Microfluidic device and related systems and methods

Abstract

A microfluidic assembly including at least one microfluidic device is provided herein. The microfluidic assembly can be applied for vascularizing multicellular aggregates such as 2D or 3D animal cells or tissue implanted in a matrix that is held within an aperture of the microfluidic device. The microfluidic device in the microfluidic assembly is connected to a primary circulation system, which allows establishment and maintenance of physiologically relevant pressure gradients across the aperture of the microfluidic device. The microfluidic apparatus allows for connection of living microvessels, such as human microvessels, to a pressurized microfluidic channel via a porous membrane, allowing support of multicellular aggregates for long term culture.

Description

MICROFLUIDIC DEVICE AND RELATED SYSTEMS AND METHODSCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 082,733, filed September 27, 2023, titled “Microfluidic Device and Related Systems and Methods”, the entire contents of which are incorporated herein by reference.Field of the Disclosure

[0002] The disclosure relates generally to microfluidic devices. In particular, the disclosure relates to a microfluidic device, a microfluidic pump unit, and a microfluidic assembly for vascularization of cultured animal cells or tissues, and to its related systems and methods.Background of the Disclosure

[0003] Current microfluidic culture systems for in vitro vascularization of tissues may be subdivided into the following categories, namely: vascularization platforms; organ system platforms; and organ-on-chip platforms. However, each one of these systems has limitations.

[0004] Vascularization platforms are designed with a tissue cavity in fluid communication with a sealed microfluidic channel to provide nutrients to living microvessels in a matrix. According to their design, such platforms provide low flow rates and therefore are unable to establish physiological vessel pressures and are not compatible with the addition of large, millimeter scale, preassembled 3D tissues to the cavity due to nutrient requirements.

[0005] Organ system platforms involve a microfluidic device or chip to shuttle a fluidic media through various tissue compartments. Organ system platforms are made of a plurality of monolayers or transwell tissue compartments, connected one to the other via a microfluidic device or chip and peripheral fluidic pump. Though these systems are designed to model blood movement through the body, they provide limited tissue-vesselinterfaces as the vessel compartment is either entirely separated or exists as an endothelial lined microchannel. Moreover, as these systems inherently rely on monolayer or transwell culture compartments, integration with current 3D tissue culture protocols remains limited. Thus, such systems lack a tissue-vessel interface and involve monolayer culture, which does not model tissue physiology.

[0006] Organ-on-a-chip platforms break down an organ into two monolayer cultures (to a single cellular interface of monolayer cultures). When deconstructing the system to its fundamental aspects, monolayer culture is still utilized. Furthermore, use of these systems to model organ physiology is restricted to organs such as the lung or gut, where a defined cellular interface defines the organ periphery. Other organ-on-a-chip platforms have used a flexible porous membrane to allow cytokine signaling between the two cellular compartments. The porous membrane includes pores, each defining a diameter of 0.5-5 pm, to prevent most movement of cells across the tissue microfluidic compartments resulting in cells existing in monolayer culture similar to a transwell design. These organ-on-a-chip platforms also lack higher-order cellular organization such as tissue-vessel interfaces comprising endothelial cells, pericytes, and a vascular basement membrane. These systems therefore lack a vascular component and do not allow use of 3D culture techniques.

[0007] Other known microfluidic culture platforms are restricted by employing closed microfluidic chip designs, a limited tissue-vessel interface, and non-physiologically relevant flow rates and pressures. Furthermore, most microfluidic culture systems utilize monolayer culture to model organ physiology, which is now known to be problematic as culture surface effects cellular phenotypes.

[0008] The above-mentioned systems are not suitable for whole tissues (3D tissues) due to their sealed microfluidic designs. As such, the systems have restricted applications and do not faithfully mimic human tissue physiology. Several limitations exist in the performance of these systems. For example, such systems do not provide connection of living microvessels to a pressurized environment. Though studies have demonstrated the possibility to generate microvessels in a matrix, such vessels were not connected to a pressured environment, which resulted in flow rates and pressures far below physiological conditions. These systems are also often limited by size. Existing vessel micro-physiological systems (MPSs) are typically sub-millimeter in size while organoids not only reach to be several millimeters, but also contain several orders of magnitude more cells. Consequently, virtually all aspects, from device size, nutrient media flowrates, and number of vessel connection points, must be increased accordingly. Anotherlimitation is an inability to create large, consistent, and controlled pressure gradients. Fluid flow is driven by pressure gradients and its generation in vessel MPSs has been restricted to gravity-based systems. Extending the previous challenge, larger tissues and devices require larger pressure gradients, which quickly become impractical to achieve with the use of hydrostatics. Gravity based systems also deliver inconsistent pressure levels over time, which deviate from physiology and complicate results interpretations. Although mechanical pumps are able to produce adequately large pressure levels, controlling the pressure gradient and directing it towards miniscule, fragile, endothelial vessels, is nontrivial and requires sophisticated design features.

[0009] The above-mentioned systems generally suffer from a lack of usability and scalability. This challenge applies to MPSs more generally as it restricts their widespread use. While commercial options exist, MPSs largely require levels of effort and meticulousness fargreaterthan standard tissue culture practices as well as have far lower throughputs and consistencies. Fluid flow has additionally become an increasingly common feature of MPSs as evident from the previous passages and with it, the requirement of elaborate pump systems. Work with MPSs has thus been mainly restricted to niche laboratories. Furthermore, in the context of creating perfused vascularized organoids, the need for ease of use is amplified. Organoid culture itself is a demanding process and as such, added complexities should be minimized. More usable systems also indirectly lead to more scalable ones. Complex experimental designs as well as drug screening applications require concurrent culture of a high number of devices. Current MPS setups, particularly pump-based ones, can only manage a handful of units and thus are drastically limited in their utility.

[0010] As such, there is clearly a need for a microfluidic device and system for vascularizing organoids (and tissued) which addresses the challenges previously outlined in the field. There is a need for such a system that is able to continuously perfuse tissue to organoids in a scalable and user-friendly format.

[0011] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.Summary of the Disclosure

[0012] According to an aspect, there is provided a microfluidic device for use with a primary circulation system, the microfluidic device comprising: (a) a microfluidic channelconfigured for being in fluid communication with the primary circulation system, the microfluidic channel having: an upstream channel for receiving a pressurized fluid from the primary circulation system, a pressure drop section in fluid communication with the upstream channel for receiving a first volume of the pressurized fluid from the upstream channel and inducing a pressure drop to produce a depressurized fluid having a lower pressure, and a downstream channel in fluid communication with the pressure drop section for receiving the depressurized fluid therefrom and for recirculating the depressurized fluid to the primary circulation system, (b) at least one porous membrane having a plurality of pores, and (c) at least one compartment that includes an aperture, the aperture of the at least one compartment being in fluid communication with the microfluidic channel via the at least one porous membrane such that when a second volume of the pressurized fluid flows from the upstream channel to the downstream channel via the at least one porous membrane and the compartment, a pressure differential is formed within the aperture.

[0013] In an embodiment, the at least one porous membrane is at least partially in fluid communication with both the upstream channel and downstream channel of the microfluidic channel.

[0014] In an embodiment, the at least one porous membrane includes a first porous membrane and a second porous membrane, and the first porous membrane is at least partially in fluid communication with the upstream channel of the microfluidic channel and the second porous membrane is at least partially in fluid communication with the downstream channel of the microfluidic channel.

[0015] In an embodiment, the microfluidic device further comprises: a bypass channel that is in fluid communication between the upstream and the downstream channels of the microfluidic channel for circulating a third volume of the pressurized fluid therebetween.

[0016] In an embodiment, the bypass channel is structured for increasing a velocity of the pressurized fluid within the upstream channel without reducing the pressure differential within the aperture of the at least one compartment.

[0017] In an embodiment, the bypass channel is structured for reducing particle sedimentation time within the aperture of the at least one compartment.

[0018] In an embodiment, the bypass channel is structured for increasing a rate of delivery of at least one compound to the aperture of the at least one compartment via the microfluidic channel.

[0019] In an embodiment, the bypass channel includes an inlet end and an outlet end; and

[0020] wherein the inlet end is provided in fluid communication with the upstream channel, and the outlet end is provided in fluid communication with the downstream channel for circulating the third volume of the pressurized fluid through the bypass channel.

[0021] In an embodiment, the upstream channel of the microfluidic channel is fluidly connected to an outlet of the primary circulation system via at least one outlet conduit, and the downstream channel of the microfluidic channel is fluidly connected to an inlet of the primary circulation system via at least one inlet conduit.

[0022] In an embodiment, the bypass channel is structured to permit increased diameters of the at least one outlet conduit and the at least one inlet conduit of the primary circulation system while substantially maintaining a velocity of both the pressurized fluid and the depressurized fluid through the at least one outlet conduit and the at least one inlet conduit.

[0023] In an embodiment, the bypass channel and upstream and downstream channels of the microfluidic channel are each sized such that the microfluidic device can accommodate an internal diameter of the at least one outlet conduit that is in a range from about 0.1 mm to about 5 mm, and an internal diameter of the at least one inlet conduit that is in a range from about 0.1 mm to about 5 mm.

[0024] In an embodiment, the inlet end of the bypass channel is connected to the upstream channel to form a first three-way node between the bypass channel, the upstream channel and the primary circulation system, and the outlet end of the bypass channel is connected to the downstream channel to form a second three-way node between the bypass channel, the downstream channel and the primary circulation system.

[0025] In an embodiment, the inlet end of the bypass channel is connected directly to the upstream channel of the microfluidic channel, and wherein the outlet end of the bypass channel is connected directly to the downstream channel of the microfluidic channel.

[0026] In an embodiment, a length of the bypass channel is in a range from about 10 mm to about 100 mm, and wherein an inner diameter of the bypass channel is in a range from about 0.1 mm to about 5 mm.

[0027] In an embodiment, the bypass channel includes: an inlet channel portion that defines the inlet end of the bypass channel, an outlet channel portion that defines the outlet end of the bypass channel, and a bypass portion which extends between the inlet and outlet channel portions. The inlet channel portion extends upwards, substantially vertically, from the upstream channel, the outlet channel portion extends upwards,substantially vertically, from the downstream channel, and the bypass portion is substantially horizontal.

[0028] In an embodiment, the bypass channel is sized such that the pressure differential across the aperture of the at least one second compartment is maximized.

[0029] In an embodiment, the bypass channel is structured such that a fluid resistance of the bypass channel is no less than five times a fluid resistance of the upstream channel, and such that the fluid resistance of the bypass channel is no less than five times a fluid resistance of the downstream channel.

[0030] In an embodiment, an internal diameter of each of the at least one outlet conduit and the at least one inlet conduit is at least 100 pm.

[0031] In an embodiment, an internal diameter of the bypass channel is at least 100 pm.

[0032] In an embodiment, at least a portion of the bypass channel is composed of a pliable material.

[0033] In an embodiment, the pliable material is sufficiently pliable such that the at least a portion of the bypass channel can be compressed on itself for substantially inhibiting a flow of the third portion of the pressurized fluid through the bypass channel.

[0034] In an embodiment, the bypass channel is sized such that a ratio of APComP / APpUmPis at least about 0.9, where APCOmp is defined as the pressure differential across the aperture of the at least one second compartment and APpUmp is defined as a pressure differential across the primary circulation system.

[0035] In an embodiment, the at least one compartment is a plurality of compartments.

[0036] In an embodiment, the plurality of compartments are sequentially arranged along the microfluidic channel such that each of the plurality of compartments is positioned at least partially over the upstream channel and at least partially over the downstream channel.

[0037] In an embodiment, the at least one porous membrane has a first porous membrane section in fluid communication with the upstream channel and a second porous membrane section in fluid communication with the downstream channel, and the aperture includes a first aperture portion that is aligned with the first porous membrane section and a second aperture portion that is aligned with second porous membrane section.

[0038] In an embodiment, each pore of the plurality of pores of the at least one porous membrane has a pore diameter ratio Perp:Para equal to or less than 1 :1.

[0039] In an embodiment, the plurality of pores of the at least one porous membrane are structured as a plurality of slit-shaped pores.

[0040] In an embodiment, each pore of the plurality of slit-shaped pores is structured such that a width of the pore is in a range from about 10 pm to about 200 pm and a length of the pore is in a range from about 50 pm to about 2000 pm .

[0041] In an embodiment, the plurality of slit-shaped pores are oriented such that a long axis of each pore of the plurality of slit-shaped pores extends substantially parallel to a long axis of the upstream channel.

[0042] In an embodiment, each pore of the plurality of slit-shaped pores is structured such that an aspect ratio of the pore is greater than or equal to 20:1 .

[0043] In an embodiment, the microfluidic device further comprises a secondary microfluidic channel for use with a secondary circulation system.

[0044] In an embodiment, the secondary microfluidic channel includes: a secondary upstream section for receiving a pressurized fluid from the secondary circulation system, a secondary pressure drop section in fluid communication with the secondary upstream section for receiving the pressurized fluid therefrom, the secondary pressure drop section being structured for inducing a pressure drop in the pressurized fluid to produce a depressurized fluid, and a secondary downstream portion in fluid communication with the pressure drop section for receiving the depressurized fluid therefrom and recirculating the second depressurized fluid to the secondary circulation system.

[0045] In an embodiment, the secondary pressure drop section comprises: a first pressure drop section in fluid communication with the secondary upstream section, the first pressure drop section being structured for inducing a first pressure drop in the pressurized fluid to produce a first depressurized fluid, a midstream section in fluid communication with the first pressure drop section for receiving the first depressurized fluid therefrom, and a second pressure drop section in fluid communication with the midstream section for receiving the first depressurized fluid therefrom, the second pressure drop section being structured for inducing a pressure drop in the first depressurized fluid to produce a second depressurized fluid.

[0046] In an embodiment, the midstream section includes a first midstream portion that is structured to direct the first depressurized fluid to flow in a first flow direction; and a second midstream portion in fluid communication with first midstream portion, the second midstream section being structured to direct the first depressurized fluid to flow in second flow direction opposite the first flow direction.

[0047] In an embodiment, the at least one porous membrane further comprises: a third portion in fluid communication with the secondary upstream section of the secondary microfluidic channel and a fourth portion in fluid communication with the secondarydownstream section of the secondary microfluidic channel. The aperture includes a third aperture portion that is aligned with the third portion of the at least one porous membrane and fourth aperture portion that is aligned with the fourth portion of the at least one porous membrane.

[0048] In an embodiment, the third and fourth portions of the at least one porous membrane are disposed in between the first volume and second volume of the porous membrane.

[0049] In an embodiment, a pressure difference defined between an inlet of the upstream channel and outlet of the downstream channel of the microfluidic channel is substantially equal to pressure difference defined between an inlet of the secondary upstream section and an outlet of the secondary downstream section of the secondary microfluidic channel.

[0050] According to another aspect, there is provided a microfluidic device system that includes the microfluidic device as disclosed herein, and that further comprises at least one controller that is configured to control the primary circulation system.

[0051] In an embodiment, the microfluidic device system further comprises at least one controller that is configured to control the primary circulation system and the secondary circulation system.

[0052] According to an aspect, there is provided a microfluidic device assembly including a plurality of the microfluidic devices, the microfluidic device assembly further comprising: an inlet channel section that is in fluid communication with an outlet of the primary circulation system for receiving pressurized fluid therefrom, and an outlet channel section in fluid communication with an inlet of the primary circulation system for returning depressurized fluid thereto. The plurality of microfluidic devices are connected between the inlet channel section and outlet channel section such that the upstream channel of each microfluidic device of the plurality of microfluidic devices is fluidly connected to the inlet channel section, and the downstream channel of each microfluidic device of the plurality of microfluidic devices is fluidly connected to the outlet channel section.

[0053] According to an aspect, there is provided a microfluidic device assembly for use with a primary circulation system, the microfluidic device assembly comprising: a plurality of microfluidic devices that are connected in series between an inlet and an outlet of the primary circulation system, each of the plurality of microfluidic devices including: a microfluidic channel that has an upstream channel for receiving a higher pressure fluid, a pressure drop section in fluid communication with the upstream channel for receiving a first volume of the higher pressure fluid and inducing a pressure drop to produce a lower pressure fluid, and a downstream channel in fluid communication with the pressure dropsection for receiving the lower pressure fluid, at least one porous membrane that is at least partially in fluid communication with both the upstream channel and downstream channel of the microfluidic channel, and a flow compartment that is positioned on top of microfluidic channel and that defines an aperture therethrough, the aperture being in fluid communication with the at least one porous membrane such that when a second volume of the pressurized fluid flows from the upstream channel to the downstream channel via the at least one porous membrane and through the flow compartment, a pressure differential is formed within the aperture. The plurality of microfluidic devices are connected in series such that the upstream channel of a first microfluidic device of the plurality of microfluidic devices is fluidly connected to the outlet of the primary circulation system. The upstream channel of each microfluidic device of the rest of the plurality of microfluidic devices is fluidly connected to a downstream channel of another of the plurality of microfluidic devices, and the downstream channel of a last microfluidic device of the plurality of microfluidic devices is fluidly connected to the inlet of the primary circulation system.

[0054] According to another aspect, there is provided at least one microfluidic device that is positioned on a base plate, at least one microfluidic pump unit that is fluidly connected to the at least one microfluidic device for circulating a fluid therethrough, the at least one microfluidic pump unit including: a pump housing, and an impeller that includes an impeller shaft and that is rotatably housed within the pump housing, the impeller shaft including at least one first magnetic element, and at least one magnetic drive system that is removably connectable to the pump housing and that includes at least one drive motor with an output shaft, the output shaft including at least one second magnetic element. The magnetic drive system is removably connectable on the pump housing in a position such that the at least one first magnetic element is magnetically coupled to the at least one second magnetic element, while being spaced apart from at least one second magnetic element, for rotatably coupling the at least one drive motor to the impeller of the at least one microfluidic pump unit.

[0055] In an embodiment, the microfluidic assembly further comprises a cover unit that is removably connectable atop the base plate.

[0056] In an embodiment, the magnetic drive system is housed within the cover unit such that when the cover unit is connected atop the base plate, the at least one first magnetic element of the impeller shaft is magnetically coupled to the at least one second magnetic element of the output shaft, and when the cover unit is disconnected from the base plate,the at least one first magnetic element is magnetically decoupled from the at least one second magnetic element.

[0057] In an embodiment, the microfluidic assembly further comprises a seal plate that is connectable on the base plate for inhibiting contamination and evaporation of the fluid from the microfluidic device and / or the microfluidic pump unit.

[0058] In an embodiment, the seal plate is connectable on base plate such that when the cover unit is connected atop the base plate, the seal plate is disposed between the microfluidic pump unit in the base plate and magnetically couplable drive unit.

[0059] In an embodiment, the seal plate is connectable on the base plate such that when the cover unit is connected atop the base plate, the seal plate is at least partially disposed between the base plate and the cover unit.

[0060] In an additional embodiment, the first magnetic element is disposed on a first side of the seal plate, and the second magnetic element is disposed on a second side of the seal plate opposite the first side.

[0061] In an embodiment, the seal plate is connectable on the base plate such that one end of the impeller shaft is rotatably supported by the seal plate when the seal plate is connected to the base plate.

[0062] In an embodiment, the seal plate is at least partially composed of at least one low friction material such that an amount of friction generated between the seal plate and the end of the impeller shaft is minimized.

[0063] In an embodiment, at least one of the pump housing and impeller of the at least one microfluidic pump unit is composed of one or more biocompatible materials.

[0064] In an embodiment, the one or more biocompatible materials comprises one or more biocompatible plastics.

[0065] In an embodiment, the at least one microfluidic device is positioned in at least one first well on the base plate, the at least one microfluidic pump unit is positioned in at least one second well on the base plate, and the at least one second well is adjacent to the at least one first well on the base plate.

[0066] In an embodiment, the fluid connection between the at least one microfluidic device and the at least one microfluidic pump unit includes at least one inlet conduit and at least one outlet conduit.

[0067] In an embodiment, the at least one microfluidic pump unit and the at least one microfluidic device are positioned within the at least one first well and at least one second well, respectively, such that an effective fluid capacitance of each of the at least one inlet conduit and at least one outlet conduit is minimized.

[0068] In an embodiment, the at least one microfluidic pump unit and the at least one microfluidic device are positioned within the at least one first well and at least one second well, respectively, such that a length of each of the at least one inlet conduit and at least outlet conduit is minimized.

[0069] In an embodiment, each of the at least one inlet conduit and the at least one outlet conduit is formed as a length of tubing that is oriented substantially vertically within the at least one first well.

[0070] In an embodiment, the at least one microfluidic pump unit is removably mounted in the at least one first well, and the at least one microfluidic device is removably mounted in the at least one second well.

[0071] In an embodiment, the base plate comprises an SBS (Society for Biomolecular Screening) tissue culture plate, and the at least one first well and the at least one second well are each structured as at least one SBS well on the SBS tissue culture plate.

[0072] In an embodiment, the base plate further comprises a base plate cover portion, and the SBS well plate is connectable on top of the base plate cover portion.

[0073] In an embodiment, the at least one first well is a plurality of first wells and the at least one second well is a plurality of second wells. The at least one microfluidic device is a plurality of microfluidic devices, each of the plurality of microfluidic devices being contained in a unique first well of the plurality of first wells, and the at least one microfluidic pump unit is a plurality of microfluidic pump units, each of the plurality of microfluidic pump units being contained in a unique second well of the plurality of second wells.

[0074] In an embodiment, the magnetic drive system includes a plurality of drive motors. Each drive motor of plurality of drive motors corresponds to one of the plurality of microfluidic pump units and is positioned on the cover unit such that the drive motor becomes rotatably coupled to the to the impeller of the corresponding one of the plurality of microfluidic pump units when the cover unit is connected to the base plate.

[0075] In an embodiment, the plurality of drive motors are arranged on the cover unit such that a spacing between each drive motor and the rest of the plurality of drive motors is maximized, and the plurality of microfluidic pump units are arranged on the base plate in a substantially identical arrangement to the plurality of drive motors on the cover unit such that each corresponding drive motor and microfluidic pump unit will be substantially aligned when the cover unit is connected to the base plate.

[0076] In an embodiment, the at least one second magnetic element is a first diametrically magnetized magnet that is connected to the output shaft proximate a freeend of the output shaft, and the at least one first magnetic element is a pair of diametrically magnetized magnets positioned on opposing sides of the impeller shaft, proximate a free end of the impeller shaft.

[0077] In an embodiment, the first diametrically magnetized magnet is oriented such that a magnetic axis thereof is substantially perpendicular to a long axis of the motor axle, and the pair of diametrically magnetized magnets are oriented such that a direction of magnetization for each of the pair of diametrically magnetized magnets is in substantially the same direction.

[0078] In an embodiment, the pump housing of the at least one microfluidic pump unit includes: a pump reservoir that is defined in a first section of the pump housing and which contains the impeller of the impeller pump unit and a volume of the fluid, and a media dampening reservoir that is defined in a second section of the main fluid housing and which also contains a volume of the fluid. The media dampening reservoir is in fluid communication with the at least one microfluidic device for receiving a flow of the fluid therefrom, and the media dampening reservoir is structured such that the flow of the fluid becomes more laminar as the flow of fluid enters the media dampening reservoir.

[0079] In an embodiment, the media dampening reservoir is fluidly connected to the pump reservoir via at least one fluid aperture, where the at least one fluid aperture defines an inlet for the impeller and the media dampening reservoir is disposed substantially atop the pump reservoir within the pump housing.

[0080] In an embodiment, the pump housing includes at least one inlet for receiving the flow of the fluid from the at least one microfluidic device. The at least one inlet is directly fluidly connected to the media dampening reservoir.

[0081] In an embodiment, the at least one inlet is spaced apart from the at least one fluid aperture such that the flow of the fluid enters the media dampening reservoir away from the at least one fluid aperture.

[0082] In an embodiment, the pump housing includes at least one outlet for providing a flow of the fluid to the at least one microfluidic device, and the at least one outlet is directly fluidly connected to the pump reservoir.

[0083] In an embodiment, the fluid connection between the at least one microfluidic device and the at least one microfluidic pump unit includes at least one inlet conduit and at least one outlet conduit. The inlet conduit is in fluid connection with the at least one inlet of the pump housing, and the outlet conduit is in fluid connection with at least one outlet of the pump housing.

[0084] In an embodiment, the media dampening reservoir is sized to generate a substantially laminar flow of the second volume of fluid contained therewithin, prior to the second volume of fluid being introduced into the pump reservoir via the at least one fluid aperture.

[0085] In an embodiment, the media dampening reservoir is an open top reservoir having a top surface that is open to the atmosphere.

[0086] In an embodiment, the second section of the pump housing is disposed vertically above the first section and is structured such that the second volume of fluid contained in the media calming reservoir can be evacuated from the media calming reservoir without evacuating the first volume fluid from the pump reservoir.

[0087] In an embodiment, the microfluidic assembly further comprises a control system, the control system being operatively connected to the at least one microfluidic pump unit for controlling the operation thereof.

[0088] According to another aspect, there is provided an in vitro system for culturing a multicellular aggregate comprising: the microfluidic assembly, and a control system in communication with the at least one microfluidic pump unit, the control system enabling pre-programmed fluid circulation schemes.

[0089] In an embodiment, the in vitro system further comprises at least one sensor in communication with the at least one microfluidic device, and / or the at least one microfluidic pump unit.

[0090] In an embodiment, the at least one sensor is adapted for monitoring pH, nutrient concentration, metabolic concentration, temperature, pressure, electrical output, or any combination thereof.

[0091] According to another aspect, there is provided a method of culturing at least one multicellular aggregate with the microfluidic assembly, the method comprising: forming a matrix seeded with the at least one multicellular aggregate within the at least one microfluidic device, operating the at least one microfluidic pump unit for perfusing a compartment of the at least one microfluidic device with a pressurized fluid having nutrients at a first flow rate so to induce the formation of a microvascular network within the matrix for vascularizing the at least one multicellular aggregate, and operating the at least one microfluidic pump unit for perfusing the compartment with the pressurized fluid at a second flow rate such that the pressurized fluid flows through the at least one microfluidic device and the matrix, the at least one multicellular aggregate is cultured. The second flow rate is selected to permit the survival of the at least one multicellular aggregate. The at least one microfluidic pump unit is operated such that when thepressurized fluid enters the at least one microfluidic device at either of the first flow rate of the second flow rate, the flow of the pressurized fluid is substantially non-turbulent.

[0092] According to another aspect, there is provided a method of culturing at least one multicellular aggregate with the microfluidic assembly, the method comprising: forming a matrix seeded with at least one multicellular aggregate within the at least one microfluidic device, operating the at least one microfluidic pump unit for perfusing the matrix with a pressurized fluid having nutrients at a first flow rate, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate, and operating the at least one microfluidic pump unit for perfusing the matrix with the pressurized fluid at a second flow rate such that the at least one multicellular aggregate is cultured, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the survival and maintenance of the multicellular aggregate.

