TITLE
[0001] Low Flow Microfluidic Device
TECHNICAL FIELD
[0002] The technical field generally relates to low flow microfluidic devices, low flow microfluidic systems and methods adapted for use in a variety of cell or tissue culture environments.
BACKGROUND
[0003] Since the widescale availability of cell culture microdevices, microscale cell culture techniques have advanced in sophistication, and their fields of use and application have become much more diverse.
[0004] Microdevices enable the manipulation of cells and their culture environments, giving rise to new therapies, products, and processes, many of which are still in their infancy. New microdevices and improvements to existing microdevices such as microfluidic devices, 'lab on a chip' and 'organ on a chip' technologies, microscaffolds and micromanipulation devices have given rise to new approaches to 3D tissue engineering, stem cell differentiation, and assisted reproductive technologies as well as advances in the success rates of these techniques.
[0005] For example, complex in vitro tissue culture was previously limited in success and application by the limited gaseous exchange possible via diffusion, however the development of microcapillary perfusion devices now enables the cultivation and proliferation of larger and more complex tissues.
[0006] With regard to stem cell culture techniques, and their applications; organ culture systems for various embryonic tissues now enable the cultivation of embryonic brain, retina, cardiac and vascular tissue, limb bud, lung, kidney, salivary gland, hair follicle, and tooth cell lines. The application of new tissue engineering techniques, and the microdevices that enable these techniques, promote new and improved approaches to a number of different therapies.
[0007] The success of culture methods is largely dependent on the level of control of the culture environment, including the rate of supply of nutrients to the cells.
[0008] Current technologies primarily rely on external pump mechanisms to drive fluid flow within the culture environment for control of culture conditions, typically through the application of pressure or mechanical force. Syringe pumps and peristaltic pumps are often used. These and other traditional pumps struggle to deliver consistent flow at very low flow rates.
[0009] As an alternative; on-chip pumps and valves have been fabricated into devices using multi-phase fabrication techniques, such as multi-layer polydimethylsiloxane (PDMS) devices, or sandwich devices with mechanically directed components embedded between two lithographically produced microfluidic chips (Clement Quintard, Emily Tubbs, Jean-Luc Achard, Fabrice Navarro, Xavier Gidrol, Yves Fouillet; Microfluidic device integrating a network of hyper-elastic valves for automated glucose stimulation and insulin secretion collection from a single pancreatic islet; Biosensors and Bioelectronics, Volume 202, 15 April 2022, 113967) (Soohong Kim, Gabriel Dorlhiac, Rodrigo Cotrim Chaves, Mansi Zalavadia and Aaron Streets, Paper-thin multilayer microfluidic devices with integrated valves, Lab on Chip, Issue 7, 2021). However, the complex multiple element nature of the assembly makes failure more likely overall.
[0010] Traditionally produced microfluidic chips, which are usually sealed systems, are difficult devices from which to recover cultured cells without causing damage to the device or to the cells therein, or to monitor the media with any analysis method that is not built into the chip itself.
[0011] Microfluidic systems utilising PDMS are also restricted by the pressure they can maintain. Monolithic microfluidic chips produced by 3D printed methods which are acrylate based allow for greater chip integrity, and therefore the successful handling of more volatile liquids.
[0012] Alternative solutions to overcome these issues are described herein which include:
• The use of integrated, pneumatically activated diaphragm pumps and squeeze valves (or similar) as a method of delivering low-volume flow to cell culture targets, such that flow may be delivered to all targets or to individual targets in isolation, as required.
• The use of dynamic flow, adjustable in real time according to automated programming, operator input or process monitoring feedback, to increase, decrease, halt or redirect flow according to the culture protocols.
• Housing of cultured cells in a 3D printed cradle, or an array of cradle chambers, which are modular and removable, interfacing with the non-sealed microfluidic system, and allowing for ease of cell recovery along with tracking of individual cells or cell groups.
• Housing of cells in a chamber consisting of a 'wine-glass' geometry, restricting the entry into the lower chamber for larger cell complexes, until modification of the complex changes its shape and allows entry.
• The use of monolithic chips to allow for the microflow of high-pressure cryogenic liquids to deliver them to culture targets for their preservation.
• Microfluidic mechanisms of extracting and analysing small aliquots of culture media from the cradle for monitoring purposes.
[0013] Accordingly, the disclosure provided herein describes a divergent approach to cell culture, which provides a number of advantages and improvements over the prior art.
[0014] Currently, nearly all cell culture products and devices occur in static conditions. However, certain functional devices utilise dynamic flow principles to automate bulk media exchange within a dish, for example, the Takasago medium replacement system utilises two reservoirs, one for the replacement medium and one as a waste reservoir. The replacement medium reservoir is connected via a series of discrete tubing to a number of pumps. The pumps, when activated, drive fluid flow from the replacement medium reservoir to individual wells contained within a multi-well plate. A pump driven drain tubing within each well, connected to a waste reservoir, pumps to the reservoir. The pumps are actuated independently from one another to fill or drain the multi-well plates at pre-determined time points (Takasago Electric Inc).
[0015] Outside of custom printed PDMS chips attached to host embedded pumps or custom fluidic systems, there is no known device to generate a dynamic flow at the microscale.
[0016] Current commercial devices rely on external pumps having low control with higher minimum flow rates. Further, fluid pumps are typically not single use devices which necessitates cleaning protocols. As described herein, embedded pumps and valves may be actuated to achieve controlled levels of fluid flow to precise locations, either to one channel in isolation or to multiple channels in parallel.
[0017] A form of single cell housing is known in the prior art which involves the injection moulding of dishes; such as the embryo 'Culture Coin". This polystyrene injected moulded dish is a variant of a standard petri-dish and comprises a series of aligned numbered (1-14) indent depressions within wells, where cells, and especially individual preimplantation embryos, can be segregated and housed apart from one another, to prevent the unintended migration of cells or embryo within the dish. The dish also contains wells adjacent to the indented wells that are for culture medium storage, possibly having a different medium formulation.
