices of various aspects of the present invention include at least one second cell carrier unit integrated with a first cell carrier unit to form a cell carrier cassette. Preferably, the cell carrier cassette may include an unlimited number of cell carrier units, each integrated with another unit. Preferably, the cell carrier cassettes will be linear in a horizontal plane, but cassettes or a mixture of both may also be formed in a vertical plane. In a preferred embodiment, the cell carrier cassettes may be stacked or joined with another cell carrier cassette to define an array of cell carrier cassettes.

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

[0054] In a preferred embodiment, the cell culture microdevice of various aspects of the present invention further includes a first cell cover unit having a first cover wall, wherein when the first cell cover unit and the first cell carrier unit are connected to form a cell culture unit base, the first cover wall is configured to cover at least a portion of the opening above the cell culture chamber.

[0055] In a preferred embodiment, the cell cover unit is shaped to surround the cell carrier unit substantially on three surfaces (including the top surface), and when located on the top surface, the cell cover unit completely covers the top opening.

[0056] In alternative embodiments, the cell cover unit is shaped 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 in a portion capable of engaging with the cell carrier unit of aspects of the application. Preferably, the portion capable of engaging with the cell carrier unit is adapted to engage with the base of the cell carrier unit, thereby forming a cell culture base. In another preferred form, the cell cover unit is configured to slidably engage with the base of the cell carrier unit.

[0058] In preferred embodiments, the first cell cover unit of aspects of the application 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 can comprise an unlimited number of cell cover units, each adapted to engage with another unit. Preferably, the cell cover array will be a linear array in a horizontal plane, but can alternatively be stacked in a vertical plane or a mixture of both. Preferably, the cell cover units will be clipped together in a horizontal plane and stacked in a vertical plane, or can be slidably engaged together in either a horizontal or vertical plane.

[0059] In preferred embodiments, the cell culture microdevice of aspects of the application further comprises at least one second cell cover unit integrated with the first cell cover unit, thereby forming a cell cover cassette. Preferably, the cell cover cassette can comprise an unlimited number of cell cover units, each integrated with another cover unit. Preferably, the cell cover cassette will be linear in a horizontal plane, but can alternatively produce a cassette in a vertical plane or a mixture of both. In preferred embodiments, the cell cover cassette can be stacked or engaged with another cell cover cassette to define a cell cover cassette array.

[0060] In preferred embodiments, the first cell cover unit and the first cell carrier unit of aspects of the application define a cell culture unit base. In preferred embodiments, the cell culture unit base is defined by the first cell cover unit and the first cell carrier unit terminating in a flat bottom surface to provide a stable support. Alternatively, the cell culture base can be defined by one of the first cell carrier unit or the first cell cover unit in one configuration and the other in the other configuration.

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

[0062] In preferred embodiments, the first cell cover unit of aspects of the application is configured to slidably engage with the first cell carrier unit. In preferred embodiments, the first cell cover unit is shaped to slidably engage with each of two sides of the first cell carrier unit perpendicular to the proximal-distal axis.

[0063] In certain embodiments, the first cell cover unit comprises a media inlet configured to engage with a tubing fitting on the first cell carrier unit and allow a perfusion tube to be connected to the tubing fitting thereon. Alternatively, the media inlet can be configured to engage with a perfusion manifold or other means of supplying perfusion media.

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

[0065] In preferred embodiments, the access aperture is the same size and shape as the top opening, such that the top opening can be accessed entirely through the access aperture. In alternative embodiments, the access aperture can be larger than the top aperture, and be of an inclined shape to direct an instrument or cells into the cell culture chamber when configured for access.

[0066] In certain embodiments, the outer surface of the chamber base of aspects of the application comprises a notch configured to accommodate a lug that protrudes outwardly from the cell carrier unit or cell cover unit.

[0067] A method of using a cell culture microdevice of aspects of the application, comprising the steps of placing at least one cell within the cell culture chamber of the cell culture microdevice and culturing the cell therein.

[0068] A method of using a cell culture microdevice of aspects of the application, comprising the steps of obtaining instructions for constructing the cell culture microdevice and executing the instructions in an additive manufacturing process.

[0069] In preferred forms of the present application, the cell culture unit comprises at least four walls and a base defining a cell culture chamber therein. The at least four walls preferably comprise a proximal wall having a curved inner surface defining the cell culture chamber, wherein the curvature provides a reference point for orienting a cell handling device, a left side wall, a right side wall, and a medial distal wall. In preferred embodiments, the medial distal wall comprises an opening formed therethrough, an inner surface defining the cell culture chamber, and an outer surface having a channel formed substantially perpendicular thereto. The channel is preferably formed by the inner surface of the left side wall and the inner surface of the right side wall, which extend distally beyond the medial distal wall and terminate substantially perpendicular to a distal outer wall having an opening formed therethrough.

[0070] In the medial distal wall, the channel and the opening formed in the distal outer 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 to an embryologist or other user to carefully introduce an instrument (e.g., a micropipette) into the cell culture chamber, thereby accessing the cells with little or no disturbance or damage to the cells.

[0071] For example, an embryologist attempting to remove an embryo from the cell culture chamber for implantation can introduce a micropipette through the distal outer wall opening, and they can move and / or cradle the tip of the micropipette over the channel until the micropipette reaches the curved inner surface of the proximal wall. The curvature of the proximal wall guides the micropipette to the center of the cell culture chamber, at which point the embryologist can gently aspirate or inject the embryo cells and media directly below the micropipette. The configuration of the cell culture carrier provides physical support for the instrument (e.g., micropipette) and guidance for placement or positioning of the instrument, reducing the impact of operator error that can harm or impede the optimal growth of the cells. Thus, the configuration of the cell culture carrier optimizes culture technique, which in turn optimizes cell viability, and reduces the impact of user error.

[0072] Preferably, the left and / or right side walls comprise an overflow opening formed therethrough. Preferably, the proximal wall comprises an inlet opening defined by an inlet fitting located on an outer surface of the proximal wall. The inlet fitting can serve as a connector for tubing or other instruments. It can connect tubing for transferring fluid into the cell culture unit for perfusion culture. The inlet fitting can also connect devices such as tubes that serve only as a reference point for the location of the cell culture unit or a fixture to maintain the cell culture unit in place.