[0093] According to another aspect, there is provided a microfluidic pump assembly that is fluidly connectable to at least one microfluidic device for circulating a fluid therethrough, the microfluidic pump assembly comprising: a pump housing, an impeller that includes an impeller shaft and that is rotatably housed within the pump housing, the impeller shaft including at least one first magnetic element, and a magnetic drive system that is connectable to the pump housing and that includes at least one drive motor with an output shaft, the output shaft including at least one second magnetic element. The magnetic drive system is removably connectable to the pump housing. The magnetic drive system is positioned relative to the at least one microfluidic pump unit such that the at least one first magnetic element is magnetically coupled to the at least one second magnetic element, while being spaced apart from at least one second magnetic element, for rotatably coupling the at least one drive motor to the impeller.Brief Description of the Drawings

[0094] Embodiments will now be described, by way of example only, with reference to the attached Figures, wherein:

[0095] Figure 1 provides a schematic illustration of the microfluidic apparatus according to an embodiment of the disclosure;

[0096] Figure 2A shows a top, perspective view of a tissue culture plate that contains an embodiment of the microfluidic device and microfluidic pump unit of the disclosure;

[0097] Figure 2B shows a perspective view of the embodiment of the microfluidic device in Figure 2A;

[0098] Figure 2C shows a top perspective view of the embodiment of the microfluidic device in Figure 2A, where a microfluidic channel of the microfluidic device is presented in a blown-up view;

[0099] Figure 2D shows a perspective view of the embodiment of the microfluidic channel in Figure 2C;

[0100] Figure 2E shows a top view of the embodiment of the microfluidic device in Figure 2A, where the microfluidic device is formed in a polymer base;

[0101] Figure 2F shows a close-up view of a number of slit-shaped pores from the embodiment of the porous membrane in Figure 2E;

[0102] Figure 2G shows a close-up view of a number of curved sections from embodiment of the serpentine pressure drop section in Figure 2E;

[0103] Figure 2H shows an exploded view of an embodiment of the microfluidic device in Figure 2A;

[0104] Figure 3A shows a top view of the embodiment of the microfluidic channel in Figure 2E, where a schematic of pressure values from a computational fluid dynamics (CFD) simulation for the microfluidic channel is superimposed over the channel;

[0105] Figure 3B shows a top view of the embodiment of the microfluidic channel in Figure 2E, where a schematic of local flow resistance values from a CFD simulation for the microfluidic channel is superimposed over the channel;

[0106] Figure 3C depicts the results of a CFD simulation across the embodiment of the microfluidic device in Figure 2B, where the results are depicted as flow streamlines and pressure values across the microfluidic device;

[0107] Figure 3D is a schematic diagram that depicts the compartment and aperture of the embodiment of the microfluidic device in Figure 2B, where a direction of fluid movement across the aperture is illustrated within the diagram;

[0108] Figure 3E is a log-log plot of the relationship between bulk permeability and compartment flow rate within the embodiment of the microfluidic device in Figure 2B;

[0109] Figure 3F is a schematic diagram that shows a computational flow streamline simulation of the path of fluid flow through the aperture of the embodiment of themicrofluidic device in Figure 2B, where the flow streamlines are shown together with fluid velocities and fluid pressures, and blown-up views of the streamlines at the porous membrane are provided;

[0110] Figure 4A shows a top view of a microfluidic channel of an embodiment of the disclosure;

[0111] Figure 4B shows a schematic of the CFD simulation for the microfluidic channel of Figure 4A;

[0112] Figure 5A shows an exploded view of a microfluidic device according to another embodiment of the disclosure;

[0113] Figure 5B shows a top plan view of the embodiment of the microfluidic device in Figure 5A;

[0114] Figure 6A shows an exploded view of a microfluidic device according to another embodiment of the disclosure;

[0115] Figure 6B shows a top perspective view of the embodiment of the microfluidic device in Figure 6A;

[0116] Figure 7A is a schematic diagram of a tissue culture plate with an embodiment of the microfluidic device and primary circulation system mounted thereon, where a blown-up view of the microfluidic device, where the microfluidic device includes a bypass channel, within the tissue culture plate is also provided;

[0117] Figure 7B shows a perspective view of an embodiment of the microfluidic device in Figure 7A, where the microfluidic device includes a bypass channel;

[0118] Figure 7C shows an exploded view of the embodiment of the microfluidic device in Figure 7B;

[0119] Figure 8A shows a top perspective view and top plan view of an embodiment of the microfluidic device in Figure 2B, where the microfluidic device has additional microfluidic channels and additional access ports;

[0120] Figure 8B shows a top perspective view and top plan view of another embodiment of the microfluidic device in Figure 2B, where the microfluidic device has additional microfluidic channels and additional access ports;

[0121] Figure 9A shows a top, perspective view of a tissue culture plate that contains an embodiment of the microfluidic device with both a primary microfluidic channel and a secondary microfluidic channel, where the primary circulation system is connected to the primary microfluidic channel and the secondary circulation system is connected to the secondary microfluidic channel;

[0122] Figure 9B shows a top, perspective view of the primary and secondary microfluidic channels of the embodiment of the microfluidic device in Figure 9A;

[0123] Figure 9C provides a schematic-cross sectional view of the porous membrane from the embodiment of the microfluidic device in Figure 9A, where blown-up views of the porous membrane at the interfaces with the primary microfluidic channel and secondary microfluidic channel are provided;

[0124] Figure 9D shows a top view of the embodiment of the primary and secondary microfluidic channels in Figure 9B, where a schematic of pressure values from a CFD simulation for the microfluidic channel is superimposed over the channels;

[0125] Figure 9E shows a top view of the embodiment of the primary and secondary microfluidic channels in Figure 9B, where a schematic of velocity values from a CFD simulation for the microfluidic channel is superimposed over the channels;

[0126] Figure 9F is a schematic diagram that shows a computational flow streamline simulation of the path of fluid flow through the aperture of the embodiment of the microfluidic device in Figure 9A, where the flow streamlines are shown together with fluid pressures;

[0127] Figure 9G is a schematic diagram that shows a computational flow streamline simulation of the path of fluid flow through the aperture of the embodiment of the microfluidic device in Figure 9A, where the flow streamlines are shown together with fluid velocities;

[0128] Figure 10A shows a top view of a tissue culture plate that includes microfluidic devices with multiple compartments connected in parallel, where a blown-up view of the microfluidic channel with the multiple compartments is also provided;

[0129] Figure 10B shows a top view of an alternate embodiment of the microfluidic device that has multiple compartments connected in parallel, where a schematic of pressure values from a CFD simulation for the microfluidic channel is superimposed over the channels;

[0130] Figure 10C shows a top view of an embodiment of the microfluidic device that has multiple compartments connected in series, where a schematic of pressure values from a CFD simulation for the microfluidic channel is superimposed over the channels;

[0131] Figure 11 shows a schematic diagram of the magnetic drive system and microfluidic pump unit according to an embodiment of the disclosure;

[0132] Figure 12 shows a perspective, partially cut-out view of the embodiment of the microfluidic pump unit in Figure 11 ;

[0133] Figure 13 provides an exploded view of the embodiment of the microfluidic pump unit in Figure 1 1 ;

[0134] Figure 14A shows a side cross-sectional view of the embodiment of the microfluidic pump unit in Figure 1 1 , where the pump reservoir and media dampening reservoir within the pump housing are shown filled with fluid;

[0135] Figure 14B shows a side cross-sectional view of the embodiment of the microfluidic pump unit in Figure 11 , where a schematic of pressure values from a CFD simulation for the microfluidic channel are superimposed over the reservoirs in the pump housing;

[0136] Figure 14C shows a side cross-sectional view of the embodiment of the microfluidic pump unit in Figure 11 , where a schematic of fluid velocity values from a CFD simulation for the microfluidic channel are superimposed over the reservoirs in the pump housing;

[0137] Figure 14D shows three top, cross-sectional views of the embodiment of the microfluidic pump unit in Figure 11 , where schematics of pressure, fluid velocity and fluid shear stress from a CFD simulation for the microfluidic channel are superimposed over the reservoirs in the pump housing;

[0138] Figure 14E shows a plot of fluid shear stress within the microfluidic channel for various impeller speeds;

[0139] Figure 15 shows a perspective view of a tissue culture plate that contains an embodiment of the microfluidic device and microfluidic pump unit of Figure 2A;

[0140] Figure 16A shows a partial exploded view of the cover unit according to an embodiment of the disclosure;

[0141] Figure 16B shows a schematic diagram of how the embodiment of the cover unit in Figure 16A can be mounted on a tissue culture plate;

[0142] Figure 16C shows a perspective view of the embodiment of the cover unit in Figure 16A mounted on a tissue culture plate;

[0143] Figure 17 shows a schematic diagram that illustrates how the first and second magnetic elements can be connected to the impeller shaft and motor output shaft of the embodiment of the magnetic drive system and microfluidic pump unit in Figure 11 ;

[0144] Figure 18A shows a schematic control diagram for the microfluidic assembly according to an embodiment of the disclosure;

[0145] Figure 18B shows a schematic diagram of the embodiment of the control system in Figure 18A, where the control system is communicatively connected to sixteen microfluidic pump units and magnetic drive systems on a tissue culture plate;

[0146] Figure 19A shows a top perspective view of a microfluidic apparatus with the assembled microfluidic device of Figure 16B, where the microfluidic device is uncoupled to a planar peristaltic pump;

[0147] Figure 19B shows a plurality of interconnected microfluidic apparatuses from the embodiment of Figure 19A;

[0148] Figure 20A shows a schematic diagram of the resistances and pressures within the microfluidic channel of the embodiment of the microfluidic device in Figure 1 B;

[0149] Figure 20B shows an analogous fluidic circuit for the microfluidic channel of the embodiment of the microfluidic device in Figure 1 B;

[0150] Figure 20C shows a prior art microfluidic channel that includes an upstream and downstream channel without a pressure drop section, where a schematic of fluid pressure from a CFD simulation for the microfluidic channel is superimposed over the microfluidic channel;

[0151] Figure 20D shows the embodiment of the microfluidic channel in Figure 2B where a schematic of fluid pressure from a CFD simulation for the microfluidic channel is superimposed over the microfluidic channel;

[0152] Figure 20E shows a plot of the influence of connecting hydrodynamic resistance on the ratio of the pressure difference across the compartment to the pressure difference between the pump inlet and pump outlet;

[0153] Figure 20F shows a plot of the influence of connecting hydrodynamic resistance on and the fluid shear stresses within the microfluidic channel;

[0154] Figure 21 A shows the vessel phenotype, and pressure distribution in the low pressure (200 Pa, at 3 pL / s flow rate) microfluidic configuration according to an embodiment of the disclosure;

[0155] Figure 21 B shows the vessel phenotype, and pressure distribution in the high pressure (2000 Pa, at 3 pL / s flow rate) microfluidic configuration according to an embodiment of the disclosure;

[0156] Figure 21 C shows the growth of multicellular aggregates in high-pressure microfluidic device designs overtime according to an embodiment of the disclosure;

[0157] Figure 21 D shows the growth of multicellular aggregates in low-pressure microfluidic device designs overtime according to an embodiment of the disclosure;

[0158] Figure 21 E shows the organization of human intestinal organoid in a hydrogel matrix by immunostaining;

[0159] Figure 21 F shows the organization of human cerebral organoid in a hydrogel matrix by immunostaining;

[0160] Figure 21 G shows the effects of cell types on the performance of a system according to an embodiment of the disclosure. FIG. 10A demonstrates a stable hydrogel and elongated cell phenotype (arrows) at 48 hours post seeding when adding endothelial cells (HUVECs) and fibroblasts (NHLFs) at a 1 :1 ratio (7.5 million cells / ml_, each cell type). It also demonstrates upon removing HUVECs (NHLFs only in hydrogel) the hydrogel shows marked degradation towards the outlet (arrows) and limited elongated cell phenotype and total hydrogel collapse upon removing NHFLs (HUVECs only in hydrogel);

[0161] Figure 22A shows a schematic diagram of fluid flow patterns and fluid recirculation vortices within the pores of the porous membrane according to an embodiment of the disclosure;

[0162] Figure 22B shows a graphical diagram of the mathematical definition of fluid recirculation zones based on integrating flow velocities from the pore ceiling;

[0163] Figure 22C shows a schematic diagram of fluid streamlines within the pores of the porous membrane for pores with an unoptimized aspect ratio and pores with an optimized aspect ratio;

[0164] Figure 22D shows a computational flow streamline simulation of the effect of the thickness of the porous membrane on flow streamlines at the microchannel level and pore level at the cross-sectional center of a single arbitrary circular pore in the inlet domain for porous membranes having a thickness of 10, 30, 50, and 70 pm;

[0165] Figure 22E shows a plot of the influence of porous membrane thickness on the size of the recirculation zones that form within the pores of the porous membrane;

[0166] Figure 22F shows a plot of the influence of pore aspect ratio on the size of the recirculation zones that form within the pores of the porous membrane;

[0167] Figure 22G shows a computational flow streamline simulation of the effect of the pore aspect ratio for the pores of the porous membrane, at the cross-sectional center of a single arbitrary circular pore in the inlet domain for pores having aspect ratios of 1 :4, 1 :1 , 4:1 , and 20:1 ;

[0168] Figure 23A shows a schematic, cross-sectional view of the microfluidic device i) when the porous interface remains intact and proper isolation of the matrix in the aperture occurs, and ii) when an instance of unwanted pore blockage has occurred;

[0169] Figure 23B shows a plot of the influence of pore aspect ratio on the quantitative metric of liquid entry pressure (LEP);

[0170] Figure 23C shows a plot of a parameter sweep for various pore aspect ratios;

[0171] Figure 23D shows finite element analysis plots from modeling the Young- Laplace equation of the effect of the pore aspect ratio for the pores of the porous membrane on pore deformation for a single arbitrary circular pore in the inlet domain, for pores having a circular cross-section and rectangular cross-sections with aspect ratios of, 1 :1 , 4:1 , and 20:1 ;

[0172] Figure 24A shows a schematic diagram of an exemplary application of the microfluidic device of the disclosure for the generation of vascular networks;

[0173] Figure 24B shows RFP-labeled endothelial cells within within the matrix located in the aperture;

[0174] Figure 24C shows the RFP-labeled endothelial vessels placed in the physical context of the tissue compartment and microchannels;

[0175] Figure 24D shows the endothelial vessels (CD31+) within the matrix located in the aperture of the microfluidic device;

[0176] Figure 24E shows an organoid that is embedded within the vascular network (CD31+) within the matrix located in the aperture;

[0177] Figure 24F shows an example process of initiating an experiment on the microfluidic device of the disclosure;

[0178] Figure 25A shows a schematic diagram of an instance of air bubble blockage within a microchannel on them microfluidic device of the disclosure;

[0179] Figure 25B shows a plot of optimized inlet and outlet conduit diameter in the context of a parameter sweep for the bubble unblocking pressure;

[0180] Figure 25C shows an analogous fluidic circuits for the microfluidic channel that includes the bypass channel;

[0181] Figure 25D shows a plot of a two-variable CFD parameter sweeps of the inlet and outlet conduits and bypass channel flow resistances;

[0182] Figure 25E shows a plot of a two-variable CFD parameter sweeps of the inlet and outlet conduits and bypass channel flow resistances;

[0183] Figure 25F shows a plot of pressure results from a CFD simulation for the microfluidic device with the bypass channel;

[0184] Figure 26A shows a top, plan view of the microfluidic device and microfluidic pump unit and indicates the concentration of compounds within the microfluidic device and microfluidic pump unit;

[0185] Figure 26B shows a plot of compound concentration within the microfluidic device and microfluidic pump unit overtime;

[0186] Figure 26C shows the results of the injection of fluorescent Dextran injection into the microfluidic device as the resulting perfusion of the fluorescent Dextran through the vascular network, where the resulting perfusion is shown during the running of the microfluidic pump unit and when the microfluidic pump unit is shut off;

[0187] Figure 26D shows a plot of CFD parameter sweeps, which involve modeling concentration dynamics;

[0188] Figure 27A shows a schematic diagram of a theoretical model that is applied to estimate the sedimentation risk within the microfluidic channel by considering bead passage through the connecting tubing;

[0189] Figure 27B shows the result of a CFD parameter sweep that was performed for the average tubing velocities within the connecting tubing;

[0190] Figure 28A shows a plot of the output flowrates provided by the microfluidic pump unit (for various impeller speeds) overtime;

[0191] Figure 28B shows a plot of the output flowrates provided by the microfluidic pump unit for various impeller speeds;

[0192] Figure 28C shows a plot of parameter sweeps for the pressure drop provided by the impeller pump;

[0193] Figure 28D shows an additional plot of parameter sweeps for the flowrate provided by the impeller pump;

[0194] Figure 28E shows another plot of parameter sweeps for the flowrate provided by the impeller pump;

[0195] Figure 28F shows a plot of the output flowrate from the impeller pump as a function of device resistance;

[0196] Figure 29A shows a plot of the output flowrate response of the impeller pump to the input of a step function;

[0197] Figure 29B shows a plot of the output flowrate response of the impeller pump to the termination of a step function;

[0198] Figure 29C shows a plot of an artificial, pulsatile flow that was generated by the impeller pump with the integrated PI controller; and

[0199] Figure 29D shows a plot of a complex waveform, in the form of a simulated blood flow patterns with dicrotic notches, that is generated through specification of the input waveforms within the control system and PI controller.Detailed Description of the Embodiments

[0200] For simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the Figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in orderto provide a thorough understanding of the embodiment or embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other instances, well- known methods, procedures, and components have not been described in detail so as not to obscure the embodiments described herein. It should be understood at the outset that, although exemplary embodiments are illustrated in the Figures and described below, the principles of the disclosure may be implemented using any number of techniques, whether currently known or not. The disclosure should in no way be limited to the exemplary implementations and techniques illustrated in the drawings and described below.

[0201] Various terms used throughout the present description may be read and understood as follows, unless the context indicates otherwise: “or” as used throughout is inclusive, as though written “and / or”; singular articles and pronouns as used throughout include their plural forms, and vice versa; similarly, gendered pronouns include their counterpart pronouns so that pronouns should not be understood as limiting anything described herein to use, implementation, performance, etc. by a single gender; “exemplary” should be understood as “illustrative” or “exemplifying” and not necessarily as “preferred” over other embodiments. Further definitions for terms may be set out herein; these may apply to prior and subsequent instances of those terms, as will be understood from a reading of the present description. It will also be noted that the use of the term “a” or “an” will be understood to denote “at least one” in all instances unless explicitly stated otherwise or unless it would be understood to be obvious that it must mean “one”.

[0202] As used herein, the terms “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms “comprises” and “comprising”, and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components.

[0203] As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions

[0204] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As used herein, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0205] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions supplement those in the art and are directed to the present application and are not to be imputed to any related or unrelated case. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the invention, particular methods and materials are described herein.

[0206] “Multicellular aggregate” refers to a cluster of cells suitable for culturing with or using a microfluidic device of the disclosure. A multicellular aggregate can be a cluster of cells which is architecturally and / or physiologically similar to an in vivo tissue or organ. Multicellular aggregates can comprise cells obtained from a cell line, primary cells isolated from an individual, or cells obtained from separation and isolation from a tissue biopsy. Multicellular aggregates can comprise one cell type or a plurality of different types providing heterogeneous organoids or spheroids. In an aspect, the multicellular aggregates are organized in a three-dimensional arrangement. Multicellular aggregates can be obtained from a suspension of cells, multicellular spheroids, organoids, or biopsies. Multicellular aggregates include without limitation, tissues, biopsies, tumor tissues, spheroids, organoids, embryoids, and the like. Examples of tissues include, but are not limited to, connective tissue, muscle tissue, genitourinary tissue, gastrointestinal tissue, pulmonary tissue, bone tissue, nervous tissue, epithelial tissue, endoderm-derived tissue, mesoderm-derived tissue, and ectoderm-derived tissue. In an embodiment, a multicellular aggregate is a spheroid, which can be a stem cell-derived spheroid, cancer cell-derived spheroid or cancer spheroid. In an embodiment, the multicellular aggregate is an organoid. In aspects of the disclosure, the multicellular aggregated is pre-cultured, i.e., cultured prior to introducing into a microfluidic device or system of the disclosure. Inaspects of the disclosure, the multicellular aggregate is a preassembled 3-dimensional tissue(s). In other aspects of the disclosure, the multicellular aggregated is not preassembled and / or pre-cultured, i.e., created using the microfluidic device or system of the disclosure.

[0207] “Organoid”

[0208] “Fluid” refers to a media capable of culturing or maintaining multicellular aggregates. A fluid can comprise any combination of cell culture medium, cells, whole blood, plasma, erythrocytes, nutrients, buffers, solutions, dyes, tracer compounds, antimicrobials, compounds not toxic to the multicellular aggregates or test analytes.

[0209] As used herein, the term “Fluid Velocity” refers to the average flow velocity across a cross section of a given channel.

[0210] As used herein, the term “Liquid Entry Pressure” refers to the pressure difference which creates the unwanted pore blockage across the porous membrane interface of the microfluidic device.[0021 1] As used herein, the term “Matrix” refers to any suitable scaffold material that supports the formation and survival of a vascular network and multicellular aggregates. In an embodiment, the matrix is a hydrogel, and in particular, a 3D matrix comprising a network of polymer chains. A hydrogel can be extracted from living organisms. A hydrogel can be derived from a synthetic polymer or biopolymer or a combination of polymers and / or biopolymers. For example, the hydrogel can be derived from collagen, gelatin, fibrin, agarose, alginate, polyacrylamide, polyethylene glycol, hyaluronic acid, cellulose derivatives, polypeptides, and mixtures of these polymers. A hydrogel can be obtained by either chemical crosslinking, physical crosslinking, or a combination of chemical and physical crosslinking. In an embodiment, the hydrogel is non-cytotoxic and has a gelation time of > 10 seconds to allow manipulation. A matrix can comprise growth factors, proteins, and / or peptide fragments to promote cell growth, survival, and transformation. In an embodiment, the preferred hydrogel is a fibrin hydrogel formed through crosslinking of a fibrinogen solution with thrombin.

[0212] As used herein, the term “Organoid” refers to a three-dimensional culture of organ-specific cell types that develop from stem cells and self-organize (or self-pattern) in a similar manner to the in vivo condition. Organoids can be prepared from adult stem cell-containing tissue samples, single adult stem cells, or through directed differentiation of pluripotent stem cells. In embodiments of the disclosure, organoids are generated from stem cells from patients. The organoid can be, but is not limited to skin, mammary glands, bone, brain, hypothalamus, pituitary gland, heart, blood vessels, larynx, trachea,bronchus, lung, lymphatic vessel, esophagus, stomach, gallbladder, liver, pancreas, small intestine, large intestine, colon, urethra, kidney, adrenal gland, ureter, bladder, fallopian tube, uterus, ovaries, testes, prostate, thyroid, parathyroid, tonsil, adenoid, thymus, and spleen. In an embodiment of the disclosure the organoid is a human cerebral organoid. In an embodiment, the organoid is a human intestinal organoid.

[0213] As used herein, the term “Test analyte” includes without limitation: a vitamin; a pharmaceutical agent; a nutraceutical agent; small molecule; macromolecule; a mineral, organic molecule; polypeptide; protein; nucleic acid; polynucleotide; nanoparticle; liposome; microparticle; microsphere; toxin; poison; a film-forming agent; a filler; a plasticizer; a taste-masking agent; a coloring agent; a solubilizing agent; an effervescent agent; an antioxidant; an absorption enhancer; a disintegrating agent; a pH modifying or buffer agent; a surfactant; a complexing agent; a bioadhesive agent; a tracking agent; antimicrobial preservative; catalyst; chelating agent; particles; flavoring agent; scent agent; identifying device; or combinations thereof.

[0214] As used herein, the term “Vascular network” refers to a network of cells or vessels that enables vascularization of multicellular aggregates, preferably precultured multicellular aggregate(s), and in particular functions similar to native vasculature. A vascular network can be formed from endothelial cells. A vascular network can be formed from endothelial cells and supporting cells such as fibroblasts, pericytes, adipocytes, chondrocytes, lymphocytes, mesenchymal stem cells, myofibroblasts, osteocytes, osteoblasts, myoblasts, myocytes, hematopoietic stem cells, macrophages, astrocytes, and any combination thereof, and the like “Precursor Vascular Cells”. Precursor Vascular Cells used to form a vascular network can be from a patient from which a multicellular aggregate has been obtained. In an embodiment, the vascular network comprises microvessels derived from endothelial cells. In an embodiment, the vascular network comprises microvessels derived from endothelial cells and fibroblast cells. Endothelial cells include cells of endothelial origin or differentiated cells that express markers of endothelial cells. Fibroblast cells include cells of fibroblast origin or differentiated cells that express markers of fibroblast cells.

[0215] Modifications, additions, or omissions may be made to the systems, apparatuses, and methods described herein without departing from the scope of the disclosure. For example, the components of the systems and apparatuses may be integrated or separated. Moreover, the operations of the systems and apparatuses disclosed herein may be performed by more, fewer, or other components and the methods described may include more, fewer, or other steps. Additionally, steps may beperformed in any suitable order. As used in this document, “each” refers to each member of a set or each member of a subset of a set.

[0216] The embodiments described herein are exemplary (e.g., in terms of materials, shapes, dimensions, and constructional details) and do not limit by the claims appended hereto and any amendments made thereto. Persons skilled in the art will appreciate that there are yet more alternative implementations and modifications possible, and that the following examples are only illustrations of one or more implementations. The scope of the disclosure, therefore, is only to be limited by the claims appended hereto and any amendments made thereto.Microfluidic device and apparatus

[0217] Referring now to the drawings and more particularly to Figure 1 , there is shown a microfluidic apparatus 10 of the disclosure, comprising a microfluidic device 100 in fluid communication with a primary circulation system 102 and a fluid source 16. The microfluidic device 100 has at least one aperture 132 configured for receiving a matrix. The matrix has been initially seeded with any combination of precursor vascular cells and multicellular aggregate 12. The fluid source 16 is adapted to contain / store a fluid which will be circulated throughout the aperture 132 to allow the formation and maintenance of a tissue culture 34 and survival and maintenance of multicellular aggregates 12. The primary circulation system 102 is connected to the fluid source 16 to cycle fluid through microfluidic device 100 at a predetermined rate. In at least some embodiments, the primary circulation system 102 can be the source of the fluid, while in other embodiments such as shown in Figure 1 , the source of fluid is the separate fluid source 16 and the fluid source 16 is fluidly connected to the primary circulation system 102.

[0218] The primary circulation system 102 allows fluid to be continuously recycled through microfluidic device 100 for establishing a pressure gradient within the aperture 132 and the matrix, thereby allowing perfusion of the fluid through the microfluidic device 100 and thus, into the living microvessels 36 of the tissue culture 34 at controlled flow rates and / or pressures that are tuned to match physiology conditions of the multicellular aggregate 12. Compounds of interest such as test analytes can be added to the fluid pumped through microfluidic device 100 at varying concentrations comparable to intravenous levels.