[0018] However, the geometries and features achievable with injection moulding do not allow for facilitating a dynamic media flow. The present disclosure provides a single-cell housing which allows for the separation, identification and tracking of single cells throughout the culture processes.
[0019] While dishes with small depressions, and tubes/straws for storing individual cells under cryogenic conditions are known in the prior art; micro-scale arrays comprising single cell compartments as described herein have not previously been disclosed. Arrays comprised of such containers are widely known to involve macro scale containers.
[0020] The present disclosure enables the user to provide multiple media treatments. Currently, the only way to achieve a certain number of different media treatments would involve the same number of different dishes, or different wells in a well plate. The disclosed cradle level control permits multiple media treatments within a single dish, loading only one set of reagents.
[0021] The microfluidic system is designed so that a specific operation of valves and onboard pumps can deliver microfluidic flow and fluids to an individual, or to multiple cell cradles. Resulting in each cradle being able to receive its own unique flow and media profile.
[0022] United States Patent Application No 20230263552A1 describes a method whereby a cumulus oocyte complex is translated by a pump through a channel with ridges to strip the cumulus layer. The present disclosure describes the loading step into a specific single cell housing case that allows for an oocyte with a cumulus layer to be stripped away, via an aperture at the bottom of a conical funnel shape (which is the nominal diameter of the oocytes).
[0023] Current monitoring of cells is limited to optical techniques or to sensors that are required to be built into the chip to perform monitoring. Aliquots cannot be extracted from PDMS sealed systems until the end of culture, at which time the fluidic chip is broken open, or from waste tubing which is an aggregate mixture. As described herein, the fluid that exits the single cell housing device may be extracted via an automated pumping mechanism or a syringe mechanism to generate a fluid sample. The fluid sample may be delivered to a device that will perform analytical tests on the media.
[0024] Current devices exist to fluidically provide cryoprotectants to cells, but this occurs at the macro scale. There are currently no microfluidics-based device known to the applicant that operates with liquified gases at high pressure. Cryoprotective fluids are more viscous than water-based media and require greater pressure or larger channels for equivalent flow rates. Microfluidic channels for delivery of cryoprotectants are optimised for the delivery of higher viscosity fluids to the culture chambers.
[0025] Finally, microfluidic handling of cells is primarily limited to bulk-sorting using aspiration based techniques or by bulk transport, and not specialised transport at the fewcell level as described herein.
[0026] Thus, the disclosure herein provides an integrated solution for cell culture microdevices and systems in which embedded valves may be actuated to achieve controlled levels of fluid flow at low flow and to precise locations, either to one cell or cell mass in isolation or to multiple cells or cell masses in parallel.
SUMMARY
[0027] In a first broad form, the present disclosure relates to a microfluidic device for modulating the flow of fluids in cell culture, the microfluidic device comprising a microfluidic support having thereon or therein; one or more fluid reservoirs for containing culture media therein, the one or more fluid reservoirs having an opening contiguous with a first opening in one or more fluid conduits, the one or more fluid conduits shaped to direct the flow of fluid therethrough, from the one or more fluid reservoirs to one or more cell culture chambers, the one or more fluid conduits having a second opening contiguous with an opening in the one or more cell culture chambers, the one or more fluid conduits comprising one or more valves therein to modulate the flow of fluid through the one or more fluid conduits.
[0028] Microfluidic supports described herein may comprise an upper microfluidic support surface and a lower microfluidic support surface and each of the one or more valves comprises a pump. A preferred pump may be a pneumatic pump.
[0029] Preferred valves of microfluidic devices described herein may be one or more squeeze valves and/or one or more diaphragm valves. [0030] The one or more fluid conduits described herein may provide fluid communication between each one of the one or more fluid reservoirs and each one of the one or more cell culture chambers, wherein the one or more valves may modulate the flow of fluid from each one of the one or more fluid reservoirs and each one of the one or more cell culture chambers.
[0031] The lower microfluidic support surface may comprise one or more inlets, each of the one or more inlets comprising an aperture through the lower microfluidic support surface contiguous with one of the one or more fluid conduits.
[0032] The one or more fluid conduits may comprise a network of tubing or channels comprising one or more pumps to drive fluid flow therethrough and one or more valves to modulate fluid flow therethrough, from the one or more fluid reservoirs to the one or more cell chambers.
[0033] Microfluidic devices of the present disclosure may be formed of biocompatible materials suitable for two-photon polymerisation fabrication.
[0034] The flow of fluid through the one or more conduits of the microfluidic devices of the present disclosure are preferably capable of modulation to a flow rate of between approximately 0.0001 and 0.004 mm/sec, between approximately 0.5 and 1.5 mm/sec, or between approximately 0.5 and 10 mm/sec.
[0035] The present disclosure relates to a microfluidic system for modulating the flow of fluids comprising the microfluidic device described herein and one or more cell cradles for maintaining a single cell or small cell mass therein, wherein the one or more cell cradles are shaped to be contained within the one or more cell culture chambers.
[0036] Such systems may comprise an incubator for modulating the temperature and concentrations of gases therein, further adapted to contain the microfluidic device therein. Preferably, any gases within the incubator are maintained under pressure. [0037] Systems of the present disclosure may comprise an optical system for visualising the single cell or cell mass during cell culture.
[0038] Systems may comprise a channel or micro-track adapted to move the one or more cell cradles on the upper surface of the microfluidic support.
[0039] Certain systems of the present disclosure may comprise the one or more fluid reservoirs containing a cryoprotective composition.
[0040] Conduits of the present disclosure may comprise one or more tubes, one or more channels, a combination thereof or any other means adapted for the communication of fluids.
[0041] Preferably, microfluidic devices described herein comprise an upper microfluidic support surface, which comprises one or more fluid reservoirs thereon, the one or more fluid reservoirs having an opening contiguous with a first opening in one or more fluid channels formed within the microfluidic support surface, the one or more fluid channels each having at least one valve and pump therethrough to modulate the flow of fluid to the one or more cell culture chambers.
[0042] Valves according to the present disclosure modulate the flow of fluid through the microfluidic device, preferably, upon the upper microfluidic support surface.
