Readily manufacturable flow channel devices

Abstract

The disclosure provides flow inserts and flow devices. The flow inserts provide a flow path over cells growing on an ultrathin membrane disposed in a microphysiological system (MPS), providing fluidic flow conditions to cells under the flow path. In some embodiments, the disclosure provides "half flow inserts" that cover cells growing on a first ultrathin membrane but not those growing on a second ultrathin membrane on the same membrane chip, allowing cells on the covered ultrathin membrane to grow under fluidic flow conditions, while cells on the second, uncovered ultrathin membrane grow in a static culture medium. The invention further provides flow devices that allow providing fluidic flow conditions on standard optically-clear laboratory equipment, such as microscope slides, that do not ordinarily have such conditions available

Description

READILY MANUFACTURABLE FLOW CHANNEL DEVICESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 523,341, filed June 26, 2023, the contents of which are incorporated herein by reference in their entirety.STATEMENT OF FEDERAL FUNDING

[0002] This invention was made with government support under UH3 TR003281 and R33 HL154249, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] For decades, scientists have studied biological functions by use of cell cultures in in vitro systems, followed by more expensive, but usually more informative, studies in animal models. Unfortunately, both cell culture systems and animal models have significant drawbacks. Cell culture systems typically use primary patient samples or cell lines of a single cell type, and by their nature are incapable of recapitulating the interactions between cell types in an organ. Animal models are not only costly, making it hard to scale them for screening, but can give information irrelevant for humans due, for example, to differences in enzymes or pathways in the animal used for the model and those present in humans. The problems presented by such systems contribute to, among other things, the loss of hundreds of millions of dollars in preclinical development and testing of potential therapeutics that then fail in clinical trials.

[0004] Over a decade ago, Huh et al. reported the development of a hybrid approach that could provide information at the level of a tissue or organ, as opposed to the cell level. See, Huh et al., Science, 2010, 328:1662-1668. Such “tissue chips” and “organs-on-a-chip” are microfabricated devices that support multicellular cultures of human cells interacting in microenvironments that more realistically resemble tissue. These tissue chip and “organ-on-a-chip” technologies (also referred to as “microphysiological systems,” or “3D cell culture”), are intended to fill some of the gaps between simple cell cultures and in vivo animal studies, addressing some of the deficiencies in both. For example, in 2015, Huh reported on developing a “lung-on-a-chip” model using “soft lithography-based microfabrication techniques to construct a compartmentalized three-dimensional microchannel system consisting of upper and lower cell culture chambers separated by a 10-pm-thick microporous elastomeric membrane made of poly-(dimethylsiloxane).” (Huh, Ann Am Thorac Soc. 2015; 12(Suppl 1): S42-S44.doi: 10.1513 / AnnalsATS.201410-442MG). Human alveolar epithelial cells were seeded into the upper chamber and pulmonary microvascular endothelial cells were seeded onto the lower chamber and both types of cells were allowed to adhere to their respective side of the membrane. Huh reported that the system allowed investigation of the interplay between the different types of cells on the two sides of the membrane when one side was exposed to a stimulus, such as the introduction of proinfl ammatory cytokines. Id.

[0005] During the decade that tissue chips and organ-on-a-chip systems have been available, they have been explored as alternatives for simple cell culture systems. While they represent an advance over single-layer cell cultures, however, several significant deficiencies have become evident.

[0006] In particular, there exists a lack of effective methods for analyzing the response of tissue chips and organ-on-a-chip systems to changes in their environment. Analysis is currently limited largely to methods such as lysing the cells on the chip or subjecting them to immunofluorescence microscopy. Immunofluorescence-based assays, such as ELISAs, however, are irreversible by nature, meaning that once the measurement is taken, the experiment is over. Thus, deciphering time courses of analyte secretion or passage through the barrier used to constrain the cells on the chip requires many resources to repeat the experiment at each time point, increasing the time, cost, and variability of such studies. Tracking changes to the tissues or organ disposed on a chip requires multiple chips run in parallel so that a chip is available to be subjected to an experiment-ending analysis at each time point for which information is desired. It would be desirable to be able to assess the behavior of the organ-on-a-chip in real time in a nondestructive manner. While some investigators have tried to address this problem by integrating sensors substantially downstream of the system under study, this creates its own problems by decoupling the sensor from the organ-on-a-chip both temporally and spatially. Additionally, studies of cellscultured in current tissue chips is hampered by the fact that it is difficult to access the cells themselves.