[0219] Referring to Figures 2E to 3E, there is provided an embodiment of the microfluidic device 100 of the disclosure. The microfluidic device 100 is generally structured for use with the primary circulation system 102. Various configurations andstructures of the microfluidic device 100 are provided within the disclosure, but generally, the microfluidic device 100 comprises (i) a microfluidic channel 1 10 that is configured for being in fluid communication with the primary circulation system 102. The microfluidic channel 110 of the microfluidic device 100 has at least an upstream channel 112 for receiving a pressurized fluid from the primary circulation system 102 as well as a pressure drop section 120 in fluid communication with the upstream channel 112. The pressure drop section 120 receives a first volume of the pressurized fluid from the upstream channel 112 and induces a pressure drop to produce a depressurized fluid having a lower pressure. The microfluidic channel 110 also includes a downstream channel 114 that is in fluid communication with the pressure drop section 120 for receiving the depressurized fluid therefrom, and for recirculating the depressurized fluid to the primary circulation system 102.

[0220] An alternate embodiment of the microfluidic device 100 of the disclosure including the pressure drop section 120 is provided in Figures 4A and 4B.

[0221] In addition to the microfluidic channel 1 10, the microfluidic device 100 includes (ii) at least one porous membrane 140 that has a plurality of pores 142, and (iii) at least one compartment 130 that includes an aperture 132. The aperture 132 of the at least one compartment 130 is provided in fluid communication with the microfluidic channel 110 via the at least one porous membrane 140 such that when a second volume of the pressurized fluid flows from the upstream channel 1 12 to the downstream channel 1 14 via the at least one porous membrane 140 and the compartment 130, a pressure differential is formed within the aperture 132.

[0222] Referring to Figures 2A to 2D, there is shown an embodiment of the microfluidic device 100 in fluid communication with the primary circulation system 102. The primary circulation system 102 is in fluid communication with upstream and downstream channels 1 12, 114 by connection at an inlet and an outlet of the primary circulation system 102. The microfluidic channel 1 10 is configured for receiving fluid from the primary circulation system 102 and for defining a precise pressure differential between the upstream and downstream channels 1 12, 114 to create regions of relatively high and low pressure within the microfluidic device 100. The functioning of the microfluidic channel 1 10 enables precise generation of high and low pressure sections while continuous circulation is maintained through the microfluidic device 100.

[0223] In the specific embodiment provided in Figures 2C and 2D, each of the upstream and downstream channels 1 12, 114 of the microfluidic channel 1 10 are formed as substantially straight channels. The porous membrane 140 interfaces the aperture 132of the at least one compartment 130 with the substantially straight upstream and downstream channels 1 12, 114. The aperture 132 in the at least one compartment 130 is generally provided on top of upstream channel 112 and downstream channel 1 14. The pressure drop section 120 of the microfluidic channel 110 is formed as a microchannel with as a serpentine channel 120a. Figure 2C provides a top view of a microfluidic device 100 having the two substantially straight channels (upstream channel 112 and downstream channel 114) joined by the serpentine channel 120a with high fluidic / hydrodynamic resistance (i.e., the pressure drop section 120). The geometry of the serpentine channel 120a creates areas of high and low pressure which are transferred through the porous membranes 140 into the aperture 132 of the at least one compartment 130.

[0224] In an additional embodiment, the microfluidic device 100 includes a plurality of the compartments 130, where each compartment 130 of the plurality of compartments 130 is in fluid communication with the upstream channel 112 and the downstream channel 114. The plurality of compartments 130 can be sequentially arranged along the microfluidic channel 1 10 such that each of the plurality of compartments 130 is positioned at least partially over the upstream channel 1 12 and at least partially overthe downstream channel 114.

[0225] In an embodiment such as shown in Figures 2A and 2C, the upstream channel 1 12 of the microfluidic channel 110 is fluidly connected to the outlet of the primary circulation system 102 via at least one outlet conduit 1 18, and the downstream channel 1 14 of the microfluidic channel 110 is fluidly connected to the inlet of the primary circulation system 102 via at least one inlet conduit 116.

[0226] In an additional embodiment, an internal diameter of each of the at least one inlet conduit 116 and at least one outlet conduit 118 is at least about 100 pm. By sizing the inlet and outlet conduits 116, 1 18 in this way, cells within the fluid may pass smoothly therethrough without experiencing unsuitably elevated shear stresses within the inlet and outlet conduits 1 16, 1 18.

[0227] In the specific embodiment provided in Figure 2A, the primary circulation system 102 comprises an impeller pump 102a connected to the microfluidic device 100 to cycle fluid through the microfluidic device 100 at a predetermined, controlled rate. Compounds of interest such as test analytes can be added to the fluid pumped through microfluidic device 100 at varying concentrations comparable to intravenous levels. The impeller pump 102a is circulatory, meaning that it reuses the fluid which perfuses throughmicrofluidic device 100. The inlet and outlet conduits 116, 118 connecting the primary circulation system 102 to the microfluidic device 100 include two lengths of plastic tubing.

[0228] The microfluidic channel 110 of the microfluidic device 100 is generally terminated at the extremities thereof with an upstream channel port 112a (on the upstream channel 112) and a downstream channel port 114a (on the downstream channel 114). The upstream and downstream channel port 112a, 114a provides a means for connecting the inlet and outlet conduits 116, 118 to the microfluidic device 100. The at least one outlet conduit 118 is connected to the upstream channel port 112a of the microfluidic channel 110 and the at least one inlet conduit 116 is connected to the downstream channel port 114a of the microfluidic channel 110.

[0229] In an embodiment such as shown in Figures 2B, 2C, and 2E, the microfluidic device 100 includes a base 134, and the microfluidic channel 110, compartment 130 and porous membrane 140 are all formed within the base 134.

[0230] Furthermore, the base 134 of the microfluidic device 100 can take any shape such as a squared shape, a rectangular shape, a circular shape, and the like. For example, the base 134 of the microfluidic device 100 can define a length of between about 20 mm and about 100 mm, and a width of between about 20 mm and about 100 mm (when the base 134 has a square or rectangular shape). In the case of a circular shape, the diameter of the base 134 of the microfluidic device 100 can be between about 20 mm and about 100 mm.

[0231] In an embodiment such as shown in Figures 2B and 2H, the base 134 of the microfluidic device 100 includes a first base piece 134a and a second base piece 134b that is mountable atop the first base piece 134a. The microfluidic channel 110 is formed in the first base piece 134a, the compartment 130, aperture 132 and upstream and downstream ports 112a, 114a are all formed in the second base piece 134b, and the porous membrane 140 is sandwiched between the first and second base pieces 134a, 134b. When the device 100 is assembled, the porous membrane 140 is positioned above the microfluidic channel 110, between the microfluidic channel 110 and the aperture 132 of the at least one compartment 130. A base cover 136 is mounted onto a top surface of the second base piece 134b, directly above the compartment 130 and the microfluidic channel 110. The upstream channel port 112a is formed as a channel in the second base portion 134b that is fluidly connected to the upstream channel 112, and downstream channel port 114a is a channel in the second base portion 134b that is fluidly connected to the downstream channel 114. The inlet and outlet conduits 116, 118 are connected tothe upstream and downstream channel ports 1 12a, 114a and are mounted onto the top surface of the second base piece 134b.

[0232] The microfluidic device 100 of the disclosure can be manufactured using or made of, without limitation, polydimethylsiloxane (PDMS), injection molded plastics with glass inserts and the like. An exemplary process for fabricating the microfluidic device 100 is described in Example 8 of the disclosure.

[0233] In the specific embodiment provided in Figure 2H, the first base piece 134a, the second base piece 134b, and the base cover 136 are each formed as individual silicone pieces. The silicone base cover 136 and silicone first and second base pieces 134a, 134b can be bonded together to form the overall structure of the base 134 of the microfluidic device 100. The inlet and outlet conduits 1 16, 118 are formed as inlet and outlet lengths of plastic tubing connected to the upstream and downstream ports 112a, 1 14a. The second base piece 134b is formed with an interiorly disposed through-opening that defines the at least one compartment 130 and forms the aperture 132 of the at least one compartment 130. The individual silicone pieces can be bonded together such that the aperture 132 is enclosed from the top by the base cover 136 and from the bottom by the porous membranes 140 and first base piece 134a. The porous membrane 140 is positioned to separate the tops of the upstream and downstream channels 1 12, 114 of the microfluidic channel 110 from the aperture 132 above.

[0234] In another additional embodiment, the microfluidic device 100 is configured such that the microfluidic channel 1 10 has an open-top configuration along a substantial portion of the length of the microfluidic channel 110, or alternatively, along at least part of the length of the microfluidic channel 110. The portion of the length of the microfluidic channel 110 that has the open-top configuration is sized so that the pressurized fluid is still able to directly interact via the porous membrane 140 towards (or away from) the matrix in at least one section of the microfluidic channel 110, and the depressurized first volume of fluid is still able to directly interact via the porous membrane 140 away from the matrix in at least one section of the microfluidic channel 110.

[0235] In an embodiment such as shown in Figure 2A, the at least one microfluidic device 100 and the primary circulation system 102 are each sized to fit within an individual well 210 of a tissue culture plate 200.

[0236] In the specific embodiment provided in Figure 2A, the tissue culture plate 200 is a standard multi-well Society for Biomolecular Screening (SBS) tissue culture plate that includes six wells 210 arranged in two rows of three. The tissue culture plate 200 holds three of the microfluidic devices 100 and three separate primary circulation systems 102,each of which correspond to one of the three microfluidic devices 100. The primary circulation systems 102and microfluidic devices 100 are arranged in an alternating pattern in the wells 210 of the tissue culture plate 200 and include pairs of connection tubing that are connected between each microfluidic device 100 and one of the primary circulation systems 102.Use for Vascularizing

[0237] In at least some embodiments of the disclosure, the microfluidic device 100 and primary circulation system 102 are collectively structured for culturing or vascularizing multicellular aggregates therewithin. The at least one aperture 132 in the at least one compartment 130 of the microfluidic device 100 generally houses the multicellular aggregates and is configured for receiving a matrix that forms the multicellular aggregate. The matrix that is provided to the aperture 132 in the at least one compartment 130 can have various compositions.

[0238] Example 4 provides a non-limiting embodiments of a method for loading the microfluidic device of the disclosure for use in culturing of vascularized tissue.

[0239] In the microfluidic device 100 as disclosed herein, the aperture 132 of the at least one compartment 130 is generally shaped and sized to provide sufficient room for the survival and maintenance of the multicellular aggregate.

[0240] In an additional embodiment such as provided in Figures 2C and 2D, the at least one compartment 130 of the microfluidic device 100 is a tissue compartment containing vascularized tissue. The vascular tissue held within the aperture 132 of the tissue compartment includes vascular networks with endothelial vessels in hydrogel scaffolds. The endothelial vessels receive substantially continuous intravascular flow from the pressure gradients that are created across the aperture 132 of the at least one compartment 130. Within the vascular tissue, vascularized organoids are housed within the aperture 132 of the at least one compartment 130 of the microfluidic devices 100 and receive fluid flow from the primary circulation system 102. The aperture 132 interfaces with microchannels via porous membranes 140. Various embodiments of applications and methods for using the microfluidic device 100 of the present disclosure are provided below in the “Applications” section of the detailed description.

[0241] In the embodiments where the aperture 132 contains the vascular network, the aperture 132 is also sized to promote the formation and maintenance of the microvascular network. Various sizes and shapes of the aperture 132 can be provided as part of the microfluidic device 100 as disclosed herein. For example, in the embodimentprovided in Figures 2A to 3D, the aperture 132 is a substantially cylindrical aperture 132 with a height between about 50 pm and about 10 mm, and a diameter between about 100 pm and about 20 mm. This particular shape and configuration of the aperture 132 can accommodate multicellular aggregates of more than about 2 mm.

[0242] In the specific embodiment provided in Figures 2C and 2D, the vascularized tissue, which is comprised of the vascular network and organoid, sits within the aperture 132 of the at least one compartment 130, on top of two microchannel segments of the upstream and downstream channels 112, 114. The upstream channel 112 has high relative pressure and the downstream channel 114 has low relative pressure. The porous membrane 140 forms the interface between the microchannel interior and the vascularized tissue such that fluid may flow between the channels 112, 114 through the slit shaped pores 142. The orientations of the pores 142 are positioned to be parallel with the direction of fluid flow. Fluid flows into the vascular network through the upstream channel 112 and fluid flows out of the vascular network through the downstream channel 114. The walls of the microfluidic channel 110 can be additionally coated with endothelial cells to promote anastomosis with the vascular network.

[0243] In an exemplary embodiment, the matrix is a matrix that has been initially seeded with any combination of precursor vascular cells and multicellular aggregate(s). The circulation of the fluid through the aperture 132 that contains the matrix will promote the formation and maintenance of a vascular network therewithin. The primary circulation system 102 allows fluid to be continuously recycled through microfluidic device 100 such as to establish a pressure gradient within the aperture 132 and the matrix allowing perfusion of external fluid (e.g. nutrient media) through the microfluidic device 100 and thus, into the living microvessels of the vascular network at controlled flow rates and / or pressures that are tuned to match physiology conditions of the multicellular aggregate.Pressure drop section

[0244] As shown in Figures 3A to 3D, when fluid moves through the microfluidic channel 110 of the microfluidic device 100, the fluid can pass through one of two paths: through the pressure drop section 120 (as the first volume of fluid) or through the porous membrane 140 and into the aperture 132 (as the second volume of fluid).

[0245] The pressure drop section 120 of the microfluidic channel 110 creates hydrodynamic resistance along the microfluidic channel 110, and this resistance produces the pressure gradient across the pressure drop section 120. The relative flow resistances along the pressure drop section 120 contribute to the pressure gradient theaperture 132 of the at least one compartment 130. The pressure drop and length of the pressure drop section 120 can be assessed using the following equations:AP = / (KH, Q, ... )RH= f (JI,L,R, ...) wherein AP is the pressuire drop, RHis the hydrodynamic resistance, Q is the flow rate, / / is the fluid viscosity, L is the length of the pressure drop section, and R is the radius of the pressure drop section.

[0246] In an embodiment, the pressure drop created by the pressure drop section 120 is between 50 Pa and 10 kPa. In further embodiments, the pressure drop is about 1 kPa and the hydrodynamic resistance within the pressure drop section 120 is between about 10 MPa*s*L1and about 100 MPa*s*L-1and preferably a width or radius of about 200 pm.

[0247] In the various embodiments of the disclosure, the sizing and structure of the pressure drop section 120 can be adjusted to provide for variations in the resistance and the resulting relative pressure differential across the aperture 132 of the at least one compartment 130. The magnitude of the pressure drop induced by the pressure drop section 120 is related to the hydrodynamic resistance within the pressure drop section 120. Fluidic / hydrodynamic resistance is a function of many variables, including channel length, number of bends, and channel width.

[0248] By varying the form and / or size of the pressure drop section 120, the ratio of fluid movement through the aperture 132 versus fluid movement through the pressure drop section 120 can be tuned. For example, by increasing the total length of the pressure drop section 120 or decreasing the width of the pressure drop section 120, the hydrodynamic resistance within the microchannel is increased leading to an increase in the ratio of fluid moving through the aperture 132 (i.e., a larger second fluid volume). Thus, although the form and the length of the pressure drop section 120 will affect the hydrodynamic resistance provided by the pressure drop section 120, the pressure drop section 120 can have various shapes and forms, so long as the pressure drop section 120 still produces a suitable pressure drop within the microfluidic channel 1 10.

[0249] Figures 3A to 3F show CFD simulation for the device 100 having the microfluidic channel 1 10 of Figures 2B to 2D with the serpentine channel 120a. Figure 3A depicts the pressure across the microfluidic channel 1 10 upon addition of flow. Arbitrary pressure units are given as the absolute pressure is a function of flow rate and fluid viscosity. Figure 3B depicts the hydrodynamic resistances across the microfluidicchannel 110. The serpentine channel 120a of the microfluidic channel 110 provides a pressure differential between the upstream channel 112 and the downstream channel 114, which is transferred through the porous membranes 140 into the aperture 132 of the at least one compartment 130 (and any vascular networks contained therewithin).

[0250] Example 1 of the disclosure presents a more detailed CFD driven process by which the geometry of the microfluidic channel 110 and pressure drop section 120 can be optimized. This process was utilized to arrive at the non-limiting serpentine channel 120a form of the pressure drop section 120.

[0251] In an embodiments such as provided in Figures 2A to 3D, the pressure drop section 120 is formed as the serpentine channel 120a and has a long and thin serpentine configuration with a plurality of undulating, curved portions 122 that are connected together.

[0252] In the specific embodiment provided in Figures 2C, 3A and 3B, the upstream and downstream channels 112, 114 are connected by the serpentine channel 120a form of the pressure drop section 120. Fluid enters at high pressure through the upstream channel 112, experiences a pressure drop across the serpentine channel 120a, and exits at low pressure through the downstream channel 114 (Figures 3A and 3B). As described previously, the amount of pressure drop along a particular microchannel segment is proportional to that segment’s hydrodynamic resistance. The serpentine channel 120a is an effective method of compacting a very long and narrow channel into a small footprint and thus experiences a very high flow resistance and high pressure drop. With its thin geometry, the high flow resistance serpentine channel 120a therefore experiences a high drop in pressure along its length. The local flow resistance of the entire microchannel is shown and defined as proportional to the local change in pressure. The high and low pressure regions of the microchannel connect with the tissue compartment 130 forming a pressure gradient as well as a second path for fluid flow. The connection of the tissue compartment 130 with the upstream and downstream channels 112, 114 transfers the high and low pressure regions to the compartment 130 (Figure 3A, 3B, 3C and 3F). Fluid may then flow either through the serpentine channel 120a, or across the tissue compartment 130

[0253] The serpentine channel 120a can have various configurations with various relative dimensions. In at least some embodiments, the microfluidic channel 110 is arranged so that the width and / or the height of the microfluidic channel 110 is greater in both the upstream and downstream channels 112, 114 than in the pressure drop section 120.

[0254] In further, exemplary embodiments, the microfluidic channel 110 has a width between about 50 pm and about 10 pm and a height between about 50 pm and about 10 pm in both upstream and downstream channels 112, 114, while the pressure drop section 120 of the microfluidic channel 110 has a width of about 10 pm and a height of about 20 pm. The length of the upstream channel 112 and downstream channel 114 is in a range from about 1 mm to about 25 mm (more particularly about 10 mm) and the length of the pressure drop section 120 is in a range from about 50 pm to about 10 mm (more particularly, about 50 mm).

[0255] In embodiments, the length of the upstream channel 112 and downstream channel 114 is in a range from about 5 mm to about 25 mm, from about 10 mm to about 20 mm, or from about 15 mm to about 20 mm, while the length of the pressure drop section 120 is in a range from about 1 mm to about 10 mm, from about 5 mm to about 10 mm, or from about 5 mm to about 8 mm.

[0256] While the above embodiments are specific to a serpentine channel 120a form of the pressure drop section 120, it will be readily understood that the pressure drop section 120 can take any size, shape and / or configuration, as long as the configuration of the pressure drop section 120 creates or generates a pressure gradient.

[0257] Referring to Figures 2, 3A and 3B, there is provided an embodiment of the microfluidic channel 110 that has a pressure drop section of different shape and / or configuration to the serpentine channel 120a. The pressure drop section 120 of this embodiment is a curved-circular pressure drop section 120b.

[0258] As shown in Figures 3A and 3B, the microfluidic channel 110 includes the upstream and downstream channels 112, 114. The upstream and downstream channels 112, 114 are joined by a length of microfluidic channel with high fluidic / hydrodynamic resistance that is formed as the curved-circular pressure drop section 120b. The microfluidic channel 110 has the upstream port 112a and the downstream port 114b for connecting the microfluidic device 110 to the primary circulation system 102. Figure 3B shows the CFD simulation for the microfluidic channel 110 having the curved-circular pressure drop section 120b of Figure 3A. For all intents and purposes, the pressure drop section 120b functions substantially the same as the serpentine channel 120a form of the pressure drop section 120. Only a length of channel with high fluidic / hydrodynamic resistance is required, being the serpentine channel 120a or other type of configuration.

[0259] In an additional embodiment, the pressure drop section 120 of the microfluidic channel 110 is composed as a multi-stage pressure drop section 120 that includes a plurality of pressure drop sub-sections.

[0260] Figures 3A to 3C and 3F show schematic illustrations of flow within a simulation of the microfluidic device 100 obtained with CFD analysis. Figure 3C depicts the bulk fluid flow from the inlet of the upstream channel 112 to the outlet of the downstream channel 114 of the microfluidic device 100. Figure 3F depicts the flow streamlines of random fluid particles within the fluid, as the fluid passes from the upstream channel 112 to the downstream channel 114 through the pores 142 of the porous membrane 140 and the aperture 132.

[0261] Referring now to Figure 3F, the functioning of the at least one porous membrane 140 is depicted. The at least one porous membrane 140 of the microfluidic device 100 functions to fluidly connect the aperture 132 of the compartment 130 with the upstream and downstream channels 112, 1 14 of the microfluidic channel 1 10. The at least one porous membrane 140 can allow connection of the aperture 132 (and therefore the delivery and return to vessels of the microvascular network) to the microfluidic channel 1 10. The porous membrane 140 supports the multicellular aggregate embedded in the matrix when the matrix is present within the aperture 132.

[0262] The porous membrane 140 is generally positioned across the overlap between the microfluidic channel 110 and the aperture 132 of the at least one compartment 130 and is at least partially in fluid communication with both the upstream channel 1 12 and downstream channel 114 of the microfluidic channel 110. In at least some embodiments of the microfluidic device 100, the porous membrane 140 is partially or totally coated with a coating comprising at least one of laminin, polydopamine, fibronectin, gelatin, collagen, Poly-L-Lysine, (3-Aminopropyl)triethoxysilane, glutaraldehyde and poly ethylene glycol.

[0263] Various structures, thicknesses and porosities of the porous membrane 140 can be provided within the microfluidic device 100 of the disclosure.

[0264] In an embodiment, the porous membrane 140 has a thickness ranging between about 1 pm and about 100 pm. In further embodiments, the porous membrane 140 may have a thickness ranging from about 5 pm to about 100 pm, from about 5 pm to about 40 pm, from about 20 pm to about 80 pm, and from about 40 pm to about 50 pm.

[0265] In a still further embodiment, the porous membrane 140 has porosity ranging between 0.1 and 0.5.

[0266] It may be beneficial in some embodiments of the microfluidic device 100 to provide a multi-section porous membranes 140 at the interface of the aperture 132 and the microfluidic channel 110. In an embodiment, the at least one porous membrane 140 has a first porous membrane portion 140a in fluid communication with the upstreamchannel 1 12 and a second porous membrane portion 140b in fluid communication with the downstream channel 114.

[0267] In then specific embodiment provided in Figures 2E, 2H, and 3F, the porous membrane 140 comprises the plurality of pores 142 and has the first porous membrane portion 140a in fluid communication with the upstream channel 112 and the second porous membrane portion 140b in fluid communication with the downstream channel 1 14. The first porous membrane portion 140a is disposed on top a section of the upstream channel 1 12 and the second porous membrane portion 140b is disposed on top a section of the downstream channel 1 14. This configuration of the porous membrane 140 and aperture 132 allows the second volume of the pressurized fluid to flow from the upstream channel 1 12 to the downstream channel 114 via the pores 142 of the porous membrane 140 and through the aperture 132 therefore forming a pressure differential within the aperture 132.

[0268] In some embodiments of the microfluidic device 100, the at least one porous membrane 140 has pores 142 that are distributed uniformly throughout a surface of the at least one porous membrane 140.

[0269] In at least some alternate embodiments, the at least one porous membrane 140 has pores 142 distributed over a plurality of portions of its surface. For example, the at least one porous membrane 140 can have pores 142 distributed over a first portion and second portion of its surface.

[0270] In a further embodiment such as provided in Figure 2F, the pores 142 of the porous membrane 140 have a dimension that is perpendicular to the flow of equal length as, or of shorter length than a dimension that is parallel to the flow.

[0271] In an additional embodiment, the microfluidic device 100 has a plurality of apertures 132 in fluid communication with the microfluidic channel 110. Each aperture 132 rests on a respective porous membrane 140 portion of the porous membrane 140 and is provided in fluid communication with corresponding sections of the upstream channel 112 and the downstream channel 114.

[0272] In an embodiment, the at least one compartment 130 is structured such that the aperture 132 includes a first aperture portion that is aligned with the first second porous membrane portion 140a and second aperture 132 portion that is aligned with second porous membrane portion 140b. In further embodiments, the first and second aperture portions define first and second openings formed along the bottom of the at least one compartment 130. The first and second openings are aligned with the respective first and second portions of the porous membrane portions 140a, 140b on the upstream anddownstream channels 112, 1 14 for fluidly connecting the aperture 132 in the at least one compartment 130 to the porous membrane 140 and upstream and downstream channels 1 12, 114.

[0273] Referring to Figure 3F, an illustration of the flow (including the pressures and velocities) of the fluid in the microfluidic device 100 through the plurality of pores 142 and the aperture 132 is provided. Pressurized fluid arrives from the primary circulation system 102 into the upstream channel 112 and leaves from the downstream channel 114. The upstream and downstream channels 112, 1 14 are connected to the tissue compartment 130 and vascularized tissue by the porous membranes 140, which consist of an arrays of pores 142. The pores 142 allow passage of fluid between the microchannel interiors and the hydrogel within the tissue compartment 130.

[0274] In the specific embodiment shown in Figure 3F, the first volume of pressurized fluid provided from the primary circulation system 102 makes a first pass underneath the first portion 140a of the porous membrane 140 that is in at least partially vertical alignment with the upstream channel 112. The first volume of fluid travels through the pores 142 and interfaces with the matrix (with delivery vessels), which is supported by the porous membrane 140. The depressurized fluid makes a second pass underneath the second, adjacent portion 140ab of the porous membrane 140 that is in substantially vertical alignment with the downstream channel 1 14, thereby allowing the fluid to flow back from the aperture 132 through the corresponding pores 142 and into the downstream channel 1 14 towards the end of the downstream channel 114. Again, the flow of the first volume of fluid across the aperture 132 and through the first and second portions of the porous membrane 140 is driven by the pressure gradient provided between the upstream channel 112 and downstream channel 114.

[0275] As captured by the streamlines in Figure 3F, fluid may either flow through the serpentine channel or through the tissue compartment 130. 130. Flow concentrates at the individual pores 142 of the porous membrane 140 interface. The exact flowrate through the aperture 132 of the compartment 130 depends on its bulk permeability and can be modeled with Darcy’s law using CFD. For the same pressure, a higher permeability will produce higher flowrates and thus provide more delivery of nutrient media. The total flowrate of fluid through the aperture 132 of the compartment 130 is dependent on the bulk permeability of the contents of the at least one compartment 130, in addition to the magnitude of the pressure gradient (see Figures 3D and 3E). The compartment flowrate dictates, at least in part, the amounts of nutrient media that the vascular network and organoid within the compartment 130 receives. For the samepressure differential, a higher bulk permeability will result in higher flowrates and nutrient media delivery. Bulk permeability is a value which depends on the hydrogel material properties as well as the degree of vascularization, in that vascularization lowers permeability by providing direct fluid paths.