[0043] Preferably the microfluidic device is adapted for culturing one or more cells thereon.
[0044] Preferably, microfluidic devices according to the present disclosure comprise a network of tubing and/or channels, one or more fluid reservoirs (of which at least one of the reservoirs comprises a fluid exhaust reservoir), one or more pumps to drive fluid flow, one or more valves to actuate flow, a control system, and (optionally) a feedback system. [0045] Additionally, the devices disclosed herein may comprise channels to deliver fluid to a reservoir, within which one or more cell cradles, or removable cell capsules, may be maintained.
[0046] The flow of fluid within the microfluidic device to cell cradles comprising one or more cells therein may be stopped at predetermined positions to allow for static (no flow in either direction) conditions as required.
[0047] Where more than one type of media is delivered by the system, microfluidic devices may comprise mixing channels or chambers to ensure that solution equilibrium is achieved. Thereafter, exposure to biological elements may commence.
[0048] Systems disclosed herein may optionally allow for the flow of media for cell culture, cleaning agents for sterilisation, or cryoprotectants to allow for cell preservation following culture, and the interchange between these fluid types.
[0049] Microfluidic devices and systems disclosed herein may comprise a network of tubing and 2PP printed channels with thin, mechanically flexible membranes spanning the channels.
[0050] Additionally, microfluidic devices and systems disclosed herein may comprise a pneumatic supply and control system.
[0051] The present disclosure relates to methods for use of microfluidic devices and systems. The timing of the repeated programmed sequences of such methods can be modified to achieve higher or lower flow rates.
[0052] The one or more valves of the microfluidic devices, systems and methods disclosed herein may actuate the flow of fluid through the microfluidic device by a singular pneumatic channel (i.e. a channel containing fluids driven by pressurised gas), allowing for multiple embedded pumps to run simultaneously on a singular, shared programmed sequence, or the cessation of fluidic flow to all single cell holding devices. [0053] Incubators used for cell culture are typically supplied with a premix of reduced air comprising various concentrations of oxygen, carbon dioxide, nitrogen or air gas (either separately or mixed in various proportions) for the purpose of modulating the level of oxygen dissolved in the media and the regulation of the pH of the media.
[0054] Gases are typically provided under pressure where they can be harnessed to drive a dedicated pump. Further, pressurised ambient gases such as incubator gases may be used to drive additional pumps and/or to actuate the valves described here. This is termed 'incubator supply'.
[0055] Incubator supply pressure can be used to drive the pumps and/or valves described herein or they can be driven by an additional source of pressurised fluid. The pressure that drives the pumps and actuates valves is the 'pneumatic supply' whether it is sourced from the 'incubator supply' or some independent source of gas pressure.
[0056] Incubator supply pressure is one source of 'pneumatic supply' which may be utilised to drive the pumps and/or actuate the valves described herein. Preferably, pneumatic supply comprises incubator supply, preventing changes in gas concentration from impacting the osmolarity or pH of the fluid or media in the event of valve pneumatic leakage (gas from pneumatic channels impacting on the fluidic channels).
[0057] The pneumatic supply of preferred methods is preferably the same as the incubator supply, preventing changes in gas concentration from impacting the osmolarity or pH of the fluid or media in the event of valve pneumatic leakage (and gas from pneumatic channels impacting on the fluidic channels).
[0058] Valves may be either closed, or open in the fluidic design to allow for fail safe or fail secure functionality. The valves described herein are open in their inactive state and can be actuated to close, however a valve that is closed in its inactive state is envisaged and is preferred. [0059] The valves described herein may be actuated by one of several means, for example, they may be actuated by solenoids.
[0060] The system may be primed by the operation of the on-board diaphragm pump(s) and squeeze valve(s), or optionally by use of manually applied driving pressure from an external system.
[0061] The design of the one or more fluid conduits and/or a network of fluid conduits of the microfluidic devices of the systems described herein comprise a large reservoir within which components of the device or system are maintained. Preferably the components within the large reservoir comprise one or more cells which may, for example, be maintained in a cell cradle or similar. The large reservoir acts as a passive sink through which media can be changed, media formulations can be altered, or samples can be taken for further testing.
[0062] The size and shape of devices and systems may be scaled to achieve a flow rate that mimics the environment of the cell or tissue type. Devices and systems may aim to replicate somatic vessels, for example blood vessels, lymphatic vessels, ventricular vessels, urinary vessels and the like, or reproductive vessels for example the fallopian tubes.
[0063] The flow rate of devices and systems configured to provide an interstitial flow rate deliver fluid at approximately 0.07 - 3.2 pL/sec. The flow rate of devices configured to provide a capillary flow rate deliver fluid at approximately 25 - 75 pL/sec. The flow rate of devices configured to provide a venule or arteriole flow rate deliver fluid at a velocity of at approximately 0.12 - 2.55 microL/sec.
[0064] The flow rate of devices and systems may be configured to provide fluids having similar physical properties to blood at an interstitial flow rate whereby fluid is delivered at a velocity of 0.0001 - 0.004 mm/sec. To provide a capillary flow rate fluid is delivered at a velocity of 0.5 - 1.5 mm/sec. To provide a venule or arteriole flow rate fluid is delivered at a velocity of 0.5 - 10 mm/sec. [0065] For oocyte or embryo culture, the flow of fluids within devices and systems described herein emulate conditions within the fallopian tube. Fluid flow rates are very similar to interstitial flow rates. Thus, the preferred flow rate of devices and systems for oocyte or embryo culture occur at a velocity of 0.0001 - 0.004 mm/sec.
[0066] Preferably, systems and devices deliver fluids in a pulsatile rhythm to mimic flow with any one of a number of somatic or reproductive conduits. Pumps and valves, such as evaporative, diaphragm or peristaltic pumps, may be selected which achieve pulsatile flow.
[0067] The very small scale of the devices and systems described herein have a lower impact on the environment that the alternatives available. The very small scale of devices means that they may be manufactured and sanitised as single use devices without the need to reuse and sanitise prior to each use.
[0068] The biocompatible materials from which systems and devices are manufactured are readily degraded and have minimal environmental impact upon disposal.
