Pneumatic microfluidic perfusion system for cell culture

Abstract

We provide a perfusion device for microfluidic cell culture. [Solution] A patterned substrate defining a plurality of receptacles, each having at least two pairs of medium exchange receptacles, each receptacle having a bed, a medium outlet on or near the bed, a closed array of at least one side wall separating the bed from a first of at least two contact surfaces, and a GCM for supplying medium into the receptacle at the opening of the GCM; and a plurality of perfusion channels for non-capillary transport of medium, each having a hydraulic diameter (dHC) of 0.1 to 1 mm, wherein the perfusion channels are fluidly connected to circulate medium between paired receptacles via interfaces with each lane of a 3-raceway cell culture layer, with a separation of at least 5 mm between the opening and the bed.

Description

Technical Field

[0001]

[0001] The present disclosure relates to organ-on-a-chip (OOC) devices. In particular, the present disclosure relates to patterned substrates for perfusing cell cultures.

Background Art

[0002]

[0002] Human and animal blood vessels form a complex network that is critical for supporting the function of every organ and tissue throughout the body by transporting blood and cells, delivering oxygen and nutrients, and removing metabolic wastes and carbon dioxide produced by cells. The inner lining of blood vessels consists of a single layer of endothelial cells that acts as a barrier between the blood flow and the surrounding tissue. Shear stress is a key factor in maintaining endothelial function, vascular homeostasis, blood flow regulation, and angiogenesis (Hui Lin Zhou et al., "Role of blood flow in endothelial functionality:a review" (2023), Front. Cell Dev. Biol. 11:1259280). The ability to precisely control and stimulate fluid flow is of great importance to mimic physiologically relevant conditions in microfluidic organ-on-a-chip (OOC) devices designed to recapitulate organ-level functions.

[0003]

[0003] Perfusion flow also plays a crucial role in organ-on-chip (OOC) devices, where two or more types of organ cells are cultured together to enable dynamic interactions between them, thereby providing more suitable conditions for human physiological functions. Active perfusion control enables more realistic intercellular, intertissue, or interorgan communication by mimicking physiological fluid flow, providing significant insights into complex biological processes. These enabled interactions allow for the study of inter-organ toxicity, gene regulation, and drug interactions between cells and tissues in a controlled microenvironment. Furthermore, such systems can simulate the effects of drugs metabolized by cells, tissues, and organs on each other and within a larger biological system, leading to more accurate predictions of systemic effects on other organs (Paul M. van Midwoud et al., Lab Chip, 2010, 10, 2778-2786, DOI: https: / / doi.org / 10.1039 / C0LC00043D; Leung et al., 2022, "A guide to the organ-on-a-chip", Nature Reviews Methods Primers 2, 33).

[0004]

[0004] Various approaches have been demonstrated on OOC platforms to mechanically simulate blood flow (Joshua Lim et al., ACS Biomater.Sci.Eng, 2024, 10, 6, 3548-3567, DOI:10.1021 / acsbiomaterials.3c01978). These include microfluidic devices in which fluid flow is driven by external forces generated by pumps or gravity, which have not been entirely successful in providing physiologically appropriate fluid flow to cell cultures.

[0005]

[0005] Syringe pump-based fluid systems utilize an operating mechanism for pushing or pulling fluid into or out of an OOC device via piping, providing a simple and efficient experimental configuration with setpoint-controlled flow rates. Small arterial segments are incorporated into the OOC platform to evaluate the structure and function of resistant arteries. Syringe pumps facilitate the delivery of culture media and enable drug testing in these configurations (Gunther et al., Lab on a chip, 2010, DOI:10.1039 / C004675B).

[0006]

[0006] The pneumatically controlled fluid system provides a stable and responsive flow because the pressure controller can establish a non-pulsating flow. This pressure-controlled system is supported by a manifold holder and can deliver multiple liquids into the OOC device, allowing for the transfer of culture medium to the other side of the microfluidic reservoir (Yasotharan et al., Lab Chip, 2015, 2660-2669; DOI:10.1039 / c5lc00021a).

[0007]

[0007] In OOC devices, gravity-driven hydrostatic flow is also frequently used, which is controlled by changing the height of the culture medium reservoir, the dimensions of the microfluidic channels, or by manipulating the tilt of a oscillating system, such as OrganoFlow® by MIMETAS (Moon et al., Lab on a chip, 2024, DOI:10.1039 / d4lc00530a).

[0008]

[0008] Conventional 24, 48, 96, and 384-well plate cell cultures offer reasonable processing capacity from a drug discovery perspective, but they have significant limitations due to physiologically inappropriate two-dimensional cell culture and non-physiological intercellular interactions. For example, hypoxia, i.e., low levels of oxygen in the cell tissue, limits the growth of the lower layers of cells that are not in direct contact with the cell culture medium.

[0009]

[0009] In three-dimensional cell culture systems, syringe pumps provide continuous flow with precise and reliable control of fluid delivery, which makes them essential for ensuring stable and consistent conditions in microfluidic OOC systems. However, the main drawback of syringe pump systems is their limited capability for multiplexing applications. Their operation typically involves challenges with tubing connections, tip-to-external bridging issues, and capacity limitations, requiring frequent manual intervention for refilling or managing multiple parallel channels. As a result, scaling up for experimental multiplexing becomes cumbersome, inefficient, time-consuming, and labor-intensive.

[0010]

[0010] Small-footprint hydrostatic flow-based OOC devices offer a compact solution for fluid delivery. However, they often face reliability issues due to changes in flow rate over time, which can lead to a drop in liquid level and inconsistent flow. Rocker systems can enable quasi-continuous flow by gently tilting the device to maintain fluid movement, but they still lack the precision required for certain applications. Furthermore, rockers occupy considerable volume space within cell culture incubators. While advances have been made to improve processing capacity, significant challenges remain, especially when managing a large number of samples or simultaneous experiments. Maintaining a consistent and reproducible flow rate across multiple different chips in high-processing-capacity configurations can be challenging.

[0011]

[0011] Microvalves are a key component of OOCs, enabling the delivery of minute amounts of fluid in a sequential and intermittent manner, which brings about the automation of assay protocols. While some microvalves may resemble microscale versions of macroscale valves, microvalve technology is generally a separate field of study, at least in part, due to surface tension dominating flow behavior at the microscale and gravity being relatively weak. Most active microvalves are integrated features into the chip in a stacked multilayer structure, which can involve opposing forces that are difficult to fabricate and assemble into devices and typically require more force and energy for deployment and control. This is particularly evident in relation to the structure and elasticity required for checking (i.e., preventing backflow). Simpler microvalve structures exist, such as siphon valves, but they require mechanisms such as centrifugation to operate.

[0012]

[0012] There is still a need for a cell culture perfusion device that is small, energy-efficient, robust, mass-producible, and modular. Furthermore, it is desirable to deliver fresh culture medium, extract the medium from three-dimensional cell cultures while minimizing interference with perfusion, and administer drugs. It is also desirable to provide physiologically appropriate unidirectional perfusion.

[0013]

[0013] This background information is provided for the purpose of presenting known information that the applicant considers to be potentially relevant to the present disclosure. It is not necessarily intended, nor should it be construed, that any of the aforementioned information constitutes prior art to the present disclosure. [Overview of the project]

[0014]

[0014] The present disclosure relates to a pneumatically based cell culture perfusion device. The perfusion device has a patterned surface that provides multiple low-pressure seals to two perfusion channels at two opposing ends of the perfusion device, such that the perfusion channels are adjacent to a central cell culture region along their respective cell culture interfaces. The perfusion device has at least first and second three-phase receptacles for each perfusion channel and a flow path that directs cell medium through the perfusion channel via the interface from the first receptacle to the second receptacle. In some embodiments, the flow path can be reversed to reuse the perfusion by directing the medium back to the first receptacle via the interface, and in other embodiments, the flow path can bypass the interface when redirecting the fluid back to the first receptacle, which represents a loop resulting in continuous unidirectional recirculation of the same medium. In some embodiments, the channel, whether inverted or continuous, has branches for either or both of the following: removal of used medium, injection of fresh medium, sampling and optional testing of the medium, or injection of drugs, adjuvants, growth regulators, CO2, O2, or other solid, liquid, or gaseous materials. The perfusion apparatus may be multiplexed. The perfusion apparatus provides a suitable low-pressure seal to allow liquid to be injected almost continuously or at moderate intervals and drawn out of the perfusion channel, but may be dischargeable from the tip to allow testing of the cell culture. The use of a three-phase chamber allows for a low-pressure (positive or negative relative to the ambient) pump to drive the perfusion, and a pressure difference of 0.5–2 psi may be used to achieve highly efficient aerated mixing of the medium, depending on the low-pressure pump, resistance to bubbles, and, if desired. Measured removal of used medium and metered injection of fresh medium can be achieved.