[0073] In another preferred form, the inlet opening, the intermediate distal end wall, the passageway and the outer distal end wall are preferably aligned to provide a line of sight from the distal end wall through the cell culture carrier and through the inlet opening. Preferably, the line of sight through the cell culture carrier is aligned to enable the culture of cells during perfusion of the 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 minimises physical disruption of the cultured cells by turbulence or currents caused by fluid perfusion.

[0074] Perfusion techniques can optimise growth conditions for certain cell types, particularly cells that are sensitive to biochemical changes in the culture medium, which can be caused by depletion of nutrients or increase in waste products in the medium, or those that can have different biochemical requirements when cultured at different growth stages. Embryo culture for IVF procedures can benefit from perfusion culture, as can 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 susceptible to tissue rejection.

[0075] Broad embodiments of the present application will now be described with reference to the accompanying drawings and examples and preferred embodiments disclosed in the detailed description. The present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided by way of example only, so that a thorough and complete disclosure of the present application can be made, and so that the full scope and breadth of the present application can be conveyed to those who are skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 A top perspective view of an assembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0077] Figure 2 A front view of an assembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0078] Figure 3 A rear view of an assembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0079] Figure 4 A bottom perspective view of an assembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0080] Figure 5 A bottom view of an assembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0081] Figure 6 A top perspective view of an unassembled cell culture array or cartridge according to an embodiment of the present application is shown.

[0082] Figure 7 A back view of an unassembled cell culture array or cartridge is shown in accordance with embodiments of the application.

[0083] Figure 8 A bottom perspective view of an unassembled cell culture array or cartridge is shown in accordance with embodiments of the application.

[0084] Figure 9a and 9b A cell cradle portion of a cell culture unit carrier is shown in accordance with embodiments of the application. Figure 9a A top perspective view is shown, Figure 9b A bottom perspective view is shown. Figure 9c 、 9d and 9e show an unassembled cell unit lid and an unassembled cell culture array. Figure 9c A back perspective view of an expandable cell unit lid is shown, Figure 9d A back perspective view of an expandable cell culture array is shown, Figure 9e A bottom back perspective view of an expandable cell culture array is shown.

[0085] Figure 10a 、 10b and 10c show a cell culture unit carrier in accordance with embodiments of the application. Figure 10a A front top perspective view is shown, Figure 10b A back top perspective view is shown. Figure 10c A front top perspective view of a cell culture unit carrier is shown in accordance with alternative embodiments. Figure 10d 、 10e and 10f provide a front top perspective view of a cell culture unit carrier in three positions for engagement with a cell culture array. Figure 10g and 10h A top perspective view of a cell culture unit carrier in two engagement positions for introducing cells into the cell culture unit carrier is shown.

[0086] Figure 11a 、 11b and 11c show a cell culture array or cartridge with a unit carrier positioned therein in accordance with embodiments of the application. Figure 11a A back view is shown, Figure 11b A front corner view is shown, Figure 11c A bottom perspective view is shown.

[0087] Figure 12 A side view of a cell culture array or cartridge is shown in accordance with embodiments of the application.

[0088] Figure 13a 、 13bFigs. 13a and 13c show side views of a cell receiving micropipette within a cell culture array or cassette according to embodiments of the application.

[0089] Figure 14 Fig. 14 shows an atomic force microscope (AFM) mounting assembly for a cell culture microdevice according to embodiments of the application.

[0090] Figure 15a 、 15b Figs. 15a and 15c provide results of a 3D printer polymer toxicity study. Figure 15a Fig. 16 shows the percentage of embryo development in the presence of a microdevice according to the application and its media, Figure 15b Fig. 17 shows the percentage of embryo development in the presence of a microdevice according to the application, Figure 15c Fig. 18 shows the percentage of DNA repair.

[0091] Figure 16 A schematic is provided illustrating tissue vascularization by using a cell culture unit according to embodiments.

[0092] Figures 17a to 17e Results of a cell culture optimization study are provided. Figures 17a to 17c Fig. 19 shows embryo development in static culture conditions from day of development to day 5 according to media type and intervention, Figure 17e Fig. 20 shows the percentage of DNA repair within the same group, Figure 17f Fig. 21 shows inner cell masses of the same treatment group.

[0093] Figures 18a to 18d Results of an oxygen optimization study are provided. Figures 18a to 18d Fig. 22 shows embryo development over time exposed to different oxygen concentrations, Figure 18e Fig. 23 shows the percentage of DNA repair within the same group.

[0094] The following examples describe several embodiments of the application. DETAILED DESCRIPTION

[0095] The cell culture microdevices described in the following examples are generally comprised of a single cell culture unit having a unit carrier and a unit cover, an array of repeating units joined to form a cell culture array having an array carrier and an array cover, or a box of repeating units integrated to form a cell culture box having a box carrier and a box cover. They are generally referred to hereinafter as "carriers" and "covers" when they can take any of the following forms, as a single unit, an array of repeating units of any shape or number, or a box of repeating units of any shape or number.

[0096] Those skilled in the art will appreciate the benefits of fabricating the "carrier" and "cover" embodiments in a unified form, in a form that can be joined in an array, and in a form that collectively comprises multiple "carriers" or "covers." While one of these forms can be referred to in each of the embodiments below, it should be understood that the other forms can be substituted in certain cases.

[0097] Example 1 - Cell Culture Array

[0098] Figure 1 An assembled cell culture array 100 is shown, having an array cover 110 placed on an array carrier 120. Figure 1 A linear cell culture array is depicted, 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 is adjacent to the next unit cover, forming a plane on the top exterior of the array cover 110, with a slight groove between each unit cover. The cover is formed of four walls, including a rectangular planar top wall 140 terminating in a planar left end wall 150 (not shown) and a planar right end wall 160, which is formed at a 90 degree angle and extends downward from the top wall 140. A series of annular openings 170 are formed through the top wall 140 across the length of the top wall; one opening is formed 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, which have left base flanges 180 (not shown) and right base flanges 190.