[0069] The present disclosure provides a single-cell housing which allows for the separation, identification and tracking of single cells throughout culture processes. Single cell 'cradles' consist of 2PP printed structures that may be connected to a microfluidic system. The structures will have geometries that support a range of single cell systems from approximately 10 pm to 500 pm. Structures may be modular, removable and are optionally compatible with multiple systems.
[0070] Any surface of the devices and systems described herein may be surface treated either chemically or physically. For example, embodiments described herein are plasma treated to modify surface wettability to emulate the natural surface of a somatic or reproductive channel. It is envisaged that surface treatment to alter surface hydrophobicity or surface tension will be necessary for certain uses. Typically, the surface modification required will be determined according to the cell type and the conditions present in the natural environment in which it occurs. [0071] Any surface of the microfluidic device may be surface treated or modified, in particular, the one or more conduits, reservoirs or cradle surfaces. Surfaces may undergo chemical or physical surface treatment or functionalisation to improve biocompatibility, material stability, cell loading and cell retrieval, as well as to facilitate connection and disconnection from a larger microfluidic system.
[0072] The one or more reservoirs described herein may comprise at least one single cell housing. The at least one single cell housing may have different geometries for connection and disconnection from a larger microfluidic system, or different geometries to help achieve desired fluid concentrations and gradients.
[0073] The microfluidic devices, systems and methods disclosed herein may comprise one or more cell cradles. Such cradles are nominally transparent to allow for monitoring through an optical system; either a microscope system or other type of camera.
[0074] Optionally, multiple single cell housing cradles may be linked in parallel to form an array of individual compartments, facilitating the ease of loading and unloading larger groups of cells at once, speeding up these processes.
[0075] Cradle arrays may also be stacked vertically to create arbitrarily large two- dimensional or three-dimensional arrays of single cell housing devices for creating large micro factories wherein each cradle contains one cell in contact with fluid flow.
[0076] The micro factories may have modular components allowing for multiple arbitrarily large arrays to be removed and substituted during operation. This may be utilised, for example, for the simultaneous culture of many hundreds of blood cells, stem cells or other cells.
[0077] One device may then allow for the contents of each single cell housing device to be tested using various monitoring techniques and selectively included or discarded. Another embodiment would allow graduated treatment to be conducted across a range of samples in order to optimise the type and dosage of a treatment. [0078] Embodiments may be formed as an array of multiple joined units of a single cell housing.
[0079] Methods may comprise the delivery of media, or other fluids to one, or many distinct large two- or three- dimensional arrays of single cell holding devices.
[0080] Where the network of tubing and printed channels feeds into one, or many distinct, large two-dimensional arrays of single cell holding devices or multi cell holding devices.
[0081] The loading of a large array of single cell holding devices is achieved by loading the array separately and docking it with a larger fluidic system and / or by delivering cells down fluidic channels that bind to specialised sites in the individual compartments of the single cell holding devices.
[0082] Embodiments may comprise a host system that allows for the live interchange of a large two-dimensional cradle array without interruption to other cradle arrays.
[0083] The host system may be informed, via operator input, or RFID communication, or some other signalling method that a new cradle array has been connected or disconnected.
[0084] Embodiments may comprise 2PP printed channels in communication with a single cell housing comprising either an embedded pump; that can optionally be addressed independently without impacting other 2PP printed channels. Further embodiments may comprise or one or more embedded valves to isolate the channel from changes in the system, or a combination thereof wherein a single embedded pump can feed multiple channels, that are independently isolated with embedded valves. Such embodiments may be formed with or without individual monitoring.
[0085] In another aspect, the present disclosure describes a feature of the loading step of use in a specific single cell housing case that allows for an oocyte with a cumulus layer to be stripped away, as the aperture at the bottom of a conical funnel shape, is the nominal diameter of the oocytes.
[0086] The cell can sit in two locations, an upper location and a lower location. While the cell has a cumulus layer, it rests in the upper location and is fluidically accessible via the lower layer, or a dedicated fluidic channel. As the cell undergoes a physiological change and the cumulus layer detaches from the oocyte, the oocyte will move, by gravity and fluidic assistance to the lower location.
[0087] As described herein, the fluid that exits the single cell housing device may be extracted via an automated pumping mechanism or a syringe mechanism to generate a fluid sample. The fluid sample may be delivered to a device that will perform analytical tests on the media, including, but not limited to UV-Vis spectroscopy, FTIR spectroscopy, High Pressure Liquid Chromatography - Mass Spectroscopy (HPLC-MS), Gas Chromatography - Mass Spectroscopy (GC-MS), Non Invasive Preimplantation Genetic Testing (NIPGT). The results of the analysis of the fluid sample may then be used to modify the programmed sequence that delivers the dynamic flow.
[0088] As described herein, the flow and flow rate of cell culture fluids may be modulated, mixed or adjusted upon operator request or based upon results of any morphological assessment, media sampling, or any sensors attached to the device. The direction and speed of the flow may be controlled to divert fluid to a premixing chamber to achieve a desired concentration gradient for delivery to one or many single cell holding devices. The tubing or channels may also undergo chemical or physical surface modification to improve flow control.
[0089] As described herein, embedded pumps and valves may be actuated to achieve controlled levels of fluid flow to precise locations, either to one channel in isolation or to multiple channels in parallel. 2PP printed structures that are actuated using pneumatic pressure, using diaphragms and squeeze valves to open and close fluidic channels. An embedded pump is a series of valves operating in a repeated programmed sequence to drive fluid flow in a direction using a peristaltic motion. [0090] Certain forms comprise a cradle with a bi-funnel shape, allowing for the cell complex to be held at the neck, and microf luidica lly accessed from the dynamic flow system, such that delivery of active biological components may be accomplished.
[0091] Several mechanisms may be individually or collectively incorporated into embodiments described herein; for example, a mechanism for extracting a sub-microlitre volume fluid sample from a location near the single cell housing, such as a syringe or thin tube connected to a pump to generate vacuum , a mechanism for delivering that fluid sample to an analytical device, such as pumping source, or robotics to move the fluid sample inside of a capsule or other vessel, a mechanism for analysing the sample and delivering the results to an operator, or as the input of an evaluation algorithm.