[0007] Earlier work by some of the present inventors reported the development of tissue chips that have on-board photonic integrated circuits, or “PIC.” See, e.g., International Patent Application No. PCT / US2021 / 058498, published as Publication No. WO 2022 / 099161. This application further disclosed the use of devices bearing on-board PICs to monitor changes to tendon cells, known as tenocytes.

[0008] The need for organs-on-a-chip and tissues-on-a-chip that recapitulate aspects of biology is only increasing. As of this year, 2023, the FDA is permitted to base an IND approval on results from methodologies, such as organs-on-a-chip, that do not use animal models. See, Wadman, M., Science, 379(66281:127-128 (2023). Accordingly, even incremental improvements that allow better monitoring of the effects of agents on tissues or organ models can have significant value in predicting the safety and efficacy of drug candidates and whether they should be advanced into clinical trials.

[0009] It would be desirable to have methods and devices that improve the ability of practitioners to determine the effects of potential therapeutic agents on cells on a tissue chip or organ-on-a-chip that are non-destructive and that do not decouple sensors in time and space from the tissue chip or organ-on-a-chip. And, it would be useful to have devices and methods that allow simulating blood or fluid flow across cells to better simulate the physiological environment in which cells exist in the body. Surprisingly, the present invention fulfills these and other needs.BRIEF SUMMARY OF THE INVENTION

[0010] In a first set of embodiments, the invention provides flow inserts for a microphysiological system (MPS) having a well in a top compartment of said MPS sized to accept a 5.4 mm x 5.4 mm membrane chip bearing one ultrathin membranes or or a pair of ultrathin membranes, said flow insert comprising: (a) a body having a top, sides, and a bottom, which bottom has a perimeter, (b) a flow channel defined by a continuous open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel, (c) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and (d) a second port fluidlyconnected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert (i) is 5.4 mm x 5.4 mm or smaller, and (ii) has disposed around its perimeter a pressure-sensitive adhesive that bonds to low surface energy materials. In some embodiments, the pressure-sensitive adhesive is covered by a protective coating. In some embodiments, the protective coating is a film. In some embodiments, the pressure-sensitive adhesive is non-toxic to human cells. In some embodiments, the human cells are selected from bronchial epithelial cells and brain endothelial cells. In some embodiments, the pressure-sensitive adhesive is a silicone pressure-sensitive adhesive. In some embodiments, the pressure-sensitive adhesive is an acrylic rubber pressure-sensitive adhesive. In some embodiments, the pressure-sensitive adhesive is a natural rubber pressure-sensitive adhesive. In some embodiments, the pressuresensitive adhesive is a synthetic rubber pressure-sensitive adhesive. In some embodiments, the membrane chip has a pair of ultrathin membranes and said flow insert is sized to cover only one of said ultrathin membranes. In some embodiments, the flow insert is sized to cover one of half of the area of said bottom of said well, ±20%. In some embodiments, the flow insert is sized to cover half of the area of said bottom of said well, ±10%. In some embodiments, the flow insert is sized to cover half of the area of said bottom of said well.

[0011] In a second group of embodiments, the invention provides flow devices to introduce fluidic flow over a top surface of a piece of laboratory equipment, said top surface of said laboratory equipment being optically clear, said flow device comprising: (a) a body having a top, sides, and a bottom, which bottom has a perimeter, (b) a flow channel defined by an open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel, (c) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and (d) a second port fluidly connected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert has disposed around said perimeter a pressure-sensitive adhesive that bonds to low surface energy material. In some embodiments, the pressure-sensitive adhesive is covered by a protective coating. In some embodiments, the pressure-sensitive adhesive is non-toxic to human cells. In some embodiments, the human cells are selected from vascular endothelial cells and brain endothelial cells. In some embodiments, the pressure-sensitive adhesive is a natural rubber pressure-sensitive adhesive. In some embodiments, the pressure-sensitive adhesive is asynthetic rubber pressure-sensitive adhesive. In some embodiments, the pressure-sensitive adhesive is an acrylic pressure-sensitive adhesive. In some embodiments, the pressuresensitive adhesive is a silicone pressure-sensitive adhesive. In some embodiments, the laboratory equipment is a glass microscope slide. In some embodiments, the laboratory equipment is a cover slip. In some embodiments, the laboratory equipment is a well of a tissue-culture plate. In some embodiments, the flow insert has a length having a maximum of 25 mm. In some embodiments, the flow channel of said flow insert has a length of no more than 23 mm.