[0099] The left and right base flanges can be configured to engage with other components, such as robotic equipment, petri dishes, additional cell culture units, or other laboratory equipment; or, as shown, they can be configured to simply provide stability to the cell culture array 100 when placed thereon. Figure 1

[0100] In addition to the top wall, left end wall, and right end wall, the array cover 110 also includes a front wall. Figure 2 ​The front wall 200 of the array cover 110 is shown. The rectangular planar front wall 200 of the array cover 110 extends downward from the top wall 140 at approximately 90 degrees and extends between the leading edges of the left end wall 150 and the right end wall 160. The bottom edge of the front wall 200 is flush with the bottom edges of the left end wall 150 and the right end wall 160. A vertical groove 210 within the front wall 200 defines the next unit cover and extends through the array cover, forming a continuous groove within the top wall. A series of annular openings 220a, 220b, 220c, 220d, and 220e are formed by the front wall 200 spanning the width of the top wall, as one opening is formed through the front wall of each unit cover. Each annular opening is defined by an annular connector 230 projecting from the surface of the front wall. The annular connector is configured to connect to any number of different devices, but most commonly a simple silicon tube connector capable of forming a fluid seal with a silicon tube.

[0101] Figure 3 The back of the opening of the array cover 110 is shown, the array cover having an array carrier 250 located therein. Vertical grooves 210 between each unit cover within the array cover extend through the array covers between inner walls 260, which complete the formation of each unit cover within the array cover. Figure 3 As shown, the inner wall 260 forming each unit cover is shaped or formed to fill the space between adjacent unit carriers to minimize the gap between the unit carrier and the unit cover. This allows the shape of the unit cover to guide the sliding placement of the unit carrier and also minimizes the gap between the outer wall surfaces of each unit cover. When formed in an array, the repeatable nature of this configuration ensures that each unit carrier of the array carrier can easily slide to the desired position relative to the array cover and be securely positioned therein.

[0102] A sliding mechanism 270 is provided at the bottom edge of each unit cover wall to reversibly and securely connect one unit cover to the next, forming an array of covers. The sliding mechanism allows individual cell culture units to be assembled in an array, but is also easy to separate from each other, so that one cell culture can be treated differently from another, with minimal damage or interference to the cells in culture (from unnecessary treatment).

[0103] pass Figure 4 Perspective view in, and Figure 5 The intuitive view in the middle, Figure 4 and Figure 5 The base of the cell culture array is shown. These figures illustrate the configuration of the bottom surface of the array carrier, showing the shape of the inner wall of the individual unit cap and the base flanges (left base flange 180 and right base flange 190). The sliding mechanism 270 extends only through a portion of the inner wall of the individual unit cap.

[0104] Figure 6 ,Figure 7 and Figure 8 showing the array cover and array carrier separated and side by side, and providing a top perspective view, a back perspective view, and a bottom perspective view. Referring to Figure 6 , the array carrier 120 is composed of a linear cell culture array carrier having five repeating unit carriers 280a, 280b, 280c, 280d, and 280e arranged side by side. Each unit carrier includes a media inlet 290 protruding from the outer front surface of the unit carrier, a cell cradle 300 formed within the unit carrier, and a guide channel 310 formed between the cell cradle and the exterior of the unit carrier 280. The media inlet 290 allows for the perfusion of liquid media (or other fluids) into or through the cell environment within the carrier. Fluids can also pass from the cell environment within the carrier through the guide channel 310 to the exterior of the unit carrier through a channel outlet 320. Figure 7 shows the base of the channel outlet 320, the bottom surface of the guide channel 310, and the alignment of the hole formed by the media inlet 290. Figure 7 shows the clear line of sight through these openings, so any turbulence caused during cell perfusion through the media inlet 290 is unlikely to affect cell growth in the cell cradle 300.

[0105] Figure 6 and Figure 7 shows the relative size of the annular connector 230 of the array cover 110 relative to the media inlet 290 of the array carrier 120. The outer surface of the media inlet 290 is configured to be slightly smaller than the inner surface of the annular connector 230. Thus, when the array cover is slid over the array carrier, the outer surface of the media inlet 290 is able to very closely match the inner surface of the annular connector 230. As a result, fluid can pass through the annular connector and media inlet without leaking fluid through these contact points.

[0106] Figure 6 and Figure 8 shows the position of the annular opening 170 of the array cover 110 relative to the array carrier 120. Turning to Figure 8Further shown is that the sliding mechanism 270 consists of slides 330 between the individual cell lids of the array lid 110, which terminate towards the back of the array lid 110 in front of a gap 340 between the individual cell lids of the array lid. The slides 330 and the gap 340 of the array lid 110 engage with the slider 350 on the array carrier 120. When placed in an overlapping manner, the slider 350 slides on the slides 330 and is guided by the space that allows the slider 350 to slide between the individual cell lids. When the slider 350 reaches the gap 340, the sliding stops and can rest in a closed position therein. In the open position, the slider 350 is in contact with the slides 330. In this position, the annular opening 170 is located above the cell cradle 300 and is properly aligned with the cell cradle 300 for optimal placement of the egg or other cell from above in the center of the cradle. In the closed position, the slider 350 is moved and stopped within the gap 340, whereby the annular opening 170 is completely located above the guiding channel 310 and covers the cell cradle 300 from above. The cradle outlet 360 is also shown in the array carrier 120. However, this feature is optional and, if it is provided, allows other channels to reach the cell cradle, enabling removal or transplantation of the embryo or its surrounding medium. Figure 8

[0107] The drawings show a linear array configuration, however, as will be apparent to the skilled person, the array configuration can be extended beyond five cells and can also easily be adapted to a non-linear array configuration. For example, the linear array configuration can easily be adapted to a circular array, which can more easily be modified to be usable for automation or robotic handling techniques. For example, the circular array can be mounted on a turntable, which can then in turn be mounted in a microscope or other visualization device. The turntable arrangement can more easily be accessed by an operator, a handheld device or a robotic device for pipetting, for introduction of a vacuum manifold, for introduction of a pump system or for implementation of a cryopreservation process.

[0108] Example 2 - Cell culture unit

[0109] Figure 9a and 9b A configuration of the cell cradle 300 is provided and shown, which is separate from the guiding channel 310 (as shown in Figure 6 ); Figure 10a and 10b An internal configuration of the complete single cell culture unit carrier 400 is shown. The cell unit carrier 400 is formed by five walls, including a rectangular planar left side wall 410 and a rectangular planar right side wall 420, each wall terminating towards the back of the unit in a planar back outlet wall 430 connected at a 90 degree angle and towards the front of the unit in a planar front inlet wall 440 connected at a 90 degree angle. Figure 9a and 9b ​The cell cradle shown measures approximately 0.23 mm at its widest point and approximately 0.23 mm at its highest point.