[0092] Aspects described herein may comprise one or more high pressure microfluidic channels capable of flowing liquified gases, and/or modified microfluidic channels capable of flowing viscous cryoprotectants. Optionally, cradles to hold oocytes and embryos that are separated from liquified gases via membranes but that are connected to the cryoprotectant microfluidic channels may also be comprised in the low flow microfluidic devices described herein.
[0093] Cells and embryos are typically moved or handled using aspiration-based techniques, or by bulk transport. Microfluidic handling of cells is primarily limited to bulk-sorting, and not specialised transport at the few-cell level.
[0094] Cryoprotective fluids are more viscous than water-based media and require greater pressure or larger channels for equivalent flow rates. Cryo-microfluidic (cryoprotectant carrying) channels may be optimised for the delivery of higher viscosity fluids to the culture chambers by increasing their diameter thereby reducing their resistance.
[0095] Methods for modulating the flow of fluids in the culture of a single cell or small cell mass, according to the present disclosure, comprising the steps of; obtaining the microfluidic device of the present disclosure, adding a cell to be cultured to a cell culture chamber, adding a culture medium to a fluid reservoir, actuating the one or more pumps, modulating the flow of fluid through the one or more fluid conduits by opening and closing the one or more valves.
[0096] The step of modulating the flow of fluid through the one or more fluid conduits by opening and closing the one or more valves in the present disclosure, may comprise the modulation of the flow of fluid through the one or more conduits to a flow rate of between approximately 0.0001 and 0.004 mm/sec, between approximately 0.5 and 1.5 mm/sec, or between approximately 0.5 and 10 mm/sec.
[0097] The step of actuating the one or more pumps according to the methods of the present disclosure may be undertaken in pulses to affect the pulsatile flow of fluid through the one or more fluid conduits.
[0098] Methods for delivery of liquid nitrogen via cryogenic liquid microfluidic channels directly to the single cell housing are described herein, which preferably allow the cryopreservation of a cell to occur in situ.
[0099] Cryoprotective fluids may require an external pumping source, as well as a dedicated cryoprotectant channel. Segmentation from the main microfluidic system would be achieved using embedded valves in a closed state, near to the single cell housing location.
[0100] According to preferred methods, cryoprotective fluids would be pumped in a gradient, rather than a sequential series of baths of decreasing hydration.
[0101] Accordingly, cryoprotectant channels may by modified to accommodate necessary adaptions, for example, they may be manufactured from different material, they may be manufactured to incorporate chemical or physical surface treatment, variations to their geometry, and/or to improve compatibility with cryoprotectants.
[0102] Preferably, liquified gases will flow in channels that are vented and highly insulated. [0103] The cryoprotection phase and the cryopreservation phase may be conducted in different zones of the device with the cradles moved mechanically during the process.
[0104] Cradles are able to be moved along channels, or micro-tracks, hydraulically using microfluidics or using some other means of driving motion such as a linear motor, in order to move them from process to process within a single device.
[0105] Broad embodiments of the invention now will be described with reference to the accompanying drawings together with the Examples and the preferred embodiments disclosed in the detailed description. The invention may be embodied in many different forms and should not be construed as limited to the embodiments described herein. These embodiments are provided by way of illustration only such that this disclosure will be thorough, complete and will convey the full scope and breadth of the invention.
DETAILED DESCRIPTION OF EMBODIMENTS
BRIEF DESCRIPTION OF THE FIGURES
[0106] Figure 1 provides a top perspective view of a low flow microfluidic device according to an embodiment of the present disclosure.
[0107] Figure 2 provides a bottom perspective view of a low flow microfluidic device according to an embodiment of the present disclosure.
[0108] Figure 3 provides a side sectional view through section A-A of Figure 1 according to an embodiment of the present disclosure.
[0109] Figure 4 provides a side sectional view through section B-B of Figure 1 according to an embodiment of the present disclosure.
[0110] Figure 5 provides a side sectional view through section C-C of Figure 1 according to an embodiment of the present disclosure. [0111] Figure 6 provides a side sectional view through section D-D of Figure 1 according to an embodiment of the present disclosure.
[0112] Figure 7 provides a side perspective view of a unit valve used in a low flow microfluidic device according to an embodiment of the present disclosure.
[0113] Figure 8 provides a side sectional view through section E-E of Figure 7 according to an embodiment of the present disclosure.
[0114] Figure 9 provides a side sectional view through section F-F of Figure 7 according to an embodiment of the present disclosure.
[0115] Figure 10 provides a side sectional view through section G-G of Figure 7 according to an embodiment of the present disclosure.
[0116] Figure 11 provides a schematic representation of a squeeze valve according to an embodiment of the present disclosure.
[0117] Figure 12 provides a top perspective view of a squeeze valve according to an embodiment of the present disclosure.
[0118] Figure 13 provides a top perspective view of a modified squeeze valve according to an embodiment of the present disclosure.
[0119] Figure 14 provides a side view of a diaphragm valve according to an embodiment of the present disclosure.
[0120] Figure 15 provides side perspective view of a housing structure of a low flow microfluidic device according to an embodiment of the present disclosure. [0121] Figure 16 provides a side perspective sectional view of a housing structure through section H-H of Figure 11 of a low flow microfluidic device according to an embodiment of the present disclosure.
[0122] Several embodiments of the invention are described in the following examples.
EXAMPLES
[0123] The present example describes low flow microfluidic devices and microfluidic systems adapted for use in the culture of a single cell or small cell mass; in particular an oocyte, blastocyst or early embryo. The following description may be readily adapted for use in the culture of different cell types by adopting modifications that would be known to and understood by persons skilled in the art. For example, compositions of various media types for the culture of different cells are well known to skilled persons, and the need for modification of media compositions for the culture of different cell types would be readily apparent to such persons.
