Microfluidic tissue biopsy and immune response drug evaluation devices and systems

This disclosure describes microfluidic tissue biopsy and immune response drug evaluation devices and systems. A microfluidic device can include an inlet channel having a first end configured to receive a fluid sample optionally containing a tissue sample. The microfluidic device can also include a tissue trapping region at the second end of the inlet channel downstream from the first end. The tissue trapping region can include one or more tissue traps configured to catch a tissue sample flowing through the inlet channel such that the fluid sample contacts the tissue trap. The microfluidic device can also include one or more channels providing an outlet.

BACKGROUND

Current technology for simulating dynamic processes involving interactions between mammalian tissue samples and cells is gated by the inability to recapitulate the tissue microenvironment and interactions between tissues, therapeutic compounds and the host immune system.

SUMMARY

One aspect of this disclosure is directed to microfluidic device comprising including a substrate. The substrate defines an inlet channel having a first end configured to receive a fluid sample optionally containing a tissue sample. The substrate defines a tissue trapping region at the second end of the inlet channel downstream from the first end. The tissue trapping region includes one or more tissue traps configured to catch a tissue sample flowing through the inlet channel such that the fluid sample contacts the tissue trap. The substrate also defines one or more channels providing an outlet.

In some implementations, at least one of the one or more tissue traps comprises an arrangement of one or more walls. In some implementations, the one or more channels providing the outlet include one or more branch channels connecting to the second end of the inlet channel where the second end of the inlet channel and the tissue trapping region converge. In some implementations, the convergence of the second end of the inlet channel and the tissue trapping region further includes a first branch channel coupled to the second end of the inlet channel at a first junction and configured to direct a first portion of the fluid sample in a first direction, and a second branch channel coupled to the second end of the inlet channel at the first junction and configured to direct a second portion of the fluid sample in a second direction, different form the first direction, wherein the tissue trap is positioned at the first junction.

In some implementations, the one or more channels providing the outlet further include one or more suction channels downstream of the one or more tissue traps and configured to hold the tissue sample in place within the one or more tissue traps. In some implementations, at least one of the one or more tissue traps includes a bottom surface positioned at a lower depth than a bottom surface of the inlet channel. In some implementations, the first branch channel and the second branch channel converge at a second junction downstream from the one or more tissue traps.

In some implementations, the microfluidic device further includes a first suction channel coupling at least one of the one or more tissue traps to the first branch channel at a third junction downstream from the second end of the inlet channel. The microfluidic device can also include a second suction channel coupling the at least one of the one or more tissue traps to the second branch channel at a fourth junction downstream from the second end of the inlet channel. In some implementations, a diameter of at least one of the one or more the tissue traps is about twice that of the inlet channel.

In some implementations, the tissue trapping region includes a ribbed channel coupling the inlet channel to the one or more channels providing the outlet. In some implementations, at least one of the one or more tissue traps can be defined by sidewalls of ribs of the ribbed channel and a bottom wall positioned at a lowest depth of the ribbed channel. In some implementations, the at least one tissue trap can further include at least a second tissue trap and a third tissue trap.

In some implementations, the tissue trapping region can include a circuitous channel having a first curved portion coupled to the second end of the inlet channel. The microfluidic device can also include at least one of the one or more tissue traps positioned at a center of the first curved portion such that the fluid sample flows along the first curved portion past the tissue trap. In some implementations, the one or more channels providing the outlet channel can include a suction channel coupling to the at least one of the one or more tissue traps and configured to carry the fluid sample downstream from the at least one of the one or more tissue traps. In some implementations, the circuitous channel can further include a second curved portion coupled to a downstream end of the first curved portion and a second tissue trap positioned at a center of the second curved portion such that the fluid sample flows along the second curved portion past the second tissue trap. In some implementations, a downstream end of the second curved portion is coupled to the one or more channels providing the outlet.

In some implementations, the microfluidic device can also include an inlet port coupled to the first end of the inlet channel and configured to deliver the fluid sample to the inlet channel. In some implementations, the inlet port can include a first threaded connector configured for attachment to a fluid line.

In some implementations, the microfluidic device can also include a bubble trapping structure coupled to the inlet channel downstream from the inlet port. The bubble trapping structure can be configured to facilitate evacuation of air bubbles from the fluid sample. In some implementations, a surface of the bubble trapping structure can have a shape defined by a parabolic function. In some implementations, the bubble trapping structure can further include a second threaded connector configured for attachment to an air release line.

In some implementations, the microfluidic device can also include an outlet port coupled to the at least one of the one or more channels providing the outlet and configured to remove the fluid sample from the microfluidic device. In some implementations, the substrate can be formed from a biocompatible material. In some implementations, the substrate can be formed from an optically transparent material, and the microfluidic device can further include an optical interface providing optical access to the tissue sample positioned within the tissue trapping region. In some implementations, the one or more tissue traps can be configured to entrain the tissue sample in place within the one or more tissue traps.

Another aspect of this disclosure is directed to a method for evaluating an interaction between a tissue sample and a fluid sample. The method can include introducing a tissue sample into a first end of an inlet channel of a microfluidic device. The method can include introducing a fluid sample into the first end of the inlet channel to cause the tissue sample to flow to a tissue trapping region at a second end of the inlet channel downstream from the first end. The tissue trapping region can include a tissue trap configured to catch the tissue sample such that at least a portion of the fluid sample contacts the tissue sample. The method can include collecting the sample fluid from at least one channel providing an outlet downstream from the tissue trapping region.