[0110] The annular protrusion from the front surface of the inlet wall 440 forms a media inlet 290 having a diameter approximately equal to (but slightly less than) the inner surface diameter of the annular connector 230( Figure 6 ). The inner surface of the front inlet wall 440 is curved horizontally to enable a pipette to be introduced through the passage outlet 320 and guide channel 310 into the cell cradle area, orienting the center point of the cell cradle area on a horizontal axis. The inner surfaces of the left side wall 410, right side wall 420, and rear outlet wall 430 are generally flat. The left side wall 410 and right side wall 420 each include an overflow hole 450 to allow media to overflow from the cell cradle 300, especially when used to perform cell perfusion. As shown, the lowest point of the overflow hole 450 is at the same height as the lowest point of the guide channel 310, which ensures that excess liquid is preferentially removed from the cell cradle area through the side of the cell culture unit rather than through the guide channel 310. Figure 9b

[0111] Example 3 - Cell Cap

[0112] Figure 9c A cell cap unit 600 is shown, configured for linear array assembly with other cell cap units and for housing a single cell cradle. This figure shows a "jigsaw puzzle" clip and lock system 610a and 610b that allows each cell cap unit to interlock with one another in an infinitely expandable linear array. Figure 9c An embodiment of a cell cap unit is also shown that does not have a hole for access to the cell culture chamber, but instead relies on a slidable engagement within the cell carrier unit to cover or uncover at least a portion of the opening from above. This embodiment of a cell cap unit shows a groove 620 for alignment with the inlet channel 470 of a cell cradle.

[0113] Figure 9d and Figure 9e Cell cap units are shown fully arrayed in a linear cell cap cassette 700, which has the ability to form a three-dimensional array. On the left and right sides of the cell cap cassette, "jigsaw puzzle" clip and lock systems 710a and 710b hold orientation with subsequent units, allowing an array to be formed in these directions. Variations of this system 720a and 720b are also shown on the front and back of the cell cap cassette, which similarly hold orientation with subsequent units and allow an array to be formed in these directions. Above the cell cap cassette is a tab 730a that is configured to fit into a reverse-shaped groove 730b below the cell cap cassette to stack in this direction.

[0114] Figure 10a ​and Figure 10b (as well as Figure 9a and 9b This shows the flow from guide channel 310 ( Figure 10a and 10b The inner wall 460 of the cell cradle 300 is positioned such that it maintains a consistent height relative to the front inlet wall 440, left side wall 410, right side wall 420, and rear outlet wall 430, so that the array cover 110 (or unit cover) remains flush with the top of the unit carrier, which would otherwise open from above. The inner wall 460 has a longitudinal inlet channel 470 formed therethrough. Figure 10a and 10b As shown, the lowest point of the inlet channel 470 is at the same level as the base of the guide channel 310. The inlet channel 470 opens towards the rear of the unit carrier to the guide channel 310 and towards the front of the unit carrier to the inner surface of the cell cradle 300. The inner surface of the cell cradle defines a cell culture chamber 510 for culturing and / or growing cells therein.

[0115] The size and shape of the cell culture chamber 510 are determined by the maximum size of the developed embryo to ensure the physical stability of the embryos contained therein. The cell cradle 300 is approximately 0.23 mm × 0.23 mm in size. The surface shape of the cell cradle is generally circular to conform to the general shape of the cell cluster. In particular, each wall gradually tapers downwards to give the cell cradle 300 a more rounded internal shape.

[0116] The cell culture chamber 510 is located away from the flow of the perfusion fluid. The orifice, defined by the media inlet, rear outlet wall, guide channel, longitudinal outlet channel, and overflow orifice, is typically horizontally aligned, thus defining the fluid path. The cell culture chamber is positioned below the fluid path to ensure that the cells in the cell cradle remain immersed in the liquid medium and are not subjected to physical agitation or other disruption from the flow along the fluid path. The walls of the cell culture chamber terminate at a cell cradle base 520, which is typically formed on a horizontal plane and positioned lower than the orifice defined by the media inlet, rear outlet wall, guide channel, longitudinal outlet channel, or overflow orifice. The cell cradle base 520 also has a cell cradle outlet 530, which is closed during use but can be released to drain fluid from within the cell cradle 300.

[0117] Figure 10cAn alternative embodiment showing features of the cell carrier unit 800, including a narrow guide channel 810, a circular channel entrance 820, and a triangular footprint cell culture chamber 840. The narrow guide channel 810 and circular channel entrance 820 work in concert to allow the guided instrument and its insertion into the cell culture chamber 840 without the need to move or exit through the narrow guide channel, but still allow for the observation of the instrument as it passes through the narrow guide channel. The triangular footprint of the cell culture chamber 840 demonstrates other shapes that can be suitable for the cell culture chamber to employ without the need for a curved proximal wall.

[0118] Example 4 - Cell Cap and Cell Carrier

[0119] Figure 10d 、 Figure 10e 、 Figure 10f 、 Figure 10g and Figure 10h illustrates how the cell cap cassette 900 according to an embodiment engages with the cell carrier unit 950 according to an embodiment. Figure 10d A rear perspective view of the cell cap cassette 900 and cell carrier unit 950 unassembled is shown, allowing for the insertion of the cell carrier unit into the center cap unit. Figure 10d Further shown is the cell culture chamber 960 open from above, and the annular opening 920 configured to allow access therethrough. A feature of the cell culture chamber 960 of this embodiment is that it has an overflow hole 965 on each side, allowing for potential overflow to occur from the cell culture chamber through similarly positioned cap overflow holes 925.

[0120] Figure 10e and Figure 10g shows the cell cap cassette 900 and cell carrier unit 950 partially engaged in a first position, where the cell culture chamber 960 is accessible from above through the annular opening 920 for cell deposition, processing, and recovery, and the guide channel 980 is accessible from above and through the channel entrance 970.

[0121] Figure 10f and Figure 10g shows the cell cap cassette 900 and cell carrier unit 950 fully engaged in a second position, where the cell culture chamber 960 is covered by the cell cap cassette 900, and the media inlet 990 is exposed through the front of the cell cap cassette. In this position, the overflow holes 965 are adjacent to the cap overflow holes 925, allowing overflow to pass therebetween. Sight lines are maintained through the channel entrance and into the cell culture chamber 960, and through the media inlet 990.

