Scaffold system for microfluidic applications and methods of using thereof

Abstract

Microfluidic scaffolds and methods of using thereof are described herein. The microfluidic scaffolds can provide pressure regulation when incorporated into microfluidic systems or platforms and can define a controlled pressure differential when fluid(s) are flowed or pumped through or around a gel module containing, for example, cells in a hydrogel.

Description

[0001] SCAFFOLD SYSTEM FOR MICROFLUIDIC APPLICATIONS AND METHODS OF USING THEREOF

[0002] CROSS-REFERENCED TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 709,164, filed October 18, 2024, which is hereby incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under EB029132 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] This invention is generally in the field of microfluidic scaffolds which can be used in microfluidic applications, where the microfluidic scaffolds can provide controllable pressure regulation.

[0008] BACKGROUND OF THE INVENTION

[0009] Currently, there is a need for tissue culture microfluidic scaffold designs that can provide a delta in pressure across the hydrogel chamber bed in a pumped configuration. For instance, there are microfluidic devices which have built-in tissue chambers with established flow channels (U.S. Patent No. 9,121,847 B2; and U.S. Publication No. 20230 / 146860 Al) where the entire flow passes through the hydrogel chamber with the delta pressure being created by the resistance in the tissue and the hydrogel itself. However, such devices take choices away from the designer in terms of being able to select or vary the pressure drop that is decoupled from the hydrogel chamber geometry. This also introduces large variability in pressure changes throughout the experiment, as the hydrogel and tissue morphology changes under flow.

[0010] Therefore, there remains a need for microfluidic scaffolds which can address the issues and limitations of current microfluidic scaffolds.

[0011] Therefore, it is the object of the present invention to provide microfluidic scaffolds which address the issues and limitations of current microfluidic scaffolds.

[0012] It is a further object of the present invention to provide methods of using such microfluidic scaffolds in various microfluidic applications.

[0013] 1

[0014] 45784491.1 SUMMARY OF THE INVENTION

[0015] Described herein are microfluidic scaffolds that provide controllable pressure regulation when used in microfluidic systems or platforms, such as for culturing cells. In one nonlimiting instance, a microfluidic scaffold includes: a pressure module: a gel module; optionally a by-pass module comprising one or more by-pass flow paths; and at least one gel loading port; where a controllable pressure differential can be induced within the gel module when a fluid, such as a culture medium, is flowed through or around the gel module.

[0016] In some instances, the microfluidic scaffold includes an imageable region which can form part of the gel module, when the scaffold is placed inside a microfluidic device, and the gel module hosts a cell-laden hydrogel for tissue engineering. The microfluidic scaffolds can provide a controlled pressure differential in use by way of a method for establishing a delta pressure across the hydrogel or tissue within the gel module. The microfluidic scaffold can be integrated into a host microfluidic device, system, or platform as an integral built-in component or as a removable slot-in module.

[0017] The microfluidic scaffold can have two flow configurations according to the direction of flow of a fluid, such as a culture medium, through the gel module or hydrogel chamber being an: 1) Axial flow; or 2) Lateral flow.

[0018] In some instances, the one or more pressure regulators include a linear pressure regulator. In some other instances, the one or more pressure regulators include a non-linear pressure regulator. In still other instances, the one or more pressure regulators include a hydrostatic pressure regulator. In some instances, the one or more pressure regulators include a ball- valve including a ball inside a retaining feature, such as a cage including one or more vent holes, and the ball is on ball seat which is opened or closed when the ball is lifted. In some instances of the ball-valve, the ball has a pressure applied thereon by a spring, such as a linear or non-linear spring which can be precompressed. In some instances, the pressure applied by the spring can prevent the fluid from flowing through the ball seat of the ball-valve when the fluid’s pressure is not sufficient to overcome the pressure applied by the spring, and optionally the ball’s weight, to permit the fluid to flow through the ball seat of the ball-valve. In some other instances, the microfluidic scaffold includes a membrane valve including: a membrane sealing feature including a ridge and an inlet via;

[0019] 2

[0020] 45784491.1 a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; and wherein the membrane includes one or more membrane vent openings.

[0021] In some instances, a flexural member backing applies a pressure on the membrane; wherein the flexural member backing includes one or more vent openings; and wherein the flexural member backing is part of the microfluidic scaffold or is separately bonded thereto.

[0022] In some other instances, the microfluidic scaffold includes a flexural member including one or more flexural member vent openings; wherein the flexural member covers a sealing feature including a ridge and an inlet via; wherein the flexural member contacts the ridge; and wherein the fluid can flow through the one or more vent openings of the flexural member when the fluid’s pressure is sufficient to lift the flexural member off the ridge to allow the fluid to flow between the ridge and the flexural member.

[0023] In yet other instances, the microfluidic scaffold includes a membrane valve including: a membrane sealing feature including a ridge; a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; wherein the membrane optionally includes one or more membrane vent openings; wherein a hydrostatic pressure is applied on the membrane by a hydrostatic pressure tube containing a working liquid and the hydrostatic pressure can be varied by the height of the working liquid present in the hydrostatic pressure tube.

[0024] The microfluidic scaffold includes one or more gaps, such as segmented gaps, that provide the one or more by-pass flow paths. In some instances, the one or more by-pass flow paths can be closed; and optionally the one or more by-pass flow paths can be closed with a by-pass flow plug. In some instances, the one or more by-pass flow paths are defined by the one or more gaps or segments of the scaffold and can be closed by placing a gasket thereon and clamping the gasket thereon, such as with a retaining ring. In some instances, the one or more gaps or segments can be closed by plugs, such as a by-pass flow plug, placed thereon.

[0025] In some instances, the by-pass module further includes a removable ball-valve or a removable hydrostatic pressure tube and can perform the function of the pressure module.

[0026] The microfluidic scaffold includes at least one gel loading port which can have various suitable designs and features as detailed herein. In some instances, the at least one gel loading port comprises a pipette tip locating feature and the gel module includes at least one gel channel having an open top. In some other instances, the at least one gel loading port comprises a pipette tip locating feature and the gel module includes at least one gel channel having closed top. In still other

[0027] 3

[0028] 45784491.1 instances, the at least one gel loading port comprises a pipette tip locating feature and the gel module includes at least one gel channel having a partially open top. In yet other instances, the at least one gel loading port is formed from a removable pipette tip adapter feature placed on the microfluidic scaffold.

[0029] The microfluidic scaffolds described herein can be integrated into a host microfluidic device, system, or platform either as an integral built-in component or as a removable slot-in module. The microfluidic scaffolds described herein can be used for various microfluidic applications, such as for cell culturing or tissue engineering when hydrogel containing cells are placed into the hydrogel chamber of the scaffold and a fluid, such as a culture medium, is flowed through. Such microfluidic scaffolds enable the creation of a well-defined delta pressure drop across the hydrogel during operation, providing biomechanical cues to the encapsulated cells to form tissues with one or more desired properties. The hydrogel channel geometry can be designed for specific tissue types, e.g. such as a vascular bed, organoid growth, etc. The delta pressure can be turned on or off during operation to simulate different physiological conditions during tissue growth.

[0030] In one non-limiting instance, a method of culturing cells includes the steps of:

[0031] (a) loading a hydrogel comprising a plurality of cells into a microfluidic device or platform, where the hydrogel comprising the plurality of cells is loaded into the gel module of a microfluidic scaffold as described herein;

[0032] (b) flowing a fluid, such as a culture medium, through the gel module; wherein a controllable pressure differential can be induced within the gel module when the fluid is flowed through the gel module.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Non-limiting embodiments are described by way of example with reference to the accompanying Figures.

[0035] Figure 1 a shows a non- limiting configuration of a spoke lateral flow microfluidic scaffold 100 including a gel module or channel (102), a pressure module (104), by-pass flow segments (106), a flow inlet (108), a flow outlet (110), and a gel loading port (112).

[0036] Figure lb shows a non-limiting side-view of spoke lateral flow microfluidic scaffold 100 where the arrows illustrate the path of the fluid flow.

[0037] Figures 2a-2c show non-limiting representations of a lateral flow microfluidic scaffold 100’ including a gel module or channel (102’), a pressure module (104’), by-pass flow segments (106’), a flow inlet (108’), a flow outlet (110’), and phase guides (114’).

[0038] 4

[0039] 45784491.1 Figures 3a-3c show non-limiting representations of a lateral flow microfluidic scaffold 100” including a gel module or channel (102”), a pressure module (104”), a by-pass flow outlet (106”), a flow inlet (108”), a flow outlet (110”), and phase guides (114”).

[0040] Figures 4a-4c show non-limiting representations of a lateral flow microfluidic scaffold 100”’ including a gel module or channel (102”’), a pressure module (104”'), a by-pass flow outlet (106”'), a flow inlet (108’”), a gel loading port (112’”), a gel flow outlet (113”’), and phase guides (114”).

[0041] Figure 5a shows non-limiting representations of a lateral flow microfluidic button scaffold 200 including a gel module or channel (202), a flow inlet (204), a flow outlet (206), a gel loading port (208), a gel vent (210), an upstream media channel (212a), a downstream media channel (212b), a flow restrictor (214), an optional notch for holding the scaffold (216), phase guides (218), and laminate layer (220).

[0042] Figure 5b shows the bonding surfaces (222) onto which the laminate layer (220) can be bonded to.

[0043] Figure 6 shows a representation of the flow with a pump with a capacitor in combination with a flow restrictor, where the capacitor and restrictor provides conditions for smooth flow through the scaffold after filtering out oscillatory pump operation.

[0044] Figure 7 shows a pressure drop numerical simulation of a lateral flow button scaffold for a steady flow rate of 1 ul / second.

[0045] Figure 8 shows the proposed configuration for a button scaffold with a resistor disc below it.

[0046] Figure 9a shows a non-limiting representation of a resistor disc 300 including an inlet (302), an outlet (304), a flow restrictor channel (306), an embossed sealing ring (308) around the outlet to guide flow into the inlet of the scaffold, and embossed contact points (310) to prevent wedge error between the resistor disc and scaffold.

[0047] Figure 9b shows non-limiting representations of resistor discs that provide varying degrees of flow resistance based on the length of the flow restrictor channel.

[0048] Figure 10 shows a fluorescence microscope image of a HUVEC and NHLF encapsulated hydrogel loaded in a gel channel of a lateral flow button scaffold, cultured in a perfusion platfomi for 7 days. Arrows show locations where beads infiltrated the endothelial cell networks indicating perfusable microvessels-like structures.

[0049] Figure 11 shows a non-limiting representation of a custom Protocol Development Platform used in Example 2, where the button scaffold evaluated is placed in the scaffold well.

[0050] Figure 12a shows a graph of the flow profile measured as a function of time for the Physiomimix scaffold having a filter paper in place with pulsatile flow exhibited as compared to the

[0051] 5

[0052] 45784491.1 flow profile when a peristaltic pump is connected to the circuit running at the same average flow rate, the flow profile becoming much smoother with no large changes in flow over a cycle.

[0053] Figure 12b shows a graph of the flow profile measured as a function of time for a button scaffold having a filter paper in place compared to the combination of a button scaffold with a resistor disc with low, medium, or high smoothing effects.

[0054] Figure 13 shows fluorescence images of results of Example 2 at day 7 with different amounts of smoothing (no, medium, and high) and the effect on vascular network formation in the gel channels.

[0055] Figure 14 shows a fluorescence image of a vascular network with the hepatocyte spheroids contained therein.

[0056] Figure 15a shows a graph of albumin secretion in collected media from the button scaffold and Physiomimix (liverchip) scaffold normalized against the number of hepatocytes.

[0057] Figure 15b shows a graph of albumin secretion for different experiments done on the button scaffold with hepatocytes.

[0058] Figure 15c shows a graph of CYP3A4 enzyme activity for different experiments done on the button scaffold with hepatocytes.

[0059] Figure 16 of a scaffold having a gel module having a radial spoke geometry used in Example 3.

[0060] Figures 17a- 17b shows fluorescence images of an experiment performed on an exemplary microfluidic scaffold where fibrin hydrogel encapsulating human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast (NHLF) cells were cultured in a perfused platform for a period of 5 days. The fluorescence microscopy images show network-like structures formed by the endothelial cells as they remodel the hydrogel molecules and rearrange themselves in response to biomolecular cues.

[0061] Figure 18a shows a non-limiting schematic of an axial flow microfluidic scaffold.

[0062] Figure 18b shows a non-limiting schematic of a lateral flow microfluidic scaffold.

[0063] Figure 19 shows a non- limiting example of a hydrostatic pressure regulator.

[0064] Figure 20 shows a non- limiting example of a variant hydrostatic pressure regulator.

[0065] Figure 21 shows a non- limiting example of ball-valve where a ball is placed inside a retaining feature such as a cage with vent holes.

[0066] Figure 22 shows a non- limiting example of ball-valve where the ball is pressed onto its seat with a spring which puts a preload on the ball to keep the flow passage closed at low flows.

[0067] 6

[0068] 45784491.1 Figure 23 shows a non- limiting example of a variant ball-valve in a sideways orientation where the weight of the ball is not acting to close the valve, the spring force alone acts on the ball to press it against the seat.

[0069] Figure 24 shows a non-limiting example of a membrane-valve.

[0070] Figure 25 a shows a non-limiting example of a stretched membrane augmented with a flexural member backing.

[0071] Figure 25b shows a non- limiting example of a membrane-valve augmented with a flexural member backing but with a non-stretched membrane.

[0072] Figure 25c shows a non-limiting example of a membrane-valve augmented with a flexural member backing where the preload on the flexural member backing can be adjusted by a linear screw.