[0012] In another group of embodiments, the invention provides systems for providing fluidic flow over a surface of a piece of laboratory equipment not having said fluidic flow, said systems comprising (a) a flow device for introducing said fluidic flow over a surface of a piece of laboratory equipment, said surface of said laboratory equipment being optically clear, said flow device comprising: (i) a body having a top, sides, and a bottom, which bottom has a perimeter, (ii) a flow channel defined by an open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel, (iii) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and (iv) a second port fluidly connected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert has disposed around said perimeter a pressure-sensitive adhesive that bonds to low surface energy material, (b) a piece of optically clear laboratory equipment used for culturing or observing cells, to which said flow device is bonded by said pressuresensitive adhesive, (c) a means of introducing fluid under a pressure into said first port of said flow device, and (d) a means of receiving fluid from said second port of said flow device. In some embodiments, the pres sure- sensitive adhesive is non-toxic to human cells. In some embodiments, the human cells are selected from vascular endothelial cells and brain endothelial cells. In some embodiments, the pressure-sensitive adhesive is an acrylic pressure-sensitive adhesive. In some embodiments, the pressure-sensitive adhesive is a silicone pressure-sensitive adhesive. In some embodiments, the piece of laboratory equipment is a glass microscope slide. In some embodiments, the piece of laboratory equipment is a cover slip. In some embodiments, the laboratory equipment is a well of a tissue-culture plate.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figs. 1 A-1C. Fig. 1 A. Fig. 1 A shows two views of a flow insert configured to fit into the open well of a top compartment of a microphysiological system (MPS), such as an organ-on-a-chip. Fig. IB. Fig. IB is an exploded view of an exemplar flow insert, showing the layers from which the insert is constructed. Layer LI is the body of the insert. L2 is a double sided piece of silicon pressure-sensitive adhesive (“PSA”) bonding layer L3 to LI. Layer L3 is a layer of a harder material, while L4 is another layer of silicone PSA to bond the flow insert to the surface to which the user wants to apply a fluid flow through the flow insert. Fig. 1C. Fig. 1C is an exploded view of an assembled flow insert, over a chip holding an ultrathin membrane. The chip, in turn, is over the well of the top compartment of a MPS. The top compartment of the MPS is shown over the bottom compartment of the MPS.

[0014] Figs. 2A-2D. Fig. 2A. Fig. 2A shows the protective coating over the pressuresensitive adhesive being peeled off. Fig. 2B. Fig. 2B is a drawing showing an exemplar flow insert before being inserted into the well of a MPS. Fig. 2C. Fig. 2C is a drawing of an exemplar flow insert in position in the well of a MPS.

[0015] Fig. 3. Fig. 3 is a drawing showing a system for providing fluidic flow to cells growing on the ultrathin membrane in the well of a MPS. In the middle of the drawing is a MPS with central well in the top compartment. A membrane chip holding an ultrathin membrane is disposed at the bottom of the well. A flow insert has been secured to the membrane chip and fluid is provided to cells growing on the membrane within the flow channel of the flow insert. Fluid is provided to the flow insert through the metal tube coming in from the left, which is connected to a hose connected to a fluid reservoir. The fluid is under a pressure sufficient to cause the fluid to flow over the cells and up the port on the right hand side, which has a second metal tube positioned to receive the medium exiting the flow insert and transfer it to a hose connected to a collection vessel or to a sink.

[0016] Fig. 4. Fig. 4 depicts a “half flow insert.”