[0276] In an embodiment where the aperture 132 of the at least one compartment 130 contains a vascular network, the pressurized fluid flowing from the segment of upstream channel 112 to the segment of downstream channel 114 via the porous membrane 140 enables the perfusion of the multicellular aggregate and thereby stimulates the formation of the vascular network from the embedded precursor vascular cells. The vascular network connects the multicellular aggregate to pores 142 of porous membrane 140. The porous membrane 140 therefore provides underlying support for the matrix, while allowing a portion of pressurized fluid to flow from the upstream channel 112 to the downstream channel 114 through the aperture 132. In this embodiment, the microfluidic device 100 is configured such that the pores 142 of the porous membrane 140 are connected to the lumens of the vascular network. With this structure, the passage of fluid across the aperture 132 of the at least one compartment 130 primarily occurs through the vascular network as the individual pores 142 of the porous membrane 140. Fluid flowing through the microfluidic channel 110 creates the pressure gradient across the aperture 132. This prompts the lumen of the microvessels to fully form connections with the pores 142 of the porous membrane 140, enabling fluid movement from the underlying microfluidic channel 110 through pores 142 and the microvessels, towards the multicellular aggerate within the aperture 132.

[0277] The pores 142 of the at least one porous membrane 140 can take any shape, size and / or configuration so long as the flow at the interface of the microfluidic channel 1 10 and porous membrane 140 is predominantly non-turbulent. By providing this predominantly non-turbulent flow, vascular networks in the matrix within the compartment 130 are able to traverse the porous membrane 140 and establish a connection with the underlying microfluidic channel 110.

[0278] In an additional embodiment, the porous membrane 140 has a thickness which minimizes the creation of turbulence at an interface of the microfluidic channel 1 10 (e.g., the upstream and downstream channels 112, 114) and the porous membrane 140. Said another way, the porous membrane 140 is structured to minimize turbulence within the pores 142 thereof.

[0279] In a first non-limiting example of the pore geometry for the plurality of pores 142, each pore of the plurality of pores 142 of the at least one porous membrane 140 has a pore diameter ratio Perp:Para equal to or less than 1 :1.

[0280] In a second non-limiting example of the pore geometry for the plurality of pores 142, the pores 142 are structured as elongated pores 142 having a diameter perpendicular to the flow of shorter length than the diameter parallel to the flow (Perp:Para < 1).

[0281] In a third non-limiting example of the pore geometry for the plurality of pores 142, the pores 142 are structured as circular pores 142 having a diameter perpendicular to the flow of equal length as the diameter parallel to the flow (Perp:Para ~ 1).

[0282] In a fourth, non-limiting example of the pore geometry for the plurality of pores 142, the pores 142 have a pore diameter ratio Perp:Para ranging between about 1 :1 and about 1 :100. In further embodiments, the pores 142 have a pore diameter ratio Perp:Pararanging between about 1 :20 and about 1 :100.

[0283] In a fifth, non-limiting example such as shown in Figure 2F, the plurality of pores 142 are structured as a plurality of slit-shaped pores 142a. Figure 2F provides a magnified top view of the porous membrane 140 depicting an array of the slit-shaped pores 142a.

[0284] In the specific embodiment provided in Figure 2F, the plurality of slit-shaped pores 142a are aligned in the direction of flow. Effectively, the plurality of slit-shaped pores 142a are oriented such that a long axis of each pore of the plurality of slit-shaped pores 142a extends substantially parallel to a long axis of the upstream and downstream channels 1 12, 114.

[0285] In an embodiment, the pores 142 are structured for minimizing fluid recirculation vortices therewithin. Example 3 of the disclosure provides a non-limiting embodiment of a process for optimizing the aspect ratio of the pores 142 of the porous membrane 140 for limiting the occurrence of recirculation vortices within the pores 142 and for providing a more robust design of the porous membrane 140.

[0286] In an additional embodiment, each pore 142a of the plurality of slit-shaped pores 142a is structured such that an aspect ratio of the pore 142a is greater than or equal to about 20:1.

[0287] In another additional embodiment, each pore 142a of the plurality of slitshaped pores 142a is structured such that a width of the pore 142a is in a range from about 10 pm to about 100 pm and a length of the pore is in a range from about 50 pm to about 200 pm.

[0288] Referring to Figures 5A and 5B, there is provided a first alternate embodiment of the microfluidic device 100 of the present disclosure, where the microfluidic device is the microfluidic device 200.

[0289] In this embodiment, the microfluidic device 200 has a first compartment 230 defining a microfluidic channel 210 configured for being in fluid communication with the primary circulation system 102 (not shown). The microfluidic channel 210 has an upstream channel 212 which receives a pressurized fluid for flow therethrough, a pressure drop section 220 in fluid communication with the upstream channel 212 for receiving the pressurized fluid and inducing a pressure drop to produce a depressurized fluid having a lower pressure, and a downstream channel 214 in fluid communication with pressure drop section 220 for receiving the depressurized fluid and returning the depressurized fluid to the circulation system. The upstream channel 212 is substantially colinear with the downstream channel 214. The upstream channel 212 extends outwards from a first end of the pressure drop section 220 and the downstream channel 214 extends out from a second end of the pressure drop section 220 that is opposite the first end.

[0290] In an additional embodiment such as shown in Figures 5A and 5B, the microfluidic channel 210 is terminated at both extremities at the upstream channel end 212a and the downstream channel end 214b. The microfluidic device 200 further comprises a porous membrane 240 which has the plurality of pores 142. The porous membrane 240 is substantially equivalent to the porous membrane 140. The microfluidic device 200 further comprises a second compartment 240 which defines an aperture 232 (also sometimes referred to as tissue cavity) therethrough and openings 228 positioned above the end of the upstream channel 212a and end of the downstream channel 214a to create the upstream port 227 and downstream port 229, which enable connection of the microfluidic device 200 to fluid connection lines such as the inlet and outlet conduits 116, 118. The second compartment 240 rests and is secured over first compartment 230 and the porous membrane 240 is positioned between the compartments 230, 240 interfacing between the microfluidic channel 210 and the aperture 232. The aperture 232 and microfluidic channel 210 interfaces with opposite sides of the porous membrane 240 and is positioned directly above the pressure drop section 220 (depicted here as a serpentine channel). This configuration creates a pressure differential across the microfluidic device 200 and allows pressurized fluid to take one of two paths; through the upstream channel 212 to the downstream channel 214 or through the aperture 232 viathe pores of the porous membrane 240. The microfluidic device 200 further comprises a lid or coverslip 234, which is adapted to seal the aperture 232..

[0291] Referring to Figures 6A and 6B, there is provided a second alternate embodiment of the microfluidic device 100 where the microfluidic device is structured as the microfluidic device 250. The microfluidic device 250 comprises a microfluidic channel 260. The microfluidic channel 260 has an upstream channel 262, and a downstream channel 264 in fluid communication with a pressure drop section 270.

[0292] In an embodiment such as provided in Figure 6BC, the microfluidic channel 260 is embedded in a first compartment comprising a circular shape containing a region with flexible microchannels 260a. The microfluidic device 250 further comprises a second compartment 280 which is supported by the first compartment, as well as the fluid source 290 which is supported by the second compartment 280. The second compartment 280 comprises an aperture 282 and openings 284 to allow fluid flow from the fluid source 290 to the microfluidic channel 260. The microfluidic device 250 further comprises the porous membrane 240, which covers part of the upstream channel 262 and part of the downstream channel 264. The microfluidic device 250 also has a lid 271 to cover the fluid source 290 to prevent contamination and evaporation. The aperture 282 is sealed by placing the fluid source 290 on top of the second compartment 280, which is designed to provide a seal to both the bottom of the fluid source 290 and the top of the aperture 282. The fluid source 290is connected to the end of the upstream channel 262a and the end of the downstream channel 264a by aligned openings 288 in the fluid source 290 and second compartment 280. This stacked configuration creates the upstream port 273 and downstream port 274 with in the microfluidic device 250.Bypass channel

[0293] Integrating the various embodiments of the microfluidic device of the disclosure with the primary circulation system 102 is non-trivial and requires balancing multiple factors in order to maximize utility. Two of the main factors to consider when integrating the microfluidic device 100 and primary circulation system 102 is the internal diameter of the inlet and outlet conduits 116, 118 that provide fluid communication therebetween, and the fluid velocities and pressure gradients within the microfluidic device 100. These two factors are, in most embodiments of the device, conflicting.

[0294] On one hand, larger diameters of the inlet and outlet conduits 116, 1 18 are generally favorable, as these larger diameters inhibit bubble obstructions and reduce pressure loss along the length of the inlet and outlet conduits 1 16, 118. Air bubbleobstructions within flow conduits (e.g., tubing) is a common issue within microfluidics. Tubing obstructions tend to stop the flow of nutrient media, therefore starving vascularized tissue within microfluidic devices. Bubble obstructions within capillary tubes, such as those illustrated in Figures 8A, have been previously described as the “Jamin Effect”, wherein entrapped bubbles produce a pressure in opposition to fluid flow. Although the tubing could be made as wide as possible to prevent these obstructions, larger diameter tubing results in lower fluid velocities within the microfluidic channel 1 10. These lower velocities in turn lead to unfavorable increases in the delivery time of compounds from the primary circulation system 102 to the aperture 132 of the at least on compartment 130. The lower velocities can also result in increased sedimentation of suspended cells and / or particles within the circulated fluid.

[0295] To address the drawbacks of lower fluid velocities while still including relatively high diameter inlet and outlet conduits 1 16, 1 18 between the microfluidic device 100 and primary circulation system 102, at least some embodiments of the microfluidic device in the disclosure include a bypass channel 310. The bypass channel 310 is fluidly connected between the upstream and downstream channels 1 12, 114 of the microfluidic device 100 and effectively “bridges” the inlet and outlet of the microfluidic device 100 for optimizing the flow conditions within the device 100.

[0296] In an embodiment such as shown in Figure 7A, 7Band 7C, the microfluidic device 100 further comprises the bypass channel 310. The bypass channel 310 is provided in fluid communication between the upstream and the downstream channels 1 12, 1 14 of the microfluidic channel 110 for circulating a third volume of the pressurized fluid therebetween. The bypass channel 310 generally provides a secondary path for fluid to flow across the ports of the microfluidic device 100. The bypass channel 310 is structured to optimize the flow conditions within the microfluidic device 100 to effectively increase fluid velocities within the microfluidic channel 1 10 and inlet / outlet conduits 116, 1 18 without substantially affecting the pressure and fluid velocity landscapes within the microfluidic device 100 itself. In at least some embodiments, the bypass channel 310 is, sized to ensure that the pressure differential across the aperture 132 of the at least one compartment 130 is maximized.

[0297] In an embodiment, the bypass channel 310 of the microfluidic is structured for increasing a velocity of the pressurized fluid within the upstream channel 1 12 without reducing the pressure differential within the aperture 132 of the at least one compartment 130. By maximizing the pressure differential across the at least one compartment 130 (via the inclusion of the bypass channel 310), the fluid velocities within the microfluidicchannel 110 can be raised without reducing the pressure differential across the aperture 132 to a pressure differential that is below a required pressure for tissue culturing within the aperture 132 of the at least one compartment 130.

[0298] In an additional embodiment, the bypass channel 310 of the microfluidic device 100 is sized such that a ratio of APCOmp / APpump is at least about 0.9. In this embodiment, APCOmp is defined as the pressure differential across the aperture 132 of the at least one compartment 130, and APpUmp is defined as a pressure differential across the primary circulation system 102.

[0299] In the microfluidic device 100 as disclosed herein, various designs, sizes and configurations of the bypass channel 310 can be provided, and the bypass channel 310 can be configured in various ways for optimizing the flow conditions within the microfluidic device 100.

[0300] In an embodiment, the bypass channel 310 is structured as an optimized bypass channel 310 based on the optimization of one or more objective variables of the bypass channel 310. Example 5 of the present application provides a non-limiting example of the optimization of the form of the bypass channel 310. When optimizing the form of the bypass channel 310, the flow and associated resistances within the microfluidic device 100 can be approximated by an analogous fluid circuit. The bypass channel 310 introduces complexity into the analogous circuit, making the formulas governing the flow in the circuit difficult to manage, and necessitating CFD optimization when optimizing the form of the bypass channel 310.

[0301] Figures 7A and 7B provide an embodiment of an optimized form of the bypass channel 310. The bypass channel 310 generally includes an inlet end and an outlet end. The inlet end is provided in fluid communication with the upstream channel 112, and the outlet end is provided in fluid communication with the downstream channel 114 for circulating the third volume of the pressurized fluid through the bypass channel 310.

[0302] In an additional embodiment such as provided in Figure 7A, the bypass channel 310 includes an inlet channel portion 31 Oa that defines the inlet end of the bypass channel 310, an outlet channel portion 310b that defines the outlet end of the bypass channel 310, and a bypass portion 310c which extends between the inlet and outlet channel portions 310a, 310b.

[0303] In the specific embodiment provided in Figure 7A, the inlet channel portion 310a extends upwards, substantially vertically from the upstream channel 112, the outlet channel portion 310b extends upwards, substantially vertically from the downstream channel 114, and the bypass portion 310c is substantially horizontal. The inlet channelportion 310a, outlet channel portion 310b and bypass portion 310c are formed by a continuous length of tubing. The tubing of the inlet and outlet channel portions 310a, 310b is mounted onto the top surface of the second base portion 134b of the microfluidic device 100, on either side of the upstream and downstream ports 1 12a, 114a of the microfluidic device 100. The tubing of the bypass portion 310c extends substantially horizontally overtop of the upstream and downstream ports 112a, 1 14a to connect the tubing of the inlet and outlet channel portions 310a, 310b.

[0304] In at least some embodiments of the disclosure, an internal diameter of the bypass channel 310 is at least 50 pm.

[0305] In an additional embodiment, the bypass channel 310 is sized such that a length of the bypass channel 310 is in a range from about 10 mm to about 50 mm, and an inner diameter of the bypass channel 310 is in a range from about 0.1 mm to about 5 mm.

[0306] In embodiments, the length of the bypass channel 310 is in a range from about 15 mm to about 50 mm, from about 20 mm to about 50 mm, from about 30 mm to about 50 mm, or from about 30 mm to about 40 mm.

[0307] The bypass channel 310 of the microfluidic device 100 device may be fluidly connected to the rest of the microfluidic channel 110 in various ways, provided that relatively non-turbulent fluid flow between the microfluidic channel 110 and the bypass channel 310 can be achieved.

[0308] In the specific embodiment provided in Figure 7C, the second base portion 134b includes bypass channel ports 135. The bypass channel 310 can be connected directly to the bypass channel ports 135. The bypass channel ports 135 are formed such that when first and second base portions 134a, 134b are mounted together, the bypass channel 310 is in fluid communication with microfluidic channel 110.

[0309] In an exemplary embodiment provided in Figures 7A, 7B, and 7C, the inlet end of the bypass channel 310 is connected to the upstream channel 112 of the microfluidic channel 110 to form a first three-way node between the bypass channel 310, the upstream channel 1 12 and the primary circulation system 102. Similarly, the outlet end of the bypass channel 310 is connected to the downstream channel 114 to form a second three-way node between the bypass channel 310, the downstream channel 1 14 and the primary circulation system 102.

[0310] In an additional embodiment such as shown in Figures 2E and 2H, each of the upstream and downstream channels 112, 114 include connection channel arms 115 that extends out from the rest of the length of the channel at a non-zero angle. The inletend of the bypass channel 310 is fluidly connected to the connection channel arm 1 15 of the upstream channel 112, and the outlet end of the bypass channel 310 is fluidly connected to the connection channel arm 115 of the downstream channel 114.[0031 1] In an alternate embodiment, the inlet end of the bypass channel 310 is connected directly to the upstream channel 1 12 of the microfluidic channel 110 along the length of the upstream channel 1 12, and the outlet end of the bypass channel 310 is connected directly to the downstream channel 1 14 along the length of the downstream channel 114.

[0312] Due to the optimization of the form of the bypass channel 310 using CFD optimization processes such as described in Example 5, a hydrodynamic resistance provided by the bypass channel 310 is also optimized. In an embodiment, the bypass channel 310 is structured such that a fluid resistance of the bypass channel 310 is no less than five times a fluid resistance of the upstream channel 112, and such that the fluid resistance of the bypass channel 310 is no less than five times a fluid resistance of the downstream channel 1 14.

[0313] The bypass channel 310 can, in at least in some embodiments of the disclosure, provide additional performance improvements to the microfluidic device 100 when the fluid is being circulated through the microfluidic device 100 via the primary circulation system 102.

[0314] In the embodiments where the connection of the microfluidic device 100 to primary circulation system 102 is via the inlet and outlet conduits 1 16, 118, the bypass channel 310 is structured to permit increased diameters of the at least one outlet conduit 1 18 and the at least one inlet conduit 1 16 of the primary circulation system 102 compared to what could be used with an embodiment of the microfluidic device 100 that does not include the bypass channel 310. The bypass channel 310 enables the use of the increased diameter conduits while substantially maintaining a velocity of both the pressurized fluid and the depressurized fluid through the at least one outlet conduit 118 and the at least one inlet conduit 1 16.

[0315] In an additional embodiment, the bypass channel 310 and upstream and downstream channels 112, 114 of the microfluidic channel 1 10 are each sized such that the microfluidic device 100 can accommodate an internal diameter of the at least one outlet conduit 1 18 that is in a range from about 0.1 mm to about 5 mm, and an internal diameter of the at least one inlet conduit 1 16 that is in a range from about 0.1 mm to about 5 mm.

[0316] In an embodiment, the bypass channel 310 of the microfluidic is structured for reducing particle sedimentation time within the aperture 132 of the at least one compartment 130. For certain biological applications, particles such as cell suspensions may be added to the fluid. These particles are at risk of undesired sedimentation in the inlet and outlet conduits 116, 118 when fluid velocities are too low. Example 7 of the disclosure demonstrates an optimized form of the bypass channel 310 which provides for reduced particle sedimentation time across the operating ranges of a particular embodiments of the primary circulation system 102.

[0317] In an embodiment, the bypass channel 310 of the microfluidic is structured for increasing a rate of delivery of at least one compound to the aperture 132 of the at least one compartment 130 via the microfluidic channel 110. Compounds added to the primary circulation system 102 must first transit through the connection tubing (inlet and outlet conduits 1 16, 118) before arriving at the tissue at least one compartment 130. Example 6 of the disclosure demonstrates an optimized form of the bypass channel 310 which provides improved delivery times for the at least one compound. Example 6 of the disclosure demonstrates that usage of the bypass channel 310 is able to reduce the compound delivery time from about ten minutes to about ten seconds by increasing average fluid velocities within the inlet and outlet conduits 1 16, 118.

[0318] In at least some embodiments of the disclosure, the bypass channel 310 is formed such that the bypass channel 310 can be physically manipulated for altering a resistance characteristic thereof.

[0319] In one such embodiment, at least a portion of the bypass channel 310 is composed of a pliable material. The pliable material of the bypass channel 310 is selected to be sufficiently pliable such that the at least a portion of the bypass channel 310 can be compressed on itself for substantially inhibiting a flow the of the third volume of the pressurized fluid through the bypass channel 310. The compression of the pliable portion of the bypass channel 310 will increase the effective hydrodynamic resistance of the bypass channel 310 and thereby inhibit the flow of the third volume of fluid through the bypass channel 310.Additional microfluidic channels

[0320] Referring to Figures 8A and 8B, there are provided two separate embodiments of the microfluidic device 110, where each microfluidic device includes additional microfluidic channels and at least one additional access port 450 in fluid communication with the aperture 132 via the additional microfluidic channel. Suchadditional access ports 450 can be used to access the sealed aperture 132 within the compartment 130 without disruption to act as a source or sink for circulating compounds, selective spatial diffusion of compounds (or cells) and / or fluid sampling.

[0321] In an embodiment, the microfluidic device 100 includes at least one additional access port 450. The at least one additional access port 450 is provided in fluid communication with the aperture 132 of the at least one compartment 130. The at least one additional access port 450 is used for selective spatial diffusion of compounds into the aperture 132 of the microfluidic device 100.

[0322] In an embodiment such as shown in Figures 9A and 9B, the microfluidic device 100 further comprises at least one secondary microfluidic channel 410 for use with a secondary circulation system 402. The at least one secondary microfluidic channel 410 is provided as part of the microfluidic device 100 for culturing a second tissue culture that may require different flow properties then those provided by the primary circulation system 102 within the microfluidic channel 110.

[0323] The at least one secondary microfluidic channel 410 is structured such that pressure outputs of the primary and secondary circulations systems 102, 402 substantially match. The matching pressure outputs of the primary and secondary circulation systems 102, 402 helps to ensure that there is substantially no net fluid flow occurs between the microfluidic channel 1 10 and the secondary microfluidic channel 410. Effectively, the presence of the secondary channels in the microfluidic device 100 does not obstruct the pressure gradient across the aperture 132 of the at least one compartment 130.

[0324] In the specific embodiment provided in Figure 9A, a pair of the microfluidic devices 100 that include both the microfluidic channel 110 and the secondary microfluidic channel 410 are provided on a six-well tissue culture plate with the primary and secondary circulation systems 102, 402 formed as primary and secondary impeller pump. The six- wall tissue culture plate includes two rows of three wells each. On each row, the microfluidic device 100 is contained in a centremost well, the primary circulation system 102 is provided in a rightmost well, and the secondary circulation system 402 is provided in a leftmost well. The primary and secondary circulation systems 102, 402 are each connected to the microfluidic device 100 by a length two lengths of tubing (forming the inlet and outlet conduits 116, 118).

[0325] In a non-limiting example, the secondary microfluidic channel 410 is applied for emulating the gastrointestinal tract in addition to vascular channels. Shear stress levels in the endothelium are higher than those found in the gut and elsewhere in thebody and thus, channel flow resistances for the secondary channel 410 are chosen accordingly.

[0326] In an embodiment such as shown in Figure 9B, the secondary microfluidic channel 410 includes a secondary upstream section 412 for receiving a pressurized fluid from the secondary circulation system 402, and a secondary pressure drop section 420 in fluid communication with the secondary upstream section 412 for receiving the pressurized fluid therefrom. The secondary pressure drop section 420 is structured for inducing a pressure drop in the pressurized fluid to produce a depressurized fluid. The secondary microfluidic channel 410 also includes a secondary downstream section 414 in fluid communication with the pressure drop section 120 for receiving the depressurized fluid therefrom and recirculating the second depressurized fluid to the secondary circulation system 402. Input pressures of the primary microfluidic channel 1 10 and secondary microfluidic channel 410 are provided as substantially equal to substantially prevent net flow between the primary and secondary microfluidic channels 110, 410. Said another way, a pressure difference defined between an inlet of the upstream channel 112 and outlet of the downstream channel 1 14 of the microfluidic channel 110 is substantially equal to pressure difference defined between an inlet of the secondary upstream section 412 and an outlet of the secondary downstream section 414 of the secondary microfluidic channel 410.

[0327] As shown in Figure 9C, the secondary microfluidic channel 410 is formed on the base 134 of the microfluidic device 100 and is positioned on the base 134 such that secondary microfluidic channel 410 is disposed below the aperture 132 of the at least one compartment 130, with the porous membrane 140 and associated pores 142 also being located between the secondary microfluidic channel 410 and the aperture 132 of the at least one compartment 130.

[0328] In an additional embodiment such as shown in Figures 9B and 9D, the secondary pressure drop section 420 of the secondary microfluidic channel 410 comprises a plurality of pressure drop sections 420 that are formed in series between the upstream and downstream sections 412, 414 of the secondary microfluidic channel 410.

[0329] In another additional embodiment, the plurality of pressure drop sections 420 include a first pressure drop section 420a in fluid communication with the secondary upstream section 412, where the first pressure drop section 420a is structured for inducing a first pressure drop in the pressurized fluid to produce a first depressurized fluid. The plurality of pressure drop sections 420 also include a second pressure drop section 420b. A midstream section 422 is provided between the first and second pressuredrop sections 420a, 420b, and is in fluid communication with the first pressure drop section 420a for receiving the first depressurized fluid therefrom. The second pressure drop section 420b is in fluid communication with the midstream section 422 for receiving the first depressurized fluid therefrom. Like the first pressure drop section 420a, the second pressure drop section 420b is structured for inducing a pressure drop in the first depressurized fluid to produce a second depressurized fluid having a pressure that is less than that of the first depressurized fluid.

[0330] In the specific embodiment provided in Figures 9A to 9E, each of first and second pressure drop sections 420a, 420b of secondary microfluidic channel 410 are serpentine pressure drop sections.

[0331] In another additional embodiment, the midstream section 422 of the secondary microfluidic channel 410 includes a first midstream section 422a that is structured to direct the first depressurized fluid to flow in a first flow direction, and a second midstream section 422b in fluid communication with first midstream section 422a. The second midstream section 422b is structured to direct the first depressurized fluid to flow in second flow direction that is substantially opposite the first flow direction.

[0332] In the specific embodiment provided in Figure 9B and 9D, the upstream channel 112 of the microfluidic channel 1 10 includes an inlet section 1 12b, a curved section 112c and a transfer section 112d. The downstream channel 1 14 of the microfluidic channel 110 also includes an inlet section 1 14b, a curved section 1 14c and a transfer section 114d. The transfers sections 112d, 1 14d of the upstream and downstream channels 112, 114 are fluidly connected to serpentine pressure drop section 120 of the microfluidic channel 110. The transfer sections 1 12d, 114d are spaced apart to define a gap therebetween. For the secondary microfluidic channel 410, the secondary upstream and downstream sections 412, 414 are short, substantially straight sections. The first pressure drop section 420a connects the upstream section 412 of the secondary microfluidic channel 410 to the first midstream section 422a, and the second pressure drop section 420b connects the downstream section 414 of the secondary microfluidic channel 410 to the second midstream section 422b. The first and second midstream sections 422a, 422b are formed as substantially straight, parallel channels with a connecting portion at one end thereof.

[0333] In an embodiment such as shown in Figures 9B and 9G, the microfluidic channel 110 and secondary microfluidic channel 410 are arranged relative to each other such that the first and second midstream sections 422a, 422b are substantially parallel to the transfer sections 112d, 1 14d.

[0334] In an additional embodiment, the first and second midstream sections 422a, 422b are disposed within the gap defined between the first and second transfer sections 1 12d, 114d. The at least one compartment 130 and aperture 132 are disposed atop the transfer sections 1 12d, 114d and the first and second midstream sections 422a, 422b.

[0335] The porous membrane 140 can be specifically configured for interfacing with the microfluidic channel 110 and the secondary microfluidic channel 410. In an embodiment such as shown in Figures 9F and 9G, the at least one porous membrane 140 further comprises a third portion 140c in fluid communication with the secondary upstream section 412 of the secondary microfluidic channel 410, and a fourth portion 140d in fluid communication with the secondary downstream section 414 of the secondary microfluidic channel 410. In this same embodiment, the aperture 132 of the at least one compartment 130 includes a third aperture portion that is aligned with the third portion 140c of the at least one porous membrane 140 and a fourth aperture portion that is aligned with the fourth portion 140d of the at least one porous membrane 140.