[0073] Figure 26 shows a non-limiting example of a valve including only a flexural member backing.

[0074] Figure 27a shows such a non-limiting variation of a membrane valve where a stretched membrane is preloaded by hydrostatic pressure through a working liquid column of a given height.

[0075] Figure 27b shows a non-limiting variation of a membrane valve without any membrane prestretch which is preloaded by hydrostatic pressure through a working liquid column of a given height.

[0076] Figure 28 shows a block of porous media as a flow conduit.

[0077] Figure 29 shows a generic and non-limiting representation of lateral flow versions of pressure regulators.

[0078] Figure 30a shows a non-limiting example of a microfluidic scaffold with a captive cage defining a hole through flow can go through the hole,

[0079] Figure 30b shows a non- limiting example of a microfluidic scaffold with a captive cage defining a hole through flow can go through the hole wherein a heavy metal ball is inserted into the cage when pressure module operation is required.

[0080] Figure 30c shows a non-limiting example of a microfluidic scaffold with a grooved hole for receiving a removable press fit hydrostatic pressure tube.

[0081] Figure 30d shows a non-limiting example of a microfluidic scaffold with a removable press fit hydrostatic pressure tube placed thereon.

[0082] Figures 31a and 31b show such a non-limiting example of a microfluidic scaffold that is made up of two parts, bottom and top plates or layers.

[0083] Figure 31c shows a non-limiting example of a manifold plate (bottom plate / layer) that has an inlet port and by-pass segments and flow guides.

[0084] 7

[0085] 45784491.1 Figure 3 Id shows a non-limiting example of a main scaffold (top plate / layer) which sits on top of a manifold plate (bottom plate / layer).

[0086] Figure 3 le shows a non-limiting representation of the fluid flow pattern (arrows) of a microfluidic scaffold that is made up of two parts, bottom and top plates or layers.

[0087] Figure 32a shows non-limiting representation of a microfluidic scaffold which is a built-in part of a microfluidic device or platform.

[0088] Figure 32b shows non-limiting representation of a drop-in microfluidic scaffold which is placed into a microfluidic device or platform and held by a gasket(s) and a retaining ring.

[0089] Figure 33a shows non-limiting representation of a multi-layer microfluidic scaffold which is a built-in part of a microfluidic device or platform.

[0090] Figure 33b shows non-limiting representation of a multi-layer drop-in microfluidic scaffold which is placed into a microfluidic device or platform and held by a gasket(s) and at least one retaining ring.

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] Microfluidic scaffolds for microfluidic applications that provide controllable pressure regulation and methods of using thereof are described herein.

[0093] I. Definitions

[0094] It is to be understood that the disclosed microfluidic scaffold, and methods of making thereof, are not limited to specific manufacturing methods, specific materials, or to particular dimensions or shapes unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms and embodiments only and is not intended to be limiting.

[0095] The term “microfluidic” typically refers to a system that involves the control and manipulation of small fluid volumes in channels with dimensions on the order of a few micrometers up to a few millimeters and total system volumes on the scale of nanoliters to a few milliliters.

[0096] “Axial,” or “lateral” are terms that refer to flow orientations of one or more fluids which are flowed or pumped through a gel module and are determined based on the direction of a pressure gradient and flow through the gel module. For example, axial is a fluid flow which is parallel to a scaffold axis and lateral is a fluid flow which is orthogonal to the scaffold axis.

[0097] “Pressure differential,” “delta pressure,” or “pressure gradient,” refers to AP which is a measurement of pressure differences between two different points (Pl) and (P2) of the gel module or channel and is calculated as AP = P1-P2. It can also refer to pressure differences between two different points (Pl) and (P2) in media being flowed around the gel module or channel.

[0098] 8

[0099] 45784491.1 “Gel" or “hydrogel” refers to gel-like structure formed of a network of hydrophilic polymers that can absorb and retain high amounts of water. Such (hydro)gels are typically soft, flexible, and highly permeable, making them useful in microfluidic applications. Biocompatible hydrogel refers to a polymer forms a gel which is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the encapsulated cells to maintain viability.

[0100] The term “media” or “medium," refers to a culture fluid that is used for cell culture and contains nutrients, growth factors, or other biomolecules that are included to grow and proliferate cells.

[0101] As used herein, the term “biodegradable”, refers to a material that will degrade or erode by enzymatic action and / or hydrolysis under physiologic conditions to smaller units or chemical species that are capable of being metabolized and / or eliminated.

[0102] The term “gasket” refers to a compressible material that when compressed between two other components makes a reliable and fluid-tight seal.

[0103] The term “bond” or “bonded” refers to the state of two materials that are joined due to covalent molecular bonds, crosslinking of polymers, or some other molecular adhesion force. A bond may be generated with solvents, surface activation using plasma, heat, pressure, and time.

[0104] The numerical ranges disclose individually each possible number that such a range could reasonably encompass, as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example, in a given range of flow rates from 0.1 to 1 ul / second the range also discloses 0.2, 7, 0.45, and 0.99 ul / second, as well as any subrange between these numbers (for example, 0.3 to 0.7 ul / second), and any possible combination of ranges possible between these values.

[0105] Use of the term "about" is intended to describe values either above or below the stated value, which the term “about” modifies, to be within a range of approximately + / - 10%. When the term "about" is used before a range of numbers (i.e., about 1-5) or before a series of numbers (i.e., about 1, 2, 3, 4, etc.) it is intended to modify both ends of the range of numbers and / or each of the numbers recited in the entire series, unless specified otherwise.

[0106] II. Microfluidic Scaffolds

[0107] Described herein are microfluidic scaffolds that provide controllable pressure regulation when used in microfluidic systems or platforms, such as for culturing cells. In tissue engineering, continuous flow and pressure delta across the tissue bed can often be beneficial for growing tissues with desired properties, over static culturing on a flat plate. Various such microfluidic scaffolds are described herein.

[0108] In one non-limiting instance, a microfluidic scaffold includes: a pressure module;

[0109] 9

[0110] 45784491.1 a gel module; optionally a by-pass module or outlet including one or more by-pass flow paths or segments; and at least one gel loading port; where a controllable pressure differential can be induced within the gel module when a fluid, such as a culture medium, is flowed through or around the gel module.

[0111] In some instances, the microfluidic scaffold includes an imageable region which can form part of the gel module, when the scaffold is placed inside a microfluidic device, and the gel module hosts a cell-laden hydrogel for tissue engineering. The microfluidic scaffolds can provide a controlled pressure differential in use by way of a method for establishing a delta pressure across the hydrogel or tissue within the gel module. The microfluidic scaffold can be integrated into a host microfluidic device, system, or platform as an integral built-in component or as a removable slot-in module.

[0112] In some instances, the gel module is a hydrogel chamber. The gel module includes one or more gel channels which can hold a cell laden hydrogel, such as for tissue culturing and / or tissue engineering. Any hydrogel can be used and can exhibit properties including: biocompatibility: supporting cell growth and enabling tissue culture integration; permeability: allowing diffusion of nutrients, waste, and small molecules; tunability: ability to adjust mechanical properties (e.g., stiffness) by altering polymer composition or crosslinking density; and / or mimicking of biological tissues for creating physiological environments. Without restriction, hydrogels used in microfluidics can include fibrin hydrogel, collagen, Matrigel™, agarose, alginate, polyethylene glycol (PEG), and gelatin. In some instances, the hydrogel is a polyethylene glycol (PEG)-based hydrogel including an eight-arm PEG with a molecular weight of, for example, about 20 kDa where vinylsulfone-based click chemistry can be used to polymerize the hydrogel to form a synthetic extracellular matrix (ECM) for cells to grow into. Other synthetic hydrogels, such as those described in PCT / US2020 / 044067 “Synthetic Hydrogels for Organogenesis” by Massachusetts Institute of Technology, may also be used. Such hydrogel matrices may or may not include additional binders.

[0113] In some instances, the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300. 200, 100, 75, 50, 25, 15, 10, 5, or 1 mmPPO between at least two different points of the gel module; in a range from between about 1 to 1500 mmI PO, 1 to 1250 mml PO, 1 to 1000 mmfEO, 1 to 750 mmI PO, 1 to 500 mmkPO, 1 to 250 mini PO. 1 to 100 mini PO, 1 to 50 mmI PO, or 1 to 25 mini PO between at least two different points of the gel module; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmH20 between at

[0114] 10

[0115] 45784491.1 least two different points of the gel module. In some other instances, the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mmlLO across the entirety of the gel module; in a range from between about 10 to 99 mmthO across the entirety of the gel module; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 nimFbO across the entirety of the gel module. Individual values and subranges contained within the aforementioned ranges are possible. a. Flow Configurations

[0116] The microfluidic scaffold can have two flow configurations according to the direction of flow of a fluid, such as a culture medium, through the gel module or hydrogel chamber being an: 1) Axial flow; or 2) Lateral flow.

[0117] In some instances, the microfluidic scaffold has an axis, such as central axis, and flow of the fluid through the gel module is parallel, or axial, to the axis. In some other instances, the microfluidic scaffold has an axis, such as a central axis, and flow of the fluid through the gel module is orthogonal, or lateral, to the axis. Such flow orientations can be detemrined based on the direction of a pressure gradient and flow through the gel module. Lor instance, Figure 18 shows an exemplary axial flow configuration that is considered axial because the flow through a gel channel of the gel module and hence the orientation of the resulting tissue growth is parallel to the axis of the microfluidic scaffold. In another instance, Figure 18b shows an exemplary lateral flow configuration that is considered lateral because the flow through a gel channel of the gel module and hence the orientation of the resulting tissue growth is orthogonal to the axis of the microfluidic scaffold.

[0118] In some instances, the microfluidic scaffold has three features that are built into the scaffold body: a pressure module, a gel module, and a bypass flow module. Total flow Q feeds the microfluidic scaffold from the microfluidic host device and splits into flows that feed each of these modules, pressure module flow = Qp, gel module flow = QG, bypass flow = QB, and additional leakage flow = QL. In such examples, AP is measured between points upstream (Pl) and downstream (P2) of the gel module. Further, as shown, the microfluidic scaffold can be held in place by placing it on a support feature of a host microfluidic device and compressing a gasket on top by a retaining ring. b. Pressure Regulators

[0119] The pressure module can include one or more pressure regulators. Non-limiting characteristics of the one or more pressure regulator(s) in the pressure module include when the bypass flow is closed. In an ideal system, the ideal pressure regulator acts either as a completely

[0120] 1 1

[0121] 45784491.1 linear or non-linear pressure regulator. It starts at a regulated non-zero pressure at a zero total flow rate Q and is either constant, linear with a fixed slope or non-linear with a slope that is a function of Q. The flow through the gel module and leaks can be approximated as linear with increasing pressure drop with a high positive slope. At the operating point, the total flow is given by the sum of these three flows. The resultant system starts from the origin, i.e. there is always a small amount of flow before the operating AP is reached. In other instances, operation of the microfluidic scaffold proceeds with the bypass flow open with an ideal pressure regulator. In this case, the bypass flow is also linear but with a small slope. Hence, the operating AP is very small for a given Q.

[0122] In some instances, the one or more pressure regulators include a linear pressure regulator. In some other instances, the one or more pressure regulators include a non-linear pressure regulator. In still other instances, the one or more pressure regulators include a hydrostatic pressure regulator. In one non-limiting example of a hydrostatic pressure regulator, the fluid media flows upwards, against gravity from the upstream side of the microfluidic scaffold at the bottom, through a tube of length h, exits at the top and either beads or sheets down the side on the downstream side of the microfluidic scaffold, as shown in Figure 19. AP is proportional to the height of the tube between two measured points. It is almost linear and constant with a small slope due to frictional losses through the tube. A variation of such a hydrostatic pressure regulator is possible where the tube is short and fully submerged in the fluid, as shown in Figure 20. Tube diameter is comparable to height. In this case, the pressure drop is mainly due to the frictional losses, as the pressure characteristics show a linear curve with a large slope.

[0123] In some instances, the one or more pressure regulators include a ball-valve including a ball inside a retaining feature, such as a cage including one or more vent holes, and the ball is on ball seat which is opened or closed when the ball is lifted. In some instances, the ball is made of material that is denser than the fluid that flows through the ball- valve. In one non-limiting example, a ballvalve includes a ball that is placed inside a retaining feature, such as a cage with vent holes, as shown in Figure 21. The ball sits on a circle of predefined geometry, such that when the ball lifts the fluid flows out through a passage with a given cross section. There can be a slight leakage through the seat at all points until the pressure is high enough to lift the ball cleanly off the seat. The slope of the line after the valve opens is typically close to zero.

[0124] In some instances of the ball-valve, the ball has a pressure applied thereon by a spring, such as a linear or non-linear spring which can be pre-compressed. In some instances, the pressure applied by the spring can prevent the fluid from flowing through the ball seat of the ball- valve when the fluid’s pressure is not sufficient to overcome the pressure applied by the spring, and optionally the ball’s weight, to permit the fluid to flow through the ball seat of the ball-valve. For example, a

[0125] 12

[0126] 45784491.1 ball- valve where the ball is pressed onto its seat with a spring puts a preload on the ball to keep the flow passage closed at low flows, as shown in Figure 22. The liquid must overcome the spring preload and the weight of the ball before the ball lifts. And afterwards the slope of the characteristic pressure curve is determined by the spring constant, i.e. it can be linear or non-linear. In another variation of this ball- valve motif, there can be a sideways orientation where the weight of the ball is not acting to close the valve, but the spring force alone acts on the ball to press it against the seat, as shown in Figure 23.