[0124] With reference to Figure 1, a low flow microfluidic device, having a width of approximately 20mm, is shown, configured for culturing a single or multiple cells therein. The design of the low flow microfluidic device is configured to mimic the flow of fluids through the fallopian tube. The size and shape of the device's components, in particular the selection of channels sizes, the configuration of channels and pumps, and the selection of pump type to deliver a low flow rate in microfluidic operation, have been selected to deliver flow rates similar to interstitial flow rates.
[0125] Microfluidic device 100 generally comprises a microfluidic support 107 having four fluid reservoirs 104 formed thereon. For the culture of embryos, each medium comprises stage of development-specific formulations, for example, for the maturation of oocytes, for the fertilisation of mature oocytes, for supporting pre compaction growth and for supporting post compaction development. Greater or fewer fluid reservoirs may be provided depending on the needs of the cell culture environment. Microfluidic device 100 further comprises one or more of fluid conduits which provide for the communication of fluids to and from fluid reservoirs 107, to and from cell culture chamber 103, and they provide for the communication of fluids between any one of fluid reservoirs 107 and cell culture chamber 103. The various fluid conduits of microfluidic device 100 comprise a combination of one or more tubes and one or more channels either formed within microfluidic support 107 or on the surface of microfluidic support 107.
[0126] Notably, the microfluidic support and components are formed by two-photon polymerisation ("2PP").
[0127] In use, each cell culture medium 101 is introduced into each 6.5mm diameter fluid reservoir 104 of reservoir array 105 via fluid channel 106 which is actuated by a series of valves and pumps 102. Each valve and pump 102 comprises an independently actuated valve 401 and an integrated pump 403.
[0128] Cell culture media 101 is, in turn, transferred to cell culture chamber 103 via fluid channel 106. The transfer of cell culture media 101 is directed from reservoir 104 to cell culture chamber 103 by one or more valves directing and/or modulating the flow of fluid along fluid channel 106. The coordination and control of valves and pumps 102 and further independently actuated valves 401 placed along various fluid conduits, including fluid channel 106, supply cell culture media 101 to a cradle array 108 containing the desired cell type for culturing.
[0129] Thus, each independently actuated valve 401 and integrated pump 403 may be operated dynamically; independently but in parallel with each other independently actuated valve 401 and integrated pump 403 within the series of valves and pumps 102, such that each cradle 604 within cradle array 108 may be provided a unique culture environment specifically adapted to the individual need of the cell or cell cluster in each cradle 604.
[0130] Individual cradles 604 (shown in Figure 6) within the cradle array 108 are fluidically connected to cell culture chamber 103 via a connector such as a barb connector (not shown). International Patent Application Number PCT/AU2020/051318 describes the form of cradles 604 and cradle arrays 108 in detail. While the low flow microfluidic device 100 may be suitable for culturing any cell type, the low flow microfluidic device described herein and depicted at 100 is adapted for culturing single cells and small cell clusters, for example embryos.
[0131] Low flow microfluidic systems and devices described in the present example deliver 20 picolitres per second per cycle and deliver fluid in a pulsatile rhythm. The exemplified systems and devices can be modulated to provide a cyclical flow rate that repeat with a frequency of as low as one cycle per minute (or even lower) to deliver 20 picolitres per minute. Similarly, cyclical flow rates can be increased in frequency by increasing the pressure driving the flow rate or by increasing the frequency of cycles.
[0132] Figure 1 shows the upper surface 109 of microfluidic support 107. Figure 2 shows the lower surface 204 of microfluidic support 107 of the low flow microfluidic device of Figure 1. Figure 1 shows that microfluidic support 107 comprised two gas inlet arrays 203 having six individual gas inlets 201 formed through the lower surface of microfluidic device 100. Fluids, including gases and liquid fluids are introduced into microfluidic device 100 via valve and pump array 102 which align with gas inlet arrays 203. Two fixing holes 202 are provided proximal to each lengthwise edge of microfluidic device 100 to fix the device in a desired static position.
[0133] Figure 3 provides a side sectional view of microfluidic device 100 through microfluidic support 107 at section A-A of Figure 1. The side sectional view of Figure 3 provides further detail of the pump array 301 and pump 303 and provides a cross sectional view through the pneumatic control line (not shown). As illustrated in Figure 1, the number of valves 401 ganged together is equal to the number of cradles 604; i.e. 16 valves and 16 cradles. Each valve 401 is provided within supply line 302 which can expand when pressurised to control the flow of fluid within the supply line 302.
[0134] Figure 4 provides a side sectional view of microfluidic device 100 through section B-B of microfluidic support 107 shown in Figure 1. The side sectional view provided at Figure 4 shows further detail of integrated pumps 303 which comprise independently actuated valves 401 each having an intermediate fluid chamber 402 and a central chamber 403 controlled by a pneumatically pressurized chamber 404, restricting the flow rate of fluid through fluid channel 405 upon actuation of valve 401.
[0135] Figure 5 provides a side sectional view of microfluidic device 100 through section C-C of microfluidic support 107 shown in Figure 1. Bifurcated supply line 503 enables the control of the flow fluids upon actuation of independently actuated valve 401. Independently actuated valve 401 may be actuated to direct and/or modulate the full or partial flow of fluid through waste line 501 and downstream to waste reservoir (not shown) to control the supply and/or circulation of cell culture media 101 through the microfluidic device.
[0136] Figure 6 provides a side sectional view of the microfluidic device 100 through section D-D of microfluidic support 107 shown in Figure 1. Bifurcated supply line 503 provides two channels through which to direct the flow of fluids; one comprising waste valve 601 to direct fluid to waste reservoir (not shown) and the other comprising supply valve 502 to direct and/or modulate the flow of cell culture media 101 to cradle 604. Waste media emanating from cradle 604 is transferred from cradle 604 through waste line 501 to waste reservoir (not shown) and fresh supply media (cell culture media 101) is directed to cradle 604 for further culture of cells therein.
[0137] Figure 7 shows independently actuated valve 401 comprising inlet port 701 provided to enable the transfer of incoming gases which pressurise valve chamber contained therein, thereby restricting the flow of fluid through neighbouring outlet channel 702.