[0122] Turning to FIG. 9, a perspective view of the cell cap cassette 900 and cell carrier unit 950 is shown, with the cell cap cassette 900 in a first position and the cell carrier unit 950 in a second position. Figure 10a and 10bThe outer shape of the unit carrier 400 shown, the left side wall 410 and the right side wall 420 are connected at about 100 degrees outward to the base wall 480, while the rear exit wall 430 and the front entrance wall 440 are connected at 90 degrees to the base wall 480. Figure 9a , Figure 9b , Figure 10a and Figure 10b The left side slide flange 490 and the right side slide flange 500 are shown protruding from the base wall 480. The left side flange 490 and the right side flange 500 are shaped or otherwise configured to secure the base of the unit carrier within or on another object, or to slide over or through another object. Figure 11a , 11b and 11c show the placement of the unit carrier 400 within the array cover 110. Figure 11a A rear view of the unit carrier 400 is provided, showing the positioning of the channel exit 320. Figure 11b A front view of the unit carrier 400 is provided, showing the positioning of the media inlet 290 within the annular connector 230 of the array cover 110. Figure 11c A bottom perspective view of the unit carrier 400 is provided, with the left side flange 490 and the right side flange 500 in slide engagement on the slide 330, respectively.

[0123] In certain embodiments, the slide 350 shown in Figure 8 may be truncated to enable a single unit carrier of the array carrier 120 to be opened or broken apart (with or without the use of specialized tools) into individual unit carriers. When the array carrier 120 is configured to be broken apart into individual unit carriers in this manner, the array is configured to provide left and right flanges upon breakage.

[0124] The ability to separate individual cell culture carriers or unit carriers from the array can provide advantages for the cryopreservation of cells. For example, individual units can be prepared in the carriers for cryopreservation and stored as small portions after the unit is separated from the array without any further physical manipulation of the cells.

[0125] The small batch production of the cell culture array described above can be performed using 3D printing technology of biocompatible polymeric materials. Certain polymers have been shown to be printable biocompatible materials and also shown to be resistant to breakage when prepared for cryopreservation, for example, nanopolymers or crystal polystyrene.

[0126] Example 5 - Use of the cell culture array

[0127] The embodiments described herein can be used for any type of cell culture, but have shown particular use in the culture of mammalian cell lines. The embodiments described herein are particularly useful for cell cultures involving embryogenesis, and in turn can be used in subsequent IVF procedures.

[0128] The examples can also be used for general cell culture, static perfusion, or active perfusion of cells in culture.

[0129] like Figure 12 As shown, a single cell culture unit can be used independently outside of an array configuration. A single silicon tube can be attached to the unit via a ring connector, which can be used to position or place the unit as needed. This tube can be used to hold the unit in place for observation of cells under a microscope, or to manipulate cells using micropipettes, etc. The tube can also be used to fix the unit's position in a liquid medium, which can be static or flowing (e.g., in a medium within a larger container where the unit is 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 medium held therein.

[0130] As shown in Figure 13, the cell culture array contains five cell culture units, with 100 μm inner diameter silicon tubes attached to three of the five units. Cell culture medium is perfused through the carrier in each of the three units, while the other two units are not perfused.

[0131] Figure 14 A component is provided for holding cell culture arrays within an atomic force microscope (AFM) mounting assembly. Variations of this component can be used when mounting cell culture arrays within automated or other equipment, for example, to tailor the mounting assembly for users of lasers, piezoelectric injection, micropumps, vision “training” robots, or other equipment, to reduce or eliminate human intervention by embryologists during cell culture.

[0132] Figure 14 The assembly shown retains a culture dish base with a glass disk of 35 mm in diameter and 1 mm in thickness for mounting cell culture arrays or cell culture units, such as... Figure 12 As shown in Figure 13. Petri dishes are specifically designed to maintain the contents on them. Figure 12 The silicon tube shown in Figure 13 can be used, or it can simply be clamped into place. The culture dish is mounted using a special support that accommodates 12mm or 25mm circular coverslips for high-NA inverted optical microscopy. This assembly allows cells maintained within an array or unit to be cultured and imaged in the dish.

[0133] Figure 14The enclosed assembly provides a fitment for a culture dish on an enclosed fluidic cell holder. The holder is sealed to a membrane threaded holder and secured to an enclosed fluidic cell culture dish. Access to the inlet / outlet to the culture dish is provided by a port plug and an inlet / outlet tube is provided that passes through the enclosed fluidic cell culture dish. The assembly is sealed to a threaded base holder that is attached to an AFM by installing an O-ring between the enclosed fluidic cell culture dish and the glass plate. It is well known to those skilled in the art that additional O-rings need to be replaced or used, or assembly tools, tweezers, and cleaning brushes are used to achieve successful installation.

[0134] The cell culture array is designed for use in addition to culture dishes, microscope slides, or other culture plates. It does not replace these devices, but is used in conjunction with these existing culture devices to position cells or cell aggregates within these devices, to manipulate or store cells without disturbing the cells, and to make it easier to work with the cells. The above assembly is exemplary in nature and can be readily adapted to the specific use of those skilled in the art. For example, some users can prefer to place microdevices on a slide that can be easily placed in the above assembly in place of a culture dish.

[0135] Figure 14 The assembly is implemented in a perfusion assembly for enclosed and sealed array perfusion. Access ports are used for liquid and gas exchange for array perfusion by syringe injection, gravity feed, and micro-pump systems. The AFM installation can be used for time-lapse microscope imaging, embryo culture (with and without perfusion), and vitrification in addition to fertilization using or not using intracytoplasmic sperm injection (ICSI). The manufacturer-recommended polymer, materials used, and properties were used (the device was microfabricated using a Nanoscribe GT Professional machine (Nanoscribe GmbH, Germany)).

[0136] Example 6 - In vitro study design

[0137] 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. Prepubertal CBA x C57B1 / 6 Fl hybrid and Swiss albino female mice (9-11 g, 3-4 weeks of age) were housed in Laboratory Animal Services (University of Adelaide, Australia) under temperature-controlled, 12-hour light-dark cycles (12 hours light: 12 hours dark), with free access to water and food.

[0138] Prepubertal female mice were given 5 IU equine chorionic gonadotrophin (eCG; Folligon, Intervet, Boxmeer, The Netherlands) intraperitoneally to induce superovulation, and 47 hours later, mice were given human chorionic gonadotrophin (hCG; Humagon, Orgenon) intraperitoneally.

[0139] Mice were then mated with males from the same strain (1 male: 1 female) and checked for copulation plugs the next morning. Mice were sacrificed by cervical dislocation 22 hours after hCG and presumptive zygotes were collected from the ampulla and randomly allocated to each treatment group.