[0015]

[0015] Accordingly, in one embodiment, a perfusion device is provided having a patterned upper surface and an opposing lower surface, the upper surface being Multiple receptacles, where each receptacle is The floor and, Exit and A plurality of receptacles comprising: at least one side wall having a check microvalve, the check microvalve having an inlet on the side wall; 0.1-1 mm hydraulic diameter (d) for non-capillary transport of liquids HC A plurality of supply channels, each having a check valve, wherein each supply channel is fluidly connected to the inlet of a check valve microvalve, comprising: The vertical clearance between the entrance and the floor is at least 5 mm. Multiple receptacles comprise a pair of first receptacles and a pair of second receptacles, and the outlet of the first receptacle of each pair can be configured to be fluidly connected to the second receptacle of each pair.

[0016]

[0016] In some embodiments, the check microvalve is a gravity check microvalve (GCM), the GCM having a deposit surface on the inlet or a side wall near the inlet, the deposit surface being fitted to induce the formation and growth of a liquid bolus and to limit the horizontal spread of the liquid beyond the deposit surface, thereby moving the center of gravity of the bolus away from the first side wall.

[0017]

[0017] In some embodiments, the outlet is located on the floor.

[0018]

[0018] In some embodiments, at least one of the outlets is fluid-connected to a vent on the bottom of the perfusion device.

[0019]

[0019] In some embodiments, the bottom surface of the perfusion device is patterned to define at least a portion of a perfusion network that is fluid-connected to at least one of the outlets.

[0020]

[0020] In some embodiments, the perfusion device is made of a material containing a thermoplastic polymer.

[0021]

[0021] In another embodiment, a device for perfusing a cell culture is provided, which device The perfusion apparatus described in the present specification, a base layer having an upper surface adapted to seal at least a portion of the bottom surface of the perfusion apparatus, wherein the base layer and the bottom surface of the perfusion apparatus together define a perfusion network in fluid connection with at least one outlet, a cover layer having a bottom surface adapted to seal at least a portion of the top surface of the perfusion apparatus, a plurality of ports configured to couple to a pressure source, wherein each of the plurality of ports is fluidly connected to a plurality of receptacles, and at least one port is disposed at each opposing end of the device. The perfusion network is configured to provide a first perfusion path and a second perfusion path between at least one port at one end of the device and at least one port at the opposing end of the device, The perfusion network is configured such that a first pair of receptacles are fluidly interconnected via the first perfusion path, and a second pair of receptacles are fluidly interconnected via the second perfusion path, The perfusion network includes a central region, and the central region has at least one central port open to the outside of the device, The perfusion network is configured to provide fluid communication between the central region and the first perfusion path at a first interface, and between the central region and the second perfusion path at a second interface, The first and second interfaces are located on opposite sides of the central region, The first interface is fluidly positioned between the first pair of receptacles, and the second interface is fluidly positioned between the second pair of receptacles.

[0022]

[0022] In some embodiments, the perfusion network is configured such that the first and / or the second perfusion path is branched.

[0023]

[0023] In some embodiments, the perfusion network is configured such that the first and / or the second perfusion path forms a loop.

[0024]

[0024] In some embodiments, the bottom surface of the cover layer is patterned, and both the top surface of the perfusion device and the bottom surface of the cover layer define an auxiliary network that is fluidly connected to the perfusion network.

[0025]

[0025] In some embodiments, the perfusion network and / or auxiliary network comprises a metering receptacle and / or a micromixing receptacle.

[0026]

[0026] In some embodiments, the perfusion device and the base layer are separate components.

[0027]

[0027] In some embodiments, the perfusion device and the base layer are integrated as a single component.

[0028]

[0028] In another embodiment, a kit comprising the device and pressure source described herein is provided.

[0029]

[0029] In another embodiment, a kit is provided comprising a plurality of devices and a pressure source as described herein, wherein the plurality of devices are configured for multiplexed operation.

[0030]

[0030] In another embodiment, a method for operating the kit described herein is provided, which is a. A step of fluidly connecting a pressure source to a port of the device, b. A step of providing cell cultures to the central region, c. The step of perfusing the cell culture, The pressure source is operated at a relative pressure of ±2 psi or less to perfuse the liquid medium along the first and second perfusion pathways during step c.

[0031]

[0031] In some embodiments, the first and second perfusion pathways are controlled independently.

[0032]

[0032] In some embodiments, the first and second perfusion paths reverse direction at least once during step c.

[0033]

[0033] In some embodiments, the first and second perfusion paths are provided in the same direction.

[0034]

[0034] In some embodiments, the pressure source operates at a relative pressure of about -0.05 to about -1 psi, preferably about -0.05 to about -0.5 psi.

[0035]

[0035] Copies of the claims at the time of filing and registration are incorporated herein by reference.

[0036]

[0036] Other aspects and features of the present disclosure will become apparent to those skilled in the art by considering the following descriptions of specific embodiments of the present disclosure.

[0037]

[0037] Embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings. [Brief explanation of the drawing]

[0038] [Figure 1A] This is a conceptual diagram of a three-lane perfusion apparatus design, in which a central lane (c) for cell culture is adjacent to two perfusion lanes (p) supporting the culture medium for perfusing the cell culture, and these lanes connect to the central lane at their respective interfaces (i). [Figure 1B] These are top, side, and bottom views of one embodiment of a cell culture perfusion apparatus 100 having a 3-lane design. [Figure 1C] Figure 1B shows two perspective views of the perfusion apparatus 100. [Figure 2A] Figures 1B-1C show a perfusion apparatus 100, which comprises a base layer 112 and an upper cover 111 fitted to seal the perfusion apparatus, and which, when assembled, provides a cell culture perfusion device, as well as a base layer 112 with a microfluidic network and a bottom cover, and an upper cover 111, in one perspective view. [Figure 2B]This is a different perspective on the same thing. [Figure 3A] These are top, elevation, and bottom views of another embodiment of a cell culture perfusion apparatus having the same configuration as those in Figures 1 and 2, in which the perfusion apparatus and microfluidic network are fabricated on the same layer or chip. [Figure 3B] This is a perspective view of the same thing. [Figure 4A] Figure 3 is a perspective view showing the layers and the bottom and top covers fitted to seal the layers, which, when assembled, provide a cell culture perfusion device. [Figure 4B] This is a different perspective on the same thing. [Figure 5A] These are top, side, and bottom views of another embodiment of a cell culture perfusion apparatus 200, in which the perfusion apparatus and microfluidic network are fabricated on the same layer, with each perfusion lane configured for unidirectional recirculation flow along the interface region 215. [Figure 5B] Figure 5A shows two perspective views of the perfusion apparatus 200. [Figure 6A] This is a schematic diagram of an embodiment of a pneumatically controlled cell culture perfusion device having a three-lane design, in which a gel acting as a seed region for cell culture is suspended in a central lane and perfused with culture medium from two adjacent perfusion lanes controlled by pneumatic pressure via ports at both ends of the device, with both perfusion lanes controlled by the same ports, one at each end. [Figure 6B] This is a schematic diagram of an embodiment of a pneumatically controlled cell culture perfusion device, also shown in Figure 3, having a three-lane design, in which a gel acting as a seed region for cell culture is suspended in a central lane and perfused with culture medium from two adjacent perfusion lanes that are pneumatically controlled via ports at both ends of the device, with each perfusion lane connected to a separate set of ports, allowing for independent pneumatic control of both lanes. [Figure 6C] This figure shows a three-dimensional rendering of the embodiment of Figure 6A, in which the outlet 302 of the first receptacle 301 is fluidly connected to the outlet 304 of the second receptacle 303. [Figure 6D] This figure shows a three-dimensional rendering of the embodiment shown in Figure 6B, with the central lane 305 marked in red. [Figure 7] This figure shows an embodiment of the working configuration illustrated in Figure 6A, in which two different cell culture media are held in each perfusion lane, and the hydrogel is suspended in the central lane. [Figure 7A] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, with the hydrogel suspended in the central lane and the media deposited in the two receptacles on the right, one for each lane. [Figure 7B] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and negative pressure draws the media from both lanes into the left receptacle. [Figure 7C] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and negative pressure draws the media from both lanes into the left receptacle. [Figure 7D] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and negative pressure draws the media from both lanes into the left receptacle. [Figure 7E] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and negative pressure draws the media from both lanes into the left receptacle. [Figure 7F] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and negative pressure draws the media from both lanes into the left receptacle. [Figure 7G]This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and the media are drawn back to the original receptacle from left to right. [Figure 7H] This figure shows an embodiment illustrated in Figure 6A in operation, in which two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and the media are drawn back to the original receptacle from left to right. [Figure 7J] This figure shows an embodiment illustrated in Figure 6A in operation, where two different cell culture media are held in each perfusion lane, a hydrogel is suspended in the central lane, and the media are drawn back into the original receptacle from left to right. This procedure may be repeated until the media have been replaced. [Figure 8A] This figure shows a 3D rendering of another embodiment of a cell culture perfusion apparatus, in which each perfusion lane is connected to a separate set of ports, allowing independent pneumatic control of both lanes, and the outlet 402 of the first receptacle 401 is fluidly connected to a check microvalve 404 of the second receptacle 403, with the check microvalve 404 forcing unidirectional flow of the perfusion lane along the interface with the central region. [Figure 8B] This is a schematic diagram of another embodiment of a cell culture perfusion apparatus, in which each perfusion lane is connected to a separate set of ports, allowing for independent pneumatic control of both lanes. [Figure 9A] This figure shows an embodiment of the working configuration illustrated in Figure 8, in which two different cell culture media are held in each perfusion lane, with the hydrogel suspended in the central lane and the media deposited in the two receptacles on the right, one for each lane. [Figure 9B] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9C]This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9D] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9E] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9F] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9G] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the lower lane into the lower left receptacle. [Figure 9H] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the upper lane into the upper left receptacle. [Figure 9I] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the upper lane into the upper left receptacle. [Figure 9J] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the upper lane into the upper left receptacle. [Figure 9K]This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the upper lane into the upper left receptacle. [Figure 9L] This figure shows an embodiment illustrated in Figure 8 in operation, in which two different cell culture media are held in each perfusion lane, the hydrogel is suspended in the central lane, and negative pressure draws the medium from the upper lane into the upper left receptacle. [Figure 10] This figure conceptually illustrates a one-way recirculation channel in the embodiment of Figure 5, in which the liquid in receptacle 205 is drawn into receptacle 206 via a central lane through interface 215, and the liquid from receptacle 206 is returned to receptacle 205 via receptacle 207. [Figure 11A] This figure conceptually illustrates a one-way recirculation channel in the embodiment of Figure 5, in which the liquid in receptacle 205 (marked in Figure 11A) is drawn into receptacle 206 (marked in Figure 11C) via interface 215 using a central lane (the interface location marked in Figure 11B), and the liquid from receptacle 206 is returned to receptacle 205 via receptacle 207 (marked in Figure 11D). [Figure 11B] This figure conceptually illustrates a one-way recirculation channel in the embodiment of Figure 5, in which the liquid in receptacle 205 (marked in Figure 11A) is drawn into receptacle 206 (marked in Figure 11C) via interface 215 using a central lane (the interface location marked in Figure 11B), and the liquid from receptacle 206 is returned to receptacle 205 via receptacle 207 (marked in Figure 11D). [Figure 11C]This figure conceptually illustrates a one-way recirculation channel in the embodiment of Figure 5, in which the liquid in receptacle 205 (marked in Figure 11A) is drawn into receptacle 206 (marked in Figure 11C) via interface 215 using a central lane (the interface location marked in Figure 11B), and the liquid from receptacle 206 is returned to receptacle 205 via receptacle 207 (marked in Figure 11D). [Figure 11D] This figure conceptually illustrates a one-way recirculation channel in the embodiment of Figure 5, in which the liquid in receptacle 205 (marked in Figure 11A) is drawn into receptacle 206 (marked in Figure 11C) via interface 215 using a central lane (the interface location marked in Figure 11B), and the liquid from receptacle 206 is returned to receptacle 205 via receptacle 207 (marked in Figure 11D). [Modes for carrying out the invention]