[0138] Figures 8, 9 and 10 provide side sectional views of microfluidic device 100 across sections E-E, F-F and G-G of independently actuated valve 401 shown in Figure 7. Figures 8- 10 show embedded channel 801 which is pressurised with gas and thereby deforms the surrounded channel 802 to restrict the flow of fluid passing therethrough.
[0139] Independently actuated valve 401 may take various forms. By way of example, Figures 11-13 illustrate squeeze valve 1101 suitable for implementation with the embodiment of Figures 1-10 and Figures 14a-b illustrate a diaphragm valve suitable for implementation with the embodiment of Figures 1-10. Squeeze Valves
[0140] Figure 11 shows the general form of squeeze valve 1101, produced and actuated with a pneumatic pump (not shown) formed from 2PP printed UpFlow; an acrylate-based resin available commercial from UpNano, Vienna, Austria. Squeeze valve 1101 consists of a thinned, rectangular central fluid chamber 1102 flanked by two rectangular pressure chambers 1103, providing two thin membranes on either side of fluid chamber 1102. The membranes provide fluid deflection when pressure is applied.
[0141] Figure 12 shows the location of each pressure chamber 1103 on microfluidic support 107 which is serviced by air line inlet 1201 and air channel 1202 for flushing, connected to the pressure chambers 1103 at the top and bottom of the pressure chambers 1103 respectively. Attachment nozzle 1203 at one end of air line inlet 1201 allows for the connection of tygon tubing (not shown) to permit the flushing the air channel 1202 and fluid channel 1207, and connection of air channel 1202 to pneumatic pump (not shown).
[0142] Air outlet 1204 may be released to enable the flushing of air channel 1202 and fluid channel 1207 but must be remain closed to pressurize air channel 1202. A connector on fluid outlet 1205 allows tubing to be held in place for flushing, and for liquid to be added into media reservoir 1206.
[0143] As illustrated in Figure 13, a second tubing connection 1301 may optionally be fitted to permit a manual valve 1302 to be added. Manual valve 1302 may be released and closed to pressurize air channel 1202 and/or fluid channel 1207.
Diaphragm Valves
[0144] Figure 14 provides a side sectional view of diaphragm valve 1301 showing its general form. Diaphragm valve 1301 provides an alternative to squeeze valve 1101 to provide a larger diameter membrane for culture conditions that may require a lower flow rate.
[0145] Diaphragm valve 1301 is implemented by replacement of squeeze valve 1101 in the microfluidic device described in Figures 11-13, with diaphragm valve 1301. Membrane 1302 is maintained in positioned between control chamber 1303 and fluid chamber 1304 as illustrated in Figure 14a. The construction of diaphragm valve 1301 is based on the work of prior authors; namely, Gong et al. Lab Chip, 2016, 16, 2450-2458.
[0146] In use, tygon tubing (not shown) is connected to the air lines inlet 1201. Tygon tubing is connected at one end to a mitos pump and at the other end it is closed with a manual diaphragm valve for modulation of flow rate. Upon actuation of diaphragm valve 1301, air flow and fluid flow are driven in parallel as shown in Figure 14b.
[0147] With reference to Figure 15, cradle 604 is shown prior to insertion into housing structure 1501 which can house multiple cradles 604. Housing structure 1501 can house multiple cradles 604 within garage 1502. This configuration maintains the position or placement of cradle 604 to provide fluidic connection with microfluidic device 100.
[0148] Such embodiments may administer an ultra-low flow rate perfusion, which enables the automation of media delivery and achieves dynamic culturing without excessive disturbance to the embryo environment. Culture conditions may be adapted or modified by persons skilled in the art to optimise fluid flow rate according to the nutritional needs of the embryo.
[0149] Optimal media flow rates for the culture of embryos, or other cell types, will typically mimic the flow rates of the environment in which these cells naturally occur. Therefore, depending on the cell type under culture, fluid flow rates may be modified to mimic interstitial flow rates, capillary flow rates, venule flow rates and arteriole flow rates.
[0150] Figure 16 illustrates the use of cradle 604 and housing structure 1501 for embryo culture. In use, an embryo is deposited into conical funnel 1601 which is immersed in culture media, and is shaped to shave embryonic cumulus cells to remove them from the surface of the embryo prior to placement into the cradle 604. Thereafter, cell culture media 101 is perfused through cradle 604 at low flow through microfluidic device 100 as described above. The passage of cell culture media 101 through microfluidic device 100 may be modulated as desired utilising integrated valves 401 and pumps 303 as described above. [0151] The methods described herein for use of the present embodiments may be adapted for housing oocytes, and then subsequently embryos, by utilising low flow microfluidic devices to mimic the fluidic conditions in vivo. The methods described herein allow for the dynamic culturing of these cells without disturbance to the local environment, while meeting their metabolic needs throughout the duration of cell culture.
[0152] For the purposes of culturing embryos, methods may provide for pulsatile fluid flow rates which are selected from a rate and frequency that mimics the ciliated lining of the fallopian tube, as well as other fluid forces unique to this environment.
[0153] Methods may be adapted to provide dynamic flow conditions which provide cell culture media 101 to cells or embryos under specific culture conditions. Integrated valves and pumps 102 may be pre-programmed or set to deliver low volume flow rates.
[0154] In certain embodiments, low flow microfluidic devices may be adapted to dynamically exchange media and cryoprotectants gradually; in a controlled manner. Integrated valves and pumps 102 may be modified to facilitate flow, in instances wherein a housing structure 1501 is being utilised to separate and identify embryos. Methods involving cryo-microfluidics may be adopted to allow for the low flow of cryopreservation fluids, optionally, together with use of single cell cradle arrays that ease the recovery of cells cultured therein.
[0155] Additional devices may be adopted that remove the dependency on operator skill for the handling of liquid nitrogen, by automating the selection of media concentrations and/or the flow rate of cryoprotectants and, thereafter, the application of liquid nitrogen. Methods that utilise such devices together with low flow microfluidic devices may result in the minimisation of oocyte and embryo handling and consequently a reduction of stress on the cultured cells.