In some implementations, the method can include priming the inlet channel with fluid prior to introducing the tissue sample into the first end of the inlet channel. In some implementations, the method can include observing an interaction between the tissue sample and the fluid sample in the tissue trapping region. In some implementations, the microfluidic device can be formed from a transparent material, and observing the interaction between the tissue sample and the fluid sample can further include positioning a lens of a microscope in proximity to the microfluidic device.

In some implementations, the tissue trap can be configured to secure the tissue sample without damaging the tissue sample. In some implementations, the method can include introducing the tissue sample via a bubble trapping structure coupled to the inlet channel, and introducing the fluid sample via an inlet port coupled to the inlet channel. The inlet port can be upstream from the bubble trapping structure. In some implementations, the method can include removing air from the fluid sample via the bubble trapping structure.

In some implementations, the method can include releasing the tissue sample from the tissue trap by introducing a second fluid sample into at least one of the one or more channels configured to provide the outlet such that the second fluid sample flows towards the inlet channel.

In some implementations, after collecting the sample fluid at least one of the one or more channels configured to provide the outlet downstream from the tissue trapping region, the method can include reintroducing the collected sample fluid into the inlet channel of the microfluidic device.

DETAILED DESCRIPTION

This disclosure aims to establish a robust platform to recapitulate the tissue microenvironment and interactions with host immune cells.

This disclosure describes devices and systems capable of recapitulating the tissue microenvironment and tissue interactions with fluid which may contain cells (such as circulating immune cells), medications, therapeutic compounds, or other components. As used herein, “fluid” can refer to fluid containing components that are intended to interact with a tissue sample (such as cells, medications, therapeutic compounds, or other substances) in order to observe a response, or can refer to fluid devoid of such components. A key challenge in this regard is the ability to maintain a tissue sample, such as a tumor biopsy, in a configuration that permits real-time observation of tumor viability and responses to therapeutic compounds, such as dynamic interactions between circulating immune cells and the tissue biopsy sample. This disclosure describes multiple novel designs capable of capturing and maintaining the position of a tissue sample in a flow field that presents cells, medications, therapeutic compounds, or other components to the tissue in a physiologically relevant manner, permitting control over perfusion rates and shear forces to ensure that results are relevant to human in vivo conditions.

Beyond the tissue trapping and flow field device, in order to fully recapitulate the dynamics of tissue interactions with cells such as immune cells, medications, therapeutic compounds, or other components, and to do so in a high throughput manner, it can be useful to integrate the device with a system capable of sustaining the tissue, maintaining control over the flow rate, viability of cells and density of circulating components, and to avoid problems common to microfluidic systems such as bubbles, debris, blockages or variability in flow rates. A key challenge is the ability to integrate these features in a manner that provides robust control over system dynamics for periods of up to one week or more.

In some implementations, the devices of this disclosure are capable of ex vivo simulation of the dynamics of tissue interactions with various fluid components, such as cells, medications, or therapeutic compounds. The devices can integrate capture regions, cell flow channels, resistance lines and fluidic connections, and bubble trapping structures. The devices described herein can permit observation and control over interactions between various types of fluid components and excised tissues such as tumor biopsy samples, skin biopsies, epithelial tissues such as gut, airway, renal or reproductive tract tissues. The figures and corresponding description below provide further detailed information regarding the design of such devices and systems. In brief, this disclosure includes various aspects, including specific designs for tissue traps, including a heart-shaped branching structure, ribbed channel bottom structure, S-curve structure, and suction port structure. Each serves as a means to precisely control and freeze the position of a tissue biopsy sample in a flow stream, and to expose the fixed tissue sample to a precisely controlled flow of fluid containing components such as cells, medications, or therapeutic compounds in order to observe interactions between the fluid components and tissue samples. This disclosure also includes aspects relating to integration of these trapping devices with other fluidic components. These additional components can include resistance channels, fluidic connectors and branch points, tissue sample loading ports, bubble trapping structures, drug dosing and media sampling ports, cell containment vessels, and manifolds that serve as distribution branches for cells and gas pressure lines.

For tissue trapping regions, other ways to address the problem include the use of V-shaped posts to trap tissues, side chamber regions, or side-to-side channels with cells flowing through one lane and tissues held in another, with a gel region in between. Additional potential designs for these systems include methods where the biopsied tissue sample is contained within a side channel or side compartment that indirectly receives flow from the main dynamic perfusion channel, methods where excised biopsy samples are contained within larger excised tissues or organs, or methods where biopsy samples are contained within constructs that are molded from mammalian tissues.

In other implementations, interactions between fluid and tissues can be mimicked by generating tissue constructs contained within gel or matrix regions. Fluid can flow through adjacent channels in which they are permitted to migrate toward the matrix-embedded tissue constructs. Some such devices and systems can utilize conventional microwell plates or transwells to contain excised tissues, as a static representation of the cell-tissue interaction.