[0336] In the specific embodiment provided in Figures 9F and 9G, the porous membrane 140 and the aperture 132 of the at least one compartment 130 are structured such that the third and fourth portions 140c, 140d of the at least one porous membrane 140 are disposed in between the first and second porous membrane portions 140a, 140b. The first and second portions of porous membrane 140a, 140b are aligned with transfer sections 112d, 1 14d such that transfer sections 112d, 114d are disposed underneath respective the first and second portions 140a, 140b of the porous membrane 140. The third and fourth portions 140c, 140d are aligned with first and second midstream sections 422a, 422b of secondary microfluidic channel 410 such that first and second midstream sections 422a, 422b are disposed underneath respective third and fourth portions 140c, 140d. The microfluidic channel 1 10 is lined with endothelial cells and the secondary microfluidic channel 410 is lined with non-endothelial cells.Multiple device configurations

[0337] An aspect of the microfluidic assembly 10 and microfluidic device 100 of the disclosure is to provide the microfluidic assembly 10 that includes a plurality of interconnected microfluidic devices 100. This embodiment of the microfluidic assembly 10 can be used, at least in some embodiments, in order to model multi-organ phenomena.

[0338] In a first embodiment of the microfluidic assembly 10 shown in Figures 10A and 10B, the plurality of microfluidic devices 100 of the microfluidic assembly 10 areconnected in parallel to the primary circulation system 102. The microfluidic assembly 10 further comprises an inlet channel 104 that is in fluid communication with the outlet of the primary circulation system 102 for receiving pressurized fluid therefrom. The microfluidic assembly 10 also comprises an outlet channel 106 in fluid communication with the inlet of the primary circulation system 102 for returning depressurized fluid thereto. Parallel connection of the plurality of microfluidic devices 100 to the inlet channel 104 distributes the same input pressure across all tissue compartments 130. Parallel connections helps to maintain proper pressure gradients across all tissue compartments 130 with no design changes to the primary circulation system 102.

[0339] Referring to Figure 10A, a simplified parallel connection design of the microfluidic assembly 10 is provided. The simplified parallel connection design includes a plurality of the compartments 130 which each include the aperture 132. A single serpentine pressure drop section 120 is shared between all of the plurality of compartments 130, and the primary circulation system 102 drives all fluid flow within the microfluidic assembly 10. The multiple compartments 130 share the same upstream channel 1 12, downstream channel 114, and serpentine pressure drop section 120, thereby creating a more minimal design of the microfluidic assembly 10. As shown in Figure 10A, the simplified parallel connection design of the microfluidic assembly 10 can be provided on a modified 4 well plate.

[0340] Referring to Figure 10B, a refined parallel connection design of the microfluidic assembly 10 is provided. The plurality of microfluidic devices 100 of the microfluidic assembly 10 are connected between the inlet channel 104 and outlet channel 106 such that the upstream channel 112 of each microfluidic device 100 of the plurality of microfluidic devices 100 is fluidly connected to the inlet channel 104, and the downstream channel 114 of each microfluidic device 100 of the plurality of microfluidic devices 100 is fluidly connected to the outlet channel 106. Figures 10A and 10B also provide the results of a CFD simulation for the simplified and refined parallel connection of the multiple microfluidic devices 100. The various arrangements of parallel connections distribute substantially the same input pump pressure across each aperture 132 of the plurality of compartments 130 .

[0341] In a second embodiment of the microfluidic assembly 10 such as shown in Figure 10C, the plurality of microfluidic devices 100 of the microfluidic assembly 10 are connected in series to the primary circulation system 102. In this embodiment, the microfluidic assembly 10 comprises the plurality of microfluidic devices 100 that are connected in series between an inlet and an outlet of the primary circulation system 102.Each of the plurality of microfluidic devices 100 includes a microfluidic channel (like the microfluidic channel 110) that has the upstream channel 112 for receiving a higher pressure fluid, the pressure drop section 120 in fluid communication with the upstream channel 112 for receiving a first volume of the higher pressure fluid and inducing a pressure drop to produce a lower pressure fluid, and the downstream channel 114 in fluid communication with the pressure drop section 120 for receiving the lower pressure fluid. Each of the plurality of microfluidic devices 100 also includes the at least one porous membrane 140, and the at least one compartment 130 that is positioned on top of microfluidic channel 1 10 and that defines an aperture 132 therethrough.

[0342] The plurality of microfluidic devices 100 are connected in series such that the upstream channel 1 12 of a first microfluidic device 100 of the plurality of microfluidic devices 100 is fluidly connected to the outlet of the primary circulation system 102. The upstream channel 112 of each microfluidic device 100 of the rest of the plurality of microfluidic devices 100 is fluidly connected to a downstream channel 114 of another of the plurality of microfluidic devices 100 at a junction 115, and the downstream channel 1 14 of a last microfluidic device 100 of the plurality of microfluidic devices 100 is fluidly connected to the inlet of the primary circulation system 102. Figure 10C also provides the results of CFD simulations for the series connections of the plurality of microfluidic devices 100. The series connection of the plurality of microfluidic devices 100 divides the input pressure from the primary circulation system 102 between compartments 130 of the microfluidic devices 100.

[0343] The microfluidic device 100 described herein allow living microvessels to connect to a pressurized microfluidic device 100 to generate a perfusable vascular bed that can support a diverse range of implanted 3D tissues, through anastomosis of the endothelial cells with the pores 142 of the porous membrane 140(s). The above described systems each involves a microfluidic channel 110 design that allows the end user to culture any tissue fragment(s) in the tissue cavity(cavities) (second compartment 130). Notably, the fluid (e.g. nutrient media) is supplied to implanted 3D tissue(s) through the living microvessel network allowing expansion to a diverse range of scientific disciplines. Ultimately, the embedded tissue(s) can be supported by nutrient media delivered through the living human microvessel network.

[0344] In an embodiment, the microfluidic device 100 of the disclosure is integrated as part of a microfluidic system with the primary circulation system 102 and / or the secondary circulation system 402. In an embodiment, the microfluidic system comprises at least one controller (not shown) that is configured to control the primary circulationsystem 102 and / or the secondary circulation system 402 for controlling the flow of the fluid(s) provided to the microfluidic device 100. The at least one controller enables the primary and / or secondary circulation systems 102, 402 to be operated based on preprogrammed fluid circulation schemes.

[0345] The microfluidic system can further include at least one sensor (not shown) operatively connected to the at least one controller. The at least one sensor can be adapted to monitor data regarding, without limitation, physical cellular processes, chemical cellular processes, electrical cellular processes, and the like, such as, without limitation, pH, nutrient concentration, metabolic concentration, temperature, pressure, electrical output, fluid velocity, and the like. This data can be relayed to the at least one controller, and a processor of the at least one controller can be programmed with computer-executable instructions for controlling the operation of the primary circulation system 102 and / or the secondary circulation systems 102, 402 based on the data monitored by the at least one sensor.Microfluidic assembly and pump unit

[0346] The disclosure provides for various configurations and arrangements of the primary circulation system 102 that is fluidly connected to the microfluidic device 100. In at least some embodiments, the primary circulation system 102 is included as part of the microfluidic assembly 10.

[0347] In at least some embodiments, the use of the primary circulation system 102 is not limited to the specific microfluidic devices 100 and / or assemblies of the disclosure.

[0348] In a first embodiment of the microfluidic assembly 10, the microfluidic assembly 10 includes at least one microfluidic device 100, and the microfluidic device 100 is positioned on a base plate 560 (such as the tissue culture plate 200). The microfluidic assembly 10 also includes at least one microfluidic pump unit 520 and at least one magnetic drive system 510. The at least one microfluidic pump unit 520 and the at least one magnetic drive system 510 form at least part of the primary circulation system 102. The at least one microfluidic pump unit 520 is fluidly connected to the at least one microfluidic device 100 for circulating the fluid therethrough.

[0349] Referring to Figures 1 1 to 14D, the at least one microfluidic pump unit 520 includes a pump housing 522, and an impeller 540 that includes an impeller shaft 542. The impeller 540 is rotatably housed within the pump housing 522. The impeller shaft 542 of the impeller 540 includes at least one first magnetic element 544 (as shown in Figure 14A).

[0350] The at least one magnetic drive system 510 is removably connectable to the pump housing 522 and includes at least one drive motor 512 with an output shaft 514. In this embodiment, the output shaft 514 includes at least one second magnetic element 516 (shown in Figure 11). Within the primary circulation system 102, the magnetic drive system 510 is removably connectable on the pump housing 522 in a position such that the at least one first magnetic element 544 is magnetically coupled to the at least one second magnetic element 516, while being spaced apart from at least one second magnetic element 516. Through the connection of the magnetic drive system 510 and pump housing 522 (and the magnetic coupling of the first and second magnetic elements 544, 516) the at least one drive motor 512 can be rotatably coupled to the impeller 540 of the at least one microfluidic pump unit 520.

[0351] The at least one microfluidic pump unit 520 of the disclosure is, in at least some embodiments, provided as at least one magnetically-actuated impeller pump 520a. In the operation of the magnetically-actuated impeller pump 520a and magnetic drive system 510, rotation of the output shaft 514 of the drive motor 512 induces a rotation in the impeller shaft 542 and the impeller 540, thus driving fluid flow out of the magnetically- actuated impeller pump 520a and through the microfluidic device 100. The action of the impeller pump 520a is through the rotating impeller 540 receiving fluid from the inlet conduits and pushing fluid through the outlet conduits. The impeller 540 rotation is magnetically actuated, with the first magnetic element 544 of the impeller shaft 542 coupling with the second magnetic element 516 attached to the spinning shaft of the magnetic drive system 510.

[0352] The working principle of the impeller pump 520a is energy transfer from the rotating blades to the fluid in order to generate pressure. As shown in Figures 12 to 14D, each microfluidic pump unit 520 includes an impeller 540 rotating within a hollow interior of the pump housing 522. The impeller 540 includes an impeller head 541 and the impeller shaft 542 that extends through the impeller head 541 and is generally arranged to be coaxial with the impeller head 541. Figures 14A to 14C provide cross sectional views of the impeller 540 mounted within the pump housing 522.

[0353] In an embodiment such as shown in Figures 12 to 14C, the pump housing 522 of the at least one microfluidic pump unit 520 includes a pump reservoir 526 that is defined in a first section of the pump housing 522 and which contains the impeller 540 of the impeller pump 520a unit and a volume of the fluid.

[0354] Figures 14B and 14C provide perpendicular cross-sectional views of an exemplary CFD simulations showing the pressure and fluid velocity distributionthroughout the pump housing 522. Figure 14D provides planar cross sections of the exemplary CFD simulations showing the same pressure and fluid velocity distribution within the pump reservoir 526. Fluid shear stresses are also shown, with peak values at the blade tips of the impeller head 541. Figure 14E provides a plot of fluid shear stress values within the pump housing 522 for an exemplary, non-limiting CFD simulation. The impeller CFD simulations were applied through frozen rotor approximation. Cross sectional view of the CFD simulations displaying pressure, fluid velocity, and streamlines are provided in Figures 14B to 14E. Significant levels of fluid shear stress are created within the pump reservoir 526 chamber, particularly at the blade tips of the impeller head 541. High fluid shear stress is deleterious to cells which may be present suspended in the nutrient media for certain experimental applications. Notably, existing spinning flask bioreactors typically have shear stresses on the order of 0.01-0.1 Pa. Fluid shear stress in physiological blood vessels are known to be on the order of 1-10 Pa. In the exemplary CFD models of the fluid shear stresses within the pump reservoir 526, it was determined that, while the shear stress distributions are on the high end of existing spinning flask bioreactors, the shear stress distributions are still far below physiological blood vessel values.

[0355] In the specific embodiment provided in Figures 11 to 14C, the pump housing 522 includes a hollow, partially cylindrical base portion 523. The base portion 523 includes a bore 525 that is centrally disposed on the base portion 523. The bore 525 is sized to receive a first end 542a of the impeller shaft 542 therewithin for rotatably connecting the impeller 540 on the base portion 523. The base portion 523 also includes a circumferential support lip 525 and the pump housing 522 includes a support frame 530. The support frame 530 has a circular base that is mounted atop the circumferential support lip 525, and the circumferential support lip 525 is spaced apart from a bottom surface of the partial-cylinder base portion 523. The first section of the pump housing 522 (which forms the pump reservoir 526) is defined in the space between the bottom surface of the partial-cylinder base portion 523 and the circular base of the support frame 530.

[0356] The support frame 530 also includes a number of support arms 534 and a fluid aperture 532 that receives the impeller shaft 542 of the impeller 540. The at least one first magnetic element 544 on the impeller shaft 542 is mounted to a free, second end 542b of the impeller shaft 542, and the free, second end 542b of the impeller shaft 542 is disposed in a space between the support arms 534 of the support frame 530. The support frame 530 also defines an inlet 536 and an outlet 537 of the pump housing 522,and the inlet and outlet conduits 116, 118 are connected to inlets 536 and outlets 537of the pump housing 522 on the support frame 530.

[0357] In an additional embodiment, the impeller 540 and the support frame 530 are each removable from the partial-cylinder base portion 523 of the pump housing 522.

[0358] In an embodiment, the microfluidic pump unit 520 includes a seal plate 550 that is connectable on the base plate 560 and / or the pump housing 522 for inhibiting contamination and evaporation of the fluid from first and second sections of the pump housing 522 of the microfluidic pump unit 520. The seal plate 550 is also provided as part of the microfluidic pump unit 520 for rotatably supporting the impeller pump 520a of the impeller 540. As shown in Figures 12 and 13, the seal plate 550 is connectable on the base plate 560 such that an end of the impeller shaft 542 is rotatably supported by the seal plate 550, when the seal plate 550 is connected to the base.

[0359] In the specific embodiment provided in Figures 11 to 14A, the seal plate 550 is a circular seal plate 550 that is supported atop the plurality of support arms 534 of the support frame 530. The seal plate 550 includes a seal plate inset 550a that is removably connectable to the rest of the seal plate 550 such that at least part of the seal plate 550 can be replaced. The first end 542a of the impeller shaft 542 is supported in the borehole 524 on the base portion 523, and the second end 542b of the impeller shaft 542 is in contact with an underside of the seal plate 550 such that the impeller shaft 542 is rotatably supported between the borehole 524 and the underside of the seal plate 550.

[0360] Friction between the seal plate 550 and the second end 542b of the impeller shaft 542 is generally undesirable in the operation of the microfluidic pump unit 520. This friction has the effect of creating “choppy” flow. Furthermore, because the impeller 540 can be arranged to spin nearly continuously for extended period of time (such as when the microfluidic assembly 10 is being used for culturing tissues over an extended period of time), any amount of friction effectively turns the second end 542b of the impeller shaft 542 into a drill bit which can damage the microfluidic pump unit 520 to the point of stalling. In an embodiment, the seal plate 550 is at least partially composed of at least one low friction material such that an amount of friction generated between the seal plate 550 and the second end of the impeller shaft 542 is minimized. For example, the seal plate 550 can be formed as borosilicate glass seal plate 550, or as a combination glass-PTFE combination seal plate 550.

[0361] In an embodiment such as provided in Figures 14Ato 14C, the pump housing 522 also includes a media dampening reservoir 528 that is defined in a second section of the pump housing 522 and which also contains a second volume of the fluid. Inoperation of microfluidic assembly 10, the media dampening reservoir 528 is provided in fluid communication with the least one microfluidic device 100 for receiving a flow of the fluid therefrom. The media dampening reservoir 528 is defined within the pump housing 522 between the inlet of the pump housing 522 and an inlet of the pump reservoir 526. As shown in Figures 14A to 14C, the flow of fluid from the media dampening reservoir 528 enters into the pump reservoir 526 near the center of the impeller head 541 , increases in pressure as it travels with the blades, and exits through tubing placed around the support arms 534 of the support frame 530.

[0362] In an embodiment, the media dampening reservoir 528 is sized such that the flow of the fluid from the inlet 536 of the pump housing 522 becomes more laminar as the fluid enters the media dampening reservoir 528. By providing a media dampening reservoir 528 that is of sufficient size so as to dampen the flow of the fluid therewithin, fluid flowing into the media dampening reservoir 528 can become substantially non- turbulent. In the embodiments where the media dampening reservoir 528 is sized such that the fluid becomes substantially non-turbulent, any fluid which is drawn through the fluid aperture 532 of the support frame 530 to be pressurized by the impeller 540 will, at least initially, be substantially non-turbulent. This allows for more effective pressurization of the fluid by the impeller 540.

[0363] In the specific embodiment provided in Figures 11 to 14C, the media dampening reservoir 528 is formed by a top surface of the support frame 530. The top surface of the support frame 530 includes a recess 527 that surrounds the through- aperture, and this recess 527 at least partially defines the media dampening reservoir 528.

[0364] In an embodiment such as shown in Figure 12 to 14C, the second section of the pump housing 522 (defining the media dampening reservoir 528) is disposed at least partially vertically above the first section of the pump housing 522. In this way, the media dampening reservoir 528 is disposed substantially atop the pump reservoir 526. The fluid aperture 532 effectively defines an inlet for the impeller 540 within the pump reservoir 526 and the media dampening reservoir 528 atop the pump reservoir 526 is fluidly connected to the pump reservoir 526 via the fluid aperture 532. By providing the media dampening reservoir 528 above the pump reservoir 526, the second volume of fluid contained in the media dampening reservoir 528 can be evacuated from the media dampening reservoir 528 without evacuating the first volume fluid from the pump reservoir 526.

[0365] In another embodiment, the media dampening reservoir 528 is an open top reservoir having a top surface that is open to atmosphere. The pump housing 522 isarranged without a closed-off top such that the media dampening reservoir 528 (formed on the top surface of the support frame 530) is effectively open to atmosphere.

[0366] In an embodiment such as shown in Figures 14A to 14C, the inlet 536 of the microfluidic pump unit 520 is provided on the pump housing 522 as is for receiving the flow of the fluid from the at least one microfluidic device 100. The pump housing 522 also includes the outlet 537 of the microfluidic pump unit, where the outlet 537 is for providing a flow of the fluid to the at least one microfluidic device 100. The inlet 536 on the pump housing 522 is generally spaced apart from the fluid aperture 532 which connects the media dampening reservoir 528 and the pump reservoir 526. By spacing the inlet 536 away from the fluid aperture 532, the flow of the fluid enters the media dampening reservoir 528 away from the fluid aperture 532 and has sufficient space for the flow of the fluid to become a less turbulent before the flow of the fluid passes through the fluid aperture 532 and into the pump reservoir 526.

[0367] In the specific embodiment provided in Figures 14A, the inlet 536 is directly fluidly connected to the media dampening reservoir 528 and the outlet 537 is directly fluidly connected to the pump reservoir 526. The inlet 536 and the outlet 537 are formed as a conduit within the support frame 530 and are each oriented substantially vertically as part of the support frame 530.

[0368] The microfluidic pump unit 520 can be fluidly connected to the microfluidic device 100 by various arrangements and configurations. In an embodiment such as provided in Figure 15, the fluid connection between the at least one microfluidic device 100 and the at least one microfluidic pump unit 520 includes the at least one inlet conduit 116 the at least one outlet conduit 118. The at least one outlet conduit 118 is connected to an inlet of the microfluidic pump unit 520 and the at least one inlet conduit 116 is connected to an outlet of the microfluidic pump unit 520.

[0369] The microfluidic assembly 10 as disclosed herein is scalable, due to its modular design and minimal use of tubing. Other designs may incorporate lengthy amounts of tubing which quickly become difficult to manage with increasing numbers. The microfluidic assembly 10 features magnetic coupling between the microfluidic pump unit 520 and magnetic drive system 510, which can be easily disconnected and reconnected. With the structure of the microfluidic pump unit 520 and magnetic drive system 510 as described herein, the engagement of the magnetic drive system 510 with the microfluidic pump unit 520 for driving the microfluidic pump unit is connector free and only requires physical placement of the magnetic drive system 510 proximate the microfluidic pump unit 520. This configuration eliminates the need for gaskets andmanually disconnecting the inlet and outlet conduits 116, 118 when interchanging elements of the microfluidic assembly 10. The inlet and outlet conduits 116, 1 18 can remain connected between the microfluidic pump unit 520 and the associated microfluidic device 100.

[0370] In an embodiment, the microfluidic pump unit 520 is sized to fit within one of the wells 562 of the base plate 560.

[0371] In one such embodiment provided in Figure 15, the base plate 560 includes the tissue culture plate 200, and the at least one microfluidic device 100 is positioned in at least one first well 562a on the tissue culture plate; and the at least one microfluidic pump unit 520 is positioned in at least one second well 562b on the tissue culture plate 200.

[0372] In some embodiments, each of the at least one microfluidic device 100 and the at least one microfluidic pump unit 520 are fixedly mounted within the respective at least one first well 562a and at least one second well 562b on the tissue culture plate. In at least some other embodiments, the at least one microfluidic pump unit 520 is removably mounted in the at least one first well 562a, and the at least one microfluidic device 100 is removably mounted in the at least one second well 562b.

[0373] While the tissue culture plate may contain many different numbers of wells 562, the choice of the at least one first well 562a and at least one second well 562b on the tissue culture plate is generally done such that the at least one second well 562b is adjacent to the at least one first well 562a on the base plate 560. In this way, the at least one microfluidic pump unit 520 and the at least one microfluidic device 100 can be positioned within the at least one first well 562a and at least one second well 562b, respectively, such that an effective fluid capacitance of each of the at least one inlet conduit and at least one outlet conduit is minimized. Similarly, the at least one microfluidic pump unit 520 and the at least one microfluidic device 100 can be positioned within the at least one first well 562a and at least one second well 562b, respectively, such that a length of each of the at least one inlet conduit and at least outlet conduit is minimized.

[0374] By providing a connection between the at least one microfluidic pump unit 520 and the microfluidic device 100 with a very low fluid capacitance (i.e., placing the impeller 540 as close as possible to the microfluidic device 100 to minimize tubing length) shorter response times for the control and variation of the flow by the at least one microfluidic pump unit 520 can be achieved. The lower fluidic capacitance here reduced lag between when a flow signal is provided to the microfluidic pump unit 520, and when the fluid actually begins to move in the microfluidic device 100.

[0375] In an embodiment such as shown in Figure 15, the at least one first well 562a is a plurality of first wells 562a and the at least one second well 562b is a plurality of second wells 562b. The at least one microfluidic device 100 is a plurality of microfluidic devices 100, and each of the plurality of microfluidic devices 100 being contained in a unique first well 562a of the plurality of first wells 562a. Similarly, the at least one microfluidic pump unit 520 is a plurality of microfluidic pump units 520, and each of the plurality of microfluidic pump units 520 is contained in a unique second well 562b of the plurality of second wells 562b.

[0376] In the specific embodiment provided in Figure 15, the base plate 560 includes an SBS tissue culture plate mounted atop the base plate cover portion 564, and the plurality of first wells 562a and the plurality of second wells 562b are each structured as a plurality of SBS wells on the SBS tissue culture plate. The SBS tissue culture plate is the six-well tissue culture plate. Three microfluidic pump units 520 are contained within three of the SBS wells (thereby defining three first wells 562a) on the tissue culture plate, and each microfluidic pump unit 520 is fluidly connected to a unique one of the three microfluidic devices 100 contained within the remaining three wells of the tissue culture plate.

[0377] The components of the microfluidic pump unit 520 may be composed of various suitable materials in the embodiments of the microfluidic assembly 10 provided herein.

[0378] In an embodiment, at least one of the pump housing 522 and impeller 540 of the at least one microfluidic pump unit 520 is composed of one or more biocompatible materials. In a further embodiment, the one or more biocompatible materials comprises one or more biocompatible plastics.

[0379] In an additional embodiment such as shown in Figure 15 where the at least one microfluidic pump unit 520 is a plurality of microfluidic pump units 520, the magnetic drive system 510 includes a plurality of drive motors 512. Each drive motor 512 of plurality of drive motors 512 corresponds to one of the plurality of microfluidic pump units 520. In a further embodiment, the microfluidic assembly 10 further comprises a base plate cover portion 564, where the base plate 560 is connectable on top of the base plate cover portion 564.

[0380] In an embodiment such as shown in Figures 16A to 16C, the microfluidic assembly 10 further comprises a cover unit 570 that is removably connectable atop the base plate 560. The magnetic drive system 510 is housed within the cover unit 570 such that when the cover unit 570 is connected atop the base plate 560, the at least one firstmagnetic element 544 of the impeller shaft 542 is magnetically coupled to the at least one second magnetic element 516 of the output shaft 514, and when the cover unit 570 is disconnected from the base, the at least one first magnetic element 544 is magnetically decoupled from the at least one second magnetic element 516.

[0381] In some embodiments of the microfluidic assembly 10 such as shown in Figures 16B and 16C, the cover unit 570 is removably connectable directly on top of the base plate 560. In at least some other embodiments of the microfluidic assembly 10, the base plate 560 further comprises a base plate 560 cover (not shown). The tissue culture plate 200 plate is connected on top of the base plate 560 cover to collectively form the base plate 560, and the cover unit 570 is removably connectable to the base plate 560 cover. When the cover unit 570 is connected atop the base plate 560, the seal plate 550 is at least partially disposed between the interior of the pump housing 522 and the cover unit 570. The first magnetic element 544 is disposed on a first side of the seal plate 550, and the second magnetic element 516 is disposed on a second side of the seal plate 550 opposite the first side.

[0382] Various arrangements of the drive motors 512 within the cover unit 570 are provided in the microfluidic assembly 10 disclosed herein. Generally, each drive motor 512 of the plurality of drive motors 512 corresponds to one of the plurality of microfluidic pump units 520 and is positioned on the cover unit 570 such that the drive motor 512 becomes rotatably coupled to the to the impeller 540 of the corresponding one of the plurality of microfluidic pump units 520 when the cover unit 570 is connected to the base plate 560.

[0383] In an embodiment, the plurality of drive motors 512 are arranged on the cover unit 570 such that a spacing between each drive motor 512 and the rest of the plurality of drive motors 512 is maximized. In this same embodiment, the plurality of microfluidic pump units 520 are arranged on the base plate 560 in a substantially identical arrangement to the plurality of drive motors 512 on the cover unit 570 such that each corresponding drive motor 512 and microfluidic pump unit 520 will be substantially aligned when the cover unit 570 is connected to the base plate 560.

[0384] In the specific embodiment provided in Figure 16A, the magnetic drive system 510 includes the plurality of drive motors 512. The cover unit 570 includes a hollow bottom housing section 574 and a top housing section 572 that is connectable onto the hollow bottom housing section 574. The bottom housing section 574 includes a plurality of mounting apertures 573 and a plurality of through apertures 575. Each drive motor 512 of the plurality of drive motors 512 is mounted to the bottom housing section 574 viafasteners that are secured in the mounting apertures 573. The mounting apertures 573 and through apertures 575 are relatively positioned such that when the drive motor 512 is mounted in place, the output shaft 514 of the drive motor 512 extends down, through the through aperture 575 and into the hollow interior of the bottom housing section 574 such that the second magnetic element 516 is disposed in the interior of the bottom housing section 574. The plurality of drive motors 512 include three drive motors 512 that are arranged in an alternating pattern to correspond to the three microfluidic pump unit 520 on the base plate 560. The plurality of drive motors 512 are all communicatively connected to a single communication line.