[0039]

[0049] One or more exemplary embodiments have been described as examples. It will be understood that the embodiments and examples are provided for illustrative purposes only and are not intended to limit those skilled in the art. All references to embodiments, examples, aspects, formulas, compounds, compositions, solutions, kits, etc., are intended to be illustrative and non-limiting.

[0040]

[0050] In one embodiment, a perfusion apparatus is provided having a patterned upper surface and an opposing lower surface, the upper surface being Multiple receptacles, each receptacle is, The floor and, Exit and A plurality of receptacles comprising: at least one side wall having a check microvalve, the check microvalve having an inlet on the side wall; 0.1-1 mm hydraulic diameter (d) for non-capillary transport of liquids HCA plurality of supply channels, each having a check valve, wherein each supply channel is fluidly connected to the inlet of a check valve microvalve, comprising: The vertical clearance between the exit and the floor is at least 5 mm. Multiple receptacles comprise a pair of first receptacles and a pair of second receptacles, and the outlet of the first receptacle of each pair can be configured to be fluidly connected to the second receptacle of each pair.

[0041]

[0051] As used herein, “patterned surface” refers to a surface having a three-dimensional relief pattern. The patterned upper surface of a perfusion device defines at least a portion of the supply channels and receptacles. The upper and lower surfaces of the lower layers of the perfusion device and any other layers disclosed herein (e.g., base layer and cover layer) may or may not be patterned. In some embodiments, whether or not the upper and lower surfaces of the perfusion device and any other layers disclosed herein (e.g., base layer and cover layer) are patterned, they may further provide a portion of vents, as well as other microfluidic channels, chambers, ports and vents, and optionally other three-phase receptacles and holds. As used herein, a “three-phase” microfluidic space is a space in which a solid substrate supports a liquid separated from a gas (e.g., air) by a free surface. These compartments are formed in or on the perfusion device and can be 3D printed, otherwise fabricated as a single piece, or joined by overmolding. In some embodiments, the perfusion device may further include, for example, other examples of the microfluidic check valves described herein (i.e., check microvalves) for supplying fluid to the same receptacle or other three-phase hold, other valves, vias for microfluidic channels (pneumatic and / or liquid), and a negative pressure source or port for coupling thereto. In some embodiments, the perfusion device is patterned on two opposing surfaces (i.e., a top and a bottom surface) having vias optionally interconnecting the two surfaces, the vias being higher than the channels on the two surfaces. HC It may have a value.

[0042]

[0052] In some embodiments, the ceiling of the receptacle may be provided by the bottom surface of a optionally patterned cover layer. In some embodiments, at least the top surface of the supply channel may be provided by the bottom surface of a optionally patterned cover layer so that the side walls of the receptacle do not completely seal the inlet. In some embodiments, a particular section of one or more networks (e.g., some or all receptacles and other three-phase structures) is formed on the perfusion device, and other sections (e.g., some or all two-phase structures) are formed on the cover layer.

[0043]

[0053] In some embodiments, the outlet of the receptacle is located on the floor of the receptacle. In some embodiments, at least one of the outlets is fluid-connected to a vent on the bottom surface of the perfusion device. In some embodiments, the bottom surface of the perfusion device is patterned to define at least a portion of a perfusion network fluid-connected to at least one of the outlets.

[0044]

[0054] In some embodiments, the perfusion device is patterned, for example, with segments of microfluidic channels, on both the top and bottom surfaces, which are adapted for sealed contact with the cover layer and base layer, respectively.

[0045]

[0055] The check microvalves provided herein allow for quantitative supply and dispensing of cell culture medium in low aliquots of approximately 25 μL per dose. Precise control over the introduction of fresh medium reduces cell shock induced by the sudden exchange of large volumes of fresh medium under conventional methods. Check microvalves can be operated using sub-atmospheric pressure or low negative pressure to perform fluid operations, including valve action. This expands the range of suitable materials for the devices and their seals described herein and simplifies their design and manufacture. In some embodiments, check microvalves are provided at the interface between the two-phase and three-phase portions of a microfluidic network within a mesoscale device (i.e., a tip less than 2 cm but with a height greater than 5 mm, ideal for three-phase microfluidics and too deep for typical efficient two-phase microfluidics). By adjusting the valve around the valve opening, it is possible to reliably backfire the flow of much smaller volumes. The cost of a deeper chamber for the three-phase network segment, in terms of additional tip material and formation time, is very small considering the efficiency and reliability of valve action, as well as the low energy of valve action. Furthermore, the amount of material in contact with the liquid during valve operation is minimal, which reduces material constraints and allows for the use of highly "tolerant" microfluidic designs, preventing many of the problems associated with capillary flow and limited contact angles in (material) processes, and avoiding reliability issues that plague other microvalves. In some embodiments, check microvalves are resistant to bubbles entering the liquid and allow for microfluidic tip designs that prevent ineffective volume, which is used to prevent bubble inclusion.

[0046]

[0056] In some embodiments, the check microvalve is a gravity check microvalve (GCM). The GCM has a deposit surface on the inlet or a side wall near the inlet, the deposit surface being fitted to induce the formation and growth of a liquid bolus and to limit the horizontal spread of the liquid beyond the deposit surface, thereby moving the center of gravity of the bolus away from the first side wall, and thereby reducing the contact area and adhesion force on the bolus.

[0047]

[0057] Compared to most pneumatically driven valves, GCMs require only a slightly larger volume of tip material and can preferably operate under low amplitude pressures such as pressure differences of less than 15 kPa (about 2 Psi) (about 15% of atmospheric pressure on Earth at sea level), preferably under negative pressure (i.e., relative to the atmosphere), and without surface activation or hydrophilic or hydrophobic coatings in the selected range. This technology provides check valve operation for small amounts of liquids such as aqueous liquids, hydrocarbons, and oils with better control than expected from such low-cost tip and low-energy processes.

[0048]

[0058] Therefore, the microfluidic device, when placed on a horizontal surface, is equipped with a gravity-operated check microvalve (GCM). During operation, as the bolus grows while the surface contact area is constrained by the deposit surface, the weight of the bolus eventually exceeds the adhesive force, and the bolus falls. In some embodiments, the volume of liquid deposited before falling is about 10 to about 500 μL.