[0140] Culture media used in the study were sourced from ART Lab Solutions (Adelaide, Australia) and included embryo flushing and cleavage culture media.

[0141] Microfabrication designs were developed in CAD and manufactured using a Nanoscribe GT Professional (Nanoscribe GmbH, Germany) using the manufacturer’s recommended polymer, material and settings for microfabrication.

[0142] All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). Statistical analyses were performed to compare embryo development in standard embryo culture and embryo development in standard culture inside Pods docked inside Garages in the presence of other variables (as described below). To determine whether parametric or non-parametric tests should be used, a normality test was first performed. Statistical significance of differences in mean values between groups was assessed using unpaired t-test (for normally distributed data) or Kruskal-Wallis test (for non-normally distributed data). P values < 0.05 were considered as significant differences, and 10% differences were considered as biologically significant differences.

[0143] Example 7 - Preliminary analysis of safety

[0144] A preliminary experiment was performed to investigate the toxicity of the 3D printable polymer provided by Nanoscribe and used to build the cell culture array and unit and determine its potential impact on embryo development.

[0145] The table below provides the results of the first repetition of the analysis of embryo development from day 1 (zygote) and day 2 (two-cell) embryos to day 5 (blastocyst) embryos.

[0146]

[0147] - In vivo: blastocysts were collected 94 hours after hCG and mating

[0148] - Treatment 1 : putative zygotes were collected and cultured in fresh cleavage medium according to standard embryo culture protocol

[0149] - Treatment 2: putative zygotes were collected and cultured in cleavage medium used for washing gadgets

[0150] - Treatment 3: putative zygotes were collected and cultured in fresh cleavage medium + gadgets

[0151] - Treatment 4: putative zygotes were collected and cultured in cleavage medium + gadgets used for washing gadgets

[0152] The table below provides the results of the second repetition of the analysis of embryo development

[0153]

[0154] - In vivo: blastocysts were collected 94 hours after hCG and mating

[0155] - Treatment 1 : putative zygotes were collected and cultured in fresh cleavage medium according to standard embryo culture protocol

[0156] - Treatment 2: Collect putative zygotes and culture in cleavage medium for cleaning of the small tool

[0157] - Treatment 3: Collect putative zygotes and culture in fresh cleavage medium + small tool

[0158] - Treatment 4: Collect putative zygotes and culture in cleavage medium for cleaning of the small tool + small tool

[0159] The following table provides the results of the third repetition of the embryo development analysis

[0160]

[0161] - In vivo: Collect blastocysts 94 hours after hCG and mating

[0162] - Treatment 1 : Collect putative zygotes and culture in fresh cleavage medium according to standard embryo culture protocol

[0163] - Treatment 2: Collect putative zygotes and culture in cleavage medium for cleaning of the small tool

[0164] - Treatment 3: Collect putative zygotes and culture in fresh cleavage medium + small tool

[0165] - Treatment 4: Collect putative zygotes and culture in cleavage medium for cleaning of the small tool + small tool

[0166] Further studies were performed inserting four carrier units into two array carriers. Embryo culture was performed in 20 μΙ_ droplets of cleavage medium. Mouse embryos from highly stimulated and mated female 4-week-old Fl CBA x C57B16 mice were cultured from zygotes for 24 hours to the 2-cell stage. Results from four replicates (40 replicates per group) of zygote culture to blastocyst in 4 days showed no statistically significant difference in viability between cells cultured in carrier units and cells cultured in culture dishes.

[0167] Results of the 3D printer polymer toxicity study are shown in Figure 15a which shows the percentage of CBA Fl mouse embryo development rate from cleavage embryos. Briefly, 10 embryos were cultured in 20 μΙ_ droplets of cleavage medium. For the treatment groups, embryos were cultured in devices produced using 3D printing technology, which included 2 array covers and 10 unit carriers, configured as a 2 x 5 array of units. The 10 embryos were placed in 20 μΙ_ droplets of cleavage medium. The droplets were covered with paraffin oil.

[0168] Embryos assigned to Group 1 were cultured in clean cleavage medium (control). Embryos assigned to Group 2 were cultured in cleavage medium prior to exposure to 10 pods and 2 reservoirs. Embryos assigned to Group 3 were cultured in new cleavage medium and co-incubated with each droplet of culture containing 10 pods and 2 reservoirs. Embryos assigned to Group 4 were cultured in clean cleavage medium and co-incubated with each droplet containing 10 pods and 2 reservoirs.

[0169] Figure 15b The percentage of CBAF1 mouse embryos developing from cleavage embryos is shown. Embryo culture was performed in 10 μL droplets of cleavage medium. Embryos assigned to the control treatment group were cultured under standard culture conditions, and embryos assigned to the study treatment group were cultured under standard culture conditions docked in pods within reservoirs. The study treatment culture group had 5 pods and 1 reservoir per droplet (mean ± SEM).

[0170] The percentage of DNA repair of CBAF1 mouse embryo development after blastocyst γH2A.X DNA repair staining for the control and study treatment groups is shown. Figure 15c Embryos assigned to the control treatment group were cultured under standard culture conditions, and embryos assigned to the study treatment group were cultured under standard culture conditions docked in pods within reservoirs (mean ± SD).

[0171] In all studies, no significant differences were observed in the treatment groups. No toxicity of the materials used in the manufacturing process was shown, indicating that the microdevice can be safe.

[0172] Example 8 - Optimization of embryo culture conditions within the cell culture unit

[0173] Figure 16 A schematic showing the vascularisation of tissue achievable by using the cell culture unit according to the embodiments is provided. It is expected that under optimal conditions, organoids will develop successfully within the cell culture chamber. The chamber coated with a Matrigel coating provides a scaffold environment to promote the proliferation of adherent cells. Directed linear growth of cell masses is provided through additional pores between the cell culture chamber and the external environment. The location and size of the pores are selected by the cell carrier and the cell cover to promote vascularisation, supporting growth in situ. These depend on the cell type and organoid type and can be determined by the person skilled in the art.

[0174] Cell culture conditions for embryo development were optimized. The optimal culture medium and air mixture were determined using a method that can be adapted for other condition optimizations. It is expected that the optimized cell culture conditions can be transferred to the proliferation of other cell types. Putative zygotes were collected and randomly assigned to five treatment groups. Embryo development was observed and recorded daily.