[0156] Integration of automated and precise tools and techniques within methods for the transfer of embryos to patients may reduce the impact of excessive handling. This, in turn, may lead to a less variable transfer process which maintain embryo viability from petri dish to implantation.
[0157] Methods may comprise additional devices that assist in reducing manual handling during the procedures described above and automating the process of extracting homologous (mother's) ovarian-stem cell sourced mitochondria. The process of injecting mitochondria from a homologous source (preferably ovarian) in conjunction with or without other "rejuvenating compounds" may additionally be automated using the devices described above together with mitochondrial "sieves".
[0158] Low flow microfluidic devices may be utilised in conjunction with the methods described herein to pass COC cells through a sorting system for visualisation, collection, handling with cell cradles to denude COC cells, to gently transfer cells or embryos, individually or with multiple other co-cultured cells. For example, visualisation technologies such as Optical Coherence Tomography may be adopted and, optionally, incorporated into oocyte retrieval equipment in conjunction with low flow culture devices to support the timely and visible collection of oocytes.
[0159] Systems and/or devices described herein may comprise a precision incubator sufficiently smaller in size to suit the cells being incubated rather than the operator; providing a higher level of precision of temperature, humidity, and gas environment. Methods for the use of low flow microfluidic devices described herein enable the delivery of controlled amounts of gas through integrated valves and pumps 102 to deliver a controlled flow at a microscale.
[0160] The devices and methods described herein may be adapted by persons skilled in the art for ART applications and may be used to make all forms of cell therapy and culturing more precise, efficient and repeatable. Such devices and methods may be adapted for cell models such as stem-cells, organoids, viruses and bacteria to automate production and improve quantity and quality of cellular output. [0161] Methods for non-ART cell therapies may be adapted to incorporate the use of low flow microfluidic devices to provide media to cells under therapy or culture. Each cradle may contain a cell, organoid or group of cells (e.g. for non-human non-ART). Integrated valves and pumps 102, described herein, may then deliver flow to an individual cradle at a desired flow rate such that the flow rate to each cradle within an array may be individually controlled.
[0162] Low flow microfluidic devices may be integrated into systems for the full automation of in vitro fertilisation procedures, to reduce the financial constraints and dependency on operator skill for successful procedures. An end-to-end system may integrate devices described herein to automate the process of growing healthy and viable embryos.
[0163] In particular, full automation may comprise low flow microfluidic devices to provide media to the cells or embryos under culture conditions, integrated valves and pumps 102 to deliver a low volume flow, housing structure 1101 may be adapted to separate and identify the embryos, cryo-microfluidic modifications may allow for flow of high viscosity fluids to preserve the embryos once fertilised, for cumulus oocyte complex removal from the oocyte, as well as to transfer the cells between stations and allow ease of cell recovery, to individually access particular embryos during culture, and to monitor metabolite and nutrient levels as well as culture progress.
[0164] Additionally, devices and systems adapted for automated in vitro fertilisation may comprise an automated injector, fixed to image-controlled single micromanipulator, and may be used to align the Z-plane, identify the sperm, complete sperm pickup, identify the oocyte in the microwell, align the injector, and fertilise the oocyte with sperm.
[0165] Throughout this specification the word "comprise", or 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. [0166] It will be understood that the terms 'fastener' or 'fastening', 'coupling' or 'sealing' when used alone or together with other terms such as 'means' or others, may be used interchangeably where interpretation of the term would be deemed by persons skilled in the art to be functionally interchangeable with another. Further, the use of one of the aforementioned terms does not preclude an interpretation when another term is included.
[0167] The various apparatuses and components of the apparatuses, as described herein, may be provided in various sizes and/or dimensions, as desired. Suitable sizes and/or dimensions will vary depending on the specifications of connecting components or the field of use, which may be selected by persons skilled in the art.
[0168] It will be appreciated that features, elements and/or characteristics described with respect to one embodiment of the disclosure may be used with other embodiments of the invention, as desired.
[0169] Although the preferred embodiments of the present disclosure have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the disclosure and accompanying claims.
[0170] It will be understood that when an element or layer is referred to as being "on" or "within" another element or layer, the element or layer can be directly on or within another element or layer or intervening elements or layers. 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.
[0171] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
[0172] It will be understood that, although the terms first, second, third, etcetera, may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.
[0173] Spatially relative terms, such as "lower", "upper", "top", "bottom", "left", "right" and the like, may be used herein for ease of description to describe the relationship of one element or feature to another element(s) or feature(s) as illustrated in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of structures in use or operation, in addition to the orientation depicted in the drawing figures. For example, if a device in the drawing figures is turned over, elements described as "lower" relative to other elements or features would then be oriented "upper" relative the other elements or features. Thus, the exemplary term "lower" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein should be interpreted accordingly.
[0174] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. 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 "including," "comprises" and/or "comprising," when used in this specification, 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.
[0175] Embodiments of the description are described herein with reference to diagrams and/or cross-section illustrations, for example, that are schematic illustrations of preferred embodiments (and intermediate structures) of the description. 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 description should not be construed as limited to the particular shapes of components illustrated herein but are to include deviations in shapes that result, for example, from manufacturing.
[0176] 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 description 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 idealized or overly formal sense unless expressly so defined herein.
[0177] 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 of the description. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with any embodiment, it is within the purview of one skilled in the art to effect and/or use such feature, structure, or characteristic in connection with other ones of the embodiments.
[0178] Embodiments are also intended to include or otherwise cover methods of using and methods of manufacturing any or all of the elements disclosed above.
[0179] While the invention has been described above in terms of specific embodiments, it is to be understood that the invention is not limited to these disclosed embodiments. Upon reading the teachings of this disclosure many modifications and other embodiments of the invention will come to the mind of those skilled in the art to which this invention pertains, and which are intended to be and are covered by both this disclosure and the appended claims.
[0180] All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. 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 invention as it existed in Australia or elsewhere before the priority date of each claim of this application.
[0181] It is indeed intended that the scope of the invention should be determined by proper interpretation and construction of the appended claims and their legal equivalents, as understood by those skilled in the art relying upon the disclosure in this specification and the attached drawings.