[0017] Fig. 5. Fig. 5 is a schematic drawing showing a flow insert (solid line) fit over a 5.4 mm x 5.4 mm membrane chip (dashed line). The bump outs on either side are created where flow inserts are attached to each other in some methods of manufacturing arrays of flow inserts and are not part of the functioning of the inserts.DETAILED DESCRIPTION

[0018] As noted in the Background, modeling the reaction of human tissues and organs to candidate therapeutic agents has traditionally been performed using cell cultures or animal models. In recent years, pressure to reduce the use of animals in pre-clinical studies and other factors has increased the need for systems, such as organs-on-a-chip (sometimes referred to herein as “OoCs”) and tissues-on-a-chip (sometimes referred to herein as “ToCs”), that can model various aspects of human biology, such as the response of organs and tissues to candidate therapeutic agents. Such model systems are sometimes referred to as microphysiological systems (sometimes referred herein to as “MPS”). For convenience, the term “MPS” will be used herein to refer to both OoCs and ToCs, unless another use is noted or required by context.

[0019] The present invention provides improvements to such microphysiological systems and other microfluidic devices, allowing them to be manufactured much more easily than prior microphysiological systems and microfluidic devices providing similar characteristics. Further, in some aspects, the improvements surprisingly augment the ability of the systems to recapitulate aspects of cell response to exogenous agents, and the interactions between cells. In some embodiments, the invention provides devices that allow the conversion of a piece of scientific equipment normally used for viewing of cells or microorganisms in a culture medium or other fluid under static conditions, such as a microscope slide or petri dish, into one that allows studying those cells or microorganisms under fluidic conditions (that is, conditions allowing the the culture medium or other fluid in which the cells or microorganisms reside to flow past those cells).

[0020] Co-owned International Patent Application PCT / US2021 / 058498 (the “UR 2021 application”), reported the development of MPSs containing two compartments, an upper compartment and a lower compartment. The upper, or apical, compartment is an open well, which has one or a pair of ultrathin membrane on its bottom. The membrane or membranes separate the upper compartment from the bottom compartment. The ultrathin membrane or pair of membranes is typically provided in a 5.4 mm x 5.4 mm chip, and the well of the upper compartment is sized to be able to tightly accept the chip so that any fluid moving from the upper compartment into the lower compartment has to pass through the ultrathin membrane. The ultrathin membranes, the exposed (freestanding) portion of which within the chip istypically 0.7 mm x 2 mm, are provided as either a single small rectangle held by the chip within the chip’s overall 5.4 mm x 5.4 mm area, or as a pair of small rectangles, positioned parallel to each other, held by the chip within that area.

[0021] While the ultra-thin membrane physically separates the two compartments, it allows soluble factors to diffuse between them. Its pores are, however, too small for cells to pass through unless they actively migrate in response to chemotactic or other factors. The lower compartment of the MPS is a flow channel, with the ultrathin membrane serving as the top of the channel. Cells that normally form barriers between the blood and the cells of an organ or a tissue, such as the tightly packed endothelial cells lining blood vessels in the brain, can be grown on the surface of the ultrathin membrane facing into the upper compartment (the “apical surface” of the membrane), while cells of a tissue or organ of interest, such as neurons, can be grown in the flow path under the membrane which, as noted, is in the lower compartment of the devices. The UR2021 application shows devices in which the fluid in the upper compartment is static, but also shows devices in which the upper compartment is provided with a flow path. In those embodiments, the flow path in the upper compartment is provided by including an additional layer in the device which has a section cut-out to define the flow path. Devices containing such a flow path cannot be converted to study static conditions. Further, such devices cannot be used to simultaneously study two cohorts of cells within the same device, one cohort being subjected to static fluid conditions and the other being subjected to fluidic conditions.

[0022] A publication in 2022 disclosed improvements in MPS systems like those disclosed in the UR 2021 application. The 2022 publication disclosed an insertable flow channel with “plug-and-play” capability that can be inserted onto the ultrathin membrane within the open, upper (“apical”) well of MPS devices, thereby subjecting some or all of the cells on top of the ultrathin membrane to a fluidic culture in which the medium or other fluid flows over the cells in place of the non-moving (static) culture medium conditions to which cells growing on the membrane are normally subjected. See, Mansouri et al., Adv Healthcare Matls., 2022, 11(21): 2200802; doi.org / 10.1002 / adhm. 202200802 (hereafter, “Mansouri 2022”).