The devices of this disclosure include innovative aspects in the nature of the tissue trapping geometry as compared to other approaches that may use V-shaped posts, side chambers, or side-to-side channels with intervening gel regions. The disadvantages of these approaches relate to the inability to precisely control the rate at which circulating fluid are presented to the tissue biopsy sample, because V-shaped post regions require dealing with a tradeoff between allowing flow around the tissue and raising the hydrostatic pressure of flow against the tissue sample. For side chambers or side-to-side channels, tissue interactions with fluid can occur via migration phenomena, which may be difficult to control in the microenvironment, or by random “strikes” of fluid traveling obliquely through the flow stream. This disclosure provides novel designs that can be used to contain tissue biopsy samples and channels for flowing fluid.

Other approaches to solving the tissue-cell interaction problem include using conventional means to contain tissues and fluid (e.g., static wells or transwells) and/or gel-matrix systems in which tissue samples are disaggregated and seeded into microfluidic devices in compartments adjacent to blood/cell-flow channels. Technical obstacles to the innovations described herein include developing designs capable of capturing tissue biopsy samples and effectively causing interaction of these captured samples with flowing media. These innovative concepts are not obvious because they include new tissue biopsy sample containment designs that overcome previous limitations. Key advantages of the devices and systems of this disclosure include designs that effectively entrain tissue biopsy samples and expose them to flowing fluid in a manner that optimizes the cross-section of interaction between the two.

FIG. 1Aillustrates a perspective view of an example microfluidic device100, according to an illustrative implementation.FIG. 1Billustrates a second perspective view of the example microfluidic device100ofFIG. 1A. Similar reference numerals inFIGS. 1A and 1Brefer to similar elements. Referring to bothFIG. 1AandFIG. 1B, the microfluidic device100can be used to simulate interactions between tumors or other tissue samples and the immune system, for example by providing a microenvironment for testing the effectiveness of immunotherapy treatments on lymphocytes and tumor biopsies taken directly from a patient. As a result, the microfluidic device100can be used to model the in vivo environment and analyze the prolonged response of a tumor and circulating lymphocytes to the controlled introduction of immunotherapy pharmaceuticals. Thus, the microfluidic device100can enables judicious administration of immunotherapy treatments by allowing medical professionals to make informed decisions regarding course of treatment for a patient based on experiments conducted using the microfluidic device100.

The microfluidic device100is formed from a substrate102. The substrate102defines a variety of structural features, including an inlet port105leading to an inlet channel115. Downstream from the inlet port105and coupled to the inlet channel115is a bubble trapping structure110. Farther downstream from the inlet channel115is a tissue trapping region120, which leads to an outlet channel125. An outlet port130is positioned at a downstream end of the outlet channel125. While only a single microfluidic device100is depicted inFIGS. 1A and 1B, it should be understood that in some implementations, multiple devices similar to the microfluidic device100can be incorporated into a single chip without departing from the scope of this disclosure.

In use, the microfluidic device100can capture a tissue sample and allow testing of the interaction of the tissue sample with various cells, medications, therapeutic compounds, or other agents or components included within a fluid sample flowing within the microfluidic device100. For example, a tissue sample, such as a portion of a tumor, can be loaded into the device via the inlet port105or via the bubble trapping structure110. After the tissue sample flows through the inlet channel115, the structural characteristics of the tissue trapping region120cause the tissue sample to become trapped. A fluid sample can then be introduced into the inlet port105and flowed through the inlet channel115, while the tissue sample remains held in place in the tissue trapping region120. At least a portion of the fluid sample (and the cells, medications, therapeutic compounds, or other components within the sample) can contact the trapped tissue sample as it flows from the inlet channel115to the outlet channel125and finally exits the microfluidic device100via the outlet port130. In some implementations, air bubbles that may be present in the fluid sample, and which may cause damage to the tissue sample or may otherwise interfere with the results of the experiment, can be removed from the microfluidic device100via the bubble trapping structure110.

It should be understood that, in the implementation shown inFIG. 1A, the outlet channel125serves as an outlet for the microfluidic device100as a whole, but not for the tissue trapping region120. Thus, in some implementations, the outlet channel125may not be an outlet channel relative to the tissue trapping region120, and therefore may be referred to by a different name. In some implementations, one or more channels may provide an outlet for fluid at or near the tissue trapping region120. For example, branching channels, suction channels, and other channels further described below may provide such an outlet. Thus, in some implementations, these channels also may be referred to as outlet channels. Various types of channels that may provide an outlet for fluid at or near the tissue trapping region120are described further below.

In some implementations, the microfluidic device100can be further configured to provide an optical interface for viewing the interaction site where the tissue sample interacts with the fluid sample. To facilitate optical access, the channels within the microfluidic device100can be configured to substantially avoid optical distortion. In some implementations, the channels can have a rounded rectangular cross-sectional shape. Such a shape exhibits smaller surface area to volume ratio than a purely rectangular channel, which can help to preserve pumping efficiency by reducing resistance in the channels. In addition, rounded rectangular channels may not produce image distortion that is characteristic of channels having circular cross-sectional shapes.

These and other aspects of this disclosure are described further below. In particular, a variety of different geometries and structural shapes can be used to implement the tissue trapping region120, and several examples of such geometries are shown in the figures. In particular,FIGS. 2A-2Fgenerally relate to a first geometry for the tissue trapping region120,FIGS. 3A and 3Bgenerally relate to a second geometry for the tissue trapping region120, andFIGS. 4A-4Dgenerally relate to a third geometry for the tissue trapping region120.