[0127] Pressure differences across in vitro microvascular capillary beds have been shown to be below 100 mini I2O (Adv. Funct. Mater. 2022, 32, 2206767). This can be achieved by deliberate selection of a combination of ball diameter, ball seat diameter, and ball material, depending on space and material constraints. Table 1 below shows a calculation for two different materials with 1.5 mm diameters balls and a seat diameter equal to half the ball diameter. Both combinations are within the range of pressures which are desired for vascular tissue engineering in microfluidic scaffolds.

[0128] Table 1.

[0129] Thus, in some instances, without limitation the ball- valve controls the controllable pressure differential based on any one of the following parameters: the ball seat having a circular geometry with a diameter which is half the diameter of the ball’ s diameter; the ball being made of sapphire or stainless steel; the ball having a density of about 3 to 9 or 4 to 8 g / cm3; and / or the ball having a weight in a range of about 0.05 to 0. 15 N.

[0130] The ball seat having a circular geometry can have any suitable diameter up to the diameter of the ball. The ball can be made of any suitable material, such as metals, minerals, plastics, or ceramics. Further, the density and weight of the ball may be chosen based on the application / pressure delta desired and the material chosen for the ball.

[0131] In some instances, the microfluidic scaffold includes a membrane valve including:

[0132] 13

[0133] 45784491.1 a membrane sealing feature including a ridge and an inlet via; a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; and wherein the membrane includes one or more membrane vent openings.

[0134] Fluid can flow through the one or more vent openings of the membrane when the fluid’ s pressure is sufficient to lift the membrane off the ridge to allow the fluid to flow between the ridge and the membrane. In one non-limiting example, a stretched membrane sealing a passage with a well-defined ridge acting as a membrane sealing feature is used, as shown in Figure 24. The membrane is bonded or clamped to the microfluidic scaffold surface. The fluid has to overcome the preload, pressure force, provided by the membrane due to membrane tension before the membrane lifts and allows fluid to flow through vent holes. This preload in the membrane can be changed by changing the membrane tension during bonding or clamping. The characteristic pressure curve shows a AP proportional to the membrane preload and the opening area. The curve is close to linear with a slope greater than zero.

[0135] In some instances, a flexural member backing applies a pressure on the membrane; wherein the flexural member backing includes one or more vent openings; and wherein the flexural member backing is part of the microfluidic scaffold or is separately bonded thereto. In one non-limiting example, a stretched membrane augmented with a flexural member backing can be used, as shown in Figure 25a. In other instances, the same configuration but with a non-stretched membrane and hence no preload where the sealing force is provided by the flexural member backing only, and the membrane acts like a gasket can be used, as shown in Figure 25b. In some other instances, the pressure applied on the membrane by the flexural member backing can be adjusted by a linear screw which is in contact with the flexural member backing. In such an instance, the preload on the flexural member can be adjusted by a linear screw, as shown in Figure 25c. The expected pressure curve of such membrane-valves is similar to the membrane valve alone with a higher operating pressure.

[0136] In some other instances, the microfluidic scaffold includes a flexural member including one or more flexural member vent openings; wherein the flexural member covers a sealing feature including a ridge and an inlet via; wherein the flexural member contacts the ridge; and wherein the fluid can flow through the one or more vent openings of the flexural member when the fluid’ s pressure is sufficient to lift the flexural member off the ridge to allow the fluid to flow between the ridge and the flexural member. A variation of this without the membrane and only with the flexural member is also possible, as shown in Figure 26. The characteristic pressure curve is similar to the membrane valve configurations.

[0137] 14

[0138] 45784491.1 In yet other instances, the microfluidic scaffold includes a membrane valve including: a membrane sealing feature including a ridge: a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; wherein the membrane optionally includes one or more membrane vent openings; wherein a hydrostatic pressure is applied on the membrane by a hydrostatic pressure tube containing a working liquid and the hydrostatic pressure can be varied by the height of the working liquid present in the hydrostatic pressure tube. The working liquid can be any suitable fluid which can provide a pressure when added to the hydrostatic pressure tube but is typically different from the fluid (culture medium) being flowed and is only used to preload the membrane valve. Typically, the fluid can flow between the ridge and the membrane when the fluid’s pressure is sufficient to lift the membrane off the ridge. In one non-limiting variation, the membrane valve where a membrane is preloaded by hydrostatic pressure through a working liquid column of a given height can be used, as shown in Figure 27a. The amount of force on the membrane can be varied by changing the working liquid column height by adding or taking away the working liquid. In one non-limiting variation, this can be achieved without any membrane pre-stretch, as shown in Figure 27b. Pressure curve characteristics are similar to the other preloaded membrane valves. In some instances, the hydrostatic pressure tube is a flow conduit including a porous medium therein. For example, a block of porous media can be used as a flow conduit with a Darcy permeability K of suitable dimensions, where the pressure drop is approximately linear through this block, as shown in Figure 28. In some cases, there is a small hydrostatic pressure head due to the height of the hydrostatic pressure tube.

[0139] Figure 29 shows a generic and non-limiting representation of lateral flow versions of pressure regulators, such as those discussed above, these can have a format which guides the flow sideways. In such cases, inflow and outflow channels are designed such that the flow of fluid is internal to the microfluidic scaffold. Such a design can be used in some of the lateral flow scaffolds, as needed. Any of the pressure regulators described herein can be adapted to a lateral flow version by guiding the flow before and after the gel module itself in lateral directions rather than axial directions. c. By-pass Flow Configurations

[0140] The microfluidic scaffold can include one or more gaps, such as segmented gaps, that provide the one or more by-pass flow paths or segments. In some instances, the microfluidic scaffold optionally includes one or more feet which can lift the microfluidic scaffold when placed on a base or platform. In some instances, the microfluidic scaffold includes one or more feet which

[0141] 15

[0142] 45784491.1 can lift the microfluidic scaffold when placed on a base or platform, where the base of platform can form part of a microfluidic device or platform into which the microfluidic scaffold is placed into.

[0143] Without limitation, a microfluidic scaffold can include bypass flow paths which are segmented around the circumference, like a gear. In an alternative scaffold design, the gaps or segments are not at the circumference, but at an internal location. The microfluidic scaffold can be placed on a circular step of a host microfluidic device or platform. The feet underneath the scaffold can lift up the scaffold from the step and create one or more bypass flow paths. The flow can come from either the top or bottom of the microfluidic scaffold and the feet and segmented gaps create the continuous bypass flow path(s). The bypass flow(s) can be closed by adding a gasket, where the gasket can be clamped down by a retaining ring to improve sealing. When the gaps or segments are not at the circumference, but at an internal location, feet are not required for the bypass flow and plugs instead of gaskets can be used.

[0144] In some instances, the one or more by-pass flow paths or segments can be closed; and optionally the one or more by-pass flow paths or segments can be closed with a by-pass flow plug. In some instances, the one or more by-pass flow paths or segments are defined by the one or more gaps or segments of the scaffold, as discussed above, and can be closed by placing a gasket thereon and clamping the gasket thereon, such as with a retaining ring. In some instances, the one or more gaps or segments can be closed by plugs, such as a by-pass flow plug, placed thereon.

[0145] In some instances, the by-pass module or outlet further includes a removable ball-valve or a removable hydrostatic pressure tube and can perform the function of the pressure module. In other words, in some instances, there is a pressure module and by-pass module or outlet but simultaneous operation of both modules may not be desirable. In such instances, the by-pass module or outlet may exclusively act as the pressure module, such that a pressure module is not needed and may be excluded from the scaffold. For example, a microfluidic scaffold with a captive cage, made of flexible plastic snap-fit structures, defining a hole can be used, as shown in Figure 30a. During bypass flow open operation, the flow can go through this hole, but when pressure module operation is required, a heavy metal ball can be inserted into the cage {see Figure 30b), the ball is retained due to the one-way design of the “teeth” and acts to create a pressure drop, as described previously. In other instances, a similar effect can be achieved with a removable press fit hydrostatic pressure tube see Figures 30c and 30d). d. Gel Modules

[0146] In some instances, the gel module of the scaffold has a radial spoke geometry; at least one gel channel; one or more discrete openings or channels, which can be closed, on top and on bottom of the at least one gel channel; and the gel module includes the at least one gel loading port. In some

[0147] 16

[0148] 45784491.1 instances of such gel modules, these can have: gel channels that are open ended and the fluid or medium, contacts the gel from the top and bottom compared to gel channels being completely closed; the fluid or medium cab be pumped flow-through the scaffold, as compared to static culture in a plate; and there are windows or openings on the bottom to retain the gel and provide biomechanical cues to cells in the gel module, such as vasculature forming cells in culture.

[0149] In some instances, the at least one gel module or channel is enclosed by one or more laminating films, such as a laminated flexible membrane, bonded to the body of the gel module or gel channels thereof. In some instances, the one or more laminating films are patterned with holes of any shape(s) that allow the fluid to flow in and out of the at least one gel channel. In some nonlimiting instances, the gel channel is enclosed on both top and bottom by laminating films that are bonded to the microfluidic scaffold body, where the films can have patterns of holes or other shapes in them to allow fluid, such as culture media, to come into contact with the hydrogel contained inside.

[0150] In some instances, the least one gel channel includes a plurality of straight channels; a plurality of circular channels; at least one serpentine channel; one circular channel. The plurality of straight channels may or may not be (inter)connected to each other.

[0151] In certain instances, the microfluidic scaffold includes a bottom plate or layer that includes the one or more by-pass flow paths or segments of the by-pass module or outlet and one or more inlet ports; and a top plate or layer that includes the gel module, the pressure module, and one or more outlet ports. In one non-limiting example, a microfluidic scaffold is made up of two parts, bottom and top plates or layers, as shown in Figures 31a and 31b. The main scaffold (top plate / layer) (see Figure 3 Id) can sit on top of a manifold plate (bottom plate / layer) (see Figure 31c) that has an inlet port which takes the fluid media flow and guides it to flow parallel to the gel channels before exiting through the pressure module that is on the main scaffold layer. The axial flow pattern is shown in Figure 31e. In some instances, the one or more inlet ports and the one or more outlet ports are staggered.

[0152] In some instances, the microfluidic scaffold includes one or more inlet ports and one or more outlet ports, such as a gel flow inlet, a gel flow outlet, or a by-pass outlet. e. Exemplary Scaffolds

[0153] Figure 1 a shows a non- limiting configuration of spoke lateral flow microfluidic scaffold having a gel module or channel (102), a pressure module (104), by-pass flow segments (106), a flow inlet (108), a flow outlet (110), and a gel loading port (112). Such a microfluidic scaffold can be a hollow disc with multiple openings on the bottom and on the top surface that are staggered from each other. For such as scaffold, fluid media flows from one side of the scaffold to the other,

[0154] 17

[0155] 45784491.1 where it flows laterally through the gel channel, as shown in Figure lb. The gel channels can be any shape, such as those discussed above. Another variation of such a design is the slat lateral flow design, where the gel is installed in multiple straight slat gel channels that are not connected to each other and the inlet and outlet ports are staggered on the top and bottom layers or plates of the microfluidic scaffold. Yet another type of design is shown in Figures 2a- 2c where the scaffold includes a gel module or channel (102’), a pressure module (104’), by-pass flow segments (106’), a flow inlet (108’), a flow outlet (110’), and phase guides (114’). Still another variation of this scaffold is shown in Figures 3a-3c where the by-pass flow is inboard and can be plugged with a plug, when desired. Figure 3a shows a scaffold including a gel module or channel (102”), a pressure module (104”), a by-pass flow outlet (106”), a flow inlet (108”), a flow outlet (110”), and phase guides (114”). Yet another lateral flow design is shown in Figures 4a-4c including a gel module or channel (102’”), a pressure module (104’”), a by-pass flow outlet (106’”), a flow inlet (108’”), a gel loading port (112’”), a gel flow outlet (113’”), and phase guides (114”). When the by-pass flow is open, most of the flow exits through the by-pass outlet. But the by-pass outlet can be plugged and then most of the flow must exit through the pressure module and gel flow outlet. The gel can be installed in the center through a gel loading or install port.

[0156] In some instances, the microfluidic scaffold further includes a laminate layer bonded thereto which can guide flow of the fluid within the scaffold. For instance, in one version of a lateral flow design, microfluidic scaffold can have a design as shown in Figure 5a, which can be denoted as a button scaffold and includes a gel module or channel (202), a flow inlet (204), a flow outlet (206), a gel loading port (208), a gel vent (210), an upstream media channel (212a), a downstream media channel (212b), a flow restrictor (214), an optional notch for holding the scaffold (216), phase guides (218), and laminate layer (220). Such a design can have a flow restricting pressure drop feature (i.e., flow restrictor) connecting two media channels flanking the gel module or channel. A flow restrictor is understood to refer to a connection that imposes a hydraulic resistance to fluid movement, similar to how a resistor limits electrical current, where by increasing the flow resistance it reduces the flow rate for a desired pressure drop. In some instances, the flow restrictor represents a narrowing of the channel between the upstream and downstream portions that produces the change in pressure. Various types of flow restrict ors / features may be used, as are known in the field of microfluidics. In some instances, such a design can consist of multiple concentric arc-like channels for gel, fluid media, and pressure drop. A laminate layer, can be bonded to the bottom of the scaffold guides the flow of media into the scaffold through the opening in the center, as shown in Figure 5b.The fluid media, after entering the scaffold from the bottom face, flows in sequence: upstream media channel -> outer flow restrictor -> downstream media channel -> outlet. The gel is

[0157] 18

[0158] 45784491.1 loaded through the gel loading port from the top surface and is held in place by the posts and phase guides. The flow restrictor’s cross section and length can be designed to achieve the desired pressure delta for a given fluid flow rate.