[0023] The flow insert has two ports at the top which fluidly connect to an open flow channel at the bottom of the insert. When the flow insert is secured on top of the ultrathin membrane of the apical well of a MPS, flowing fluid into and through a first port of the flow insert causes the fluid to flow through the flow channel at the bottom of the flow insert,where it flows over cells growing on the apical surface of the ultrathin membrane within the area bounded by the open flow channel of the flow insert, and then enters the second port of the insert and exits the second port from the top of the insert. As noted above, the ultrathin membrane is provided as a rectangle, which inherently has a length longer than its width. The flow insert is positioned on top of the ultrathin membrane so that the flow channel of the insert aligns with the length of the rectangle of the ultrathin membrane.

[0024] The flow module, or insert, disclosed in Mansouri 2022 was configured with a magnetic latching assembly to secure the flow insert to the bottom of the interior of the open well of the MPS. One advantage intended by using a magnetic seal for the flow insert was to provide a releasable hold so that the MPS could be transitioned from culturing the cells on the membrane under static conditions to culturing them under fluidic conditions, and back again, allowing the practitioner to model chemical signaling, response to drugs, and response of the cells to shear stresses, thereby more closely recapitulating physiological conditions.

[0025] Since the publication of the 2022 publication, the present inventors have continued working with the Mansouri 2022 flow insert. Unfortunately, they found problems with the flow inserts described in that publication. First, the need to latch magnetically to the bottom of the well of a flow insert requires tight manufacturing tolerances on the parts used, which makes it difficult to manufacture the inserts at scale. Not only does the insert have to be able to accept and hold magnets itself, but the magnets on the flow insert also have to align precisely to magnets on the surface of the apical face of the well. Any misalignment causes the magnets in the flow insert and those on the surface of the well to not be in full contact, and reduces the force of the magnetic attachment holding the flow insert to the apical side of the well.

[0026] Second, magnetic force is inversely related to distance; halving the distance between the magnets on the flow insert and those on the surface of the well therefore increases the magnetic force between them by four times. If the flow insert is not carefully handled as the insert is lowered towards the apical surface of the well, the increase in magnetic force as the magnets in the insert approach the magnets in the well can pull the insert abruptly onto the membrane, causing an sudden increase in air pressure on the membrane that can disrupt the physical integrity of the membrane (the disruption of the membrane is sometimes referred to in the art as “popping” the membrane). The need to carefully control the placement of theflow insert in the well bottom and the possibility of rupturing the membrane complicates automating the manufacturing of MPS devices with pre-installed flow inserts.

[0027] Third, since it is impractical to have magnets disposed continuously around the perimeter of the insert or of the device onto which the insert is being positioned, the insert is not secured to the device onto which the insert is being positioned with equal force at both the positions at which the magnets are present, and those at which it is not. This difference in how strongly the insert is held against the device onto which it has been positioned creates points at which the insert is held less strongly than others, creating the opportunity for leaks to occur at those points.

[0028] Given these problems with the usability of the Mansouri 2022 flow insert, the present inventors experimented with other methods of making flow inserts that could be manufacted and attached to a MPS. Microfluidic chips are commonly made of poly(dimethylsiloxane), often referred to as “PDMS.” PDMS chips are routinely made open-faced and sealed by bonding the PDMS to glass or another impermeable substance by covalent attachment, such as by air plasma. (See, e.g., Gonzalez-Gallardo et al., Microfluid Nanofluid, 2021, v. 25, article no. 20; doi.org / 10.1007 / sl0404-021-02420-3; Sevli and Sahan, protocols. io, dx.doi.org / 10.17504 / protocols.io.byzepx3e).

[0029] The present inventors experimented with bonding PDMS flow inserts to MPS devices by plasma bonding, a process which requires the use of plasma generators. Plasma generators are typically the size of a countertop microwave and can only accommodate one or a few inserts at a time. Further, the time needed to prepare the very clean surfaces needed for successful plasma bonding, and for the plasma bonding itself, makes this technique less suitable for “plug-and-play” uses and less suitable for production at a scale that would permit commercial sale. Bonding flow inserts to MPS or other surfaces by air plasma bonding is therefore more suitable when manufacturing devices with such inserts individually. Further constraints were added by the need to have the flow insert fit into the well of a MPS sized for a typical 5.4 mm x 5.4 mm nanomembrane chip.