FIG. 2Aillustrates a cross-sectional view of a portion of an example microfluidic device200that can be used to implement the microfluidic device100ofFIG. 1A, according to an illustrative implementation. The features of the microfluidic device200generally correspond to the features of the microfluidic device100, and like reference numerals refer to like elements. For example, the microfluidic device200includes an inlet channel215, a tissue trapping region220, and an outlet channel225that can carry fluid out of the microfluidic device200.FIG. 2shows the structural details of the tissue trapping region220, which in this example includes a tissue trap (also referred to as a tissue trapping zone or trapping zone)235positioned at a downstream end of the inlet channel215, as well as two branch channels240aand240bbranching off from the inlet channel215in opposing directions at a junction near the tissue trap235.

As described above, the tissue trapping region220is configured to trap a tissue sample in a fixed location while a fluid sample is flowed through the microfluidic device200. For example, in some implementations, the tissue trapping region220is shaped such that, when the fluid sample flows through the microfluidic device200, a stagnation zone exists in at least a portion of the area of the tissue trap235, causing the tissue sample to become trapped in the tissue trap235.FIG. 2Billustrates a tissue sample239within the microfluidic device200ofFIG. 2A, according to an illustrative implementation. It should be noted thatFIG. 2Bshows the microfluidic device200in a reversed orientation relative to that shown inFIG. 2A, such that fluid flows from right to left in the depiction of the microfluidic device200ofFIG. 2B. As shown, the tissue sample239becomes trapped in the tissue trap235in a manner that allows the fluid sample to continue flowing through the inlet channel215to the branch channels240aand240b, while a portion of the fluid sample contacts the tissue sample239as it flows.

In some implementations, the tissue trap or trapping zone235can have a bottom wall that is positioned at a lower depth than the bottom of the inlet channel215that leads up to it. That is, the tissue trap235can be stepped down relative to the bottom surface of the inlet channel215. Thus, the tissue trap235can serve as a pocket for catching, trapping, holding, immobilizing, or securing the tissue sample239. In some implementations, the shape of the tissue trapping region220, including the tissue trap235, is selected to catch or otherwise facilitate trapping of the tissue sample239while the fluid sample passes through the microfluidic device200. For example, the tissue trap235can have a diameter that is larger than that of the inlet channel215. In some implementations, the tissue trap235can have a diameter that is about twice that of the inlet channel215.FIG. 2Cis a visual depiction252of the flow characteristics of the microfluidic device200ofFIG. 2A, according to an illustrative implementation. The shading within the channels shows the velocity of the streamlines within the device. When the streamlines bend at the branch channels240aand240b, the inertia of the tissue sample can overcome the viscous forces and can become lodged in the tissue trap235.

Referring again toFIG. 2B, the trapping of the tissue sample239in a manner that allows the fluid sample to continue flowing through the device while contacting the tissue sample239can allow the interactions between the tissue sample239and agents within the fluid sample. For example, in some implementations fluorescent materials can be added to either the fluid sample or the tissue sample239, and the visual characteristics of the tissue sample239and the fluid sample can be observed over time. To facilitate such observation, the microfluidic device200can be formed from a material that is transparent and optically clear, at least in the region of the device near the tissue trap235. This area can serve as an optical interface that can be examined by an optical instrument, such as a camera or a microscope, that is brought into proximity with the microfluidic device200.

FIG. 2Dillustrates a first arrangement201of the microfluidic device ofFIG. 2Ahaving suction channels, according to an illustrative implementation. Components shown in the arrangement201are substantially similar to the components shown inFIG. 2A, and like reference numerals refer to like elements. However, the arrangement201ofFIG. 2Ddiffers from that shown inFIG. 2Ain that the arrangement201includes a suction channel245. The suction channel245is coupled between a downstream end of the tissue trap235and the outlet channel225. Thus, the suction channel245can provide an outlet for fluid in the tissue trap235, and therefore may sometimes itself be referred to as an outlet channel. Similarly, the microfluidic device201also includes branch channels240aand240bthat can provide an outlet for fluid near the tissue trap235, and therefore the branch channels240aand240bmay also be referred to as outlet channels240aand240b. Furthermore, it should be understood that the outlet channel provides an outlet of the microfluidic device201(i.e., it is configured to carry fluid out of the microfluidic device201), but does not couple to the tissue trap235and therefore does not serve as an outlet for fluid from the tissue trap235. In some implementations, the suction channel245can be configured to facilitate trapping of the tissue sample within the tissue trap235. For example, as the fluid sample flows from left to right in the depiction ofFIG. 2D, through the branch channels240aand240band into the outlet channel225, the suction channel245can create a pressure drop or suction effect that tends to cause the tissue sample to be forced towards the right-hand side of the tissue trap235, thereby becoming lodged within the tissue trap235more forcefully.