[0175] Embryos were divided into five groups, each receiving a different media treatment. For embryos assigned to Group 1, embryos that developed on time were moved to a new cleavage culture drop in the same dish. Embryos for Group 2 were placed in cleavage medium and then moved to a new dish containing a new cleavage culture drop on day 3. Embryos for Group 3 were placed in G1+ medium and viable embryos were moved to a new G1+ culture drop in the same dish. Embryos for Group 4 were placed in G1+ medium and then moved to a new G1+ culture drop on day 3. Embryos for Group 5 were placed in G1+ medium and then moved to a new dish containing a G2+ culture drop. Embryo culture was performed in a humidified incubator at 6% CO2, 5% O2, and 37°C.

[0176] Figure 17 shows the percentage of embryo development for embryos cultured in standard 10 pL culture drops with oil overlay in a dish. Embryos for Group 1 were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 5. Embryos for Group 2 were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 3 and then embryos were moved to new ART Lab Solutions embryo cleavage medium and cultured to day 5. Embryos for Group 3 were cultured in Vitrolife G1+ medium from day 1 to day 5. Embryos for Group 4 were cultured in Vitrolife G1+ medium from day 1 to day 3 and then embryos were moved to new Vitrolife G1+ medium and cultured to day 5. Embryos for Group 5 were cultured in Vitrolife G1+ medium from day 1 to day 3 and then moved to new Vitrolife G2+ medium and cultured to day 5.

[0177] Embryo development was recorded daily; the percentage of embryo development for each treatment group is shown in Figure 17. Figure 17a showing the percentage of embryo development from day 1 to day 2, Figure 17b showing the percentage of embryo development from day 1 to day 4, Figure 17c showing the percentage of embryo development from day 1 to day 5 (mean ± SD). By day 5, embryos cultured in Vitrolife G1+ medium that were subsequently cultured in Vitrolife G2+ medium starting at day 3 showed a significant improvement in development.

[0178] Figure 17dResults of further studies are provided in which cells were cultured within cell unit carriers and covered by a cell unit lid. Group 1 embryos were cultured in Vitrolife G1+ medium from day 1 to day 5. Group 2 embryos were cultured in Vitrolife G1+ medium from day 1 to day 3, then the embryos were moved to new Vitrolife G1+ medium and cultured to day 5. Group 3 embryos were cultured in Vitrolife G1+ medium from day 1 to day 3, then moved to new Vitrolife G2+ medium and cultured to day 5. Figure 17d Embryo development percentages from day 1 to day 5 for each treatment group are shown when cells were cultured in a device according to the present application.

[0179] Figure 17e Percent intensity of gH2a.x staining is shown, which illustrates DNA repair of embryos cultured in standard 10 μL droplets of culture medium with oil overlay in culture dishes (control is in vivo blastocyst). Group 1 embryos were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 5. Group 2 embryos were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 3, then transferred to new ART Lab Solutions embryo cleavage medium and cultured to day 5. Group 3 embryos were cultured in Vitrolife G1+ medium from day 1 to day 5. Group 4 embryos were cultured in Vitrolife G1+ medium from day 1 to day 3, then moved to new Vitrolife G1+ medium and cultured to day 5. Group 5 embryos were cultured in Vitrolife G1+ medium from day 1 to day 3, then moved to new Vitrolife G2+ medium and cultured to day 5 (mean ± SD).

[0180] Figure 17fThe percentage of inner cell mass (ICM) to total cell number (TCN) for embryos cultured in standard 10 μL droplets of culture medium overlaid with oil in a petri dish is shown (control is in vivo blastocyst). Embryos in group 1 were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 5. Embryos in group 2 were cultured in ART Lab Solutions embryo cleavage medium from day 1 to day 3, then moved to fresh ART Lab Solutions embryo cleavage medium and cultured to 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 moved to fresh Vitrolife G1+ medium and cultured to day 5. Embryos in group 5 were cultured in Vitrolife G1+ medium from day 1 to day 3, then moved to fresh Vitrolife G2+ medium and cultured to day 5 (mean ± SD).

[0181] DNA repair and percentage of inner cell mass were improved for all treatment groups of moved embryos compared to non-moved embryos in the same medium. For DNA repair and percentage of inner cell mass results, embryos cultured in Vitrolife G1+ medium followed by Vitrolife G2+ medium exhibited improved development compared to embryos transferred to Vitrolife G1+ medium at day 3.

[0182] The problem of poor development of embryos cultured in Vitrolife G1+ medium followed by Vitrolife G1+ medium was addressed by conducting further studies with the same interventions on embryos cultured in cell unit carriers and cell unit lids. While Vitrolife G1+ medium followed by Vitrolife G2+ medium still exhibited the best growth medium conditions, cells cultured in cell unit carriers and cell unit lids also showed improved development with a change in medium compared to no change in medium. Results showed that cell unit carriers and cell unit lids maintained the same medium for the 5-day growth period improved the growth deficiency.

[0183] Figures 18a to 18d Percentage of embryo development results for embryos cultured in standard 10 μL droplets of culture medium overlaid with oil in a petri dish in air mixtures of 6% CO2, 5% O2, 89% N2 or 6% CO2, 20% O2, 74% N2 and in humidified conditions at 37°C are shown. Figure 18a Percentage of embryo development from day 1 to day 2 is shown, Figure 18b Percentage of embryo development from day 1 to day 3 is shown, Figure 18cEmbryo development percentage from day 1 to day 4 is shown, Figure 18d Embryo development percentage from day 1 to day 5 (mean ± SD) is shown. Figure 18e Percentage intensity of gH2a.x staining is shown, which illustrates DNA repair in embryos cultured in standard 10 μL droplets of culture medium overlaid with oil in the culture dish, with or without increased oxygen-air mixture (control is live blastocyst).

[0184] When embryos were cultured in 20% O2rather than 5% O2, there was a slight decrease in embryo development percentage by day 5, but this result was not consistent throughout the culture period. However, when cells were cultured in the presence of 20% O2, there was a significant improvement in the percentage of DNA repair by day 5.

[0185] These results show that, although cells are traumatized when they are disturbed during development, the replenishment of the culture medium can significantly improve the growth and viability of the cultured cells. This improvement is expected to increase by employing continuous perfusion to introduce fresh culture medium, and when the cells are not disturbed during perfusion. The inconsistency in the development and viability results over time reflects the different needs of the embryos during development at each growth stage. Optimizing the growth medium and other culture conditions for static perfusion at each growth stage is expected to further improve the growth results.