[0049]

[0059] Therefore, one critical limitation of GCM is the minimum amount of liquid that must be delivered into the receptacle for check valve action to occur without the risk of liquid backflow through the supply channel after delivery. The minimum amount of liquid is related to the critical bolus size, which is suspended above the floor for GCM valve action. In some embodiments, the vertical clearance between the outlet and the floor of the receptacle below the slide is about 5 mm or more. In some embodiments, the vertical clearance is at least about 5.5 mm, at least about 6 mm, at least about 6.5 mm, at least about 7 mm, at least about 7.5 mm, at least about 8 mm, at least about 9 mm, or at least about 10 mm. In some embodiments, the vertical separation ranges from approximately 5 to 20 mm, 5 to 12 mm, 5 to 10 mm, 6 to 10 mm, 7 to 10 mm, 8 to 10 mm, 9 to 10 mm, 5 to 9 mm, 6 to 9 mm, 7 to 9 mm, 8 to 9 mm, 5 to 8 mm, 6 to 8 mm, 7 to 8 mm, 5 to 7 mm, 6 to 7 mm, or 5 to 6 mm. In some embodiments, the plenum of the supply channel is filled with liquid, and a vertical separation of at least 5 mm allows for three-phase microfluidic behavior within the receptacle.

[0050]

[0060] The supply channel is fluidly connected to a check-valve through which the liquid exits the supply channel into the receptacle. The supply channel may locally have an axis that is vertical, horizontal (i.e., perpendicular to the first sidewall), or at any angle between them. In some embodiments, the angle is lower than horizontal, which may reduce the amount of splatter that accumulates on the ceiling of the receptacle at the end of delivery. In some embodiments, any one or more supply channels or any one or more other channels in the microfluidic network may be approximately 0.1 mm to approximately 1 mm, approximately 0.2 mm to approximately 1 mm, approximately 0.3 mm to approximately 1 mm, approximately 0.4 mm to approximately 1 mm, approximately 0.5 mm to approximately 1 mm, approximately 0.6 mm to approximately 1 mm, approximately 0.7 mm to approximately 1 mm, approximately 0.8 mm to approximately 1 mm, approximately 0.9 mm to approximately 1 mm, 0. 1mm to approximately 0.9mm, approximately 0.2mm to approximately 0.9mm, approximately 0.3mm to approximately 0.9mm, approximately 0.4mm to approximately 0.9mm, approximately 0.5mm to approximately 0.9mm, approximately 0.6mm to approximately 0.9mm, approximately 0.7mm to approximately 0.9mm, approximately 0.8mm to approximately 0.9mm, 0.1mm to approximately 0.8mm, approximately 0.2mm to approximately 0.8mm, approximately 0.3mm to approximately 0.8mm, approximately 0.4mm to approximately 0.8mm, approximately 0.5mm m~approx. 0.8mm, approx. 0.6mm~approx. 0.8mm, approx. 0.7mm~approx. 0.8mm, 0.1mm~approx. 0.7mm, approx. 0.2mm~approx. 0.7mm, approx. 0.3mm~approx. 0.7mm, approx. 0.4mm~approx. 0.7mm, approx. 0.5mm~approx. 0.7mm, approx. 0.6mm~approx. 0.7mm, 0.1mm~approx. 0.6mm, approx. 0.2mm~approx. 0.6mm, approx. 0.3mm~approx. 0.6mm, approx. 0.4mm~approx. The hydraulic diameters are approximately 0.6 mm, 0.5 mm to 0.6 mm, 0.1 mm to 0.5 mm, 0.2 mm to 0.5 mm, 0.3 mm to 0.5 mm, 0.4 mm to 0.5 mm, 0.1 mm to 0.4 mm, 0.2 mm to 0.4 mm, 0.3 mm to 0.4 mm, 0.1 mm to 0.2 mm, 0.1 mm to 0.3 mm, or 0.1 mm to 0.2 mm. In some embodiments, any one or more supply channels or any one or more other channels in the microfluidic network have a hydraulic diameter of approximately 0.1 mm, approximately 0.2 mm, approximately 0.3 mm, approximately 0.4 mm, approximately 0.5 mm, approximately 0.6 mm, approximately 0.7 mm, approximately 0.8 mm, approximately 0.9 mm, or approximately 1 mm.The supply channel may comprise any number of segments having different orientations along its length, and in some embodiments, the supply channel includes at least one via segment connecting the patterning on opposing sides of the perfusion device and, in some embodiments, the two patterned sides of the substrate. Preferably, one of the two patterned sides is joined to a bottom cover defining the floor or is within 10% of the tip thickness from the depth of the floor, and the other provides an upper contact surface for the top cover. In some embodiments, the supply channel includes at least one meandering channel segment (horizontal or vertical) to slow the flow of liquid through the supply channel (i.e., increase its flow resistance). In some embodiments, two or more supply channels are fluidly connected to the same inlet of a single receptacle. In some embodiments, for formation, the minimum dimensions of the supply channel are desired d. HC If it is larger than d, the segment junction will be smaller than desired for the supply channel. HC It may have via segments (such as those formed by pins with a larger diameter).

[0051]

[0061] The liquid may be supplied to the supply channel by any suitable means. In some embodiments, the liquid is supplied through the inlet by the action of positive relative pressure (relative to the surroundings or to the supply pressure at the opposing end). In some embodiments, the liquid is supplied through the inlet by the action of negative pressure. In some embodiments, the perfusion device further comprises a supply chamber fluidly connected to the supply channel for storing aqueous liquid upstream of the supply channel. In some embodiments, the supply chamber is provided with a vent. In some embodiments, the supply channel extends between the floor of the vented supply chamber or an outlet near it and the top of the receptacle or an inlet near it. The floors of the supply chamber and the receptacle are preferably less than 10% different in thickness from the perfusion device.

[0052]

[0062] The receptacle has a floor and at least a first side wall. In some embodiments, the receptacle defined by the substrate has an open top. In some embodiments, the receptacle has a ceiling, or the perfusion device is covered to provide a ceiling. In some embodiments, the ceiling has an underside to a cover that covers the perfusion device. In some embodiments, the perfusion device comprises a plurality of microfluidic check valves and respective supply channels for supplying a plurality of fluids to a receptacle common to the plurality of microfluidic check valves. In some embodiments, two or more of the plurality of microfluidic check valves open into a receptacle with the same side wall. In some embodiments, two or more of the plurality of microfluidic check valves open into receptacles with different side walls. In some embodiments, the perfusion device comprises a plurality of receptacles, each having a GCM. In some embodiments, the receptacles are interconnected by microfluidic channels.

[0053]

[0063] In some embodiments, one or more of the receptacles are fluidly connected to a port configured to connect to a pressure source. In some embodiments, a perfusion device, or a device comprising a perfusion device, comprises a plurality of ports configured to connect to a pressure source, each of which is fluidly connected to a plurality of receptacles, and at least one port is located at each opposing end of the perfusion device or device. In some embodiments, the plurality of ports are capable of cooperating with a pressure source to apply a negative relative pressure of ±2 psi or less (more preferably -0.03 to -0.7 psi) to control the fluid displacement through the perfusion device or device, for example, to perfuse a liquid culture medium along first and second perfusion paths. In some embodiments, either or both perfusion paths fluidly interconnect a plurality of receptacles, e.g., 2, 3, 4, 5, 6, 7, or 8 receptacles, each receptacle being fluidly connected to a corresponding port. In some embodiments, one or both perfusion paths fluidly interconnect multiple receptacles in a loop that can be unidirectional (i.e., allowing only one-way flow) or bidirectional (allowing two-way flow), for example, 2, 3, 4, 5, 6, 7, or 8 receptacles, depending on the configuration of check valves within the perfusion path. In some embodiments, the perfusion device comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 receptacles. In some embodiments, the perfusion device comprises 4-10, 4-8, or 4-6 receptacles. In some embodiments, the perfusion device comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ports. In some embodiments, the perfusion device comprises 2-10, 2-8, 2-6, or 2-4 ports.

[0054]

[0064] In some embodiments, one or more of the receptacles have a depth-to-width aspect ratio of less than 2:1, preferably less than 1:1, and more preferably less than 0.5:1. In some embodiments, one or more of the receptacles have an average depth of at least about 5 mm, at least about 5.5 mm, at least about 6 mm, at least about 6.5 mm, at least about 7 mm, at least about 7.5 mm, at least about 8 mm, at least about 9 mm, or at least about 10 mm. In some embodiments, one or more of the receptacles have an average depth of about 20 mm or less, about 19 mm or less, about 18 mm or less, about 17 mm or less, about 16 mm or less, or about 15 mm or less. In some embodiments, the receptacle may define a volume in the shape of a cuboid, prism, cylinder, disc, cone, or ellipsoid, or a frustum of any of the aforementioned shapes. If the check valve is a GCM, it is preferable that the dimensions of the receptacle accommodate the critical volume of the bolus so that, after the bolus falls, the inlet is at least twice the radius of the bolus having a threshold weight and volume, away from the free surface of the receptacle, and backflow through the supply channel from the receptacle is prevented.

[0055]

[0065] The gas chamber chamber (GCM) works in conjunction with a housing upstream and / or downstream of the supply channel, which is adapted to allow the pressure difference on either side of the housing to deliver the liquid through the supply channel to the receptacle inlet. In some embodiments, for example, when positive pressure is used to push the liquid into the microvalve, the housing may be located only upstream of the GCM. In some embodiments, negative pressure is used to pull the liquid, which may improve control. In some embodiments, the housing includes at least a portion of the supply channel and extends over the receptacle in at least one operating state of the device, with a vent located upstream of the supply channel.