[0385] Various arrangements of the first and second magnetic elements 544, 516 of the microfluidic pump unit 520 and magnetic drive system 510 are provided in the microfluidic assembly 10 of the disclosure.

[0386] In an embodiment such as provided in Figure 17, the at least one second magnetic element 516 is a first diametrically magnetized magnet 516a that is connected to the output shaft 514 of each drive motor 512 proximate a free end of the output shaft 514. The at least one first magnetic element 544 is a pair of diametrically magnetized magnets 544a positioned on opposing sides of the impeller shaft 542, proximate a free end of the impeller shaft 542.

[0387] In the specific embodiment provided in Figure 17, the output shaft 514 includes a disk-shaped end piece 584. The single diametrically magnetized magnet 516a is mounted on a flat bottom of the disk-shaped end piece 584. The impeller shaft 542 includes a main shaft 542 and a shaft casing 586 with a through-bore. The main shaft 542 is mounted through the through-bore such that the main shaft 542 and shaft casing 586 rotate together. The shaft casing 586 includes a pair of recess 588 formed proximate the second end 542b of the impeller shaft 542, and one of the pair of diametrically magnetized magnets 544a is held within each recess 588. In this embodiment, the first diametrically magnetized magnet 516a and pair of diametrically magnetized magnets 544a can be said to be on the “end faces” of the output shaft 514 and impeller shaft 542. Having magnets mounted on the “end faces” of the output shaft 514 and impeller shaft 542 provides for more forgiving tolerances in aligning the magnets. The magnetic drive system 510 can still function to rotate the impeller 540 of the microfluidic pump unit 520 to workable speeds even if the alignment of the shafts and / or magnets is offset (i.e., off- centre) by a couple of millimeters.

[0388] In an additional embodiment, the first diametrically magnetized magnet 516a is oriented such that a magnetic axis thereof is substantially perpendicular to a long axisof the output shaft 514 of the drive motor 512. Similarly, the pair of diametrically magnetized magnets 544a are oriented such that a direction of magnetization for each of the pair of diametrically magnetized magnets 544a is in substantially the same direction.

[0389] By arranging the first diametrically magnetized magnet 516a and the pair of diametrically magnetized magnets 544a in this way, an advantageous arrangement of magnetic fields from magnets is provided. This arrangement of the magnets will, in some embodiments, maximize the torque transferred into the impeller shaft 542 from the drive motor 512. This arrangement will also reduce the “pulling up force” on the impeller shaft 542 and impeller 540. If the magnets were rotated by 90 degrees (such that the direction of magnetization for each of the pair of diametrically magnetized magnets 544a is not substantially the same direction), then a much greater amount of the magnet’s force would “pull up” on the impeller shaft 542 instead of actually rotating it. By reducing this “pulling up force” frictional wear on the components of the microfluidic pump unit 520 can be better managed. This orientation of the magnets also provides for easier integration with existing tissue culture plates.Control System

[0390] In an embodiment such as shown in Figures 1 and 18A, the microfluidic assembly 10 further comprises a control system 590. The microfluidic pump unit 520 and the magnetic drive system 510 are communicatively connected to a control system 590, and the control system 590 includes at least one processor and memory. The memory of the control system 590 is programmed with computer-executable instructions that can be executed by the at least one processor for controlling the operation of at least the microfluidic pump unit 520 and the magnetic drive system 510. The control system 590 also includes at least one control sensor that is communicatively connected to the processor.

[0391] In some embodiments, the at least one controller and at least one sensor associated with the primary and / or secondary circulation systems 102, 402 are integrated as part of the control system 590, while in some other embodiments, the at least one controller and at least one sensor are separate but communicatively connected to the control system 590. In still other embodiments, the at least one controller and at least one sensor operate substantially independently from the control system 590.

[0392] In an embodiment, the memory of the control system 590 is programmed with computer executable instructions which, when executed by the at least one processor, provide velocity setpoints for each of the plurality of drive motors 512 of the magneticdrive system 510. The velocity setpoints can be communicated to each of the plurality of drive motors 512 via pulse width modulation (PWM) of other suitable communication processes. In the embodiments where the velocity setpoints are communicated via PWM, the control system 590 can include at least one signal generator (see Figure 19A), where the at least one signal generator is configured for generating PWM signals to send to the plurality of drive motors 512

[0393] In an additional embodiment, the control system 590 includes at least one proportional integral (PI) controller or other suitable controller (such as a PID controller). The memory of the control system 590 is programmed with computer executable instructions which, when executed by the at least one processor, provide velocity setpoints for specific rotational speeds of the plurality of drive motors 512 via integrated proportional integral control. The PI controller can, in some embodiments, allow the microfluidic pump unit 520 to generate programmed dynamic waveforms as inputs to the microfluidic device.

[0394] In the specific embodiment provided in Figure 18A, the at least one signal generator is a multichannel signal generator, and the at least one PI controller is a plurality of PI controllers that are integrated within each of the plurality of drive motors together with the at least one control sensor. The processor communicates with a multichannel signal generator via I2C, or other suitable communication protocol (such as SPI or UART protocols), to independently control each magnetic drive system 510. The multichannel signal generator sends PWM set points to the drive motors of the magnetic drive system as directed by the processor. The drive motors 512, with integrated PI controllers and control sensors, translate the set points to rotational speeds. Negative feedback from the control sensors provides substantially consistent rotational speeds and thus, substantially consistent output flowrates from the microfluidic pump unit 520.

[0395] While the control system 590 is shown as a single physical control system, it will be appreciated that the control system 590 can include two or more physical control systems in communication with each other. Accordingly, while the embodiment shows the various components of the control system 590 residing on the same physical system, those skilled in the art will appreciate that the components can reside on separate physical systems.

[0396] An exemplary performance of the microfluidic pump unit 520 with an embodiment of the control system 590 and connected magnetic drive system 510 is provided in Example 9 of the disclosure. An exemplary performance of the microfluidicpump unit 520 with an embodiment of the control system 590 having integrated PI controllers is provided in Example 10 of the disclosure.

[0397] The impeller pump 520a system is expandable, with the ability to control a multitude of drive unit filled incubators with a single interface. In an embodiment such as shown in Figure 18B, the microfluidic assembly 10 includes the plurality of magnetic drive systems 510, and the drive motor 512 of each of the plurality of magnetic drive systems 520 is communicatively connected to the control system 590 and the signal generator of the control system 590. The plurality of magnetic drive systems 510 are mounted atop a plurality of the microfluidic pump units 520, and each microfluidic pump unit 520 is held in a well. Each of the plurality of magnetic drive systems 510 are magnetically coupled to a unique one of the plurality of microfluidic pump units 520.

[0398] In the specific embodiment provided in Figure 18B, an array of sixteen magnetic drive systems 510 and corresponding microfluidic pump units 520 are placed within a typical tissue culture incubator, with each controlled by the control system 590. Engagement of each magnetic drive system 510 with a corresponding pump unit 520 (which are situated in a standard well of a tissue culture plate) is contactless and does not require the connection of tubing. While the embodiment provided in Figure 18B is specific to a single tissue culture plate with sixteen microfluidic pump units 520 and associated magnetic drive systems 510 other types of implementations will occurto those of skill in the art. For example, multiple tissue culture plates can be communicatively connected to a single control system 590 and the control system 590 can be configured for simultaneously controlling the operation of some or all of the magnetic drive systems 510 on the tissue culture plates.

[0399] Referring to Figures 19A and 19B, there is provided an alternate embodiment of the primary circulation system 102 within the microfluidic apparatus 10. In this embodiment, the primary circulation system 102 includes a separate primary circulation system 102 corresponding to each microfluidic device 250 within the microfluidic apparatus 10. In this embodiment, the microfluidic device is configured as the microfluidic device 250. Each separate primary circulation system 102 includes a horizontal peristaltic drive train 630. As shown in Figures 19A and 19B, the microfluidic device 250 is mounted on top of the horizontal peristaltic drive train 630. Fluid is driven through the microfluidic channels 260 by pressing flexible microchannels 260a in the microfluidic device 250 into the horizontal peristaltic drive train 630 to create a planar peristaltic system.

[0400] Figure 19B illustrates a plurality of interconnected devices 250 and peristaltic drive trains 630 (communicatively connected to control system 590) working together forvascularizing a plurality of implanted 3D tissues. In this embodiment, it is possible to support large scale tissue harvest and further, to sustain 3D tissues with the purpose of future transplantation into human hosts. It is further possible to connect multiple tissues on the same microfluidic circuit that can be controlled individually or in parallel to allow modular system operation.Applications

[0401] The microfluidic device 100 and / or the microfluidic apparatus 10 of the disclosure may be used to vascularize or culture multicellular aggregates, and can be used for applications in, without limitation, disease modeling (disease model, drug discovery model), pharmacokinetic characterization drug screening systems (pharmacokinetic model), toxicity screening and the like.

[0402] In an application, the microfluidic device 100 and / orthe microfluidic apparatus 10 described herein can be used to examine early stage vascularized human cerebral organoid (CO) colonization by cancer cells to model metastasis. Most cancers or cancer subtypes metastasize to specific organs, such as triple-negative breast cancers (TNBC), which preferentially colonize the brain. Brain metastases are difficult to treat, and little is understood of the molecular drivers of the human metastatic cascade due to inadequate in vitro models, and the reliance on in vivo mouse models that are expensive, laborious and lack species specificity. The microfluidic device 100 and / orthe microfluidic apparatus 10 described herein can be used to examine early stage vascularized human CO colonization by cancer cells and to investigate mechanisms that promote brain metastasis. For example, the microfluidic devices and systems disclosed herein can be used to examine adhesion and extravasation through the Blood Brain Barrier (BBB) and subsequent seeding within the normal cerebral organoid tissue using brain-metastasizing variants of breast cancer cells with growth of live or fixed tumor cells monitored. For example, morphological changes in individual tumor cells during the early phases of adhesion and extravasation can be monitored with counterstaining for endothelial cells (CD31), pericytes (PDGFRB), astrocytes (GFAP) and neurons (DCX). The metastatic capacity of a tumor is critically influenced by tumor cell-host organ interactions. This model of brain metastasis together with single cell sequencing can also be used to explore how tumor-vhCO cross-talk alters each other’s gene expression patterns to drive extravasation and tumor expansion.

[0403] In another application, the microfluidic device 100 and / or the microfluidic apparatus 10 described herein can be used to model Hereditary HemorrhagicTelangiectasia (HHT) and Cerebral Cavernous Malformations (CCM). These disorders are characterized by abnormal blood vessel formation, fragility, and increased risk of bleeding. This makes it challenging to study them in standard cell cultures or animal models. The microfluidic device 100 and / or the microfluidic apparatus 10 would provide a controlled, 3D environment under physiological pressure that better mimics the in vivo conditions. Using the the microfluidic device 100 and / or the microfluidic apparatus 10, precise control of the vascular microenvironment may be achieved for more realistic modeling of vascular defects. This could be done with patient-derived iPSCs (with the mutations) in combination with fibroblasts and pericytes.

[0404] In another application, the microfluidic device 100 and / or the microfluidic apparatus 10 described herein can be used to screen for Blood-Brain Barrier (BBB) permeable drugs and neurotoxicity. The BBB is formed by interactions between endothelial cells, pericytes, and astrocyte end-feet that form a semi-permeable barrier that restricts access to the Central Nervous System (CNS) for >90% of all drugs. The BBB can thus block effective therapeutics from accessing the CNS, but also protect the CNS from environmental and therapeutic neurotoxins [Fritsche, E., et al., OECD / EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. ALTEX, 2017. 34(2): p. 311-315], The BBB is poorly modeled in non-human animals [Crofton, K.M., et al., ALTEX, 2011. 28(1): p. 9-15], For example, BIA-10-2474, tested successfully in 4 species, caused tragic CNS toxicity, including death, in a phase I clinical trial [Kiss, L.E., et al., ChemMedChem, 2018. 13(20): p. 2177- 2188; Moore, N., BMJ, 2016. 353: p. i2727. Co-cultured astrocytes, endothelial cells and fibroblasts self-assemble BBBs [Cho, C.F., et al., Nat Commun, 2017. 8: p. 15623; Bergmann, S., et al., Nat Protoc, 2018. 13(12): p. 2827-2843], suggesting that the vascularized hCOs can also possess BBBs. Once validated with known BBB permeable and impermeable drugs, the systems disclosed herein can be implanted with human Cerebral Organoids. To assess neurotoxicity, drugs can be perfused through the vasculature, penetration into the tissue determined and hCO viability and growth assessed. A model of BBB assembly and neurotoxicity will provide a human pre-clinical model for development of neurological therapeutics and an important new tool for testing toxicity in humans

[0405] In another application, microfluidic device 100 and / or the microfluidic apparatus 10 described herein can be used for the generation and growth of any type of organoid or tissue.

[0406] In a non-limiting, exemplary application, the microfluidic device 100 is used to generate models fortesting intestinal drug toxicity. Understanding drug toxicity properties in the gut is a key aspect of the drug development pipeline that represents an important adverse event limiting drug dosing schedules. As much as 40% of patients undergoing cytotoxic chemotherapy report significant intestinal toxicity and dosing of receptor tyrosine kinase inhibitors in cancer is limited by Gl toxicity [Rodrigues, D., et al., Cancer Treat Rev, 2019. 77: p. 57-66], Therapy-induced toxicity is typically manifested as damage to the epithelial layer that causes mucositis and disruption of the epithelial barrier function. The microfluidic apparatus 10 disclosed herein can be used to test whether human Intestinal organoids (hlOs) display toxic responses to chemotherapeutics from the clinic. To more closely recapitulate gut specific vasculature, Human Intestinal Microvascular Endothelial Cells (HIMECs) can be used with stem-cell derived hlOs implanted onto a system of the disclosure. Vascularization of the mesenchymal layer underlying the epithelium can be confirmed by confocal and multiphoton microscopy. hlOs can then be perfused with drugs of interest and the effects on tissue morphology and epithelial integrity can be assessed.

[0407] In an application the microfluidic device 100 and / or the microfluidic apparatus 10 described herein comprises a human model of stroke.

[0408] In an aspect, a method of culturing at least one multicellular aggregate comprising:

[0409] (a) inserting a precursor solution of a matrix into the aperture 132 of the microfluidic device 100 of the disclosure;

[0410] (b) seeding the precursor solution of the matrix with Precursor Vascular Cells;[0041 1] (c) inserting at least one multicellular aggregate within the precursor solution of the matrix;

[0412] (d) treating the precursor solution to form the matrix;

[0413] (e) perfusing the aperture 132 with a pressurized fluid having nutrients throughout the microfluidic device 100 at a first flow rate so that the fluid flows through the aperture 132 and the matrix to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate; and

[0414] (f) culturing the at least one multicellular aggregate by perfusing the aperture132 with the pressurized fluid throughout the microfluidic device 100 at a second flow rate so that the pressurized fluid flows through the aperture 132 and the matrix to allow the survival of the at least one multicellular aggregate,

[0415] wherein the first flow rate and second flow rate of the pressurized fluid entering the aperture 132 are predominately non-turbulent.

[0416] In an embodiment, the step of seeding the precursor solution can be performed prior to inserting the precursor solution into the aperture 132. In an embodiment, the step of inserting at least one multicellular aggregate can be performed prior to inserting the precursor solution into the aperture 132. Alternatively, the step of inserting at least one multicellular aggregate can be performed after inserting the precursor solution into the aperture 132. In another embodiment, the step of inserting at least one multicellular aggregate can be performed after the formation of the matrix.

[0417] In an aspect, the first flow rate and the second flow rate are substantially equivalent to the flow rate encountered in vivo in an animal and more specifically, the first flow rate and second flow rate can be between the range of 0.1 pl / s and 10 ml / s. In an aspect, the first flow rate and the second flow rate are substantially similar. Alternatively, in an aspect, the first flow rate and the second flow rate are substantially distinct.

[0418] In an aspect, a method of culturing at least one multicellular aggregate comprising:

[0419] (a) forming a matrix seeded with Precursor Vascular Cells and at least one multicellular aggregate;

[0420] (b) perfusing the matrix with a pressurized fluid having nutrients at a first flow rate, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 10 kPa to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate; and

[0421] (c) culturing the at least one multicellular aggregate by perfusing the matrix with the pressurized fluid at a second flow rate, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the pressure differential between the first pressure and the second pressure is between the range of 50 Pa and 10 kPa to allow the survival and maintenance of the multicellular aggregate.

[0422] In an embodiment, the pressure differential for the first flow rate and for the second flow rate can be about 1 kPA, respectively.

[0423] In an embodiment, the first flow rate and second flow rate are predominantly non-turbulent. In an embodiment, the first flow rate and the second flow rate can be substantially equivalent to the flow rate encountered in vivo in an animal and more particularly the first flow rate and the second flow can be between the range of 0.1 pl / sand 10 m / s. In an embodiment, the first flow rate and the second flow rate are substantially similar. Alternatively, in an embodiment, the first flow rate and the second flow rate are substantially distinct. In an embodiment, the first flow rate and second flow rate can have a flow relative pulsation less than 0.1 , where relative pulsation is calculated as the standard deviation of the flow rate profile divided by the mean fluid flow rate.

[0424] In an embodiment, the disclosure provides multicellular aggregates produced by a method of the disclosure.

[0425] In an aspect, the disclosure provides multicellular aggregates such as organoids, produced by a method of the disclosure. In an embodiment, the organoid is a patient-specific organoid model. In an embodiment, the organoid is a human cerebral organoid. In another embodiment, the organoid is a human intestinal organoid.

[0426] In an aspect, the method for culturing can further comprise the step of removing the cultured multicellular aggregate from the matrix. Multicellular aggregates can be removed from the microfluidic device or system using different means. For example, the multicellular aggregates can be removed by enzymatic or chemical- mediated hydrogel digestion / lysis, using mechanical hydrogel disruption or hydrogel photodegradation. Spheroids or organoids can be removed by inducing liquefaction of a temperature-responsive hydrogel at a reduced physiologically acceptable temperature.

[0427] In an aspect, a method of vascularizing at least one multicellular aggregate comprising :

[0428] (a) inserting a precursor solution of a matrix into the aperture 132 of the microfluidic device 100 of the disclosure;

[0429] (b) seeding the precursor solution of the matrix with Precursor Vascular Cells

[0430] (c) inserting at least one multicellular aggregate within the precursor solution of the matrix;

[0431] (d) treating the precursor solution to form the matrix; and

[0432] (e) perfusing the tissue compartment with a pressurized fluid having nutrients throughout the microfluidic device 100 at a flow rate so that the fluid flows through the aperture 132 and the matrix to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate,

[0433] wherein the flow rate of the pressurized fluid entering the aperture 132 is predominately non-turbulent.

[0434] In an embodiment of this method, the step of seeding the precursor solution can be performed priorto inserting the precursor solution into the aperture 132. In another embodiment, the step of inserting at least one multicellular aggregate can be performedprior to inserting the precursor solution into the aperture 132. Alternatively, in another implementation, the step of inserting at least one multicellular aggregate can be performed after inserting the precursor solution into the aperture 132. In another embodiment, the step of inserting at least one multicellular aggregate can be performed after the formation of the matrix.

[0435] In an embodiment of this method, the flow rate can be substantially equivalent to the flow rate encountered in vivo in an animal and more specifically, the flow rate can be between the range of 0.1 pl / s and 10 ml / s.

[0436] In an aspect, a method of vascularizing at least one multicellular aggregate comprising :

[0437] (a) forming a matrix seeded with Precursor Vascular Cells and at least one multicellular aggregate; and

[0438] (b) perfusing the matrix with a nutrient fluid at a flow rate, the fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the pressure differential between the first pressure and the second pressure is between the range of 50 Pa and 10 kPa to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate.

[0439] In an embodiment, the pressure differential for the flow rate can be about 1 kPa.

[0440] In an embodiment, the flow rate can be predominantly non-turbulent. In an embodiment, the flow rate can be substantially equivalent to the flow rate encountered in vivo in an animal and more particularly the flow rate can be between the range of 0.1 pl / s and 10 ml / s. In an embodiment, the first flow rate and the second flow rate are substantially similar. Alternatively, in an embodiment, the first flow rate and the second flow rate are substantially distinct. In an embodiment, the flow rate can have a flow relative pulsation less than 0.1 , where relative pulsation is calculated as the standard deviation of the flow rate profile divided by the mean fluid flow rate.

[0441] The microfluidic devices and systems described herein can be used in an assay in which a test analyte is introduced in the fluid and the effects of the test analyte on the multicellular aggregates are observed. The microfluidic devices and systems can be particularly useful in personalized medicine and establishing screening assays to determine patient responses to drugs.

[0442] In an aspect, the disclosure relates to a test system comprising the microfluidic device 100 and / orthe microfluidic apparatus 10 of the disclosure with cultured multicellular aggregates. In a particular embodiment, a test system is provided comprisingthe microfluidic device 100 and / or the microfluidic apparatus 10 of the disclosure with cultured organoids.

[0443] In an aspect, a method of screening a test analyte is provide by adding the following steps to the method of culturing at least one multicellular aggregate:

[0444] (i) exposing the multicellular aggregate to a test analyte by adding the test analyte to the pressurized fluid or tissue compartment; and

[0445] (ii) determining the effect of the test analyte on the multicellular aggregates.

[0446] In one example, the multicellular aggregates can be obtained or derived from patients. In another embodiment, the multicellular aggregates and Precursor Vascular Cells can be obtained or derived from patients. In another embodiment, the multicellular aggregates and vascular network can be obtained or derived from patients.

[0447] In an embodiment, the test analyte is a pharmaceutical agent, and the responses of the multicellular aggregate and / or vascular network are analyzed. In an embodiment, the responses from a patient to the test analyte can be extrapolated / predicted from the response of the multicellular aggregate.

[0448] In an embodiment, the matrix or fluid can further comprise immune cells from patients, including without limitation, T cells, B cells, lymphocytes, lymphoid progenitors, myeloid progenitors, mast cells, monocytes, eosinophils, basophils, neutrophils, natural killer cells, dendritic cells, microglia, and macrophages.

[0449] The disclosure also provides kits for using the microfluidic devices and systems of the disclosure. In an embodiment, a kit comprises the microfluidic device 100 of the disclosure and instructions for using the microfluidic device 100. In an embodiment, a kit comprises the microfluidic device 100 of the disclosure, a matrix and Precursor Vascular Cells for forming the vascular network in the matrix. In an embodiment, a kit comprises the microfluidic device 100 of the disclosure and a matrix implanted with Precursor Vascular Cells for forming a vascular network and multicellular aggregates. The kits can additionally comprise a container containing the fluid to introduce into the microfluidic device.

[0450] In an embodiment, there is provided an in vitro system for culturing a multicellular aggregate comprising the microfluidic assembly 10 and the control system 590 in communication with the at least one microfluidic pump unit, where the control system enables pre-programmed fluid circulation schemes to be run on the at least one microfluidic pump unit.

[0451] In an embodiment, the there is provided a method of culturing at least one multicellular aggregate with the microfluidic assembly 10, the method comprising forminga matrix seeded with the at least one multicellular aggregate within the at least one microfluidic device 110, operating the at least one microfluidic pump unit 520 for perfusing a compartment 130 of the at least one microfluidic device 110 with a pressurized fluid having nutrients at a first flow rate so to induce the formation of a microvascular network within the matrix for vascularizing the at least one multicellular aggregate, and operating the at least one microfluidic pump unit 520 for perfusing the compartment 130 with the pressurized fluid at a second flow rate such that the pressurized fluid flows through the at least one microfluidic device and the matrix, the at least one multicellular aggregate is cultured. In this embodiment, the second flow rate is selected to permit the survival of the at least one multicellular aggregate. The at least one microfluidic pump unit 520 is operated such that the when the pressurized fluid enters the at least one microfluidic device 110 at either of the first flow rate of the second flow rate, the flow of the pressurized fluid is substantially non-turbulent.

[0452] In an embodiment, there is provided a method of culturing at least one multicellular aggregate with the microfluidic assembly 10, the method comprising forming a matrix seeded with at least one multicellular aggregate within the at least one microfluidic device 110, operating the at least one microfluidic pump unit 520 for perfusing the matrix with a pressurized fluid having nutrients at a first flow rate, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the formation of a microvascular network within the matrix vascularizing the at least one multicellular aggregate. The method also comprises operating the at least one microfluidic pump unit 520 for perfusing the matrix with the pressurized fluid at a second flow rate such the at least one multicellular aggregate is cultured, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the survival and maintenance of the multicellular aggregate.Examples

[0453] The following examples are presented to enable those skilled in the art to understand and to practice embodiments of the disclosure. They should not be considered as a limitation on the scope of the disclosure, but merely as being illustrative and representative thereof.

[0454] At least some of the various examples provided below utilized CFD simulations and display the resulting pressure and fluid velocity distributions for the microfluidic device 100. 3-dimensional models were created using Autodesk Fusion360 and exported as CAD files. The microfluidic channel 110 and other aspects of the microfluidic device 100 were modeled using Navier-Stokes for incompressible fluids. The physical parameters of water were utilized for all simulations, with the no-slip wall boundary condition. CFD software involved one of openFOAM, SimScale, and COMSOL Multiphysics,, with additional visualizations and analyses using ParaView. Specifically, analysis of vorticity metrics such as the Q-criterion were done using ParaView.Example 1 - Flow optimization in microfluidic channel

[0455] The following studies were conducted to assess the effects of various conditions on the microfluidic device in order to arrive at a system that mimics physiological blood pressure across the aperture of the at least one compartment.

[0456] Figure 20A shows a schematic diagram of some relevant parameters to consider when optimizing the design of the microfluidic channel. Figure 20A shows the resistances and pressures within the microfluidic channel 1 10. The relevant parameter for the serpentine configuration of the pressure drop section 120 (i.e., channel connecting the upstream and downstream channels 1 12, 1 14) is solely its flow resistance, with the exact method of achieving such flow resistance being irrelevant. For maximizing the pressure gradient magnitude across the tissue compartment 130, the flow resistance of the serpentine channel should be chosen to be much greater than the sum of the flow resistances for the upstream and downstream channels (shown as Riniet and Routiet in Figure 6A). Additionally, the amount of wall shear stress within the channel is inversely proportional to the sum of all microchannel resistances

[0457] Figure 20B shows an analogous fluidic circuit, where pressure is analogous to voltage, flowrate is analogous to current, and flow resistance is analogous to electrical resistance. The analogous fluidic circuit analogy is applied to guide the design of the microfluidic channel 1 10. Microchannel design may then be simplified by applying and repurposing existing methods for analyzing electrical circuits.