[0159] In some non-limiting instances, a microfluidic scaffold includes: a laminate comprising a media inlet port; a scaffold body including: a media inlet and media outlet: a gel module or channel: at least one gel loading port; optionally a gel vent opening; at least one media channel for flowing at least one fluid medium from the media inlet to the media outlet; wherein the at least one media channel surrounds the gel module or channel; wherein the at least one media channel comprises at least one flow restrictor to provide a controllable pressure differential within the gel module or channel when the at least one fluid medium is flowed from the media inlet to the media outlet; wherein the media inlet port is located over the media inlet; wherein the scaffold body includes laminate bonding surfaces to which the laminate can be bonded to.

[0160] In some instances, the at least one media channel forms a shape, such as a concentric shape, that surrounds the gel module or channel. When the microfluidic scaffold has a round or circular shape it is considered a button scaffold. The microfluidic scaffold can have any other suitable shape without particular restriction.

[0161] In some instances, the at least one media channel surrounding the gel module or channel has an upstream section or portion closer to the media inlet and a downstream section or portion closer to the media outlet and the at least one flow restrictor is between the upstream section or portion and the downstream section or portion of the media channel. In other words, one can consider that a single media channel exists between the media inlet to the media outlet which has separate sections or portions, such as an upstream section closer to the media inlet and a downstream section closer to the media outlet and the at least one flow restrictor is in between to provide a change or differential in the pressure of the media flowed through the media channel before and following passage through the flow restrictor creating the controllable pressure differential. One can also consider that the gel module or channel experiences the controllable pressure differential as the media flows through or around it.

[0162] 19

[0163] 45784491.1 The controllable pressure differential can be produced when the at least one fluid medium is flowed results from the upstream section or portion having a higher pressure than the downstream section or portion due to pressure of the at least one medium being reduced by the flow restrictor. In some instances, the controllable pressure differential between the upstream section or portion and the downstream section or portion is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mmFbO; in a range from between about 10 to 100 mmHzO; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmFbO. In some instances, the controllable pressure differential between the upstream section or portion and the downstream section or portion is less than about 1500. 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mrnl fO between at least two different locations of the gel module or channel; in a range from between about 10 to 99 mini F0 between at least two different locations of the gel module or channel; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmHjO between at least two different locations of the gel module or channel.

[0164] For instance, such a flow restriction feature can be built into the microfluidic scaffold in the fluid media flow path, before the fluid media encounters the hydrogel. In this instance, the sequential media flow is: Media inlet -> Flow restriction -> upstream media channel -> delta p channel -> downstream media channel -> media outlet. The first flow restriction can be designed to work in tandem with the capacitor in a pumping platform to provide a time constant of flow circuit that results in a smooth flow after filtering out oscillatory pump operation. See Figure 6.

[0165] For example, Figure 7 shows a numerical simulation of a button scaffold for a steady flow rate of lul / second and the pressure differential experienced by the gel. The pressure drop mainly happens along the flow restrictor with negligible drop in the wide media channels.

[0166] In some instances, the scaffold body further includes a flow resistor that eliminates or reduces flow fluctuations, oscillations, or pulsations when flowing the at least one fluid medium from the media inlet to the media outlet through the at least one media channel; and wherein the flow resistor is located in between the media inlet and the at least one media channel. In such instances, the flow resistor can reduce the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the scaffold body does not include the flow restrictor.

[0167] In some instances, the gel module, or gel channels, includes at least one gel channel wherein edges of the at least one gel channel includes features selected from a coiled serpentine fiber, a

[0168] 20

[0169] 45784491.1 mesh or a filter- like structure, vertical posts, and / or tapered posts. Such additional features can be incorporated into the gel channel to improve gel loading ease, encourage encapsulated cells to grow vasculature through different gel orientation, and / or provide biomechanical cues, etc. For example, the edges of the gel can be confined by a coiled serpentine fiber, a preexisting mesh or filter-like structures, phase guides, vertical posts, or tapered posts. The vertical post design can also be implemented in a two-piece assembly where the posts and the media flow inlet are in the bottom piece and the outlets and the gel loading port are in the top piece; and wherein both pieces have a corresponding clocking feature to align them for lateral flow. f. Gel Loading Features

[0170] The microfluidic scaffold includes at least one gel loading port which can have various suitable designs and features as detailed below.

[0171] In some instances, the at least one gel loading port includes a pipette tip locating feature and the gel module includes at least one gel channel having an open top. For example, an axial flow microfluidic scaffold with a central pipette tip locating feature for loading gel(s) can be located where the spokes meet at the center with a step at the tip of each spoke. This allows a micropipette tip to be inserted into the center and it is stopped by the step, still leaving some vertical gap for the gel to flow out of.

[0172] In some other instances, the at least one gel loading port includes a pipette tip locating feature and the gel module includes at least one gel channel having a closed top. In such instances, a microfluidic scaffold can also have a central pipette tip locating feature for gel loading feature in the form of a hole with a tapered wall and where the gel channel is closed on both the top and bottom.

[0173] In still other instances, the at least one gel loading port includes a pipette tip locating feature and the gel module includes at least one gel channel having a partially open top. Such a variation of this gel loading port can have a partially open top design which helps both the pipette tip to be located with the tapered wall in the center as well as help the gel to flow out and spread smoothly due to the surface tension of a gel channel that is confined on the top and bottom sides, in contrast to a channel that is open on the top.

[0174] In yet other instances, the at least one gel loading port is formed from a removable pipette tip adapter feature placed on the microfluidic scaffold. For instance, gel loading in open top channels with a removable pipette tip adapter that fits into a groove created by features on the microfluidic scaffold can be used. The pipette tip adapter can be sized for different pipette tip brands’ pipettes. The pipette tip is placed into the pipette tip adapter after the gel is already aspirated into the tip. Then the tip with the pipette tip adapter is placed into the scaffold groove, the

[0175] 21

[0176] 45784491.1 gel is pipeted out into the channel, and finally the pipette tip is withdrawn and the adapter comes away with it, leaving only the scaffold in place with the loaded gel.

[0177] Gel loading can be achieved by an operator with a micropipette by directly accessing the microfluidic scaffold’s gel loading port features, and a pressure drop can be established by directing the pumped fluid (culture media) to flow from the host device through the microfluidic scaffold’s pressure regulator features. In some instances, the one or more by-pass flow paths or segments direct the bulk of the fluid (culture media) to flow around the pressure regulator resulting in continuous flow but an insignificant pressure drop across the hydrogel, such as when operation in a pressure difference-free mode is desired. g. Microfluidic Devices or Platforms including Microfluidic Scaffold(s) Therein

[0178] As previously noted, the microfluidic scaffolds described herein can be integrated into a host microfluidic device, system, or platform either as an integral built-in component or as a removable slot-in module. Any suitable host microfluidic device, system, or platform can be used, such as known microfluidic devices, systems, or platforms which are described, for example, in International Publication No. WO 2005 / 123950 and International Publication No. WO 2017 / 176357. In some instances, the microfluidic scaffolds described herein can be used in commercially available platforms, such CN Bio’ s PhysioMimix® OOC Microphysiological Systems, including single-organ and multi-organ systems. The microfluidic devices, systems, or platforms can be microfluidic tissue chips. The term “chip” refers to the component where microfluidic fluid manipulation occurs, where a chip may be made of a wide variety of materials and can be of different sizes.

[0179] In some instances, the microfluidic scaffold can be dropped or slotted into the microfluidic device or platform; and a gasket holds the microfluidic scaffold therein wherein optionally the gasket can be held or clamped by a retaining ring. Thus, the scaffold can be a built-in part of the microfluidic platform, as shown in Figure 32a. In this case, there is no need for feet to be part of the design, and no retaining ring is needed. By-pass flow(s) can be plugged with a plug, such as squishy Plug-

[0180] Alternatively, the microfluidic platform can be a slot-in type piece, as shown in Figure 32b. In this case, feet and a retaining ring are typically present. A gasket can be clamped by the retaining ring or it can be left to be just a push-in type.

[0181] In some instances, the microfluidic device or platform includes more than one of the microfluidic scaffold, such as two microfluidic scaffolds therein, or more. For example, multiple scaffolds can be incorporated into a single device or platform via a stacking approach, as shown in

[0182] 22

[0183] 45784491.1 Figures 33a and 33b. In such instances, different fluid media flows can be brought in to feed the different microfluidic scaffolds directly before the media mixes at the outlet. When the scaffolds are dropped in, they can be loaded externally and installed stacked in the platform, with a single retaining ring at the top to keep them retained in place (see Figure 33b).

[0184] Various types of gaskets and retaining rings are suitable for holding the microfluidic scaffold in a microfluidic device or platform. Thus, in some instances, the gasket is a single-piece gasket; the gasket is a multi-piece gasket: and / or the retaining ring is a threaded retaining ring optionally including one or more feet features having axial and radial gaps providing by-pass flow pathways. For example, for a microfluidic scaffold design where the edges of the scaffold are in a single plane, i.e. the by-pass segments and the rest of the top of the scaffold are in a single plane a simple rectangular cross section gasket can be used to block the by-pass flow by clamping down with a retaining ring.

[0185] In some instances, the multi-piece gasket includes a top piece and a bottom piece; and the top piece includes one or more projections which can block by-pass flow paths or segments and the bottom piece optionally includes one or more holes which can hold feet when present on the microfluidic scaffold. For example, for a microfluidic scaffold design where the edges of the scaffold are not in a single plane, i.e. the by-pass segments and the rest of the top of the scaffold are at different heights, a simple rectangular cross section gasket cannot be used to block the by-pass flow by clamping down with a retaining ring. Thus, a custom gasket can be designed with projections that block each segment of the by-pass flow path. This can be used in conjunction with a bottom gasket that the microfluidic scaffold fits into, which has holes for the feet of the scaffold. The assembly can be clamped down with a retaining ring.

[0186] In some instances, a by-pass plug can seal by-pass segments in a scaffold which are internal. In this case, the microfluidic scaffold can be clamped down by a retaining ring on the outer periphery, but the ring does not have to clamp the by-pass plug. The by-pass plug can be oversized and due to the material from which it is made, can be squeezed into place to seal the by-pass segments.

[0187] In some instances, it is desirable to prevent excessive absorption of small molecules from the fluid media into the structure of the microfluidic scaffold. For instance, (soft) elastomeric gaskets can be good absorbers and hence their use can be avoided in case the by-pass flow paths or segments are on the inside of the microfluidic scaffold and converted into a pressure module. A similar approach can be used even in cases when the by-pass flow segments are on the outside, by turning up the clamping force on the retaining ring and strengthening the scaffold edges. By avoiding gaskets made of absorbent materials, such as (soft) elastomeric materials, it is possible to

[0188] 23

[0189] 45784491.1 reduce small molecule absorption by the gasket at or near the gel module and the cell culture region found therein.

[0190] In certain instances, microfluidic device or platform further includes a resistor disc which is in contact with the microfluidic scaffold, such as shown in Figures 9a and 9b. The resistor disc includes an inlet and an outlet with a flow restrictor channel in between the inlet and outlet that provides flow resistance that eliminates or reduces flow fluctuations, oscillations, or pulsations of the at least one fluid medium when flowed from the inlet to the outlet; and the outlet of the resistor disc is fluidically connected to the media inlet of the scaffold body of the microfluidic device to provide the flow of the at least one fluid medium thereto. In some instances, the use of the resistor disc reduces flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%. 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the microfluidic device or platform does not include a restrictor disc present. h. Vascular Networks

[0191] As noted, the microfluidic scaffolds described herein can be used to provide a controllable pressure differential within the gel module when a fluid, such as a culture medium, is flowed through or around the gel module or channel. The controllable pressure differential is understood to refer to a difference in pressure for the media fluid flowing around the gel module or channel such that there is a difference in relative pressure. Such an example is shown for the button scaffold in Figure 7 where a flow restrictor provides an upstream region of the media channel with a pressure that is higher than a downstream section of the media channel which results in the gel module or channel experiencing the difference due to being surrounded by the media channel and fluid flowing therethrough. Such a controllable pressure differential can influence the formation of cellular or vascular networks containing microvessel-like structures in the gel module or channel.

[0192] In addition, control of the pulsatility of the media fluid flowing through the media channel can also influence the formation of such cellular or vascular networks in the gel module or channel. For example, high amounts of smoothing to eliminate or reduce (by greater than 80%, 85%, 90%, 95%, or more reduction in pulsations) media flow pulses (i.e., changes in the media flow rate, as shown in Figure 12a) can provide for cellular or vascular networks that are uniformly oriented and show no specific directionality. By contrast, less amounts of smoothing (less than 60%, 50%, 40%, 30%, 20%, 10%, or less) or no smoothing (i.e., media fluid flow is pulsatile) can provide for more directionality of the cellular or vascular networks formed in the gel module or channel. As demonstrated in Example 2, pulsatile flow influences microvascular network morphology, leading to densely populated gel interfaces and fully perfusable networks. In contrast, smoother flow

[0193] 24

[0194] 45784491.1 conditions, such as those from use of resistor discs, resulted in sparser cell densities and partial or no perfusability, thus identifying pulsatility as a relevant factor for functional microvascular network formation. i. Manufacturing Microfluidic Scaffolds

[0195] The microfluidic scaffolds and other microfluidic components, as described herein, can be manufactured according to suitable methods and using suitable materials known to the person of ordinary skill in the field of microfluidics. For instance, suitable manufacturing techniques can include without limitation, soft lithography, 3D printing, micro-machining, and injection molding. Suitable materials can include without limitation, PDMS (Polydimethylsiloxane), polycarbonate, PMMA (Polymethyl Methacrylate), biodegradable polymers (such as PLA (Polylactic Acid) or PCL (Polycaprolactone) suitable for biocompatible scaffolds).