[0056]

[0066] The GCM described herein locally resembles a waterfall and may be formed as a component of a perfusion apparatus, which may be manufactured as a single layer. Its structure may appear waterfall-like on its surface, given special attention to its front, but it never resembles a waterfall in operation.

[0057]

[0067] To perform valve operation, the housing requires seals that overcome inertia and fluid resistance as it moves from the supply chamber to the receptacle, allowing the receptacle to have a lower air pressure than the supply chamber for a sufficient period to allow the liquid to grow on the deposit surface. Capillary effect is secondary in the operation of the GCM.

[0058]

[0068] The cover layer, more specifically its bottom surface, may provide a ceiling for the receptacle. If so, the inlet of the side wall of the receptacle is preferably sufficiently lower than the ceiling to ensure that the bolus does not come into contact with the ceiling during deposition, or is partially defined by the ceiling so that the ceiling is involved in the formation of the supply channel, at least at the inlet. Various formation pathways make the formation of channels at the interface between the substrate and the cover very efficient. Undefined portions of the ceiling may be involved in the deposition process, and as a result, the bolus spreads over the ceiling during deposition. Generally, constraints imposed by the deposition surface act to limit horizontal spreading at the side walls, and these constraints prevent spreading in the same vector direction at the ceiling, but in contrast, there are generally no constraints on spreading in the orthogonal direction toward the center of the receptacle. Thus, the liquid tends to spread toward the center of the receptacle, which further increases the pull of liquid away from the first side wall. The restriction of horizontal spread restricts flow in two directions, so the bolus is encouraged to grow in the other direction, which in turn pulls the bolus towards the center of the receptacle and vertically up and down (or horizontally away from the wall). Thus, the increased contact surface area provided by the ceiling increases the adhesion of the bolus (which requires a greater mass before falling), and undesirably the edges are included in the contact surface area, but the strong restriction of vertical upward movement and the attractive force that moves directly away from the periphery of the upper edge of the receptacle ultimately increases the distance of the bolus's center of gravity from the first side wall, promoting bolus detachment (i.e., falling). Furthermore, locating the inlet at the interface between the cover layer and the perfusion device may be preferable for low-cost tip formation and assembly.

[0059]

[0069] A GCM is a one-way valve between a two-phase supply channel and a three-phase receptacle, where liquid from the supply channel is conditionally delivered and cannot be returned after delivery. The valve is formed into the perfusion device at the mouth of the supply channel, and at least three of its faces are surrounded by a deposit surface that receives aqueous liquid from the supply channel during deposit. The liquid grows to form a bolus on the deposit surface, but the pressure difference causes the bolus to grow. The bolus grows within the deposit volume, partially bounded by the deposit surface. By limiting the spread of the bolus across the sidewalls, the bolus is forced to bulge outward, which facilitates the bolus's fall, primarily by reducing the threshold bolus size required to overcome the attractive force to the deposit surface by decreasing the contact surface area together and thus reducing the adhesive force, and by increasing the suspension volume and moving the center of gravity of the bolus further away from the sidewalls and outlet.

[0060]

[0070] The deposition surface is a single (i.e., connected) surface of the sidewall that functions to define and restrict the growth direction of the liquid bolus at the mouth of the supply channel with respect to the direction in which growth is promoted or inhibited. Various methods for inducing surface migration of liquid are known in the art, but these typically require micro-surface patterning, selective surface activation, or microfluidic inclusions, all of which are possible, but each has drawbacks with respect to the speed and cost of manufacturing, as well as reliability, particularly reliability after long-term storage under low-cost storage conditions. In some embodiments, a substantially large structural feature in the form of one or more (preferably two) limiting edges defines the deposition surface, and the deposition surface defines its structural features. A limiting edge is a step or recess formed by a second surface that is in contact with the deposition surface at the limiting edge. The limiting edge reduces the tendency for liquid to spread from the deposition surface to the second surface. Therefore, the angle between the depositional surface and the second surface should have an interior angle of less than 160°, preferably less than 145° (i.e., measured within the sidewall rather than within the hollow space of the receptacle). In some embodiments, the interior angle is about 90° to about 145°. In some embodiments, the interior angle is a dominant angle, for example, about 225°. The margin formed in this way does not provide a growth-limiting effect. It will be understood that no formation method produces a mathematically ideal structure, and the margin is often the most imperfectly formed feature. For this purpose, the sharper the limiting margin, the shorter the distance the depositional surface needs to project from the sidewall, and the less material is needed to project the depositional surface into the receptacle. The limiting margin always has some measure of roundness, which can be specified by the nominal radius of curvature. The smaller the radius, the sharper the limiting margin. To limit the spread, the limiting edge preferably has a radius of curvature smaller than 1 / 4 of the projection distance (i.e., the distance the limiting edge protrudes from the sidewall into the receptacle). Thus, the deposition surface induces bolus growth and formation by allowing the liquid from the supply channel to spread horizontally along its entire (but limited) extent on the deposition surface without texturing or selectively coating the sidewall portion.In some embodiments, limiting edges are provided on both sides of the entrance to fix the bolus to the deposit surface and restrict any horizontal spread, thereby enabling the bolus to be trapped.

[0061]

[0071] Any limiting edge tends to increase the bolus bulge and, therefore, should increase the tendency for the bolus to fall in a smaller volume. In some embodiments, the limiting edge is located at a certain distance from the inlet (e.g., at least 3 μm, more preferably at least 10 μm) to allow the initial bolus to grow to a certain size before encountering a physical limit (thus the deposit surface surrounds at least three faces of the inlet). However, the greater the distance of the limiting edge from the outlet, the larger the threshold bolus size (i.e., the bolus size at which weight overcomes the adhesive force). When the limiting edge is 4-5 mm away from the outlet, there is little to no pinching effect for most aqueous liquids. Therefore, in some embodiments, the limiting edge is located less than 2.5 mm, more preferably less than 0.75 mm, from the center of the outlet, allowing bolus growth until it reaches the limiting edge. The limiting edge extends vertically or substantially vertically, limiting the horizontal spread of the bolus.

[0062]

[0072] The deposit surface may be flat, essentially flat, simply curved, or curved in two directions, for example, concave to reduce the interior angle of the limiting edge. Any curvature of the deposit surface is preferably shallow such that the radius of curvature at any point on the surface is greater than the perimeter of the entrance, which is preferred to avoid the liquid adhering to the deposit surface after the bolus falls. Regardless of such slight curvature, the deposit surface has an average orientation defined by a normal. The normal may be horizontal, in which case the deposit surface is perpendicular to the first sidewall or protrudes from the first sidewall by a bulge. Hereinafter, a draft angle of 0.25–5°, commonly used in some molding processes to facilitate demolding, is considered perpendicular. In some embodiments, the normal of the deposit surface has a negative inclination. In this context, a negative angle refers to an orientation in which a half-line coinciding with the normal intersects the horizontal plane of the floor at a fairly acute or oblique angle. In other words, the normal is angled below horizontal. A vertical or negative angle is preferred and can usually be easily formed, but in some embodiments, a positive angle is preferred, which causes the normal to be somewhat above the horizontal. In some embodiments, the positive angle is 45° or less, 40° or less, or 30° or less.

[0063]

[0073] In some embodiments, the two limiting edges are formed by ridges defined by the deposit surface and an adjacent second surface, the ridges causing the entrance to project into the receptacle. The ridges are formed such that their horizontal cross-section is an acute trapezoid (a trapezoid with an acute angle at its long base), oriented so that the long base is in contact with the first sidewall, and the acute trapezoid may be symmetrical (isopodous) to provide equal resistance to the flow on both sides.

[0064]

[0074] In some embodiments, the ridge extends to the floor only up to the deposit surface; in these embodiments, the bottom edge of the deposit surface is adjacent to a slide that extends to the floor of the receptacle. In some embodiments, the receptacle is narrowed below the deposit surface to avoid the risk associated with the wall edges touching the floor of the receptacle and the associated dead volume. The ridge may taper gradually or abruptly below the deposit surface. Alternatively, the deposit surface may be a non-separable portion of the same ridge surface (e.g., the short bottom surface in the case of a trapezoid) that provides a slide from the deposit surface to the floor.

[0065]

[0075] When the perfusion device is placed on a horizontal plane, the slide is oriented within 30° of the vertical. In some embodiments, the slide is curved. The slide extends from the deposit surface to the floor of the receptacle. When the bolus depositing on the deposit surface becomes large enough to overcome the adhesive force holding the bolus, the bolus separates and drops the slide to the floor of the receptacle. In some embodiments, the slide extends to the floor of the receptacle. In some embodiments, the slide extends to approximately the level of liquid filling the receptacle. In some embodiments, the slide is 1–3 mm wide on average. In some embodiments, the deposit surface includes vertically oriented trenches in projections for guiding the bolus to follow the slide, and the trenches may be V-shaped grooves that gradually narrow towards the floor, with the V-shape having an angle of 20°–150°.