FIG. 2Eillustrates a second arrangement202of the microfluidic device ofFIG. 2Ahaving suction channels, according to an illustrative implementation. Components shown in the arrangement202are substantially similar to the components shown inFIG. 2A, and like reference numerals refer to like elements. However, the arrangement202ofFIG. 2Ediffers from that shown inFIG. 2Ain that the arrangement202includes two suction channels245aand245b. The suction channels245aand245bare coupled between a downstream end of the tissue trap235and the branch channels240aand240b, respectively. In some implementations, the suction channels245aand245bcan be configured to facilitate trapping of the tissue sample within the tissue trap235, in a manner similar to that of the suction channel245shown inFIG. 2D. For example, as the fluid sample flows from left to right in the depiction ofFIG. 2E, through the branch channels240aand240b, the suction channels245aand245bcan create a pressure drop or suction effect that tends to cause the tissue sample to be forced towards the right-hand side of the tissue trap235, thereby becoming lodged within the tissue trap235more forcefully. In addition, because the suction channels245aand245bcouple directly to a downstream end of the tissue trap235, the suction channels245aand245bcan provide an outlet for fluid in the tissue trap235. Therefore, in some implementations the suction channels245aand245bmay sometimes be referred to as outlet channels.

Similarly,FIG. 2Fillustrates a third arrangement203of the microfluidic device ofFIG. 2Ahaving suction channels245aand245b, according to an illustrative implementation. The arrangement203ofFIG. 2Fis similar to the arrangement202ofFIG. 2E, with the exception that the suction channels245aand245bin the arrangement203couple to a junction of the branch channels240a,240b, and the outlet channel225. However, the suction channels245aand245bin the arrangement203serve a similar purpose to that described above in connection withFIG. 2E. That is, as the fluid sample flows from left to right in the depiction ofFIG. 2F, through the branch channels240aand240band into the outlet channel225, the suction channels245aand245bcan create a pressure drop or suction effect that tends to cause the tissue sample to be forced towards the right-hand side of the tissue trap235, thereby becoming lodged within the tissue trap235more forcefully. The suction channels245aand245bcouple directly to a downstream end of the tissue trap235, thereby providing an outlet for fluid in the tissue trap235. Therefore, in some implementations the suction channels245aand245bmay sometimes be referred to as outlet channels.

FIG. 3Aillustrates a cross-sectional view of a portion of an example microfluidic device300that can be used to implement the microfluidic device ofFIG. 1A, according to an illustrative implementation.FIG. 3Billustrates a perspective view of the portion of the microfluidic device300shown inFIG. 3A. The features of the microfluidic device300generally correspond to the features of the microfluidic device100, and like reference numerals refer to like elements. For example, the microfluidic device300includes an inlet channel315, a tissue trapping region320, and an outlet channel325.FIGS. 3A and 3Bshow the structural details of the tissue trapping region320, which in this example includes a ribbed channel coupled between the inlet channel315and the outlet channel325. The ribbed channel includes ribs, such as the ribs355a-355c(generally referred to as ribs355), that project into the ribbed channel. The ribbed channel also defines tissue traps335a-335c(generally referred to as tissue traps335).

In general, each of the tissue traps335has sidewalls defined by a subset of the ribs355. As shown, the bottom wall of each tissue trap335is positioned at a lowest depth of the ribbed channel, which is lower than the bottom wall of the inlet channel315and the outlet channel325. While the depiction ofFIG. 3Ashows the ribbed channel defining three tissue traps355, it should be understood that, in other implementations, the ribbed channel may include any number of ribs355defining any number of tissue traps335without departing from the scope of this disclosure.

Similar to the tissue trapping region220shown inFIG. 2A, the tissue trapping region320(including the tissue traps335) can be configured to trap a tissue sample in a fixed location while a fluid sample is flowed through the microfluidic device300. For example, the tissue trapping region320is shaped such that, when the fluid sample flows through the microfluidic device300, the tissue sample becomes trapped in the tissue traps335. In some implementations, a separate tissue sample can become trapped in each of the tissue traps335. In some other implementations, one or more of the tissue traps335may remain unused for a given experiment.

In some implementations, the ribbed shape of the tissue trapping region320, including the tissue traps335, is selected to facilitate trapping of a tissue sample while the fluid sample passes through the microfluidic device300.FIG. 3Cis a visual depiction352of the flow characteristics of the microfluidic device300ofFIG. 3A, according to an illustrative implementation. The shading within the channels shows the velocity of the streamlines within the microfluidic device300. Generally, a tissue sample will be larger and heavier than other particles that flow through the device300within the fluid sample. As a result, the tissue sample will tend to sink within the flow due to gravity. Thus, positioning the tissue traps335at the lowest depth of the ribbed channel, which includes small obstructing ribs355, can help to cause the tissue sample to become trapped within one of the tissue traps335.

It should be understood that the microfluidic device300can include any of the features and functionality described above with respect to the microfluidic device100and the microfluidic device200shown inFIGS. 1A and 2A, respectively. For example, the microfluidic device300can be formed from a material that is transparent and optically clear in the region of the device near the tissue traps335, which can serve as an optical interface that can be examined by an optical instrument brought into proximity with the microfluidic device300. As a result, the tissue samples and the fluid sample in the tissue traps335can be observed optically over time.

It should be understood that, in the implementation shown inFIG. 3A, the outlet channel325serves as an outlet for the microfluidic device100as a whole, and also for the tissue trapping region120. In some implementations, although not illustrated inFIG. 3A, the microfluidic device300also may include one or more additional channels that serve as outlets for fluid at or near the tissue trapping region320, which may also be referred to as outlet channels. For example, such channels may be branch channels or suction channels similar to those described above in connection withFIGS. 2D-2F.