[0458] Figure 20C shows an illustration of the pressures within the microfluidic channel 1 10 when the pressure drop section 120 does not have a serpentine configuration. In the optimization of the flow within the microfluidic channel 110, the primary design objective is to maximize the magnitude of the pressure gradient across the tissue compartment 130 for a certain pump pressure. Ideally, all of the pump pressureis transferred across the tissue compartment 130, thus maximizing the device’s efficiency. Suboptimal efficiencies, as demonstrated with an unoptimized straight channel design, occur when unwanted pressure losses occur in the microchannel prior to the tissue compartment 130.

[0459] Figure 20D shows the pressures within the microfluidic channel 110 when the pressure drop section 120 has a serpentine configuration. In other non-limiting embodiments, the serpentine configuration of the pressure drop section 120 in the microfluidic channel 110 can be replaced by a straight, narrow channel with a narrowing width. For all intents and purposes, this design of the pressure drop section 120 would function substantially similarly to the serpentine configuration. Optimization of the serpentine channel involves two design objectives. In these scenarios, the impeller pump 102a must either be larger or rotate faster in order to compensate, thus unnecessarily increasing device size or component wear. Figure 20E shows a plot of the influence of connecting hydrodynamic resistance on the ratio of the pressure different across the compartment to the pressure difference between the pump inlet and pump outlet.

[0460] Figure 20F shows a plot of the influence of connecting hydrodynamic resistance on wall shear stress for the microfluidic channel 100. An additional design objective for the optimizing the form of the microfluidic channel 110 is to tune the fluid wall shear stress within the microchannels, onto which endothelial cells are seeded. This objective is highly contextual to what applied shear stress levels are desired for specific experimental setups. Generally, the wall shear stress should be low enough to minimize cell detachment from the underlying channel surface (shown in Figure 20F). To further help selection of specific values, a systematic parameter sweep was performed with CFD (Figure 20F). The flow resistance of the serpentine channel is selected to maximize the tissue compartment 130 pressure in a region of diminishing returns. Exact fluid wall shear stress values are additionally calculated. For the range of operating pressures, no cell detachment is observed from the microchannel for these shear stress values.Example 2 - Influence of microfluidic channel properties on matrix and vessel formation

[0461] The results of studies that were conducted to assess the effects of various conditions on the microfluidic device in order to arrive at a system that mimics physiological blood pressure across a hydrogel seeded with human umbilical endothelial cells (HUVECs) and primary fibroblasts as support cells are shown in Figures 21 A to 21 G.

[0462] The effect of pressure on vessel formation is shown in Figures 21 A and 21 B. Figure 21 A shows the effect of the pressure with a microfluidic device having an aperture above the pressure drop section. Figure 21 B shows the effect of the pressure with microfluidic device having the aperture above the upstream and downstream channels and distant from the pressure drop section. Images were acquired using bright field microscopy on day 14 post tissue embedding in the microfluidic device. Pressure gradients and simulations illustrated how pressure is generated in the low-pressure and high-pressure microfluidic device designs.

[0463] The tissue growth in low-pressure and high-pressure device designs over time is illustrated in Figures 21 C and 21 D, respectively. Images were acquired in live tissue using bright field microscopy every 48 hours over the course of 14 days. Figure 21 C shows cerebral organoid (mini-brain) embedded in a low-pressure microfluidic device design, and the graph shows the quantification of the circumference at the medial Z plane. The initial increase in size is due to tissue growth in the device from the original nutrients. 96 hours after implantation, the decrease in tissue size is due to the implanted tissue lacking nutrient connection to the external media source. As the vessels begin to supply the tissue with nutrients and the tissue grafts with the device, an increase in the size of the tissue is observed after 288 hours. Figure 21 D shows the cerebral organoid (mini-brain) embedded in a high-pressure microfluidic device and the graph. Quantification of the circumference at the medial Z plane. Tissue growth was for 96 hours from introduction of original nutrients. 96 hours after implantation, the decrease in tissue size is due to the depletion of nutrients. In the high-pressure design, tissue growth resumes at 240 hours suggesting the tissue successfully grafts with the microfluidic device. The arrows in the high-pressure panel denote the formation of new structures visible under bright field microscopy. A first structure was observed at 192 hours, while a second structure was observed at 288 hours.

[0464] Figures 21 E and 21 F show the influence of flow conditions on cell organization within the matrix in the aperture 132. The organization of cell types in the hydrogel by immunostaining (images to scale) is illustrated in Figures 21 E and 21 F. The images were acquired using a point scanning confocal microscope (Pinhole = 1.0). Figure 21 E shows endothelial cells wrapping around a human intestinal organoid cultured in a low-pressure microfluidic device. Figure 21 F shows endothelial cells wrapping around and penetrating an implanted human cerebral organoid cultured in a high-pressure microfluidic device.

[0465] Systematic removal of endothelial cells and fibroblasts demonstrated that each cell type is required for performance of the system (shown in Figure 21 G). The images were obtained using bright field microscopy in live tissue. The presence of Human Umbilical Vein Endothelial Cells (HUVECs) and Normal Human Lung Fibroblasts (NHLFs) lead to maintenance of the hydrogel and vessel formation in the hydrogel after 48 hours. Removal of the HUVECs lead to no vessel formation in the hydrogel after 48 hours, while removal of the NHLFs lead to gel failure 48 hours post seeding.Example 3 - Pore optimization

[0466] The following studies were conducted to assess the effects of various conditions on the porous membrane of the microfluidic device in order to arrive at a system that mimics physiological blood pressure across the aperture of the at least one compartment. Figures 22A to 22F shows an exemplary process for optimization of the porous membrane, where the porous membrane is optimized based a first design objective: the minimization of fluid recirculation vortices within the pores of the porous membrane.

[0467] Figure 22A shows a schematic diagram of fluid recirculation vortices that can occur within unoptimized pores of the porous membrane. These vortices, which are known to be deleterious to endothelial cells, occur at the pore’s leading and trailing edge due to the membrane’s sharp geometries. Prior art works have highlighted the effects of flow vortices on endothelial cells and thus their unwanted and potentially deleterious presence should be minimized. In physiological contexts, vessels walls are curved, and recirculation vortices are mainly associated with pathological conditions such as atherosclerosis. Microfabrication techniques on the other hand, such as the current method of fabricating the porous membrane, typically produce sharp corners which are prone to vortex formation. In particular, recirculation vortices are formed when the fluid flows over the pore lips. Vortices on these scales are laminar, as evident by very low Reynold’s numbers, and consequently their interpretation should focus on the recirculation zones that are created and not possible turbulent effects.

[0468] Figure 22B shows a graphical diagram of the mathematical definition of fluid recirculation zones based on integrating flow velocities from the pore ceiling. To systematically assess the formation of recirculation vortices, a mathematical definition and / or descriptor was defined by extending previous literature involving integrating flow velocities on a path normal to the wall surface (see, for example, the method discussed in Zhou et al., Mass Transfer Between Recirculation and Main Flow Zones: Is PhysicallyBased Parameterization Possible? Water Resources Research 55, 345-362 (2019)). This mathematical integration descriptor allows quantification of the recirculation vortices while also facilitating visualization of recirculation vortices. The commonly used Q-criterion, although able to detect vortex centers, was not ultimately applied because it poorly detected the recirculation zone boundaries. Using the integration descriptor, two design parameters were identified which minimize recirculation vortices: porous membrane thickness and pore aspect ratio.

[0469] Figure 22C shows a CFD simulation comparing a simple unoptimized circular pore to an exemplary optimized, slit-shaped pore. Both pores have the same area (0.008 mm2) while the slit-shaped pore has much smaller recirculation zones that form and the corners of the slit-shaped pore.

[0470] Figure 22D shows the impact of porous membrane thickness on the size of the recirculation zones within each pore of the porous membrane. Figure 22C provides four different cross-sectional views of CFD simulations showing the volume and strength of recirculation zones for porous membrane thicknesses of 10 pm, 30 pm, 50 pm and 70 pm. This membrane thickness parameter sweep reveals less fluid recirculation with thinner membranes. Figure 22E provides a plot of the thickness parameter sweeps shown in Figure 21 C.

[0471] Figure 22F shows the impact of pore aspect ratio on the size of the reticulation zones within each pore of the porous membrane. Pore aspect ratio is defined as the ratio between the parallel and perpendicular dimensions of the pore with respect to the flow direction. Rectangular pores are considered for simplicity. Figure 22G provides four different cross-sectional views of CFD simulations showing the volume and strength of recirculation zones for pore aspect ratios of 1 :4, 1 :1 , 4:1 , and 20:1 . The pore aspect ratio parameter sweep shows less fluid recirculation with higher aspect ratios for the same pore area. Figure 21 E provides a plot of the pore aspect ratio in the context of the parameter sweep results.

[0472] From the results obtained in Figures 22A to 22G, it was determined that the membrane should be made as thin as possible while staying within manufacturing limitations in order to decrease the heights of the pore lips. These results were substantiated with CFD parameter sweeps of varying membrane thicknesses which show a positive correlation between membrane thickness and recirculation zone volume. For the current material of PDMS, 30 pm was chosen as membrane manufacturing became less robust with lower thicknesses. Secondly, the pore aspect ratio should be increased as much as possible to form slits parallel to the flow direction in order to decrease thewidths of the pore lips Notably, the simple circular pore has an aspect ratio of 1 and thus contains a much larger recirculation vortices compared to a slit pore of the same area. Results are likewise substantiated with CFD parameter sweeps of pore aspect ratios which show an overall negative correlation between pore aspect ratio and recirculation zone volume. The ultimate pore aspect ratio was chosen to be 20:1 such that the minimum width of the pores, at 20 pm, was sufficiently wide to allow passage of potential cells in suspension.

[0473] Figures 23Ato 23D show an exemplary process for optimization of the porous membrane, where the porous membrane is further optimized based on a second design objective: optimal the pore arrangements for robust device loading.

[0474] Figure 23A shows a schematic diagram of the porous membrane when the porous interface remains intact and proper isolation of the matrix in the aperture occurs. Figure 23A also shows a schematic diagram of an instance of unwanted pore blockage. The diagram provided in Figure 23A contrasts when the device has been correctly loaded, and when the device has been incorrectly loaded. Prior to hydrogel polymerization, surface tension at the pore interface prevents hydrogel seepage into the microchannel below where its presence through rough handling may create unwanted blockages. Robust device loading is critical for usability as it minimizes both the handling expertise that is required as well as minimizes failure rates.

[0475] Figure 23B shows a plot of the influence of pore aspect ratio on the quantitative metric of liquid entry pressure. The quantitative metric of liquid entry pressure was identified to facilitate design for this objective. The liquid entry pressure refers to the pressure differential required between the top and bottom sides of the membrane for the surface tension barrier to be broken. The criteria for this design objective is therefore to have as high a liquid entry pressure as possible. Using the Young-Laplace equation for surface tension coupled with finite element analysis (FEA), the liquid entry pressure can be estimated for any pore geometry.

[0476] Figure 23C shows a plot of a parameter sweep for various pore aspect rations. The parameter sweep identified a positive correlation between pore aspect ratio and liquid entry pressure Therefore, higher pore aspect ratios have the additional benefit of contributing to robust device loading. Smaller pores also correspond to higher liquid entry pressures. However, the current pore design was not made any smaller in order to enable the passage of potential cells in suspension.

[0477] Figure 23D shows finite element analysis (FEA) plots of the effect of the pore aspect ratio for the pores of the porous membrane on pore deformation for a singlearbitrary circular pore in the inlet domain, for pores having a circular cross-section and rectangular cross-sections with aspect ratios of, 1 :1 , 4:1 , and 20:1. The FEA simulation results illustrate the pore deformation that results due to pore blockages in these pores of varying aspect ratios (the extent of the deformation is exaggerated). This FEA parameter sweep of the pore aspect ratios demonstrates that higher aspect ratios lead to stronger surface tension effects.Example 3 - Use of the microfluidic device

[0478] Figure 24A shows an exemplary application of the microfluidic device 100 for the generation of vascular networks. Culturing on the device involves combining endothelial cells, fibroblasts, pericytes, a hydrogel matrix, and an organoid within the microfluidic devices. This mixture is then implanted onto the devices and cultured with continuous perfusion. The inclusion of an organoid, for organoid vascularization, is optional and may be excluded in order to culture vascular networks alone for modeling vascular related phenomena. The implanted tissue is also not limited to organoids and may be any tissue with a maximum thickness of ~1 mm and up to ~3-4 mm in width. Vascular networks arise through de novo vessel formation facilitated by standard tissue culture incubation environments and continuous perfusion of nutrient media. The addition of the organoid, or any appropriately sized 3D tissue which fit within the dimensions of the tissue compartment, is optional. The endothelial vessels alone allow modeling of vascular related phenomena, and the inclusion of the organoid enables organoid vascularization. Figures 23B to 23E shows the result of the tissue implantation and culturing within the matrix located in the aperture.

[0479] Figure 24B shows RFP-labeled endothelial cells within within the matrix located in the aperture. The form of the endothelial cells demonstrates the coalescence of singularized cells into vessels over a 4 day time period.

[0480] Figure 24C shows the RFP-labeled endothelial vessels placed in the physical context of the tissue compartment and microchannels (dashed white lines).

[0481] Figure 24D shows the endothelial vessels (CD31+) within the matrix located in the aperture of the microfluidic device. The endothelial vessels display key characteristics including deposition of basement membrane proteins (i.e., laminin and collagen IV) as well as presence of lumen through intravascular perfusion of fluorescently labeled dextran.

[0482] Figure 24E shows an organoid that is embedded within the vascular network (CD31+) within the matrix located in the aperture. Here the organoid is of the humancerebral cortex and marked by SOX2 for the progenitors, GFAP for glial, and MAP2 for neurons.

[0483] Figure 24F shows an example process of initiating an experiment on the microfluidic device 100. All views depict cross sections of the tissue compartment and surrounding microchannels. (A) The microfluidic devices is assembled without the compartment lid, forming a circular well. (B) The interior of the tissue compartment and adjoining surfaces are coated with polydopamine in order to promote hydrogel adhesion. (C) Cells and organoid are suspended in unpolymerized hydrogel and transferred into the tissue compartment. (D) Following placement of the compartment lid on top of the tissue compartment, the hydrogel polymerizes thus fixing the cells and organoid in place. (E) The microchannel is coated with laminin to promote cell adhesion. (F) Endothelial cells are injected into the microchannel at high density followed by incubation at 37°C and 5% CO2 for 1 hour to form a cell monolayer. Flow can then be initiated with continuous perfusion provided by the primary circulation system.Example 5 - Bypass channel optimization

[0484] A goal of the bypass channel 310 is to increase tubing flow velocities without diminishing the pressure gradient across the tissue compartment 130. A first design consideration is the prevention of bubble blockages within the tubing. Figure 25A shows a schematic diagram of an instance of air bubble blockage within a microchannel. For a certain tubing diameter such as those shown in Figure 25A, the pressure required to unblock a bubble can be estimated through the capillary form of the Young-Laplace formula and the material values for advancing and receding contact angles.

[0485] Figure 25B shows a plot of optimized inlet and outlet conduit diameter in the context of a parameter sweep for the bubble unblocking pressure. Within this plot and the other exemplary embodiments of the disclosure, Pbrefers to the pressure across the bubble. To facilitate the optimization of the form of the bypass channel for optimal diameter of the inlet and outlet conduits, bubble pressure is utilized as the quantitative metric and modeled using the Young-Laplace formula combined with known material values for advancing and receding contact angles, as well as a parameter sweep, the bubble pressure is inversely proportional to the tubing diameter. Moreover, the bubble pressure for the chosen tubing diameter is lower than the pump pressure for much of its operating range. Consequently, air bubbles which accidentally appear within the tubing will automatically be unclogged from the pressure of the impeller pump 102a. This isfurther substantiated by practical use where minimal to no bubble obstructions are observed with the optimized design.

[0486] Figure 25C shows an analogous fluidic circuits for the microfluidic channel 1 10 that includes the bypass channel. The expressions governing the circuit become intractable, thus demonstrating the particular need for CFD modeling and parameter sweeps. Similar to the serpentine channel, a parameter of interest for the bypass channel 310 is its flow resistance, which relates to its length and diameter. Tubing flow velocities are dependent on both flowrate and tubing diameter. Tubing diameter is further related to its flow resistance as described by the Hagen-Poiseuille equation. Channel flowrates from the fluidic circuit analogy are furthermore proportional to the average tubing fluid velocities by the tubing cross-sectional area.

[0487] Figure 25D and 25E show a two-variable CFD parameter sweeps of the inlet and outlet conduits and bypass channel flow resistances. To optimize the bypass channel 310 using CFD, a 2-variable parameter sweep is performed while varying both the bypass channel 310 flow resistance and the flow resistance of the inlet and outlet tubing. Two objective variables are considered, one being the compartment pressure and the other being the average tubing fluid velocity. The optimized values are selected which simultaneously maximizes both of these variables. Ultimately, the specific embodiment of the bypass channel 310 shown in the drawings is able to increase the average tubing fluid velocities by a factor of ~70 with minimal decreases to the compartment 130 pressure gradient.

[0488] Figure 25F shows a plot of pressure results from a CFD simulation for the microfluidic device with the bypass channel. The results of this plot demonstrate that the bypass channel 310 is able to drastically increase tubing velocities with negligible decreases to tissue compartment 130 pressures.Example 6 - Optimizing compound delivery time

[0489] Figure 26A shows a top, planar view of the microfluidic device and microfluidic pump unit and indicates the concentration of compounds within the microfluidic device and microfluidic pump unit. This diagram illustrates the path that compounds added to the media reservoir of the microfluidic pump unit must first transit through to arrive in the aperture of the at least one compartment. Low compound delivery times are required to interrogate dynamic processes where compounds must reach maximal concentration as fast as possible. Compounds added to the media reservoir of the impeller pump must first transit through the tubing before arriving at the microfluidic device and vascularizedtissue. This lag is formulated as the compound delivery time and defined as the time after compound addition for the tissue concentration to reach 90% of the reservoir concentration. The compound delivery time is roughly inversely proportional to the average tubing flow velocity. To facilitate the design of the microfluidic device 100 and bypass channel 310 to optimize compound delivery time, the compound delivery time is defined quantitatively as the time between compound addition and the compound concentration at the tissue compartment reaching a value of 90% the reservoir concentration.

[0490] Figure 26B shows a plot of compound concentration within the microfluidic device and microfluidic pump unit over time. Note that the value of compound delivery time is inversely proportional to the average tubing fluid velocity through the connection tubing.

[0491] Figure 26C shows the results of the injection of fluorescent Dextran injection into the microfluidic device as the resulting perfusion of the fluorescent Dextran through the vascular network during the running of the microfluidic pump unit and when the microfluidic pump unit is shut off. As a directly applicable example application, a precise method of measuring vessel permeabilities was developed as part of the optimization of the microfluidic device 100 and bypass channel 310. To facilitate this optimization, the compound delivery time is defined quantitatively as the time between compound addition and the compound concentration at the tissue compartment 130 reaching a value of 90% the reservoir concentration. Roughly, this value is inversely proportional to the average fluid velocity through the inlet and outlet conduits 116, 118. Vessel permeability is a relevant measure of a vessel’s function and is related to numerous physiological and pathological processes. This method of measuring vessel permeabilities involves quickly pulsing the vascular network for 10 seconds with nutrient media containing fluorescently labeled dextran. After the dextran pulse, the impeller pump 102a is left off such that only diffusion acts within the tissue compartment 130 and no convective flows are present. Overtime, fluid will transit from within the vessels into the interstitial space in accordance with flux from high concentration to low concentration regions. Vessel permeabilities may be calculated by imaging the dextran transit using florescent microscopy and considering the rate at which this transit occurs. A mass balance for the dextran is then created in order to fit an exponential model to the data and extra permeability values. Notably, the devised method is most accurate when quick pulses of dextran are possible and when the timescale of the dextran transit is much higher than the compound delivery time.

[0492] Figure 26D shows a plot of CFD parameter sweeps, which involve modeling concentration dynamics. The plot displays compound delivery times both in the presence and absence of the bypass channel for various operating input pump pressures. The results of the CFD parameter sweep demonstrates that usage of the bypass channel is able to drastically reduce the compound delivery time from ~10 min to ~10 sec by increasing average tubing fluid velocities.Example 7 - Sedimentation optimization

[0493] Figure 27A shows a schematic diagram of a theoretical model that is applied to estimate the sedimentation risk within the microfluidic channel 110 by considering bead passage through the connecting tubing. For certain biological applications, particles such as cell suspensions may be added to the nutrient media. These particles are at risk of undesired sedimentation in the connecting tubing when fluid velocities are too low. For the theoretical model, the Reynold’s number is low within the tubing, and thus turbulent mixing is negligible, and beads travel in straight diagonal paths in the tubing. The downwards sedimentation velocity can be estimated using Stoke’s settling law. Sedimentation risk is defined as the case when the bead sediments downwards over the length of the tubing a distance equal to half the tubing’s diameter. Notably, this method is a rule of thumb estimation as actual sedimentation is much more complex.

[0494] Figure 27B shows the result of a CFD parameter sweep that was performed for the average tubing velocities within the connecting tubing in order to assess the settling risk across the operating range of pump input pressures both in the presence and absence of the bypass channel 310. These results demonstrate that the use of the bypass channel 310 drastically decreases sedimentation risk with practically no pump pressure within the operating ranges predicted to undergo sedimentation.Example 8 - Fabrication

[0495] Microfluidic device 100 components were generated in AutoCAD and were then transferred onto chrome photomasks using a uPG Mask Writer followed by photomask development. Depending on desired film thickness, either Su-8-50 or Su-8- 100 photoresist was spin-coated onto centimeter diameter silicone wafers. UV exposure using the transferred the designs from the photomask onto the photoresist. Removal of uncured photoresist produced wafer molds that are later used in downstream steps.

[0496] Microfluidic channels 1 10 and tissue compartments 130 were fabricated from polydimethylsiloxane (PDMS, Sylgard , Dow). Component base 134 and curing agents were mixed 10:1 by weight and poured onto wafer molds. Following degassing undervacuum, the mixture was left to cure overnight at 50°C. Specific geometries were cut using scalpels and appropriately sized biopsy punches.

[0497] The porous membranes 140 of the microfluidic device 100 was similarly fabricated from PDMS. Wafer molds are created featuring posts which correspond to the pores 142 of the porous membrane 140. These molds were first silanized with trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane through overnight deposition in a vacuum chamber followed by a 10min incubation at °C. A 1 mm “sacrificial” slab of cured PDMS the size of the wafer was then treated in a plasma chamber for 5min. Immediately afterwards, a 2.5% solution of polyvinyl alcohol (PVA) was spin coated onto the sacrificial slab. The coated slab was left in a 50C oven overnight. Mixed but unpolymerized PDMS was then poured onto the wafer followed by degassing. The sacrificial slab was treated a second time in a plasma chamber for 5min and then used to press the unpolymerized PDMS onto the wafer. The PVA coated side is facing down towards the unpolymerized PDMS. Air bubbles in the unpolymerized PDMS are removed followed by the addition of 30lbs of weights onto the sacrificial slab. Care is taken to ensure even distribution of the weights. The assembly is left at room temperature for 1 hr followed by overnight incubation in a 50C oven. After incubation, the sacrificial slab along with the cured porous membrane 140 is peeled off the wafer and excised into appropriately sized pieces with a scalpel. These pieces may then be directly plasma bonded onto corresponding microchannel pieces. To remove the sacrificial slab, pieces are incubated in ultrapure water overnight at 70C to dissolve the PVA layers.

[0498] Assembly of microfluidic devices 100 consisted of multiple plasma bonding steps. Surfaces were activated in a plasma chamber (Harrick Plasma) for 2 min, aligned manually with a stereomicroscope (Leica), and incubated overnight at 70°C to facilitate bonding. The aperture 132 of the at least one compartment 130 was of the assembled microfluidic devices 100 were furthermore coated with a 2 mg / ml solution of dopamine hydrochloride dissolved in 10 mM Tris-HCI buffer for 2 hrs. to promote hydrogel adhesion. The aperture 132 of the at least one compartment 130 was washed 3 times with ultrapure water to remove residual coating solution and left to air dry. Coated microfluidic devices 100 were stored at room temperature and used the next day.

[0499] 3D printed components were produced using polylactic acid (PLA) filament using a fused deposition modeling (FDM) 3D printer. All printed components were immersed in 70% ethanol overnight and then left to air dry.Example 9 - Characterization of impeller pump performance.

[0500] In the operation of the impeller pump 520a embodiment of the microfluidic pump unit 520, the performance of the pump 520a was estimated based on an analogous fluidic circuit. The impeller pump 520a is analogous to an electrical battery with the microfluidic device 100 serving as a load resistor. Similar to an electrical battery, the output flowrates (i.e., currents) and pressure drops (i.e., voltages) are dependent on the load resistance. Figures 28A to 28F show plots of various pump parameters for differing flow conditions within the pump.

[0501] Figure 28A shows a plot of the output flowrates provided by the microfluidic pump unit (for various impeller speeds) over time. These results demonstrate that, using negative feedback, the control system is able to precisely control the drive motors and ensure consistent rotational speeds, and thus consistent flowrates out of the microfluidic pump unit throughout the operation time of the microfluidic pump unit.

[0502] Figure 28B shows a plot of the output flowrates provided by the microfluidic pump unit for various impeller speeds. The correlation between channel flowrate and impeller speed is quadratic. Measured flowrates between devices have < 5% + / - deviation Using the negative feedback loop as described above, the control system is able to precisely control the drive motors and ensure consistent and precise rotational speeds, and thus consistent and precise flowrates out of the microfluidic pump unit.

[0503] Figures 28C shows a plot of parameter sweeps forthe pressure drop provided by the impeller pump 520a (into the microfluidic device 100) as a function of the impeller speed. The pressure drop provided by the pump 520a has a relatively low dependance on the device resistance. As shown in this Figure, a higher device 100 resistance leads to higher flowrates and lower pressure drops. For a given impeller 540 speed, the impeller 540 pressure and flowrate depend on the load flow resistance in a manner analogous to electrical voltage and current. Increases in load (i.e., microfluidic chip) flow resistance increases the pressure and decreases the flowrate.

[0504] Figures 28D shows a plot of parameter sweeps for the flowrate provided by the impeller pump 520a (into the microfluidic device 100) as a function of the impeller speed. The flowrate provided by the pump 520a has a relatively high dependance on resistance. As shown in this Figure, a lower device 100 resistance leads to higher flowrates and lower pressure drops. For a given impeller 540 speed, the impeller 540 pressure and flowrate depend on the load flow resistance in a manner analogous to electrical voltage and current. Increases in load (i.e., microfluidic chip) flow resistance increases the pressure and decreases the flowrate.