[0066]

[0076] In some embodiments, the inlet is equipped with a nozzle for modifying the velocity profile of the liquid passing through it, which may be particularly useful when the last portion of the liquid in the supply channel enters the receptacle through the inlet. The nozzle may constrict or widen the inlet in either or both directions (i.e., width and depth).

[0067]

[0077] In some embodiments, the deposit surface completely encloses the exit, and the inlet is positioned at a certain distance (e.g., about 4 mm) below the top of the receptacle to avoid interaction between the bolus and the ceiling of the receptacle. In some embodiments, the inlet is positioned at the top of the side wall so as to define a portion of the deposit surface when the ceiling of the receptacle is present (e.g., provided by a cover layer).

[0068]

[0078] In some embodiments, the portion of the deposition surface defined by the ceiling is not modified relative to the rest of the receptacle surface. In some embodiments, the portion of the deposition surface defined by the ceiling is patterned or otherwise modified. The ceiling allows the bolus to spread during deposition and increases the surface adhesion of the bolus, but this ultimately increases the distance of the bolus's center of gravity from the first sidewall, in conjunction with the clamping effect of the limiting edge. The limiting edge prevents the bolus from spreading horizontally along the first sidewall, and the ceiling prevents upward spreading, so the bolus spreads freely along the ceiling toward the center of the receptacle and also spreads downward, but the spreading toward the sidewall is much smaller than in the absence of the limiting edge.

[0069]

[0079] Bolus growth tends to pull the liquid away from the first sidewall. It is possible to form a fairly robust bolus at the inherent contact angle of the material typically used to form the receptacle and ceiling. Both pinching by the limiting edge and spreading along the ceiling pull the bolus's center of gravity away from the deposition surface. The bolus will fall when it has deposited enough liquid for its weight to exceed the adhesive force.

[0070]

[0080] In another embodiment, a device for perfusing a cell culture is provided, which device The perfusion apparatus described herein, A base layer having an upper surface adapted to seal at least a portion of the bottom surface of a perfusion device, wherein the base layer and the bottom surface of the perfusion device together define a perfusion network fluidly connected to at least one outlet, A cover layer having a bottom surface adapted to seal at least a portion of the top surface of the perfusion device, A plurality of ports configured to be coupled to a negative pressure source, each of which is fluidly connected to a plurality of receptacles, and at least one port is located at each of the opposing ends of the device, comprising: The perfusion network is configured to provide a first perfusion path and a second perfusion path between at least one port on one end of the device and at least one port on the opposite end of the device. The perfusion network is configured such that a first pair of receptacles is fluid-connected via a first perfusion pathway, and a second pair of receptacles is fluid-connected via a second perfusion pathway. The perfusion network includes a central region, which has at least one central port open to the outside of the device. The perfusion network is configured to provide fluid communication between the central region and the first perfusion path at the first interface, and between the central region and the second perfusion path at the second interface. The first and second interfaces are located on opposite sides of the central region. The first interface is fluidly positioned between the first pair of receptacles, and the second interface is fluidly positioned between the second pair of receptacles.

[0071]

[0081] The microfluidic devices described herein may have one or more advantages, including easier design, no need for surface activation, suitability for large-scale manufacturing, reliable check-stop valve operation, and high backflow resistance with very little energy or control equipment. During operation, GCMs are effective in removing air bubbles mixed into the supplied liquid and can achieve very small dead volume losses.

[0072]

[0082] Figure 1B shows one embodiment of the perfusion apparatus 100 described herein having a three-lane design, through which each perfusion lane may be controlled independently. A central channel interconnects a pair of central ports 105 that open on the top surface of the perfusion apparatus, through which gels and / or cells may be introduced for culture. One perfusion lane is defined between a pair of ports 101 configured to be coupled to a pressure source. Each port 101 is fluidly connected to its respective receptacle 104 in a check microvalve 102 via a supply channel (not shown). The channel (not shown) connects each outlet 103 of each receptacle 104 to an interface (not shown) with the central channel located within the observation window 107. Similarly, another perfusion lane is defined between a pair of ports 106 configured to be coupled to a pressure source. Each port 106 is fluidly connected to its respective receptacle 110 in a check microvalve 108 via a supply channel (not shown). Channels (not shown) connect each outlet 109 of each receptacle 110 to an interface (not shown) with a central channel located within the observation window 107. Grooves for the channels may be patterned on a separate base layer 112 (Figure 2) or on the bottom surface of the perfusion device (Figures 3 and 4). The perfusion device 100 may be sealed and bonded with the base layer 112 and upper cover 111 to seal the microfluidic network and provides a cell culture perfusion device that can be operated pneumatically with a pressure source connected to port 101 to push fluid through the upper perfusion lane and to port 106 to independently push fluid through the lower perfusion lane.

[0073]

[0083] Figure 5 shows another embodiment of the perfusion apparatus 200, which also has a three-lane design, with two perfusion paths configured for unidirectional recirculation flow along an interface with a central channel located within the observation window 215. As shown in Figures 10 and 11 (see brief description of the drawings), each perfusion lane connects receptacles 205, 206, and 207 in a unidirectional loop, with the interface with the central channel located between receptacles 205 and 206. Each independently operable port 201 is fluidly connected to the respective receptacles 205, 206, and 207 at a check microvalve 202 via a supply channel (not shown). In the upper perfusion lane, a first channel (not shown) connects the outlet 208 of receptacle 205 to the central channel located within the observation window 215 via an interface (not shown) and connects to receptacle 206 at a check microvalve 209. A second channel (not shown) connects the outlet 210 of receptacle 206 to receptacle 207 in a check microvalve 211. A third channel connects the outlet 212 of receptacle 207 to receptacle 205 in a check microvalve 213, closing the loop. The lower perfusion lane also houses three similarly configured receptacles in a loop, each receptacle independently fluid-connected to one of the ports 203.

[0074]

[0084] In some embodiments, the device further comprises additional layers for different functions. In some embodiments, the device further comprises one or more insert members between the perfusion device and either or both of the base layer and the cover layer, such as a Luer lock, a reading zone, and an inspection area.

[0075]

[0085] The entire device, or either the perfusion apparatus or other layers (e.g., base layer or cover layer) described herein, may define one or more microfluidic networks, such as a perfusion network or auxiliary network comprising one or more check microvalves (e.g., GCMs), receptacles, ports, vents, channels, and other microfluidic mechanisms. One or more microfluidic networks may comprise channels that interconnect various chambers and / or are open to the outside environment, piping to other sources of supply, or connectors to devices such as loading pins, micropipettes, or syringes. In some embodiments, the channels are located at the d of the supply channel. HC d within 10% of the value HC The system comprises multiple microfluidic channels having values. Each channel may interconnect at least one chamber or receptacle to another chamber or receptacle, or to a port on a tip. In some embodiments, each port provides one or more functions of a liquid supply or extraction port, a pressurized gas supply or extraction port, or a passive vent open to the atmosphere. In some embodiments, the same port can be used for two or more of these three uses at different points in time during operation. In some embodiments, the majority of the microfluidic channel is defined by segmented portions including at least one segment that is a via extending through the perfusion device or other layer to connect two patterned surfaces on either side of the perfusion device or other layer.

[0076]

[0086] In some embodiments, the base layer is patterned to define a perfusion network comprising microfluidic channels (along with the unpatterned bottom surface of the perfusion device), and the perfusion network optionally does not contain vias. In some embodiments, the perfusion device and the cover layer are coupled together or adapted to be coupled together to partially or completely seal one or more microfluidic networks, such as perfusion or auxiliary networks. In some embodiments, the perfusion device, base layer and / or cover layer are separate components that may be reversibly attached to and removed from each other. In some embodiments, the perfusion device is integrated with the base and / or cover layer as a single component, for example, they are manufactured as a single chip. In some embodiments, the perfusion device and the base layer are integrated as a single component.

[0077]

[0087] To increase the number of fluid operations on the liquid within the receptacle, in some embodiments the receptacle is completely sealed, and its housing is defined by a seal between the top surface of the perfusion device and the bottom surface of the device's cover layer. In some embodiments, one or more ports of the device are provided to be coupled to a positive or negative pressure source, for example, to move liquid through a valve, to mix the liquid within the receptacle, or to remove liquid from the receptacle. In some embodiments, the housing covers the receptacle and the pressure port that is in fluid communication with the receptacle, and the pressure port is fluid-coupleable to or coupled to a negative pressure source (relative to atmospheric pressure). In some embodiments, applying negative pressure to the pressure port causes liquid to enter the receptacle through a valve, to enable bubble mixing of the liquid within the receptacle, to exit the receptacle, or any combination thereof.

[0078]

[0088] The perfusion apparatus and other layers described herein may be composed of any suitable material. In some embodiments, the material is or includes a metallic or polymer material, which may be hydrophobic in nature and may be left untreated. In some embodiments, the material is or includes a thermoplastic polymer such as a thermosetting resin or a thermoplastic elastomer. In some embodiments, the material is or includes a thermoplastic polymer. In some embodiments, the material is or includes a thermoplastic polymer. The material may be selected for biocompatibility, moderately low reactivity with the liquid of interest, and preferably not gas permeable. A wide variety of polymers useful for microfluidics are well known. Glass and transparent plastics are particularly used, at least for the cover, to facilitate observation of the liquid during displacement and, in some cases, to facilitate reading and inspection. A wide variety of polymers useful for microfluidics are well known. In some embodiments, the thermoplastic polymer is polycarbonate or a cyclic olefin polymer (e.g., ZEONOR®). In some embodiments, the perfusion apparatus or any other layer of the device, such as the base layer or cover layer, is manufactured by injection molding or 3D printing. In some embodiments, an adhesive is provided for sealing and bonding two layers to either a perfusion device or another layer of a device, such as a base layer or a cover layer.