FIG. 4Aillustrates a cross-sectional view of a portion of an example microfluidic device400that can be used to implement the microfluidic device100ofFIG. 1A, according to an illustrative implementation. The features of the microfluidic device400generally correspond to the features of the microfluidic device100, and like reference numerals refer to like elements. For example, the microfluidic device400includes an inlet channel415, a tissue trapping region420, and an outlet channel425.FIG. 4Ashows the structural details of the tissue trapping region420, which in this example includes a circuitous channel coupled between the inlet channel415and the outlet channel425. The circuitous channel includes a first curved portion460aand a second curved portion460b(generally referred to as curved portions460). The curvature of the first curved portion460ais opposed to the curvature of the second curved portion460b. The first curved portion460aincludes a first tissue trap435apositioned at its center. The second curved portion460bis coupled to a downstream end of the first curved portion460a, and includes a second tissue trap435bpositioned at its center. The first tissue trap435aand the second tissue trap435bare generally referred to as tissue traps435in this disclosure. The downstream end of the second curved portion460bis coupled to the outlet channel425.

While the depiction ofFIG. 4Ashows the circuitous channel as including two curved portions460aand460b, it should be understood that, in other implementations, the circuitous channel may include any number of curved portions each defining a respective tissue trap435without departing from the scope of this disclosure. For example, the circuitous channel may include only a single curved portion (i.e., the first curved portion460a), or may include three or more curved portions.

Similar to the tissue trapping regions220shown inFIG. 2A and 320shown inFIG. 3A, the tissue trapping region420(including the tissue traps435) can be configured to trap a tissue sample in a fixed location while a fluid sample is flowed through the microfluidic device400. For example, the tissue trapping region420is shaped such that, when the fluid sample flows through the microfluidic device400, a respective tissue sample can become trapped in the tissue traps435. In some implementations, a separate tissue sample can become trapped in each of the tissue traps435. In some other implementations, one or more of the tissue traps435may remain empty.

In some implementations, the circuitous shape of the tissue trapping region420, including the tissue traps435, is selected to facilitate trapping of a tissue sample while the fluid sample passes through the microfluidic device300.FIG. 4Bis a visual depiction452of the flow characteristics of the microfluidic device400ofFIG. 4A, according to an illustrative implementation. The shading within the channels shows the velocity of the streamlines within the microfluidic device400. Generally, a particle (such as a tissue sample) in the fluid sample will tend to follow the streamline located at its center of mass. If the Reynolds number of the tissue sample is sufficiently large, the inertia of the particle will overcome the viscous forces when the streamlines bend along the circuitous path including the curved portions460of the tissue trapping region420. As a result, the tissue sample will tend to become secured within the one of the tissue traps435.

FIG. 4Cillustrates a first arrangement401of the microfluidic device400ofFIG. 4Ahaving a suction channel, according to an illustrative implementation. Components shown in the arrangement401are substantially similar to the components shown inFIG. 4A, and like reference numerals refer to like elements. However, the arrangement401ofFIG. 4Cdiffers from that shown inFIG. 4Ain that the arrangement401includes only a single tissue trap435a, as well as a suction channel465. The suction channel465is coupled between the tissue trap435aand the outlet channel425. In some implementations, the suction channel465can be configured to facilitate trapping of the tissue sample within the tissue trap435a. For example, as the fluid sample flows from left to right in the depiction ofFIG. 4D, into the outlet channel425, the suction channel465can create a pressure drop or suction effect that tends to cause the tissue sample to be forced towards the right-hand side of the tissue trap435a, thereby becoming lodged within the tissue trap435amore forcefully.FIG. 4Dillustrates the flow characteristics of the microfluidic device401ofFIG. 4C, according to an illustrative implementation. As described in the flow characteristic figures above, the shading inFIG. 4Cshows the velocity of the streamlines within the microfluidic device401. In addition, because the suction channel465couples directly to a downstream end of the tissue trap435a, the suction channel465can provide an outlet for fluid in the tissue trap435a. Therefore, in some implementations the suction channel465may sometimes also be referred to as an outlet channel.

FIG. 5illustrates a bubble trapping structure110that can be included in the microfluidic device100ofFIG. 1A, according to an illustrative implementation. Generally, the bubble trapping structure110can help to facilitate the capture of air bubbles from within the fluid sample that flows through the microfluidic device100, whose presence may be undesirable. Bubbles can be introduced into the microfluidic device100, for example, during the tissue loading process or via the incoming flow of the fluid sample. In some implementations, bubbles can negatively impact experimental outcomes. Therefore, it may be desirable to prevent air bubbles from entering the system, or to remove them before they reach the tissue sample downstream. Incorporation of an in-line bubble trapping structure110into the microfluidic device100allows for easy removal of air introduced by either mechanism

As shown, the microfluidic device100is coupled to a ceiling of the inlet channel115. The bubble trapping structure110includes sidewalls that curve inwards toward each other in a direction away from the inlet channel115. As shown inFIG. 1A, the bubble trapping structure110can be positioned downstream from the inlet port105, such that air bubbles introduced through the inlet port105can be removed via the bubble trapping structure110before they reach the tissue trapping region120. In some implementations, the shape of the sidewalls of the bubble trapping structure110can be defined by a parabolic function. The microfluidic device100also includes a threaded connector510. The threaded connector510can be configured for attachment to an air line, through which air bubbles can be removed from the device after being captured by the bubble trapping structure110.