[0196] III. Methods of Using the Microfluidic Scaffolds

[0197] The microfluidic scaffolds described herein can be used for various microfluidic applications, such as for cell culturing or tissue engineering when hydrogel containing cells are placed into the hydrogel chamber of the scaffold and a fluid, such as a culture medium, is flowed through.

[0198] The microfluidic scaffolds described can provide for a desired pressure difference across the gel module, where hydrogel encapsulated cells can be loaded and cultured inside a host microfluidic platform / device that keeps a continuous flow of fluid media, such as by pumping. Such microfluidic scaffolds enable the creation of a well-defined delta pressure drop across the hydrogel during operation, providing biomechanical cues to the encapsulated cells to form tissues with one or more desired properties. The hydrogel channel geometry can be designed for specific tissue types, e.g. as vascular bed, organoid growth, etc. The delta pressure can be turned on or off during operation to simulate different physiological conditions during tissue growth.

[0199] In one non-limiting instance, a method of culturing cells includes the steps of:

[0200] (a) loading a hydrogel including a plurality of cells into a microfluidic device or platform, where the hydrogel including the plurality of cells is loaded into the gel module or channel of a microfluidic scaffold as described herein;

[0201] (b) flowing a fluid, such as a culture medium, through and / or around the gel module; wherein a controllable pressure differential can be induced within the gel module when the fluid is flowed through or around the gel module.

[0202] In some instances, the cells of the plurality are encapsulated by the hydrogel. In some instances, the controllable pressure differential provides a delta pressure across the hydrogel including the cells which can provide biomechanical or biomolecular cues to the plurality of cells.

[0203] 25

[0204] 45784491.1 In some instances, the hydrogel is a fibrin hydrogel. In some instances, the cells of the plurality are selected from human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF). Such cells can be used to form cellular or (micro)vascular networks in the gel module or channel. In some instances, the hydrogel further includes spheroids, such as hepatocyte spheroids, allowing for formation of 3D organ models therein. In some instances, the method further includes a step of imaging the cells of the plurality, such as by fluorescence microscopy, via an imageable region of the microfluidic scaffold.

[0205] In some instances, the cells of the plurality form a vascular network within the gel module or gel channel. In some instances, the vascular network is perfusable. In some instances, the vascular network is a microvascular network.

[0206] In some instances of the method, the microfluidic scaffold further includes a flow resistor that eliminates or reduces flow fluctuations, oscillations, or pulsations when flowing the at least one fluid medium from the media inlet to the media outlet through the media channel; and wherein the flow resistor is located in between the media inlet and the at least one media channel. In some instances, the flow resistor reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the scaffold does not include the flow restrictor.

[0207] In some instances of the method, a resistor disc is present and in contact with the microfluidic scaffold and the resistor disc includes an inlet and an outlet with a flow restrictor channel in between the inlet and outlet that provides flow resistance that eliminates or reduces flow fluctuations, oscillations, or pulsations of the fluid medium when flowed from the inlet to the outlet; and the outlet of the resistor disc is connected to the media inlet of the scaffold to provide the flow of the at least one fluid medium thereto.

[0208] In some instances, the resistor disc reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the microfluidic device or platform does not include a restrictor disc present.

[0209] In some instances, the fluid is flowed at a flow rate of at least about 1 ul / second.

[0210] In some instances, the fluid is flowed for a period of time of at least about 1 day up to about 6 months, 1 day up to about 5 months, 1 day up to about 4 months, 1 day up to about 3 months, 1 day up to about 2 months, 1 day up to about 1 month, 1 day up to about 25 days, 1 day up to about 20 days, 1 day up to about 15 days, 1 day up to about 10 days, 1 day up to about 7 days, or 1 day up to

[0211] 26

[0212] 45784491.1 about 5 days. Individual time values and sub-ranges contained within the aforementioned ranges are possible.

[0213] Any suitable fluid can be flowed the microfluidic scaffolds and through and / or around the gel module or channel(s) therein. Such a fluid may be any suitable cell culture medium / media known. In some instances, the fluid contains immune cells which allows for immune cell interactions with the cells hosted in the gel module / channel.

[0214] In some instances, the microfluidic device or platform includes a pump, such as a recirculating or peristaltic pump, that provides a constant or smooth flow when the fluid is flowed through or around the gel module. This is desirable because a characteristic of pneumatic membrane micropumps is that the logic used to operate the pump strokes are step functions and as a result the flow profile can be highly “spiky” instead of a smooth flow, which is observed more in a peristaltic or syringe pump (Inman, et al., J. Micromech. Microeng. 17 (2007) 891-899). As a result, the instantaneous flow rate, pressure drop and shear stress on tissue can exceed the average reported values by orders of magnitude. To avoid this, microfluidic platforms have been designed with on chip membrane capacitors (Domansky, et al.. Lab on a Chip, (2010), 10(1), 51-58). These circuits include an additional resistor component to passively filter out flow fluctuations. Such a resistive component can be built into the microfluidic scaffolds described herein, or it can be a separate unit, such as a resistor disc, that is fluidically coupled to the microfluidic scaffold.

[0215] The disclosed microfluidic scaffolds and device and methods of using thereof can be further understood through the following numbered paragraphs:

[0216] Paragraph 1. A microfluidic scaffold comprising: a pressure module; a gel module; optionally a by-pass module or outlet comprising one or more by-pass flow paths or segments; and at least one gel loading port; wherein a controllable pressure differential can be induced within the gel module when a fluid, such as a culture medium, is flowed through or around the gel module.

[0217] Paragraph 2. The microfluidic scaffold of paragraph 1, wherein the microfluidic scaffold comprises an imageable region.

[0218] Paragraph 3. The microfluidic scaffold of any one of paragraphs 1-2, wherein the gel module is a hydrogel chamber.

[0219] Paragraph 4. The microfluidic scaffold of any one of paragraphs 1-3, wherein the gel module comprises one or more gel channels.

[0220] 27

[0221] 45784491.1 Paragraph 5. The microfluidic scaffold of any one of paragraphs 1-4, wherein the microfluidic scaffold has an axis and flow of the fluid through the gel module is parallel or axial to the axis.

[0222] Paragraph 6. The microfluidic scaffold of any one of paragraphs 1-4, wherein the microfluidic scaffold has an axis and flow of the fluid through the gel module is orthogonal, or lateral, to the axis.

[0223] Paragraph 7. The microfluidic scaffold of any one of paragraphs 1-6, wherein the pressure module comprises one or more pressure regulators.

[0224] Paragraph 8. The microfluidic scaffold of paragraph 7, wherein the one or more pressure regulators comprise a linear pressure regulator.

[0225] Paragraph 9. The microfluidic scaffold of paragraph 7, wherein the one or more pressure regulators comprise a non-linear pressure regulator.

[0226] Paragraph 10. The microfluidic scaffold of paragraph 7, wherein the one or more pressure regulators comprise a hydrostatic pressure regulator.

[0227] Paragraph 11. The microfluidic scaffold of paragraph 7, wherein the one or more pressure regulators comprise a ball-valve comprising a ball inside a retaining feature, such as a cage including one or more vent holes, and the ball is on ball seat which can be opened or closed when the ball is lifted.

[0228] Paragraph 12. The microfluidic scaffold of paragraph 11, wherein the ball is made of material that is denser than the fluid that flows through the ball- valve.

[0229] Paragraph 13. The microfluidic scaffold of paragraph 11, wherein the ball has a pressure applied thereon by a spring, such as a linear or non-linear spring which can be pre-compressed.

[0230] Paragraph 14. The microfluidic scaffold of paragraph 13, wherein the pressure applied by the spring can prevent the fluid from flowing through the ball seat of the ball-valve when the fluid’s pressure is not sufficient to overcome the pressure applied by the spring, and optionally the ball’s weight, to permit the fluid to flow through the ball seat of the ball-valve.

[0231] Paragraph 15. The microfluidic scaffold of any one of paragraphs 11-14, wherein the ballvalve controls the controllable pressure differential based on any one of the following parameters: the ball seat having a circular geometry with a diameter which is half the diameter of the ball’s diameter; the ball being made of sapphire or stainless steel; the ball having a density of about 3 to 9 or 4 to 8 g / cm3; and / or the ball having a weight in a range of about 0.05 to 0.15 N.

[0232] 28

[0233] 45784491.1 Paragraph 16. The microfluidic scaffold of any one of paragraphs 1-15, wherein the one or more by-pass flow paths or segments can be closed; and optionally wherein the one or more by-pass flow paths or segments can be closed with a by-pass flow plug.

[0234] Paragraph 17. The microfluidic scaffold of any one of paragraphs 1-16, wherein the microfluidic scaffold comprises a membrane valve comprising: a membrane sealing feature comprising a ridge and an inlet via; a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; and wherein the membrane comprises one or more membrane vent openings.

[0235] Paragraph 18. The microfluidic scaffold of paragraph 17, wherein the fluid can flow through the one or more vent openings of the membrane when the fluid’s pressure is sufficient to lift the membrane off the ridge to allow the fluid to flow between the ridge and the membrane.

[0236] Paragraph 19. The microfluidic scaffold of any one of paragraphs 17-18, wherein a flexural member backing applies a pressure on the membrane; wherein the flexural member backing comprises one or more vent openings; and wherein the flexural member backing is part of the microfluidic scaffold or is separately bonded thereto.

[0237] Paragraph 20. The microfluidic scaffold of paragraph 19, wherein the pressure applied on the membrane by the flexural member backing can be adjusted by a linear screw which is in contact with the flexural member backing.

[0238] Paragraph 21. The microfluidic scaffold of any one of paragraphs 1-16, wherein the microfluidic scaffold comprises a flexural member comprising one or more flexural member vent openings; wherein the flexural member covers a sealing feature comprising a ridge and an inlet via; wherein the flexural member contacts the ridge; and wherein the fluid can flow through the one or more vent openings of the flexural member when the fluid’ s pressure is sufficient to lift the flexural member off the ridge to allow the fluid to flow between the ridge and the flexural member.

[0239] Paragraph 22. The microfluidic scaffold of any one of paragraphs 1-16, wherein the microfluidic scaffold comprises a membrane valve comprising: a membrane sealing feature comprising a ridge; a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; wherein the membrane optionally comprises one or more membrane vent openings; wherein a hydrostatic pressure is applied on the membrane by a hydrostatic pressure tube containing a working liquid and the hydrostatic pressure can be varied by the height of the working liquid present in the hydrostatic pressure tube.

[0240] 29

[0241] 45784491.1 Paragraph 23. The microfluidic scaffold of paragraph 22, wherein the hydrostatic pressure tube is a flow conduit comprising a porous medium therein.

[0242] Paragraph 24. The microfluidic scaffold of any one of paragraphs 22-23, wherein the fluid can flow between the ridge and the membrane when the fluid’s pressure is sufficient to lift the membrane off the ridge.

[0243] Paragraph 25. The microfluidic scaffold of any one of paragraphs 1-24, wherein the microfluidic scaffold comprises one or more gaps, such as segmented gaps, providing the one or more by-pass flow paths or segments; and optionally one or more feet which can lift the microfluidic scaffold when placed on a base.

[0244] Paragraph 26. The microfluidic scaffold of paragraph 25, wherein the one or more gaps can be closed by placing a gasket thereon and clamping the gasket thereon, such as with a retaining ring.

[0245] Paragraph 27. The microfluidic scaffold of paragraph 25, wherein the one or more gaps can be closed by plugs placed thereon.

[0246] Paragraph 28. The microfluidic scaffold of any one of paragraphs 1-27, wherein the by-pass module or outlet further comprises a removable ball-valve or a removable hydrostatic pressure tube and can perform the function of the pressure module.

[0247] Paragraph 29. The microfluidic scaffold of any one of paragraphs 1-28, wherein the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mini bO; in a range from between about 10 to 99 mmlfcO; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmfl20.

[0248] Paragraph 30. The microfluidic scaffold of any one of paragraphs 1-28, wherein the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mini bO between at least two different points of the gel module; in a range from between about 10 to 99 mini bO between at least two different points of the gel module; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85. 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 nimH20 between at least two different points of the gel module.

[0249] Paragraph 31 . The microfluidic scaffold of any one of paragraphs 1-28, wherein the gel module has a radial spoke geometry; at least one gel channel; one or more discrete openings or channels, which can be closed, on top and on bottom of the at least one gel channel; and the gel module comprises the at least one gel loading port.

[0250] 30

[0251] 45784491.1 Paragraph 32. The microfluidic scaffold of paragraph 31, wherein the at least one gel channel is enclosed by one or more laminating films bonded to the body of the gel module.

[0252] Paragraph 33. The microfluidic scaffold of paragraph 31, wherein the one or more laminating films are patterned with holes of any shape(s) that allow the fluid to flow in and out of the at least one gel channel.

[0253] Paragraph 34. The microfluidic scaffold of any one of paragraphs 31-33, wherein the least one gel channel comprises a plurality of straight channels; a plurality of circular channels, at least one serpentine channel; or one circular channel; and optionally the plurality of straight channels are not connected to each other.