[0079]

[0089] In some embodiments, the perfusion network is configured such that the first and / or second perfusion paths branch. The branching allows for the incorporation of additional microfluidic compartments and networks for auxiliary functions into the device, as will be discussed later. In some embodiments, the perfusion network is configured such that the first and / or second perfusion paths form a loop, thereby enabling recirculation flow through the device.

[0080]

[0090] In some embodiments, the bottom surface of the cover layer is patterned, and the top surface of the perfusion device and the bottom surface of the cover layer together define an auxiliary network fluidly connected to the perfusion network. The auxiliary network may provide two-phase or three-phase microfluidic channels, chambers, and receptacles for auxiliary functions that may require precisely metered volume control, such as injecting fresh medium, removing used medium, testing and sampling cell media, reporting accumulated target species, and injecting drugs, adjuvants, growth regulators, or other solids, liquids, or gases such as oxygen or carbon dioxide.

[0081]

[0091] In some embodiments, the perfusion network and / or auxiliary network comprises a metering receptacle and / or a micromixing receptacle. One or more receptacles of the device may incorporate metering capabilities. Consistent metering of spent medium allows cells to avoid shocks, such as undesirable spikes in glucose levels, each time fresh medium is introduced. Retaining a metered portion of spent medium each time fresh medium is introduced may be preferable for cell proliferation. Consistent small-quantity metering also allows for the removal of small aliquots of spent medium for off-tip analysis. One or more receptacles of the device may incorporate micromixing capabilities. Bubble mixing allows for the distribution of dissolved oxygen and carbon dioxide at physiologically appropriate levels than those provided by conventional gas diffusion-based methods. Such metering and micromixing capabilities may be particularly useful for controlling liquid flow in the multiplexing operation of these devices.

[0082]

[0092] In another embodiment, a kit comprising the devices and pressure source described herein is provided. The pressure source may be a positive pressure, negative pressure, or both positive and negative pressure source. In some embodiments, the pressure source provides both positive and negative pressure. In some embodiments, the pressure source is a positive pressure source. In some embodiments, the pressure source is a negative pressure source. In some embodiments, the pressure source is a pump, such as a microdiaphragm pneumatic pump. In some embodiments, the pressure source is low power (e.g., 2-20 mW) and portable. In some embodiments, the pressure source is coupled to multiple ports using tubing, such as laboratory-grade silicone tubing. In some embodiments, the tubing is equipped with filters, such as HEPA filters, to prevent contamination of off-device elements of the system.

[0083]

[0093] In some embodiments, the pressure source can operate at relative pressures of approximately ±1.5 psi or less, approximately ±1.0 psi or less, or approximately ±0.5 psi or less. Operating the devices and kits described herein at these lower pressures reduces the required coupling strength and alleviates the need for strong seals to prevent leakage during operation.

[0084]

[0094] In some embodiments, the negative pressure source is capable of operating at relative pressures of approximately -0.05 to approximately -2 psi, -0.05 to approximately -1.5 psi, -0.05 to approximately -1 psi, approximately -0.05 to approximately -0.95 psi, approximately -0.05 to approximately -0.9 psi, approximately -0.05 to approximately -0.85 psi, approximately -0.05 to approximately -0.8 psi, approximately -0.05 to approximately -0.75 psi, approximately -0.05 to approximately -0.65 psi, approximately -0.05 to approximately -0.6 psi, approximately -0.05 to approximately -0.55 psi, or approximately -0.05 to approximately -0.5 psi. In some embodiments, the negative pressure source is capable of operating at relative pressures of approximately -0.05 to approximately -0.5 psi.

[0085]

[0095] In some embodiments, the positive pressure source is capable of operating at relative pressures of approximately 0.05 to 2 psi, 0.05 to 1.5 psi, 0.05 to 1 psi, 0.05 to 0.95 psi, 0.05 to 0.9 psi, 0.05 to 0.85 psi, 0.05 to 0.8 psi, 0.05 to 0.75 psi, 0.05 to 0.65 psi, 0.05 to 0.6 psi, 0.05 to 0.55 psi, or 0.05 to 0.5 psi. In some embodiments, the positive pressure source is capable of operating at relative pressures of approximately 0.05 to 0.5 psi.

[0086]

[0096] In some embodiments, the kit comprises multiple devices described herein, and the multiple devices are configured for multiplexing operation. As will be readily apparent to those skilled in the art, multiple copies of the devices described herein may be fabricated on a microplate along with receptacles arranged on a multiwell plate in a manner similar to conventional wells. For example, 24, 36, 48, or 96 devices may be incorporated into a single plate, depending on the volume of cell medium required for each device. Those skilled in the art will understand that any number of devices on such a plate may be connected to one another for multiplexed unidirectional or bidirectional two-lane perfusion cell culture, with each perfusion lane being controlled individually. The precision of control over the flow of medium provided by the multiplexing operation of the disclosed pneumatic devices is preferable to current multiplexing systems based on gravity-driven hydrostatic flow. The multiplexing operation of the devices disclosed herein may be adapted for automated liquid handling systems for high-throughput drug screening.

[0087]

[0097] In another embodiment, a method for operating the kit described herein is provided, which is: a. A step of fluidly connecting a pressure source to a port of the device, b. A step of providing cell cultures to the central region, c. The step of perfusing the cell culture, The pressure source is operated at a relative pressure of ±2 psi or less to perfuse the liquid medium along the first and second perfusion pathways during step c.

[0088]

[0098] Positive and negative pressure may be used to simulate different physiological processes. For example, negative pressure may be used to simulate hemodynamics and their effects on cells. The pneumatic control of the devices of this disclosure allows physiological fluid flow to be more closely mimicked than gravity-based flow, enabling a more realistic simulation of intercellular, intertissue, or interorgan interactions in cultures.

[0089]

[0099] In some embodiments, the cell culture perfusion device disclosed herein has a three-lane design (Figure 1A) in which a gel serving as a seed region for cell culture is suspended in a central lane or region, and the central lane is perfused with a liquid (e.g., cell medium) from two perfusion lanes at both ends of the device, which are pneumatically controlled via ports. The two perfusion lanes define first and second perfusion pathways for the liquid to flow through a network from one side of the device to the other. The perfusion network is configured to provide fluid communication between the central region and the first perfusion pathway at a first interface, and between the central region and the second perfusion pathway at a second interface. The first and second interfaces are located on opposite sides of the central region. In other words, the first and second perfusion lanes are located on either side of the central lane or region. The first interface is fluidically located between a first pair of receptacles, and the second interface is fluidically located between a second pair of receptacles. This allows the fluid flow at the interface of each perfusion path to be controlled by pneumatic pressure.

[0090]

[0100] In some embodiments, the perfusion paths are controlled from the same ports, one at each end. In some embodiments, the first and second perfusion paths are controlled independently, and each perfusion lane is connected to a separate set of ports, which in some embodiments can simultaneously apply different pressures.

[0091]

[0101] In some embodiments, the first and second perfusion paths reverse direction at least once during perfusion. This allows for the simulation of reverse flow or oscillating flow.

[0092]

[0102] In some embodiments, the first and second perfusion pathways are provided in the same direction. A consistent unidirectional flow is desirable in many perfusion applications. The perfusion network may be configured such that the first and / or second perfusion pathways form a loop, allowing for a continuous flow of medium in the same direction above the cell culture during perfusion. In some embodiments, the first and second perfusion pathways are provided in opposite directions.

[0093]

[0103] This disclosure is further illustrated in the following embodiments. [Examples]

[0094]

[0104] Example 1: Demonstration of perfusion flow in a cell culture microfluidic device with a 3-lane design

[0105] Figure 7 shows the perfusion flow in a cell culture microfluidic device with a three-lane design. The central lane opens to the top surface of the device through two ports, one at each end of the lane, through which hydrogel and cells may be introduced. Figure 7 shows the hydrogel suspended in the central lane. Adjacent to the central lane are two perfusion lanes connected to ports on both sides of the device. Each perfusion lane connects two three-phase receptacles, with the interface to the central lane located at the middle window. The ports are connected to pneumatic lines. A: Cell medium is placed in the two receptacles on the right, one in each perfusion lane. A cover is placed to seal the top surface of the device, including the receptacles and the central lane port. BF: Negative pressure is applied to the left pneumatic line, and the right pneumatic line functions as a vent. This draws the medium in both lanes from the right receptacle to the left receptacle. GJ: The configuration of the pneumatic lines is reversed, with negative pressure applied to the right-hand pneumatic line and the left-hand pneumatic line acting as a vent. Thus, the culture medium is drawn back to the right-hand receptacle. This procedure may be repeated as needed to simulate oscillating flow.