The bubble trapping structure110is incorporated directly into the microfluidic device100. This design eliminates the need for an external air removal device, thereby reducing the number of required connections. Additionally, inclusion of the bubble trapping structure110within the microfluidic device100can reduce the overall fluid volume requirement. In some implementations, the bubble trapping structure110can be configured to produce limited disruption of the primary flow path of the fluid sample through the inlet channel115. For example, the parabolic curvature of the bubble trapping structure110can encourage the gentle removal of bubbles from the flow, and the threaded connector510, which can couple to an air line or a syringe, allows evacuation of air from the chimney as needed.

In some implementations, the bubble trapping structure110also can be configured to serve as the loading port for the tissue sample. For example, the opening of the bubble trapping structure110can be configured to accommodate a pipette tip through which the tissue sample is introduced into the microfluidic device100. In some implementations, the tissue sample can be injected through the bubble trapping structure110, which may include a valve that can be closed after that tissue sample is injected. Flow of the fluid sample from the inlet port105can then cause the tissue sample to flow towards the tissue trapping region120, where it becomes secured in place as described above.

FIG. 6illustrates a flowchart of a method600for evaluating an interaction between a tissue sample and a fluid sample, according to an illustrative implementation. In some implementations, the method600can be carried out using a microfluidic device such as the microfluidic device100shown inFIG. 1A. In brief overview, the method600can include introducing a tissue sample into an inlet channel of a microfluidic device (step605), introducing a fluid sample into the inlet channel to cause the tissue sample to flow to a tissue trapping region of the microfluidic device (step610), collecting the sample fluid from one or more channels providing an outlet downstream from the tissue trapping region (step615), and observing an interaction between the tissue sample and the fluid sample in the tissue trapping region (step620).

Referring again toFIG. 6, the method600can include introducing a tissue sample into an inlet channel of a microfluidic device (step605). In some implementations, the tissue sample can be or can include a portion of a tumor or other cancerous cells whose reaction to an immunotherapy is of interest. The tissue sample can be injected into the microfluidic device, for example via a port configured to serve as a bubble trapping structure similar to that shown inFIG. 5. In some implementations, the inlet channel can first be primed with a fluid before the tissue sample is introduced. This can allow the tissue sample to be introduced directly into a fluid, which may help to better preserve the tissue sample for experimentation.

The method600also can include introducing a fluid sample into the inlet channel to cause the tissue sample to flow to a tissue trapping region of the microfluidic device (step610). In some implementations, the fluid sample can include cells, medications, therapeutic compounds, or other components. In some implementations, the fluid sample can be introduced at an area of the inlet channel upstream from the area where the tissue sample was introduced. For example, referring to the microfluidic device100ofFIG. 1A, the tissue sample can be introduced via the bubble trapping structure110, and the fluid sample can be introduced at the inlet port105, upstream from the bubble trapping structure110. This tissue sample and fluid sample introduction technique can help to ensure that the fluid sample is able to carry the tissue towards the tissue trapping region, which can be downstream from the areas in which both the fluid sample and the tissue sample are introduced.

In some implementations, the tissue trapping region can include at least one tissue trap configured to trap the tissue sample. The tissue trap can include an intersection or junction of one or more fluidly connected channels, cavities, spaces, or chambers. In some implementations, the geometry of the tissue trap can result in a stagnation zone configured such that the fluid flow characteristics in the stagnation zone are relatively stagnant (i.e., fluid velocity is lower, and in some cases may be zero) as compared with the fluid flow characteristics of other portions of the microfluidic device.

In some implementations, the tissue trap can be positioned at an intersection of a relatively large inlet channel and one or more relatively smaller branching channels that carry fluid away from the tissue trap to an outlet channel, for example as illustrated by the tissue trap235shown inFIG. 2A. Other structural features also may contribute to the functionality of the tissue trap. For example, in some implementations the tissue trap can include an elevation change relative to the channels that couple to it, such that tissue trap serves as a sunken pocket for receiving and securing the tissue sample. As a result, in some implementations, the tissue trap may sometimes be referred to as a tissue trapping pocket. In some implementations, other walls of the tissue trap also may be stepped away, stepped up or stepped down from the walls of channels that lead to them. For example, a ceiling of the tissue trap may be positioned at an elevated height relative to the ceiling of the inlet channel, and the sidewalls of the tissue trap may be farther apart from one another than the sidewalls of the inlet channel.

In addition, the branching channels carrying fluid away from the tissue trap, as well as the outlet channel, can have a size that helps to trap the tissue sample within the tissue trap. For example, the branching channels and the outlet channel can be sized such that tissue samples larger than about 300 microns cannot progress to the outlet of the microfluidic device from the tissue trap. Thus, the tissue sample can become secured within the tissue trap, such that the cells in the fluid sample can contact the tissue sample as the fluid sample flows through the microfluidic device.