[0254] Paragraph 35. The microfluidic scaffold of any one of paragraphs 1-34, wherein the microfluidic scaffold comprises a bottom plate or layer that comprises the one or more by-pass flow paths or segments of the by-pass module or outlet and one or more inlet ports; and a top plate or layer that comprises the gel module, the pressure module, and one or more outlet ports.

[0255] Paragraph 36. The microfluidic scaffold of paragraph 35, wherein the one or more inlet ports and the one or more outlet ports are staggered.

[0256] Paragraph 37. The microfluidic scaffold of any one of paragraphs 1-34, wherein the microfluidic scaffold comprises one or more inlet ports and one or more outlet ports and at least one media channel is in between the inlet and outlet ports for flowing the fluid therethrough.

[0257] Paragraph 38. The microfluidic scaffold of any one of paragraphs 1-37, wherein the microfluidic scaffold further comprises a laminate layer bonded thereto which can guide flow of the fluid within the scaffold.

[0258] Paragraph 39. The microfluidic scaffold of any one of paragraphs 1-38, wherein the gel module comprises at least one gel channel wherein edges of the at least one gel channel comprise features selected from a coiled serpentine fiber, a mesh or a filter- like structure, phase guides, vertical posts, and / or tapered posts.

[0259] Paragraph 40. The microfluidic scaffold of any one of paragraphs 1-39, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having an open top.

[0260] Paragraph 41. The microfluidic scaffold of any one of paragraphs 1-39, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having closed top.

[0261] Paragraph 42. The microfluidic scaffold of any one of paragraphs 1-39, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having a partially open top.

[0262] 31

[0263] 45784491.1 Paragraph 43. The microfluidic scaffold of any one of paragraphs 1-39, wherein the at least one gel loading port is formed from a removable pipette tip adapter feature placed on the microfluidic scaffold.

[0264] Paragraph 44. The microfluidic scaffold of any one of paragraphs 1-43, wherein the microfluidic scaffold comprises one or more feet which can lift the microfluidic scaffold when placed on a base or platform.

[0265] Paragraph 45. A microfluidic device or platform comprising the microfluidic scaffold of any one of paragraphs 1 -44.

[0266] Paragraph 46. The microfluidic device or platform of paragraph 45, wherein the microfluidic device or platform is built into the microfluidic device or platform.

[0267] Paragraph 47. The microfluidic device or platform of any one of paragraphs 45-46, wherein the microfluidic scaffold can be dropped or slotted into the microfluidic device or platform; and a gasket holds the microfluidic scaffold therein wherein optionally the gasket can be held or clamped by a retaining ring and the retaining ring is optionally a threaded retaining ring.

[0268] Paragraph 48. The microfluidic device or platform of any one of paragraphs 45-47, wherein the microfluidic device or platform comprises more than one of the microfluidic scaffold, such as two microfluidic scaffolds therein.

[0269] Paragraph 49. The microfluidic device or platform of paragraph 47, wherein: the gasket is a single-piece gasket; the gasket is a multi-piece gasket; and / or the retaining ring is a threaded retaining ring optionally comprising one or more feet features having axial and radial gaps providing by-pass flow pathways.

[0270] Paragraph 50. The microfluidic device or platform of paragraph 49, wherein the multi-piece gasket comprises a top piece and a bottom piece; and the top piece comprises one or more projections which can block by-pass flow paths or segments and the bottom piece optionally comprises one or more holes which can hold feet when present on the microfluidic scaffold.

[0271] Paragraph 51. The microfluidic device or platform of any one of paragraphs 45-50, wherein the microfluidic device or platform comprises pump, such as a recirculating pump, pneumatic pump, or peristaltic pump, that provides a flow when the fluid is flowed through or around the gel module.

[0272] Paragraph 52. A method of culturing cells comprising the steps of:

[0273] (a) loading a hydrogel comprising a plurality of cells into the microfluidic device or platform of any one of paragraphs 45-51, wherein the hydrogel comprising the plurality of cells is loaded into the gel module of the microfluidic scaffold;

[0274] 32

[0275] 45784491.1 (b) flowing a fluid, such as a culture medium, through and / or around the gel module; wherein a controllable pressure differential is induced within the gel module when the fluid is flowed through or around the gel module.

[0276] Paragraph 53. The method of paragraph 52, wherein the cells of the plurality are encapsulated by the hydrogel.

[0277] Paragraph 54. The method of any one of paragraphs 52-53, wherein the controllable pressure differential provides a delta pressure across the hydrogel comprising the cells which can provide biomechanical or biomolecular cues to the plurality of cells.

[0278] Paragraph 55. The method of any one of paragraphs 52-54, wherein the hydrogel is a fibrin hydrogel.

[0279] Paragraph 56. The method of any one of paragraphs 52-55, wherein the cells of the plurality are selected from human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF).

[0280] Paragraph 57. The method of paragraph 56, wherein the cells of the plurality form a vascular network within the gel module or gel channel.

[0281] Paragraph 58. The method of paragraph 57, wherein the vascular network is perfusable.

[0282] Paragraph 59. The method of any one of paragraphs 52-58, wherein the hydrogel further comprises one or more spheroids, such as hepatocyte spheroids.

[0283] Paragraph 60. The method of any one of paragraphs 52-59, wherein the fluid comprises immune cells.

[0284] Paragraph 61. The method of any one of paragraphs 52-60, wherein the method comprises a step of imaging the cells of the plurality, such as by fluorescence microscopy, via an imageable region of the microfluidic scaffold.

[0285] Paragraph 62. The method of any one of paragraphs 52-61, wherein the fluid is flowed at a flow rate of at least about 1 ul / second.

[0286] Paragraph 63. The method of any one of paragraphs 52-62, wherein the fluid is flowed for a period of time of at least about 1 day up to about 6 months, 1 day up to about 5 months, 1 day up to about 4 months, 1 day up to about 3 months, 1 day up to about 2 months, 1 day up to about 1 month, 1 day up to about 25 days, 1 day up to about 20 days, 1 day up to about 15 days, 1 day up to about 10 days, 1 day up to about 7 days, or 1 day up to about 5 days.

[0287] The disclosed microfluidic scaffolds and device and methods of using thereof can also be understood through the following numbered paragraphs:

[0288] Paragraph 1’. A microfluidic scaffold comprising: a laminate comprising a media inlet port; a scaffold body comprising:

[0289] 33

[0290] 45784491.1 a media inlet and media outlet; a gel module or channel; at least one gel loading port; optionally a gel vent opening; at least one media channel for flowing at least one fluid medium from the media inlet to the media outlet; wherein the at least one media channel surrounds the gel module or channel; wherein the at least one media channel comprises at least one flow restrictor to provide a controllable pressure differential within the gel module or channel when the at least one fluid medium is flowed from the media inlet to the media outlet; wherein the media inlet port is located over the media inlet; wherein the scaffold body comprises laminate bonding surfaces to which the laminate can be bonded to.

[0291] Paragraph 2'. The microfluidic scaffold of paragraph 1’, wherein the at least one media channel forms a concentric shape that surrounds the gel module or channel.

[0292] Paragraph 3’ . The microfluidic scaffold of any one of paragraphs l’-2’, wherein the microfludic scaffold has a round or circular shape.

[0293] Paragraph 4’. The microfluidic scaffold of any one of paragraphs l’-3’, wherein the gel module or channel is a hydrogel chamber or channel.

[0294] Paragraph 5’. The microfluidic scaffold of any one of paragraphs l’-4’, wherein the at least one media channel surrounding the gel module or channel has an upstream section or portion closer to the media inlet and a downstream section or portion closer to the media outlet and the at least one flow restrictor is between the upstream section or portion and the downstream section or portion of the media channel.

[0295] Paragraph 6’. The microfluidic scaffold of paragraph 5’, wherein the controllable pressure differential produced when the at least one fluid medium is flowed results from the upstream section or portion having a higher pressure than the downstream section or portion due to pressure of the at least one medium being reduced by the flow restrictor.

[0296] Paragraph 7’ . The microfluidic scaffold of paragraph 6’, wherein the controllable pressure differential between the upstream section or portion and the downstream section or portion is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mini PO; in a range from between about 10 to 100 mini I20; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55. 60, 65, 70, 75, 80, 85, 90, 100. 200, 300, 400, 500, 600, 700, 800, 900, 1000. 1250, 1500 mmH20.

[0297] 34

[0298] 45784491.1 Paragraph 8'. The microfluidic scaffold of paragraph 6’, wherein the controllable pressure differential between the upstream section or portion and the downstream section or portion is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50. 25, 15, 10, 5, or 1 mml PO between at least two different locations of the gel module or channel; in a range from between about 10 to 99 mnd 00 between at least two different locations of the gel module or channel; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mini PO between at least two different locations of the gel module or channel.

[0299] Paragraph 9’. The microfluidic scaffold of any one of paragraphs T-8’, wherein edges of the gel module or gel channel comprise features selected from phase guides, vertical posts, and / or tapered posts.

[0300] Paragraph 10’. The microfluidic scaffold of any one of paragraphs 1 ’ -9’, wherein the scaffold body comprises a notch which is optionally for handling the microfluidic scaffold, such as with tweezers.

[0301] Paragraph 11’. The microfluidic scaffold of any one of paragraphs l’-10’, wherein the at least one gel loading port comprises a pipette tip locating feature.

[0302] Paragraph 12’. The microfluidic scaffold of any one of paragraphs 1 ’ -9’, wherein the gel vent opening is present.

[0303] Paragraph 13’. The microfluidic scaffold of any one of paragraphs 1 ’ - 12’ , wherein the at least one gel loading port comprises a pipette tip locating feature.

[0304] Paragraph 14’. The microfluidic scaffold of any one of paragraphs 1 ’ -13, wherein the scaffold body further comprises a flow resistor that eliminates or reduces flow fluctuations, oscillations, or pulsations when flowing the at least one fluid medium from the media inlet to the media outlet through the at least one media channel; and wherein the flow resistor is located in between the media inlet and the at least one media channel.

[0305] Paragraph 15’. The microfluidic scaffold of paragraph 14’, wherein the flow resistor reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%. 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%. as compared to when the scaffold body does not include the flow restrictor.

[0306] Paragraph 16’. The microfluidic scaffold of any one of paragraphs 1 ’ -14’ , wherein the microfluidic scaffold comprises an imageable region allowing for visualization of at least a portion of the gel module or channel.

[0307] Paragraph 17’. A microfluidic device or platform comprising the microfluidic scaffold of any one of paragraphs T - 16’ .

[0308] 35

[0309] 45784491.1 Paragraph 18’. The microfluidic device or platform of paragraph 17', wherein the microfluidic scaffold can be dropped or slotted into the microfluidic device or platform; and a gasket holds the microfluidic scaffold therein wherein optionally the gasket can be held or clamped by a retaining ring and the retaining ring is optionally a threaded retaining ring.

[0310] Paragraph 19’. The microfluidic device or platform of any one of paragraphs 17’ -18’, wherein the microfluidic device or platform comprises more than one of the microfluidic scaffold, such as two microfluidic scaffolds therein.

[0311] Paragraph 20’. The microfluidic device or platform of any one of paragraphs 17’ -19’, wherein a resistor disc is present and in contact with the microfluidic scaffold; wherein the resistor disc comprises an inlet and an outlet with a flow restrictor channel in between the inlet and outlet that provides flow resistance that eliminates or reduces flow fluctuations, oscillations, or pulsations of the at least one fluid medium when flowed from the inlet to the outlet; and wherein the outlet of the resistor disc is connected to the media inlet of the scaffold body of the microfluidic device to provide the flow of the at least one fluid medium thereto.

[0312] Paragraph 21 ’. The microfluidic device or platform of paragraph 20’, wherein the resistor disc reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the microfluidic device or platform does not include a restrictor disc present.

[0313] Paragraph 22’. The microfluidic device or platform of any one of paragraphs 17’ -21 ’, wherein the microfluidic device or platform further comprises a pump, such as a recirculating pump, pneumatic pump, or peristaltic pump, that provides flow of the at least one fluid medium through the microfluidic scaffold.

[0314] Paragraph 23’. A method of culturing cells comprising the steps of:

[0315] (a) loading a hydrogel comprising a plurality of cells into the microfluidic device or platform of any one of paragraphs 17’-22’, wherein the hydrogel comprising the plurality of cells is loaded into the gel module or gel channel of the microfluidic scaffold;

[0316] (b) flowing a fluid, such as a culture medium, from the media inlet to the media outlet and through and / or around the gel module or gel channel; wherein a controllable pressure differential is induced within the gel module when the fluid is flowed through or around the gel module or gel channel.

[0317] Paragraph 24’. The method of paragraph 23’, wherein the cells of the plurality are encapsulated by the hydrogel.

[0318] 36

[0319] 45784491.1 Paragraph 25’. The method of any one of paragraphs 23 ’-24', wherein the controllable pressure differential provides a delta pressure across the hydrogel comprising the cells which can provide biomechanical or biomolecular cues to the plurality of cells.

[0320] Paragraph 26’. The method of any one of paragraphs 23’-25’, wherein the cells of the plurality are selected from human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF).

[0321] Paragraph 27’. The method of paragraph 26’, wherein the cells of the plurality form a vascular network within the gel module or gel channel.

[0322] Paragraph 28’. The method of paragraph 27’, wherein the vascular network is perfusable.

[0323] Paragraph 29’. The method of any one of paragraphs 23 ’-26’, wherein the hydrogel further comprises one or more spheroids, such as hepatocyte spheroids.

[0324] Paragraph 30’. The method of any one of paragraphs 23 ’-29’, wherein the scaffold body further comprises a flow resistor that eliminates or reduces flow fluctuations, oscillations, or pulsations when flowing the at least one fluid medium from the media inlet to the media outlet through the at least one media channel; and wherein the flow resistor is located in between the media inlet and the at least one media channel.