[0095]

[0106] Example 2: Demonstration of independent control of two perfusion lanes in a cell culture microfluidic device with a 3-lane design.

[0107] Figure 9 shows the perfusion flow in a modified cell culture microfluidic device that provides one set of two sets of ports for each perfusion lane, enabling independent pneumatic control for each lane. A check microvalve is also provided at the inlet of the second (left) receptacle of each perfusion lane. In the illustrated configuration, the outlet of the second receptacle is inoperable. A: Cell culture medium is placed in the two receptacles on the right, one for each perfusion lane. A cover is placed over the top of the device, including the receptacles and the central lane port, to seal it. BG: Negative pressure is applied to the lower left pneumatic line, and the lower right pneumatic line functions as a vent. This draws the culture medium in the lower perfusion lane from the lower right receptacle through the interface with the central lane and through the check microvalve to the lower left receptacle. HJ: Negative pressure is applied to the upper left pneumatic line, and the upper right pneumatic line functions as a vent. As a result, the culture medium in the upper perfusion lane passes through the interface with the central lane from the upper right receptacle, and is drawn through a check microvalve to the upper left receptacle.

[0096]

[0108] All publications and patent applications cited herein are incorporated herein by reference in such a manner that it is specifically and individually indicated that each individual publication or patent application is incorporated by reference. In the event of any conflict between the documents cited herein or incorporated herein and the present disclosure, the present disclosure shall prevail. No citation of any publication is intended for disclosure prior to the filing date and should not be construed as acknowledging that the present invention does not have priority over that publication on the grounds that it is a prior invention.

[0097]

[0109] While the aforementioned inventions are described in some detail as examples and illustrations for clarity, it will be readily apparent to those skilled in the art that certain modifications and alterations may be made in light of the teachings of the present invention without departing from the scope of the appended claims. Such modifications and alterations are intended to be included in the disclosed subject matter and / or the following claims. All examples, embodiments, aspects, methods and products disclosed herein are illustrative and are not intended to be limiting.

[0098]

[0110] It should be understood that all numerical values ​​inherently contain some error, which inevitably arises from the standard deviation observed in each test measurement. Furthermore, as used herein, the term “approximately” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “approximately” means within the acceptable standard error of the mean, as considered by those skilled in the art. Unless otherwise indicated, the numerical parameters described in this disclosure and the appended claims are approximations that may vary as desired. At a minimum, each numerical parameter should be interpreted by applying common rounding techniques in light of the number of significant figures reported.

[0099]

[0111] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include references to the plural unless the context explicitly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Where used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, i.e., elements that exist in some cases constitutively and in other cases disjunctively. Any multiple elements enumerated by “and / or” should be interpreted similarly, i.e., “one or more” of the elements thus combined. In addition to the elements specifically identified by the “and / or” clauses, other elements may optionally exist, whether related to those specifically identified elements or not. Therefore, as a non-restrictive example, when a reference to "A and / or B" is used in combination with non-restrictive words such as "equipped with," in one embodiment it may refer to A only (optionally including elements other than B), in another embodiment it may refer to B only (optionally including elements other than A), and in yet another embodiment it may refer to both A and B (optionally including other elements), and so on.

[0100]

[0112] Where used herein and in the claims, “or” should be understood to encompass the same meaning as “and / or” as defined above. For example, when separating items within a list, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of the number or list of elements, but also including two or more, and optionally, additional unlisted items. Where used herein, in both the main text of the specification and the accompanying claims, transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” and “involving” should be understood as inclusive or open-ended (i.e., including but not limiting) and do not exclude unlisted elements, materials, or method steps. Only the transitional phrases “consisting of” and “consisting essentially of” are restrictive or semi-restrictive transitional phrases relating to the claims and exemplary embodiment paragraphs herein, respectively. The transitional phrase "consisting of" excludes elements, steps, or components that are not specifically listed. The transitional phrase "consisting essentially of" limits the scope to specific elements, materials, or steps that do not substantially affect the fundamental characteristics of the subject matter disclosed and / or claimed herein.

Claims

1. A perfusion apparatus comprising a relief-patterned substrate having at least two contact surfaces for sealing with a cover, wherein the relief patterning is At least two pairs of culture medium exchange receptacles, each receptacle comprising: a bed; a culture medium outlet located near the bed; a closed array of at least one sidewall separating the bed from a first of the at least two contact surfaces; and a gravity check microvalve (GCM) for supplying culture medium into the receptacle at the opening of the GCM. A hydraulic diameter of 0.1 to 1 mm (d) for non-capillary transport of the culture medium. ＨＣ A plurality of perfusion channels, each having ), wherein the perfusion channels are fluidly connected to each lane of a three-raceway cell culture layer, either as part of the perfusion device or designed for sealing with the perfusion device, to circulate the culture medium between the paired receptacles, and the perfusion channels are defined as a plurality of perfusion channels, each having ), A perfusion device in which the distance between the opening and the floor is at least 5 mm.

2. The perfusion apparatus according to claim 1, wherein the GCM has a deposit surface on one of the at least one sidewalls or on an internal column of the receptacle, the deposit surface surrounding at least three sides of the opening and being adapted to induce the formation and growth of the bolus of the culture medium, to limit the horizontal spread of the culture medium beyond the deposit surface, and to draw the center of gravity of the bolus away from the first sidewall.

3. The perfusion apparatus according to claim 1, further comprising a gas inlet connecting one of the paired receptacles to a valved vent, the gas inlet for introducing bubbles into the culture medium when the valve is open to the vent.

4. The perfusion apparatus according to claim 1, wherein the substrate is made of a thermoplastic polymer and has a relief pattern that is consistent with injection molding.

5. The perfusion apparatus according to claim 1, wherein the substrate further comprises at least a portion of a pneumatic supply line connecting a suction port to the GCM in order to drive the culture medium through the circulation.

6. The perfusion apparatus according to claim 5, wherein the substrate is attached to the three-raceway cell culture layer, and generates a perfusion network for circulating the culture medium between the paired receptacles, thereby causing the culture medium to move back and forth through the lanes within one period of circulation, or to circulate the culture medium through the lanes only when flowing from the first receptacle of the paired receptacles to the second receptacle of the paired receptacles, and following a different path from the second receptacle to the first receptacle.

7. The perfusion apparatus according to any one of claims 1 to 6, wherein the bottom surface of the substrate is relief-patterned to define at least a portion of a perfusion network fluidly connected to at least one of the outlets.

8. A kit comprising a perfusion apparatus according to any one of claims 1 to 6, together with a three-raceway cell culture substrate having a set of relief-patterned surfaces and ports for coupling with the perfusion apparatus to complete perfusion channels between paired receptacles.

9. The kit according to claim 8, further comprising one or more pneumatic supply units for coupling to a port on the substrate, or a controller for controlling one or more pneumatic supply units.

10. A device for perfusing cell cultures, A perfusion apparatus according to any one of claims 1 to 6, A base layer having an upper surface adapted to seal at least a portion of the bottom surface of the perfusion apparatus, wherein the base layer and the bottom surface of the perfusion apparatus together define a perfusion network fluidly connected to at least one of the outlets, A cover layer having a bottom surface adapted to seal at least a portion of the upper surface of the perfusion device, A plurality of ports configured to be coupled to a pressure source, each of the plurality of ports being fluidly connected to the plurality of receptacles, and at least one port being located at each of the opposing ends of the device, The perfusion network described above, The device is configured to provide a first perfusion path and a second perfusion path between the at least one port at one end of the device and the at least one port at the other end of the device. The first pair of receptacles are fluidly interconnected via the first perfusion pathway, The second pair of receptacles are fluidly interconnected via the second perfusion pathway, The device comprises a central region having at least one central port, A device configured to enable fluid communication between the central region and the first perfusion path in the first interface, and between the central region and the second perfusion path in the second interface.

11. The device according to claim 10, wherein the perfusion network is configured such that the first perfusion pathway and / or the second perfusion pathway branch or form a loop.

12. The device according to claim 10, wherein the bottom surface of the cover layer is patterned, and the top surface of the perfusion device and the bottom surface of the cover layer together define an auxiliary network fluidly connected to the perfusion network, or the device comprises at least one of a metering receptacle and a micromixing receptacle.

13. A kit comprising the device according to claim 10 and a pressure source adapted to apply a maximum relative pressure of about -0.05 to about -1 psi, preferably about -0.05 to about -0.5 psi.

14. A method for perfusing cell cultures, a. The step of fluidly connecting two pairs of three-phase receptacles to a two-phase non-capillary channel and a pneumatic supply line for a microfluidic tip, b. Providing at least two perfusion lanes to the cell culture and connecting each perfusion lane to the respective pair of receptacles, c. The step of perfusing the cell culture, A method wherein the pressure source is operated at a relative pressure of ±2 psi or less to perfuse the liquid medium along the first and second perfusion pathways during step c.

15. The first and second perfusion pathways are controlled independently, The first and second perfusion paths are reversed in direction at least once during step c, The first and second perfusion pathways are provided in the same direction, or The method according to claim 14, wherein the pressure source is operated at a relative pressure of about -0.05 to about -1 psi, preferably about -0.05 to about -0.5 psi.

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