In some implementations, the tissue trap or trapping zone can have a geometry that is selected and/or arranged to trap the tissue sample without damaging the tissue sample. The tissue trap or trapping zone may be formed in any geometrical shape or combination of geometries. The tissue trap may be formed as a chamber or portion of a chamber and in some implementations may be referred to as a trapping chamber. The tissue trap may be formed as any type of pocket, such as a partial pocket or a covered pocket, and in some implementations may be referred to as a trapping pocket. The tissue trap may be formed as any type of cavity and may be referred to as a trapping cavity in some implementations. The tissue trap may be designed, configured and formed such as to provide a pressure drop or suction effect with respect to fluid sample flows traversing an opening of the tissue trap and in some implementations, may be referred to as a pressure drop trap, suction trap or tissue pressure drop zone or tissue suction zone.

The tissue trap may be formed as an arrangement of one or more walls. The one or more walls may be selected designed or configured with predetermined heights and/or lengths and/or widths, such as in relation to any of the dimensions of the device comprising the tissue trap. The one or more walls may be formed to meet at predetermined angles and/or predetermined points, such as in relation to any of the dimensions or geometries of the device comprising the tissue trap. The one or walls may be formed to be at predetermined orientations with respect to other walls and/or other walls of the device comprising the tissue trap. For example, the tissue trap can include one or more walls configured to secure the tissue sample. The walls may be formed from the edges of channels that are in fluid communication with the tissue trap, or may be formed from the edges of the tissue trap itself. In some implementations, a wall included in a tissue trap can be a sidewall, a bottom surface, or a ceiling. In some implementations, the tissue trap may include a curved wall, or may include two or more substantially flat walls that couple to one another at an edge. A wall included in a tissue trap can be configured to restrict the motion of a tissue sample without shearing, tearing, or otherwise damaging the tissue sample, in contrast to other types of structures that may be designed to trap a tissue sample. For example, while a series of narrow posts may be used to secure a tissue sample at a particular point within a microfluidic device, the relatively small width of such posts relative to the width of the tissue sample can cause the tissue sample to become torn by the posts as fluid pressure is exerted on the tissue sample by the fluid flowing through the device. Because a wall has a larger surface area than such a post, the tissue traps described in this disclosure can secure a tissue sample while substantially reducing the risk that the tissue sample will become torn or damaged.

In some implementations, a tissue trap also may include one or more channels, such as suction channels, that exit from a rear surface of the tissue trap and join with branching channels and or an outlet channel downstream from the tissue trap. Examples of such suction channels are illustrated in by the suction channels240aand240bofFIGS. 2D-2Fand the suction channel465ofFIG. 4C. As fluid flows through the microfluidic device, such suction channels can cause a pressure drop or other suction force to more securely trap a tissue sample within the tissue trap. Thus, in some implementations, the tissue trap may be referred to as a suction trap. Examples of suitable geometries for such a tissue trap have been described above, for example in connection withFIGS. 1A, 2A, 3A, and 4A.

The method600also can include collecting the sample fluid from one or more channels providing an outlet downstream from the tissue trapping region (step615). In some implementations, the microfluidic device can include an outlet port coupled to an outlet channel and configured to allow the fluid sample to be collected. For example, the outlet port can include a threaded connector, which can be coupled to a fluid line or a syringe to extract the fluid sample. In some implementations, the air bubbles also can be extracted from the fluid sample. For example, air bubbles can be extracted via a bubble trapping structure such as the bubble trapping structure110shown inFIG. 5. In some implementations, the bubble trapping structure can be positioned upstream from the tissue trapping region, such that air bubbles can be extracted from the fluid sample before they reach the tissue trapping region.

In some implementations, the method600also can include reintroducing the collected sample fluid into the inlet channel of the microfluidic device. That is, the fluid sample can be recirculated one or more times through the microfluidic device. For example, the fluid sample can be introduced into the microfluidic device at step610and can be collected at step615. Then, the same fluid sample can be recirculated through the microfluidic device by reintroducing the fluid sample back into the inlet channel of the microfluidic device, and again collecting the fluid sample from the one or more channels providing the outlet. In some implementations, steps610and615of the method600can be iterated any number of times.

The method600also can also include observing an interaction between the tissue sample and the fluid sample in the tissue trapping region (step620). Because the microfluidic device as described in this disclosure can be configured to simulate the dynamics of tissue-cell interactions that occur in vivo, the observation of the interaction between the tissue sample and the fluid sample can provide valuable insights into the way in which a patient will respond to a particular immunotherapy. In some implementations, the microfluidic device can be formed form a transparent and/or optically clear material, and can be sufficiently thin to permit observation of the interaction between the tissue sample and the fluid sample by external equipment. For example, the microfluidic device can include an optical interface positioned near the tissue trapping region, to allow a microscope, camera, or other optical equipment to be used to observe the interaction that takes place in the tissue trapping region from outside of the microfluidic device. In some implementations, at least one the tissue sample and the fluid sample can include fluorescent particles that may be observed by such optical equipment.

In some implementations, the method600also can include releasing the tissue sample from the tissue trap. To release the tissue sample, in some implementations a second fluid sample can be introduced into the one or more channels providing the outlet. This can cause the second fluid sample to flow towards the inlet channel. This reverse flow of fluid can exert fluid forces on the tissue sample within the tissue trap that tend to dislodge the tissue sample from the tissue trap. In some implementations, the tissue sample may be brought to an inlet port of the microfluidic device in this manner, and may be collected and removed from the device at the inlet port.