[0325] Paragraph 31’. The method of paragraph 30’, wherein the flow resistor reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the scaffold body does not include the flow restrictor.

[0326] Paragraph 32 ’.The method of any one of paragraphs 23 ’-31’, wherein a resistor disc is present and in contact with the microfluidic scaffold; wherein the resistor disc comprises an inlet and an outlet with a flow restrictor channel in between the inlet and outlet that provides flow resistance that eliminates or reduces flow fluctuations, oscillations, or pulsations of the at least one fluid medium when flowed from the inlet to the outlet; and wherein the outlet of the resistor disc is connected to the media inlet of the scaffold body of the microfluidic device to provide the How of the at least one fluid medium thereto.

[0327] Paragraph 33’. The method of paragraph 32’, wherein the resistor disc reduces the flow fluctuations, oscillations, or pulsations of the at least one fluid medium by at least about 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, or 1 to 10%, as compared to when the microfluidic device or platform does not include a restrictor disc present.

[0328] 37

[0329] 45784491.1 Paragraph 34’. The method of any one of paragraphs 23’-33’, wherein the fluid comprises immune cells.

[0330] Paragraph 35’. The method of any one of paragraphs 23 ’-34’, wherein the method further comprises a step of imaging the cells of the plurality, such as by fluorescence microscopy, via an imageable region of the microfluidic scaffold.

[0331] Paragraph 36’. The method of any one of paragraphs 23’-35’, wherein the fluid is flowed at a flow rate of at least about 1 ul / second.

[0332] Paragraph 37’. The method of any one of paragraphs 23’-36’, wherein the fluid is flowed for a period of time of at least about 1 day up to about 6 months, 1 day up to about 5 months, 1 day up to about 4 months, 1 day up to about 3 months, 1 day up to about 2 months, 1 day up to about 1 month, 1 day up to about 25 days, 1 day up to about 20 days, 1 day up to about 15 days, 1 day up to about 10 days, 1 day up to about 7 days, or 1 day up to about 5 days.

[0333] The present invention will be further understood by reference to the following non-limiting examples.

[0334] EXAMPLES

[0335] Example 1:

[0336] Scaffolds, with a design analogous to Figure 5a, were loaded with a PEG-based hydrogel laden with human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF) cells in densities of 18 million / ml gel and 2.57 million / ml gel, respectively. Hydrogel - laden scaffolds were installed in a perfusion platform and cultured by circulating media in a closed loop by pumps at a rate of 1 ul / second for a period of 7 days. The HUVECs used expressed green fluorescent protein to help with imaging the cellular network development. On day 7, pumping was paused, red fluorescent polystyrene beads of 1 micron diameter were added to the circulating media and pumping was resumed for 3 hours before scaffolds were imaged in a fluorescence microscope. Images showed growth of green HUVEC networks into microvessel-like structures in the gel channel, the dots (indicated by arrows) show that beads are able to flow inside the networks, indicating their perfusible nature. See Figure 10.

[0337] Example 2:

[0338] Button scaffolds with a design as shown in Figure 5a were evaluated where the button scaffold has a gel volume (~10ul), a media flow rate (lul / s) and pressure drop (~25mmH2O) in a disc form that is only 12.5mm in diameter. The button scaffold is made up of two layers, a scaffold body and a laminate. The flow of media enters the button scaffold through an inlet port in the laminate and leaves through an outlet port on the top surface of the scaffold itself. The other difference is the shape of the gel channel and the media channel(s), these are concentric circular

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[0340] 45784491.1 segments, curved to maximize the usable space inside a small, rounded geometry. In such a button scaffold, the flow enters through the port / hole on the laminate, navigates through one media channel where flow restrictor changes the pressure between the upstream section of the media channel and the downstream section of media channel, which are separated by the flow restrictorbefore leaving through the outlet port on the scaffold body, as shown in Figure 7. The button scaffolds described herein are suitable for use in the CN BioInnovations Physiomimix platform in place of the LC12 (LiverChip 12) culture plates, and are typically held in place therein by a gasket and retaining ring. The typical LC12 (LiverChip 12) culture plates include a scaffold, filter, and a structural support for the filter, that sits below it and keeps the filter in contact with the scaffold. In contrast, the design of the button scaffolds does not include or require any filter be used which opens up the opportunity to add circulating immune cells to the flow loop of media for more detailed immune-host interaction studies using the button scaffold design, which would otherwise be removed when a filter is present.

[0341] An additional feature was added to the flow circuit for some of the experiments which allowed control over the flow pulsatility in the flow loop. The pneumatic micropumps typically used in the Physiomimix platform operate on a cycle and use solenoid valves operating on step functions to dispense fluids at a fixed rate. This necessarily leads to a very pulsatile flow where the instantaneous flow rate rises up to 25ul / second. A strategy to mitigate this pulsatility is to use fluidic capacitors and resistors to smooth out the flow like an RC low-pass filter in electrical circuits. While the Physiomimix LC12 culture plate has the necessary capacitance, the default configuration with the scaffold and filter paper does not have the resistance necessary to filter out the spikes in the flow rate. By contrast, with the button scaffold configuration, a second resistor disc can be added to the circuit in series with the button scaffold and placed such that the flow of media goes from the pump -> membrane capacitor -> resistor disc -> button scaffold -> oxygenator -> pump, in a loop. The membrane capacitor is a large diameter unsupported membrane that can balloon out to store fluid coming in from the pump. Figure 8 shows a non-limiting illustration of a button scaffold and resistor disc.

[0342] As shown in Figure 12a, the flow profile was measured as a function of time in the LC- 12 plate including the default scaffold and filter paper in place. The results showed that the on-board pumps drive the flow above 1500ul / minute or 25ul / s momentarily during flow. The flowrate drops down to zero between the pulses, giving an average flowrate over the cycle equal to 60ul / minute or lul / s. When the onboard pumps are disconnected and a peristaltic pump is connected to the circuit running at the same average flow rate, the flow profile is much smoother with no large changes in flow over a cycle.

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[0344] 45784491.1 As shown in Figure 12b, the default scaffold and filter paper can be switched out for the combination of button scaffold with a resistor disc or button scaffold-only. Three different resistor discs were used, with a low, a medium, or a high amount of resistance which provided different maximum flow rates during flow spikes. Three different resistor discs are shown in Figure 9b, where the bottom surface is laminated with an offset hole in the lamination that forms the inlet to the resistor disc and the outlet of the disc is on the top surface, with a raised bump surrounding the outlet that seals around the inlet of the button scaffold forming a continuous flow path, avoiding leaking flow to the interstitial space between the two discs.

[0345] The button scaffold can also be placed in other platforms than the Physiomimix platform. A limitation of the Physiomimix platform is the inability to image during an experiment, because the LC12 plate is placed on top of a separate pneumatic dock that drives the pumps. To mitigate this issue, a custom platform such as the Protocol Development Platform (PDP), shown in Figure 11, can be used which has the same flow channels and oxygenator dimensions as those found in the Physiomimix platform, except the onboard pumps. An external peristaltic pump is used to drive the flow through the circuit of the PDP. The lid keeps the PDP protected from airborne contamination in an incubator.

[0346] Evaluation of Buton Scaffolds:

[0347] For biological validation of the button scaffold, for vascularization experiments, Human umbilical vein endothelial cells (HUVECs; Angioproteomie, cAP-0001) and normal human lung fibroblasts (NHLFs: Lonza, CC-2512) were cultured in VascuLife® VEGF Endothelial Medium Complete Kit (LifeLine, LL-0003) and FibroLife S2 Fibroblast Medium Complete Kit (LifeLine, LL-0011), respectively. For experiments involving real-time visualization of microvascular network formation, GFP- and RFP-expressing HUVECs were employed (Angioproteomie, cAP-0001GFP and cAP-OOOIRFP). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2, with medium replaced every other day. HUVECs and NHLFs were enzymatically dissociated using TrypLE (ThermoFisher) and utilized for experiments between passages 5 and 9.

[0348] The hydrogel used for encapsulation was a polyethylene glycol (PEG)-based hydrogel. The PEG molecule used was an eight-ami PEG with 20kDa molecular weight. Vinylsulfone-based click chemistry was used to polymerize the gel to form a synthetic extracellular matrix (ECM) for cells to grow into.

[0349] For experiments with liver spheroids, primary human hepatocytes and Kupffer cells were obtained either as donor-matched isolates from a single human donor (LifeNet Health LifeSciences) or purchased separately from ThermoFisher. Hepatocytes were thawed in Cryopreserved Hepatocyte Recovery Medium (CHRM; ThermoFisher), pelleted by centrifugation at 100g for 8

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[0351] 45784491.1 minutes, and resuspended in a custom physiological William’s E / VascuLife® medium formulation. Kupffer cells were thawed in ice-cold William’s E medium, centrifuged at 450g for 5 minutes, and resuspended in the same physiological medium. Both cell types were thawed immediately prior to spheroid assembly. Spheroids were generated using alginate microwells. Prior to seeding, microwells were incubated with 5% BSA for a minimum of 2 hours at 37°C. A cell suspension containing 120,000 primary human hepatocytes, 60,000 HUVECs, 12,000 Kupffer cells, and 12,000 NHLFs was prepared in a total volume of 75ul per well. Following removal of the BSA solution, microwells were equilibrated with 75ul of culture medium, after which the cell mixture was added dropwise. Plates were centrifuged at 50g for 2 minutes and subsequently overlaid with an additional 150ul of culture medium. Medium was replaced after 24 hours. For experiments involving live-cell imaging, hepatocytes were labeled with CellTracker Deep Red (ThermoFisher, C34565) at a concentration of luM immediately after thawing and prior to seeding, following the manufacturer’s instructions.

[0352] For each button scaffold, lOul of the PEG gel-cell suspension described above was dispensed into the central gel channel using a positive displacement micropipette. The final seeding densities were 18xl06HUVECs / mL, 2.57xl06NHLFs / mL, and approximately 150 liver spheroids per scaffold. In certain scaffolds, only HUVECs and NHLFs were included to serve as “vascular network alone” controls. Following seeding, scaffolds were incubated in a humidified chamber at 37°C for 30 minutes to allow gel polymerization, after which culture medium was added to the flanking media channels surrounding the gel channel. For microvascular-only experiments VascuLife® media was used in the Physiomimix platform plates and PDP devices.

[0353] Time progression of vascular network development (for days 0, 1, 2, 3, 4, 5, and 7) in the button scaffold was monitored visually by the green colorization from the GFP in the HUVEC cells, imaged with an inverted fluorescence microscope in the PDP device. There were differences observed in the network morphologies between the two culture methods depending on the platform used, Physiomimix or PDP, where the primary difference between the experimental conditions was the pulsatility of the Physiomimix platform plates on-board pumps versus the smooth flow of the off-board peristaltic pumps in the PDP. The PDP networks were found to be more crowded near the centerline of the gel channel with a sparsely populated region just inside the interface between the gel and the surrounding media channels. Also, there was a layer of cells at the interface. By contrast, for the Physiomimix platform the gel channel did not have a region of sparsely populated networks, the gel interfaces are densely populated with cells, which continues into the gel eventually getting into skinny networks at the center. This morphology suggested that a continuous

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[0355] 45784491.1 perfusion path from one media channel to another through the HUVEC networks was possible and thus was more preferable than the PDP-based networks. lOum diameter polystyrene beads with a red fluorescent tag were pumped through the circuit to demonstrate through-gel perfus ability. In the PDP networks, the beads were not observed to penetrate inside the networks indicating partial or no perfusability, but they were observed to penetrate the Physiomimix platform networks, indicating full perfus ability. This again indicated that the differences in the pulsatility of the Physiomimix and PDP platforms was a factor in the network morphology produced.

[0356] To confirm the hypothesis about pulsatility being a factor in network morphology, experiments were performed in the Physiomimix platform plate with different resistor discs. As shown in Figure 13, the results at day 7 of the experiment, where “smoothing” is analogous to the resistor value in the disc, showed that with a high amount of smoothing, scaffolds demonstrated sparser gel-media interface cell densities, similar to the PDP from the previous figure. Also, of note, the networks were more uniformly oriented, without a specific directionality. With medium and no smoothing, the networks had a more directional nature, i.e. they connect the two media channels, with large amounts of cells forming a layer at the interfaces. However, this effect was almost overpronounced in the no smoothing case, where the network structure was lost in segments where large clumps of cells from the two opposite interfaces ingress and touch each other.

[0357] For hepatocyte spheroid culture in HUVEC, the media formulation was changed to a 50%- 50% mixture of VascuLife® with hepatocyte maintenance media. Networks formed and connected with the hepatocyte spheroids, as shown in Figure 14. However, the networks were not perfusable in the initial pilot. A new media formulation was explored where the endothelial growth factors in the final media was increased to in lx concentration (up from 0.5x in the previous media) and FBS was increased to 3.5% (up from 2%). The interface was seen to have a dense layer of HUVECs, and the close-up further showed networks connecting to hepatocyte spheroids and beads which indicated perfusability as well. This experiment was done without any resistor discs.

[0358] Albumin secretion in the collected media is a common hepatocyte function test. The results shown in the graph in Figure 15a are normalized against the number of hepatocytes present in each button scaffold. The button scaffold (denoted novel scaffold) performed slightly higher than historical data on the default Physiomimix platform liverchip scaffold, indicating the culture method was viable.