[0186] In this specification, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0187] The various devices and device components described herein can be provided in various sizes and / or dimensions as desired. Suitable sizes and / or dimensions will vary depending on the size of the connection assembly or field of use, which can be selected by one of skill in the art.

[0188] It will be appreciated that features, elements and / or characteristics described in relation to one embodiment of the application can be used in conjunction with other embodiments of the application, as desired.

[0189] While the preferred embodiments of the application have been disclosed for purposes of illustration, it will be understood that various modifications, additions and substitutions can be made without departing from the scope and spirit of the application and the appended claims.

[0190] It will be understood that when an element or layer is referred to as being "on" or "within" another element or layer, it can be directly on or within the other element or layer or intervening elements or layers can also be present. In contrast, when an element is referred to as being "directly on" or "directly within" another element or layer, there are no intervening elements or layers present.

[0191] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0192] It will be understood that, although the terms first, second, third, etc. can be used herein to describe various elements, components, regions, layers and / or sections, these

[0193] For the purposes of this description, spatially relative terms, such as "lower," "upper," "top," "bottom," "left," "right," and the like, can be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the structure in use or operation in addition to the orientations depicted in the figures. For example, if a device is turned over, elements described as "lower" relative to other elements or features would then be oriented "upper" relative to the other elements or features. Thus, the exemplary term "lower" can encompass both an orientation of "lower" relative to other elements or features and "upper" relative to other elements or features. The device can be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0194] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" "comprising," "includes" and / or "including" when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0195] Embodiments of the present specification are described herein with reference to cross-sectional illustrations that are schematic illustrations of preferred embodiments (and intermediate structures) of the present specification. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and / or tolerances, are to be expected. Thus, embodiments of the present specification should not be construed as limited to the particular shapes of the regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. Thus, the shapes of the regions illustrated in the figures are schematic and their shapes in use can differ from that illustrated.

[0196] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this specification belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealised or overly formal sense unless expressly so defined herein.

[0197] Any reference in this specification to "one embodiment", "an embodiment", "example embodiment" etc. means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase in various places in the specification is not necessarily all referring to the same embodiment. Further, where a particular feature, structure, or characteristic is described in connection with any embodiment, it is submitted that it is within the purview of one skilled in the art to effect such feature, structure, or characteristic in connection with other

[0198] Embodiments also encompass and otherwise contemplate methods of using and methods of making any or all of the elements described above.

[0199] While the application has been described in terms of specific embodiments, it is to be understood that the disclosure need not be limited to the specifics of the embodiments. Rather, several modifications across the application, and in the constructions and methods set forth, can occur to those skilled in the art upon reading the teachings of the present specification and are to be incorporated within the scope of the application. Particularly, it is to be understood that the application is not to be limited to the particular embodiments illustrated herein, but is amenable to any modifications and / or enhancements which could be made to the methods and apparatus of the present application by those skilled in the art without departing from the scope and nature of the present application.

[0200] All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present application. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present application as it existed anywhere before the priority date of each claim of this application.

[0201] The scope of the application should be determined solely by a proper interpretation and understanding of the following claims and legal equivalents thereof and summarized description herein.

[0202] Citation

[0203] 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).

[0204] 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 therein, comprising: A cell culture unit having at least one first cell carrier unit, the first cell carrier unit defining a cell culture chamber formed therein. The first cell carrier unit is formed from at least the following: The chamber base, shaped as cells supported thereon, and One or more chamber walls, having one or more chamber wall surfaces surrounding the chamber boundary to enclose the cell culture chamber. The first cell carrier unit also provides a guiding surface to guide instruments or fluids to enter the cell culture chamber through pores in the chamber wall. The width, height, and length of the cell culture microdevice are between 0.1 µm and 1000 µm. The cell culture microdevice is configured to be maintained in a liquid medium within a larger container and is capable of passive exchange of liquid fluid with the larger container. The cell culture microdevice is configured to enclose a single cell or cell cluster therein.

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

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

4. The cell culture microdevice of claim 1, wherein the one or more chamber walls comprise: The proximal wall has a curved inner wall surface configured to guide the placement of instruments within the culture chamber. The distal wall defines the boundary of the distal chamber and has an opening formed therein through the chamber wall, the opening defining an opening communicating with an elongated guide portion projecting outward from the cell culture chamber, the elongated guide portion having a channel formed therein, the channel providing a guide surface to guide instruments or fluids into the cell culture chamber.

5. The cell culture microdevice of claim 4, wherein the one or more chamber walls comprise at least one left side wall and at least one right side wall, each side wall having a left side hole and a right side hole formed therethrough.

6. The cell culture microdevice of claim 4, wherein the proximal wall has a proximal pore formed therethrough, the proximal pore being configured to be horizontally aligned with the guide surface to slow the flow of fluid through the cell culture chamber between the pore and the proximal pore.

7. The cell culture microdevice of claim 6, wherein the proximal wall includes an infusion inlet opening adapted to pass through it for fluid perfusion, and a fitting configured for engagement of the perfusion tube with the infusion inlet opening.

8. The cell culture microdevice according to claim 1 or 2, wherein the first cell carrier unit includes a cell chamber wall having an outer wall coupling member adapted to engage with a corresponding outer wall coupling member on at least one second cell carrier unit to form a cell carrier array.

9. The cell culture microdevice according to claim 1 or 2, further comprising at least one second cell carrier unit integrated with the first cell carrier unit to form a cell carrier cassette.

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

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

12. The cell culture microdevice of claim 11, further comprising at least one second cell cap unit, the second cell cap unit being integrated with the first cell cap unit to form a cell cap box.

13. The cell culture microdevice of claim 12, wherein the first cell cap unit further includes an access hole formed therethrough, wherein the first cell cap unit is configured to allow access from above to the opening in a first position and to cover at least a portion of the opening from above in a second position, and is adapted to slidably engage the first cell carrier unit in both the first and second positions.

14. The cell culture microdevice according to claim 1 or 2, wherein the outer surface of the chamber base includes a groove configured to receive a lug projecting outward from the cell carrier unit or cell cap unit.

15. A method of using the cell culture microdevice according to any one of claims 1 to 14, comprising the following steps: At least one cell is placed in the cell culture chamber of the cell culture microdevice and cultured therein.

16. A method of using the cell culture microdevice according to any one of claims 1 to 14, comprising the following steps: Obtain instructions for constructing cell culture microdevices and execute those instructions during additive manufacturing.

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