[0505] Figures 28E shows a plot of parameter sweeps for the flowrate provided by the impeller pump 520a (into the microfluidic device 100) as a function of the pressure drop across the device. This plot was produced by measuring flowrates and pressure drops while varying load resistance and impeller speed. Lines of best fit connect multiple device resistances at the same impeller speed. These parameter sweeps reveal that within operating ranges, the impeller pump 520a primarily functions as a constant pressure pump with minimal decreases to pressure with increasing flowrates. Thus, a relevant parameter of interest for a particular impeller 540 speed is not the flowrate, but the pressure generated. This is also reflected in other parameter sweeps shown in Figures 28C and 28D, where pressure is the changing variable.

[0506] Figure 28F shows a plot of the output flowrate from the impeller pump as a function of device resistance. The resistance of the optimized microfluidic device is placed in the context of a device resistance parameter sweep. This plot depicts an exemplary value of an optimized device resistance. The plot also illustrates the expected pump unit output flowrates for the resistance optimized device across various impeller speeds. Using a plot such as shown in Figure 28F, output flowrates can also be predicted for the impeller pump 520a if the load resistance is knownExample 10 - Impeller pump performance with PI parameter tuning.

[0507] The performance of the impeller pump 520a version of the microfluidic pump unit 520 can be further optimized by the inclusion of the above-described PI controller. The integration of the PI controller facilitates the dynamic tunability of the impeller pump. Figures 29Ato 29D demonstrate the improved control of the impeller pump 520a when a PI controller has been integrated into the control system.

[0508] Figure 29B shows a plot of the output flowrate response of the impeller pump to the input of a step function. In this example, the controller proportional and integral constants are tuned to optimize the step responses. PI parameter tuning results in fast response times with rise and fall times of ~ 0.25 sec and low deviation between measured and target flowrates

[0509] Figure 29B shows a plot of the output flowrate response of the impeller pump to the termination of a step function. In this example, the controller proportional and integral constants are tuned to optimize the step responses. PI parameter tuning results in fast response times with rise and fall times of ~ 0.25 sec and low deviation between measured and target flowrates.

[0510] Figure 29C shows a plot of an artificial, pulsatile flow that was generated by the impeller pump 520a with the integrated PI controller. Thus, artificial pulsatile flow can be generated for up to 5 Hz in frequency. The low response times of the impeller pump 520a that are achieved by the integration of the PI controller enable controlled generation of pulsatile waveforms over a wide range of frequencies.

[0511] Figure 29D demonstrates a complex waveform, in the form of simulated blood flow patterns with dicrotic notches, that is generated through specification of the input waveforms within the control system 590 and PI controller. With the integration of the PI controller, dynamic tunability can be extended to complex waveforms such as arterial blood flow with simulated dicrotic notches as well as more exotic patterns.

[0512] In this exemplary embodiment, the time between a flow command towards maximal response is less than 0.25 seconds. By providing a connection between the impeller 540 and the microfluidic device 100 with a very low “fluidic capacitance” (i.e., placing the impeller 540 as close as possible to the microfluidic device 100 to minimize tubing length) in tandem with integrating the PI controller, a very short response time can be achieved. The lower fluidic capacitance here reduced lag between when a flow signal is given and when the fluid actual begins to move in the microfluidic device 100.

[0513] The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the above-described embodiments are intended to be examples of the disclosure and alterations and modifications may be affected thereto, by those of skill in the art, without departing from the scope of the disclosure that is defined solely by the claims appended hereto.

Claims

What is claimed is:1 . A microfluidic device for use with a primary circulation system, the microfluidic device comprising:(a) a microfluidic channel configured for being in fluid communication with the primary circulation system, the microfluidic channel having: an upstream channel for receiving a pressurized fluid from the primary circulation system; a pressure drop section in fluid communication with the upstream channel for receiving a first volume of the pressurized fluid from the upstream channel and inducing a pressure drop to produce a depressurized fluid having a lower pressure; and a downstream channel in fluid communication with the pressure drop section for receiving the depressurized fluid therefrom and for recirculating the depressurized fluid to the primary circulation system;(b) at least one porous membrane having a plurality of pores; and(c) at least one compartment that includes an aperture, the aperture of the at least one compartment being in fluid communication with the microfluidic channel via the at least one porous membrane such that when a second volume of the pressurized fluid flows from the upstream channel to the downstream channel via the at least one porous membrane and the compartment, a pressure differential is formed within the aperture.

2. The microfluidic device of claim 1 , wherein the at least one porous membrane is at least partially in fluid communication with both the upstream channel and downstream channel of the microfluidic channel.

3. The microfluidic device of claim 1 , wherein the at least one porous membrane includes a first porous membrane and a second porous membrane; and wherein the first porous membrane is at least partially in fluid communication with the upstream channel of the microfluidic channel and the second porous membrane is at least partially in fluid communication with the downstream channel of the microfluidic channel.

4. The microfluidic device of any one of claims 1 to 3, further comprising:a bypass channel that is in fluid communication between the upstream and the downstream channels of the microfluidic channel for circulating a third volume of the pressurized fluid therebetween.

5. The microfluidic device of claim 4, wherein the bypass channel is structured for increasing a velocity of the pressurized fluid within the upstream channel without reducing the pressure differential within the aperture of the at least one compartment.

6. The microfluidic device of claim 4 or 5, wherein the bypass channel is structured for reducing particle sedimentation time within the aperture of the at least one compartment.

7. The microfluidic device of any one of claims 4 to 6, wherein the bypass channel is structured for increasing a rate of delivery of at least one compound to the aperture of the at least one compartment via the microfluidic channel.

8. The microfluidic device of any one of claims 4 to 7, wherein the bypass channel includes an inlet end and an outlet end; and wherein the inlet end is provided in fluid communication with the upstream channel, and the outlet end is provided in fluid communication with the downstream channel for circulating the third volume of the pressurized fluid through the bypass channel.

9. The microfluidic device of any one of claims 3 to 8, wherein the upstream channel of the microfluidic channel is fluidly connected to an outlet of the primary circulation system via at least one outlet conduit; and wherein the downstream channel of the microfluidic channel is fluidly connected to an inlet of the primary circulation system via at least one inlet conduit.

10. The microfluidic device of claim 9, wherein the bypass channel is structured to permit increased diameters of the at least one outlet conduit and the at least one inlet conduit of the primary circulation system while substantially maintaining a velocity of both the pressurized fluid and the depressurized fluid through the at least one outlet conduit and the at least one inlet conduit.

11. The microfluidic device of claim 9 or 10, wherein the bypass channel and upstream and downstream channels of the microfluidic channel are each sized such that the microfluidic device can accommodate an internal diameter of the at least one outlet conduit that is in a range from about 0.1mm to about 5 mm, and an internal diameter of the at least one inlet conduit that is in a range from about 0.1 mm to about 5 mm.

12. The microfluidic device of claim 9, wherein the inlet end of the bypass channel is connected to the upstream channel to form a first three-way node between the bypass channel, the upstream channel and the primary circulation system; and wherein the outlet end of the bypass channel is connected to the downstream channel to form a second three-way node between the bypass channel, the downstream channel and the primary circulation system.

13. The microfluidic device of any one of claims 8 to 12, wherein the inlet end of the bypass channel is connected directly to the upstream channel of the microfluidic channel, and wherein the outlet end of the bypass channel is connected directly to the downstream channel of the microfluidic channel.

14. The microfluidic device of claim 8, wherein a length of the bypass channel is in a range from about 10 mm to about 100 mm, and wherein an inner diameter of the bypass channel is in a range from about 0.1 mm to about 5 mm.

15. The microfluidic device of any one of claims 3 to 17, wherein the bypass channel includes: an inlet channel portion that defines the inlet end of the bypass channel an outlet channel portion that defines the outlet end of the bypass channel; and a bypass portion which extends between the inlet and outlet channel portions; and wherein the inlet channel portion extends upwards, substantially vertically, from the upstream channel, the outlet channel portion extends upwards, substantially vertically, from the downstream channel, and the bypass portion is substantially horizontal.

16. The microfluidic device of any one of claims 3 to 15, wherein the bypasschannel is sized such that the pressure differential across the aperture of the at least one second compartment is maximized.

17. The microfluidic device of any one of claims 3 to 16, wherein the bypass channel is structured such that a fluid resistance of the bypass channel is no less than five times a fluid resistance of the upstream channel, and such that the fluid resistance of the bypass channel is no less than five times a fluid resistance of the downstream channel.

18. The microfluidic device of any one of claims 9, 10, and 11 , wherein an internal diameter of each of the at least one outlet conduit and the at least one inlet conduit is at least 100 pm.

19. The microfluidic device of any one of claims 9, 10, and 11 , and 18, wherein an internal diameter of the bypass channel is at least 100 pm.

20. The microfluidic device of any one of claims 3 to 19, wherein at least a portion of the bypass channel is composed of a pliable material.

21. The microfluidic device of claim 20, wherein the pliable material is sufficiently pliable such that the at least a portion of the bypass channel can be compressed on itself for substantially inhibiting a flow of the third portion of the pressurized fluid through the bypass channel.

22. The microfluidic device of any one of claims 3 to 21 , wherein the bypass channel is sized such that a ratio of APCOmp / APpump is at least about 0.9; wherein APcompis defined as the pressure differential across the aperture of the at least one second compartment; and wherein APpUmp is defined as a pressure differential across the primary circulation system.

23. The microfluidic device of any one of claims 1 to 22, wherein the at least one compartment is a plurality of compartments.

24. The microfluidic device of claim 23, wherein the plurality of compartments aresequentially arranged along the microfluidic channel such that each of the plurality of compartments is positioned at least partially over the upstream channel and at least partially over the downstream channel.

25. The microfluidic device of any one of claims 1 to 24, wherein the at least one porous membrane has a first porous membrane section in fluid communication with the upstream channel and a second porous membrane section in fluid communication with the downstream channel; and wherein the aperture includes first aperture portion that is aligned with the first porous membrane section and second aperture portion that is aligned with second porous membrane section.

26. The microfluidic device any one of claims 1 to 25, wherein each pore of the plurality of pores of the at least one porous membrane has a pore diameter ratio PerP:Para equal to or less than 1 :1.

27. The microfluidic device of any one of claims 1 to 26, wherein the plurality of pores of the at least one porous membrane are structured as a plurality of slit-shaped pores.

28. The microfluidic device of claim 27, wherein each pore of the plurality of slitshaped pores is structured such that a width of the pore is in a range from about 10 pm to about 200 pm and a length of the pore is in a range from about 50 pm to about 2000 pm .

29. The microfluidic device of claim 27 or 28, wherein the plurality of slit-shaped pores are oriented such that a long axis of each pore of the plurality of slit-shaped pores extends substantially parallel to a long axis of the upstream channel.

30. The microfluidic device of any one of claims 27 to 29, wherein each pore of the plurality of slit-shaped pores is structured such that an aspect ratio of the pore is greater than or equal to 20: 1 .

31. The microfluidic device of any one of claims 1 to 30, further comprising a secondary microfluidic channel for use with a secondary circulation system.

32. The microfluidic device of claim 30 or 31 , wherein the secondary microfluidic channel includes: a secondary upstream section for receiving a pressurized fluid from the secondary circulation system, a secondary pressure drop section in fluid communication with the secondary upstream section for receiving the pressurized fluid therefrom, the secondary pressure drop section being structured for inducing a pressure drop in the pressurized fluid to produce a depressurized fluid; and a secondary downstream portion in fluid communication with the pressure drop section for receiving the depressurized fluid therefrom and recirculating the second depressurized fluid to the secondary circulation system.

33. The microfluidic device of claim 32, wherein the secondary pressure drop section comprises: a first pressure drop section in fluid communication with the secondary upstream section, the first pressure drop section being structured for inducing a first pressure drop in the pressurized fluid to produce a first depressurized fluid; a midstream section in fluid communication with the first pressure drop section for receiving the first depressurized fluid therefrom; and a second pressure drop section in fluid communication with the midstream section for receiving the first depressurized fluid therefrom, the second pressure drop section being structured for inducing a pressure drop in the first depressurized fluid to produce a second depressurized fluid.

34. The microfluidic device of claim 33, wherein the midstream section includes a first midstream portion that is structured to direct the first depressurized fluid to flow in a first flow direction; and a second midstream portion in fluid communication with first midstream portion, the second midstream section being structured to direct the first depressurized fluid to flow in second flow direction opposite the first flow direction.

35. The microfluidic device of any one of claims 32 to 34, wherein the at least one porous membrane further comprises: a third portion in fluid communication with the secondary upstream section of the secondary microfluidic channel and a fourth portion in fluid communication with thesecondary downstream section of the secondary microfluidic channel; wherein the aperture includes a third aperture portion that is aligned with the third portion of the at least one porous membrane and fourth aperture portion that is aligned with the fourth portion of the at least one porous membrane.

36. The microfluidic device of claim 35, wherein the third and fourth portions of the at least one porous membrane are disposed in between the first volume and second volume of the porous membrane.

37. The microfluidic device of any one of claims 32 to 36, wherein a pressure difference defined between an inlet of the upstream channel and outlet of the downstream channel of the microfluidic channel is substantially equal to pressure difference defined between an inlet of the secondary upstream section and an outlet of the secondary downstream section of the secondary microfluidic channel.

38. A microfluidic device system including the microfluidic device of any one of claims 1 to 37, further comprising at least one controller that is configured to control the primary circulation system.

39. A microfluidic device system including the microfluidic device of any one of claims 29 to 37, further comprising at least one controller that is configured to control the primary circulation system and the secondary circulation system.

40. A microfluidic device assembly including a plurality of the microfluidic devices of claim 1 , the microfluidic device assembly further comprising: an inlet channel section that is in fluid communication with an outlet of the primary circulation system for receiving pressurized fluid therefrom; and an outlet channel section in fluid communication with an inlet of the primary circulation system for returning depressurized fluid thereto; wherein the plurality of microfluidic devices are connected between the inlet channel section and outlet channel section such that the upstream channel of each microfluidic device of the plurality of microfluidic devices is fluidly connected to the inlet channel section, and the downstream channel of each microfluidic device of the plurality of microfluidic devices is fluidly connected to the outlet channel section.

41. A microfluidic device assembly for use with a primary circulation system, the microfluidic device assembly comprising: a plurality of microfluidic devices that are connected in series between an inlet and an outlet of the primary circulation system, each of the plurality of microfluidic devices including: a microfluidic channel that has an upstream channel for receiving a higher pressure fluid, a pressure drop section in fluid communication with the upstream channel for receiving a first volume of the higher pressure fluid and inducing a pressure drop to produce a lower pressure fluid, and a downstream channel in fluid communication with the pressure drop section for receiving the lower pressure fluid; at least one porous membrane that is at least partially in fluid communication with both the upstream channel and downstream channel of the microfluidic channel; and a flow compartment that is positioned on top of microfluidic channel and that defines an aperture therethrough, the aperture being in fluid communication with the at least one porous membrane such that when a second volume of the pressurized fluid flows from the upstream channel to the downstream channel via the at least one porous membrane and through the flow compartment, a pressure differential is formed within the aperture; wherein the plurality of microfluidic devices are connected in series such that the upstream channel of a first microfluidic device of the plurality of microfluidic devices is fluidly connected to the outlet of the primary circulation system; wherein the upstream channel of each microfluidic device of the rest of the plurality of microfluidic devices is fluidly connected to a downstream channel of another of the plurality of microfluidic devices; and wherein the downstream channel of a last microfluidic device of the plurality of microfluidic devices is fluidly connected to the inlet of the primary circulation system.

42. A microfluidic assembly, comprising: at least one microfluidic device that is positioned on a base plate; at least one microfluidic pump unit that is fluidly connected to the at least one microfluidic device for circulating a fluid therethrough, the at least one microfluidic pump unit including: a pump housing; andan impeller that includes an impeller shaft and that is rotatably housed within the pump housing, the impeller shaft including at least one first magnetic element; and at least one magnetic drive system that is removably connectable to the pump housing and that includes at least one drive motor with an output shaft, the output shaft including at least one second magnetic element; wherein the magnetic drive system is removably connectable on the pump housing in a position such that the at least one first magnetic element is magnetically coupled to the at least one second magnetic element, while being spaced apart from at least one second magnetic element, for rotatably coupling the at least one drive motor to the impeller of the at least one microfluidic pump unit.

43. The microfluidic assembly of claim 42, further comprising a cover unit that is removably connectable atop the base plate.

44. The microfluidic assembly of claim 43, wherein the magnetic drive system is housed within the cover unit such that when the cover unit is connected atop the base plate, the at least one first magnetic element of the impeller shaft is magnetically coupled to the at least one second magnetic element of the output shaft, and when the cover unit is disconnected from the base plate, the at least one first magnetic element is magnetically decoupled from the at least one second magnetic element.

45. The microfluidic assembly of any one of claims 42 to 44, further comprising: a seal plate that is connectable on the base plate for inhibiting contamination and evaporation of the fluid from the microfluidic device and / or the microfluidic pump unit.

46. The microfluidic assembly of claim 45, wherein the seal plate is connectable on base plate such that when the cover unit is connected atop the base plate, the seal plate is disposed between the microfluidic pump unit in the base plate and magnetically couplable drive unit.

47. The microfluidic assembly of claim 45 or 46, wherein the seal plate is connectable on base plate such that when the cover unit is connected atop the base plate, the seal plate is at least partially disposed between the base plate and the coverunit, the first magnetic element is disposed on a first side of the seal plate, and the second magnetic element is disposed on a second side of the seal plate opposite the first side.

48. The microfluidic assembly of any one of claims 46 to 47, wherein the seal plate is connectable on the base plate such that an end of the impeller shaft is rotatably supported by the seal plate when the seal plate is connected to the base plate.

49. The microfluidic assembly of claim 48, wherein the seal plate is at least partially composed of at least one low friction material such that an amount of friction generated between the seal plate and the end of the impeller shaft is minimized.

50. The microfluidic assembly of any one of claims 42 to 49, wherein at least one of the pump housing and impeller of the at least one microfluidic pump unit is composed of one or more biocompatible materials.51 . The microfluidic assembly of claim 50, wherein the one or more biocompatible materials comprises one or more biocompatible plastics.

52. The microfluidic assembly of any one of claims 42 to 51 , wherein the at least one microfluidic device is positioned in at least one first well on the base plate; wherein the at least one microfluidic pump unit is positioned in at least one second well on the base plate; and wherein the at least one second well is adjacent to the at least one first well on the base plate.

53. The microfluidic assembly of any one of claims 42 to 52, wherein the fluid connection between the at least one microfluidic device and the at least one microfluidic pump unit includes at least one inlet conduit and at least one outlet conduit.

54. The microfluidic assembly of claim 53, wherein the at least one microfluidic pump unit and the at least one microfluidic device are positioned within the at least one first well and at least one second well, respectively, such that an effective fluidcapacitance of each of the at least one inlet conduit and at least one outlet conduit is minimized.

55. The microfluidic assembly of claim 53, wherein the at least one microfluidic pump unit and the at least one microfluidic device are positioned within the at least one first well and at least one second well, respectively, such that a length of each of the at least one inlet conduit and at least outlet conduit is minimized.

56. The microfluidic assembly of claim 53, wherein each of the at least one inlet conduit and the at least one outlet conduit is formed as a length of tubing that is oriented substantially vertically within the at least one first well.

57. The microfluidic assembly of claim 52, wherein the at least one microfluidic pump unit is removably mounted in the at least one first well; and wherein the at least one microfluidic device is removably mounted in the at least one second well.

58. The microfluidic assembly of claim 52 or 57, wherein the base plate comprises an SBS (Society for Biomolecular Screening) tissue culture plate; and wherein the at least one first well and the at least one second well are each structured as at least one SBS well on the SBS tissue culture plate.

59. The microfluidic assembly of any one of claims 52, 57 or 58, wherein the base plate further comprises a base plate cover portion; and wherein the SBS well plate is connectable on top of the base plate cover portion.

60. The microfluidic assembly of any one of claims 52 and 57 to 59, wherein the at least one first well is a plurality of first wells and the at least one second well is a plurality of second wells; wherein the at least one microfluidic device is a plurality of microfluidic devices, each of the plurality of microfluidic devices being contained in a unique first well of the plurality of first wells; andwherein the at least one microfluidic pump unit is a plurality of microfluidic pump units, each of the plurality of microfluidic pump units being contained in a unique second well of the plurality of second wells.61 . The microfluidic assembly of claim 42, wherein the magnetic drive system includes a plurality of drive motors; and wherein each drive motor of plurality of drive motors corresponds to one of the plurality of microfluidic pump units and is positioned on the cover unit such that the drive motor becomes rotatably coupled to the to the impeller of the corresponding one of the plurality of microfluidic pump units when the cover unit is connected to the base plate.

62. The microfluidic assembly of claim 61 , wherein the plurality of drive motors are arranged on the cover unit such that a spacing between each drive motor and the rest of the plurality of drive motors is maximized; and wherein the plurality of microfluidic pump units are arranged on the base plate in a substantially identical arrangement to the plurality of drive motors on the cover unit such that each corresponding drive motor and microfluidic pump unit will be substantially aligned when the cover unit is connected to the base plate.

63. The microfluidic assembly of claim 42, wherein the at least one second magnetic element is a first diametrically magnetized magnet that is connected to the output shaft proximate a free end of the output shaft; and wherein the at least one first magnetic element is a pair of diametrically magnetized magnets positioned on opposing sides of the impeller shaft, proximate a free end of the impeller shaft.

64. The microfluidic assembly of claim 63, wherein the first diametrically magnetized magnet is oriented such that a magnetic axis thereof is substantially perpendicular to a long axis of the motor axle; and wherein the pair of diametrically magnetized magnets are oriented such that a direction of magnetization for each of the pair of diametrically magnetized magnets is in substantially the same direction.

65. The microfluidic assembly of claim 42, wherein the pump housing of the at least one microfluidic pump unit includes: a pump reservoir that is defined in a first section of the pump housing and which contains the impeller of the impeller pump unit and a volume of the fluid; and a media dampening reservoir that is defined in a second section of the main fluid housing and which also contains a volume of the fluid; wherein the media dampening reservoir is in fluid communication with the at least one microfluidic device for receiving a flow of the fluid therefrom; and wherein the media dampening reservoir is structured such that the flow of the fluid becomes more laminar as the flow of fluid enters the media dampening reservoir.

66. The microfluidic assembly of claim 65, wherein the media dampening reservoir is fluidly connected to the pump reservoir via at least one fluid aperture; wherein the at least one fluid aperture defines an inlet for the impeller; and wherein the media dampening reservoir is disposed substantially atop the pump reservoir within the pump housing.

67. The microfluidic assembly of claim 66, wherein the pump housing includes at least one inlet for receiving the flow of the fluid from the at least one microfluidic device; wherein the at least one inlet is directly fluidly connected to the media dampening reservoir.

68. The microfluidic assembly of claim 67, wherein the at least one inlet is spaced apart from the at least one fluid aperture such that the flow of the fluid enters the media dampening reservoir away from the at least one fluid aperture.

69. The microfluidic assembly of claim 67 or 68, wherein the pump housing includes at least one outlet for providing a flow of the fluid to the at least one microfluidic device; and wherein the at least one outlet is directly fluidly connected to the pump reservoir.

70. The microfluidic assembly of claim 69 wherein the fluid connection between the at least one microfluidic device and the at least one microfluidic pump unit includes at least one inlet conduit and at least one outlet conduit; wherein the inlet conduit is in fluid connection with the at least one inlet of the pump housing; and wherein the outlet conduit is in fluid connection with at least one outlet of the pump housing.71 . The microfluidic assembly of any one of claims 65 to 70, wherein the media dampening reservoir is sized to generate a substantially laminar flow of the second volume of fluid contained therewithin, prior to the second volume of fluid being introduced into the pump reservoir via the at least one fluid aperture.

72. The microfluidic assembly of any one of claims 65 to 71 , wherein the media dampening reservoir is an open top reservoir having a top surface that is open to atmosphere.

73. The microfluidic assembly of claim 72, wherein the second section of the pump housing is disposed vertically above the first section and is structured such that the second volume of fluid contained in the media calming reservoir can be evacuated from the media calming reservoir without evacuating the first volume fluid from the pump reservoir.

74. The microfluidic assembly of claim 42, further comprising a control system, the control system being operatively connected to the at least one microfluidic pump unit for controlling the operation thereof.

75. An in vitro system for culturing a multicellular aggregate comprising: the microfluidic assembly of any one of claims 42 to 74; and a control system in communication with the at least one microfluidic pump unit, the control system enabling pre-programmed fluid circulation schemes.

76. The in vitro system of claim 75, further comprising at least one sensor in communication with the at least one microfluidic device, and / or the at least onemicrofluidic pump unit.

77. The in vitro system of claim 76 wherein the at least one sensor is adapted for monitoring pH, nutrient concentration, metabolic concentration, temperature, pressure, electrical output, or any combination thereof.

78. A method of culturing at least one multicellular aggregate with the microfluidic assembly of any one of claims 42 to 74, the method comprising : forming a matrix seeded with the at least one multicellular aggregate within the at least one microfluidic device; operating the at least one microfluidic pump unit for perfusing a compartment of the at least one microfluidic device with a pressurized fluid having nutrients at a first flow rate so to induce the formation of a microvascular network within the matrix for vascularizing the at least one multicellular aggregate; and operating the at least one microfluidic pump unit for perfusing the compartment with the pressurized fluid at a second flow rate such that the pressurized fluid flows through the at least one microfluidic device and the matrix, the at least one multicellular aggregate is cultured; wherein the second flow rate is selected to permit the survival of the at least one multicellular aggregate; and wherein the at least one microfluidic pump unit is operated such that when the pressurized fluid enters the at least one microfluidic device at either of the first flow rate of the second flow rate, the flow of the pressurized fluid is substantially non- turbulent.

79. A method of culturing at least one multicellular aggregate with the microfluidic assembly of any one of claims 42 to 74, the method comprising : forming a matrix seeded with at least one multicellular aggregate within the at least one microfluidic device; operating the at least one microfluidic pump unit for perfusing the matrix with a pressurized fluid having nutrients at a first flow rate, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the formation of a microvascular network within thematrix vascularizing the at least one multicellular aggregate; and operating the at least one microfluidic pump unit for perfusing the matrix with the pressurized fluid at a second flow rate such that the at least one multicellular aggregate is cultured, the pressurized fluid flowing in the matrix at a first pressure and flowing out the matrix at a second pressure and the differential between the first pressure and the second pressure is between the range of 50 Pa and 420 kPa to allow the survival and maintenance of the multicellular aggregate.

80. A microfluidic pump assembly that is fluidly connectable to at least one microfluidic device for circulating a fluid therethrough, the microfluidic pump assembly comprising: a pump housing; an impeller that includes an impeller shaft and that is rotatably housed within the pump housing, the impeller shaft including at least one first magnetic element; and a magnetic drive system that is connectable to the pump housing and that includes at least one drive motor with an output shaft, the output shaft including at least one second magnetic element; wherein the magnetic drive system is removably connectable to the pump housing; and wherein the magnetic drive system is positioned relative to the at least one microfluidic pump unit such that the at least one first magnetic element is magnetically coupled to the at least one second magnetic element, while being spaced apart from at least one second magnetic element, for rotatably coupling the at least one drive motor to the impeller.

Images