[0359] Figures 15b and 15c show functional results from a different experiment done on the button scaffold without resistor discs, with hepatocyte spheroids from a new donor. In this instance, control scaffolds without HUVECs or NHEFs, and static hydrogel droplets with all cell types were

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[0361] 45784491.1 also cultured side-by-side with the button scaffolds. Albumin secretion was clearly higher in the button scaffold compared to the other two methods. CYP3A4 enzyme activity is another functional metric that was measured. In this case, the CYP3A4 activity was highest in the button scaffold as well.

[0362] In summary, this example describes a button scaffold suitable for use with the Physiomimix LC12 platform, replacing the existing scaffold, filter, and support assembly to enable the creation of microvascularized 3D liver models. This button scaffold design, featuring concentric circular segments for the central hydrogel channel (housing HUVECs and NHLFs) flanked by media channels, maximizes usable space and removes the filter, opening opportunities for immune -host interaction studies. In addition, it was possible to address the highly pulsatile flow generated by the Physiomimix platform's on-board pneumatic micropumps by incorporating a resistor disc in series with the button scaffold, effectively mitigating flow pulsatility, like an RC low-pass filter. Experiments demonstrated that this pulsatile flow significantly influences microvascular network morphology, leading to densely populated gel interfaces and fully perfusable networks connecting the media channels. In contrast, smoother flow conditions, such as those from the PDF device or when using high resistance discs, resulted in sparser cell densities and partial or no perfus ability, thus identifying pulsatility as an important factor for functional microvascular network formation. Furthermore, the integration of hepatocyte spheroids with these microvascular networks was achieved using a custom media formulation, with functionality tests showing the button scaffold culture method was viable and demonstratesd higher albumin secretion and CYP3A4 enzyme activity compared to historical data or control scaffolds lacking non-parenchymal cells. In conclusion, the development of the button scaffold represents a significant advancement towards creating more physiologically relevant pre-clinical models by incorporating microvasculature and enabling pertinent paracrine signaling, with insights into the role of flow dynamics.

[0363] Example 3:

[0364] Scaffolds, with a design analogous to that shown in Figure 16, were loaded with a fibrin hydrogel laden with human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF) cells in densities of 10 million / ml gel and 1.42 million / ml gel, respectively. Hydrogel-laden scaffolds were installed in a perfusion platform and cultured by circulating media in a closed loop by pumps at a rate of 1 ul / second for a period of 5 days. The HUVECs used expressed green fluorescent protein to help with imaging the cellular network development. Fluorescent images taken on day 5 show growth of HUVEC networks into microvessel-like structures in the openings of the scaffold. See Figures 17A and 17B.

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[0366] 45784491.1 Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific instances of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0367] 45784491.1

Claims

CLAIMSWe claim:

1. A microfluidic scaffold comprising: a pressure module; a gel module; optionally a by-pass module or outlet comprising one or more by-pass flow paths or segments; and at least one gel loading port; wherein a controllable pressure differential can be induced within the gel module when a fluid, such as a culture medium, is flowed through or around the gel module.

2. The microfluidic scaffold of claim 1, wherein the pressure module comprises one or more pressure regulators.

3. The microfluidic scaffold of claim 2, wherein the one or more pressure regulators are selected from a linear pressure regulator; a non-linear pressure regulator; a hydrostatic pressure regulator; a ball- valve comprising a ball inside a retaining feature, such as a cage including one or more vent holes, and the ball is on ball seat which can be opened or closed when the ball is lifted; or combinations thereof.

4. The microfluidic scaffold of any of one claims 1-3, wherein the microfluidic scaffold comprises an imageable region.

5. The microfluidic scaffold of any one of claims 1-3, wherein the gel module is a hydrogel chamber.

6. The microfluidic scaffold of any one of claims 1-3, wherein the gel module comprises one or more gel channels.

7. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold has an axis and flow of the fluid through the gel module is parallel or axial to the axis.

8. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold has an axis and flow of the fluid through the gel module is orthogonal, or lateral, to the axis.

9. The microfluidic scaffold of claim 2, wherein the ball is made of material that is denser than the fluid that flows through the ball-valve.

10. The microfluidic scaffold of claim 2, wherein the ball has a pressure applied thereon by a spring, such as a linear or non-linear spring which can be pre-compressed.

11. The microfluidic scaffold of claim 10, wherein the pressure applied by the spring can prevent the fluid from flowing through the ball seat of the ball-valve when the fluid’s pressure is not4545784491.1sufficient to overcome the pressure applied by the spring, and optionally the ball’s weight, to permit the fluid to flow through the ball seat of the ball-valve.

12. fhe microfluidic scaffold of claim 2, wherein the ball- valve controls the controllable pressure differential based on any one of the following parameters: the ball seat having a circular geometry with a diameter which is half the diameter of the ball’ s diameter; the ball being made of sapphire or stainless steel; the ball having a density of about 3 to 9 or 4 to 8 g / cm3; and / or the ball having a weight in a range of about 0.05 to 0.15 N.

13. The microfluidic scaffold of any one of claims 1-3, wherein the one or more by-pass flow paths or segments can be closed; and optionally wherein the one or more by-pass flow paths or segments can be closed with a by-pass flow plug.

14. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises a membrane valve comprising: a membrane sealing feature comprising a ridge and an inlet via; a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; and wherein the membrane comprises one or more membrane vent openings.

15. The microfluidic scaffold of claim 14, wherein the fluid can flow through the one or more vent openings of the membrane when the fluid’s pressure is sufficient to lift the membrane off the ridge to allow the fluid to flow between the ridge and the membrane.

16. The microfluidic scaffold of claim 14, wherein a flexural member backing applies a pressure on the membrane; wherein the flexural member backing comprises one or more vent openings; and wherein the flexural member backing is part of the microfluidic scaffold or is separately bonded thereto.

17. The microfluidic scaffold of claim 16, wherein the pressure applied on the membrane by the flexural member backing can be adjusted by a linear screw which is in contact with the flexural member backing.

18. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises a flexural member comprising one or more flexural member vent openings; wherein the flexural member covers a sealing feature comprising a ridge and an inlet via; wherein the flexural member contacts the ridge; and wherein the fluid can flow through the one or more vent openings of the flexural member when the fluid’s pressure is sufficient to lift the flexural member off the ridge to allow the fluid to flow between the ridge and the flexural member.4645784491.

119. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises a membrane valve comprising: a membrane sealing feature comprising a ridge: a membrane which is bonded or clamped to the microfluidic scaffold and covers the membrane sealing feature and contacts the ridge; wherein the membrane optionally comprises one or more membrane vent openings; wherein a hydrostatic pressure is applied on the membrane by a hydrostatic pressure tube containing a working liquid and the hydrostatic pressure can be varied by the height of the working liquid present in the hydrostatic pressure tube.

20. The microfluidic scaffold of claim 19, wherein the hydrostatic pressure tube is a flow conduit comprising a porous medium therein.21 . The microfluidic scaffold of claim 19, wherein the fluid can flow between the ridge and the membrane when the fluid’s pressure is sufficient to lift the membrane off the ridge.

22. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises one or more gaps, such as segmented gaps, providing the one or more by-pass flow paths or segments; and optionally one or more feet which can lift the microfluidic scaffold when placed on a base.

23. The microfluidic scaffold of claim 22, wherein the one or more gaps can be closed by placing a gasket thereon and clamping the gasket thereon, such as with a retaining ring.

24. The microfluidic scaffold of claim 22, wherein the one or more gaps can be closed by plugs placed thereon.

25. The microfluidic scaffold of any one of claims 1-3, wherein the by-pass module or outlet further comprises a removable ball-valve or a removable hydrostatic pressure tube and can perform the function of the pressure module.

26. The microfluidic scaffold of any one of claims 1-3, wherein the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mmH20; in a range from between about 10 to 99 mmH20; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmH20.

27. The microfluidic scaffold of any one of claims 1-3, wherein the controllable pressure differential induced is less than about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 75, 50, 25, 15, 10, 5, or 1 mmH20 between at least two different points of the gel module; in a range from between about 10 to 99 mmH20 between at least two different points of the gel module; or is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200,4745784491.1300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500 mmH2O between at least two different points of the gel module.

28. 1’he microfluidic scaffold of any one of claims 1-3, wherein the gel module has a radial spoke geometry; at least one gel channel; one or more discrete openings or channels, which can be closed, on top and on bottom of the at least one gel channel; and the gel module comprises the at least one gel loading port.

29. The microfluidic scaffold of claim 28, wherein the at least one gel channel is enclosed by one or more laminating films bonded to the body of the gel module.

30. The microfluidic scaffold of claim 28, wherein the one or more laminating films are patterned with holes of any shape(s) that allow the fluid to flow in and out of the at least one gel channel.31 . The microfluidic scaffold of claim 28, wherein the least one gel channel comprises a plurality of straight channels; a plurality of circular channels, at least one serpentine channel; or one circular channel; and optionally the plurality of straight channels are not connected to each other.

32. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises a bottom plate or layer that comprises the one or more by-pass flow paths or segments of the by-pass module or outlet and one or more inlet ports; and a top plate or layer that comprises the gel module, the pressure module, and one or more outlet ports.

33. The microfluidic scaffold of claim 32, wherein the one or more inlet ports and the one or more outlet ports are staggered.

34. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises one or more inlet ports and one or more outlet ports and at least one media channel is in between the inlet and outlet ports for flowing the fluid therethrough.

35. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold further comprises a laminate layer bonded thereto which can guide flow of the fluid within the scaffold.

36. The microfluidic scaffold of any one of claims 1-3, wherein the gel module comprises at least one gel channel wherein edges of the at least one gel channel comprise features selected from a coiled serpentine fiber, a mesh or a filter-like structure, phase guides, vertical posts, and / or tapered posts.

37. The microfluidic scaffold of any one of claims 1-3, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having an open top.4845784491.

138. The microfluidic scaffold of any one of claims 1-3, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having closed top.

39. The microfluidic scaffold of any one of claims 1-3, wherein the at least one gel loading port comprises a pipette tip locating feature and the gel module comprises at least one gel channel having a partially open top.

40. The microfluidic scaffold of any one of claims 1-3, wherein the at least one gel loading port is formed from a removable pipette tip adapter feature placed on the microfluidic scaffold.

41. The microfluidic scaffold of any one of claims 1-3, wherein the microfluidic scaffold comprises one or more feet which can lift the microfluidic scaffold when placed on a base or platform.

42. A microfluidic device or platform comprising the microfluidic scaffold of any one of claims 1-3.

43. The microfluidic device or platform of claim 42, wherein the microfluidic device or platform is built into the microfluidic device or platform.

44. fhe microfluidic device or platform of claim 42, wherein the microfluidic scaffold can be dropped or slotted into the microfluidic device or platform; and a gasket holds the microfluidic scaffold therein wherein optionally the gasket can be held or clamped by a retaining ring and the retaining ring is optionally a threaded retaining ring.

45. The microfluidic device or platform of claim 42, wherein the microfluidic device or platform comprises more than one of the microfluidic scaffold, such as two microfluidic scaffolds therein.

46. The microfluidic device or platform of claim 44, wherein: the gasket is a single-piece gasket: the gasket is a multi-piece gasket; and / or the retaining ring is a threaded retaining ring optionally comprising one or more feet features having axial and radial gaps providing by-pass flow pathways.

47. The microfluidic device or platform of claim 46, wherein the multi-piece gasket comprises a top piece and a bottom piece; and the top piece comprises one or more projections which can block by-pass flow paths or segments and the bottom piece optionally comprises one or more holes which can hold feet when present on the microfluidic scaffold.

48. The microfluidic device or platform of claim 42, wherein the microfluidic device or platform comprises pump, such as a recirculating pump, pneumatic pump, or peristaltic pump, that provides a flow when the fluid is flowed through or around the gel module.4945784491.

149. A method of culturing cells comprising the steps of:(a) loading a hydrogel comprising a plurality of cells into the microfluidic device or platform of claim 42, wherein the hydrogel comprising the plurality of cells is loaded into the gel module of the microfluidic scaffold;(b) flowing a fluid, such as a culture medium, through and / or around the gel module; wherein a controllable pressure differential is induced within the gel module when the fluid is flowed through or around the gel module.

50. The method of claim 49, wherein the cells of the plurality are encapsulated by the hydrogel.

51. The method of claim 49, wherein the controllable pressure differential provides a delta pressure across the hydrogel comprising the cells which can provide biomechanical or biomolecular cues to the plurality of cells.

52. The method of claim 49, wherein the hydrogel is a fibrin hydrogel.

53. The method of claim 49, wherein the cells of the plurality are selected from human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblast cells (NHLF).

54. The method of claim 53, wherein the cells of the plurality form a vascular network within the gel module or gel channel.

55. The method of claim 54, wherein the vascular network is perfusable.

56. The method of claim 49, wherein the hydrogel further comprises one or more spheroids, such as hepatocyte spheroids.

57. The method of claim 49, wherein the fluid comprises immune cells.

58. The method of claim 49, wherein the method comprises a step of imaging the cells of the plurality, such as by fluorescence microscopy, via an imageable region of the microfluidic scaffold.

59. The method of claim 49, wherein the fluid is flowed at a flow rate of at least about 1 ul / second.

60. The method of claim 49, wherein the fluid is flowed for a period of time of at least about 1 day up to about 6 months, 1 day up to about 5 months, 1 day up to about 4 months, 1 day up to about 3 months, 1 day up to about 2 months, 1 day up to about 1 month, 1 day up to about 25 days,1 day up to about 20 days, 1 day up to about 15 days, 1 day up to about 10 days, 1 day up to about 7 days, or 1 day up to about 5 days.5045784491.1

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