[0030] Surprisingly, the present invention provides a solution allowing the ready manufacture of flow inserts that can be used in “plug-and-play” embodiments. The invention avoids the need to carefully align magnets, as with the Mansouri 2022 flow inserts, and the need for plasma bonding often used for bonding PDMS to glass or other impermeable surfaces. It is expected that the inventive inserts can be readily manufactured, in quantitiespermitting commercial sale rather than the bespoke use of previous inserts. The flow inserts of the invention are expected to be particularly useful in modeling physiological fluid flow, which is important in barrier tissue dysfunction, which is present in conditions such as inflammation, sepsis, and fibrotic disease.

[0031] The present disclosure provides flow inserts that secure to the apical surface of a well in a MPS using a pressure-sensitive adhesive, or “PSA.” When the flow insert is created, the PSA is covered by a peelable protective coating. When the protective coating is peeled off, the PSA remains on the flow insert. A MPS with an open well bearing an ultra-thin membrane can then be positioned with its open well facing the now-exposed PSA on the bottom surface of the flow insert, and the flow insert then pressed onto the apical surface of the open well, over the ultra-thin membrane. This causes the PSA to adhere to the apical surface, thereby attaching the flow insert to the MPS and creating a flow channel over the ultra-thin membrane. When the PSA is placed around the perimeter of the bottom of the insert (except, of course, for the open area defining the flow channel between the two ports), it acts not only as an adhesive but also as a seal, preventing leaks of fluid flowing through the flow channel created by the sealing of the flow insert against the apical surface.

[0032] Use of an adhesive to connect two items is of course a common practice. Use of a PSA was not, however, believed to be practical for use in a flow insert. As noted above, the initial use for the flow inserts was to provide a fluidic flow over cells disposed on an ultrathin membrane in the well of a MPS. As noted, the wells are sized to hold a typical 5.4 mm x 5.4 mm chip holding one or a pair of ultrathin membranes. To obtain a flow channel with a length of, for example, 4.4 mm within a well with dimensions of 5.4 mm x 5.4 mm, there is only 0.5 mm of edge material available on either side of the membrane on which adhesive could be positioned. To provide flow of the culture medium over the cells to provide fluidic conditons, a pressure is exerted on the fluid being introduced into a flow insert to drive the fluid over the cells within the flow channel and then back up the exit port of the flow insert. It was believed that the limited surface area available on which to apply an adhesive to a flow insert sized to fit onto a membrane chip would simply not be enough to bond the flow insert to the membrane chip without fluid leaking from the insert under the pressure needed to create the fluidic conditions. That expectation is indeed why the more cumbersome methods of magnetic latching and plasma bonding were tried before bonding the flow insert to the well of a MPS was attempted. Only once those methods were found to have significant drawbacks was the use of an adhesive attempted. To our surprise, PSA was found not only tosecure the inserts to the membrane chip, but also to do so without leaks when PSA that bonds to low surface energy materials was used.

[0033] While flow inserts for use on the apical surface of the well of a MPS needs to fit into the well, and thereby be pressed onto the surface on which it is going to form the flow channel, the tolerances for manufacturing such inserts do not need to be as tight as those needed to align magnets in embodiments in which magnets provide the force holding the flow inserts to the surface of the well. Looser tolerances, lower complexity, and fewer parts (no magnets are needed in either the insert or in the well), results in reducing the cost of making the flow inserts and the time required to make them compared to the magnetic connections of the Mansouri 2022 inserts and that can be made without having to use the plasma generating equipment needed for plasma bonding PDMS inserts to a MPS. The “peel and stick” devices of the invention are therefore expected to allow the easy conversion of the static medium in the open well of a MPS into a fluidic channel, enabling the introduction of perfusion and fluidic shear stress to cells cultured on the ultrathin membrane.

[0034] As noted above, the ultrathin membranes available for use in microphysiological systems come on chips that are 5.4 mm x 5.4 mm, and may bear one rectangluar ultrathin membrane in the middle of the chip, or two rectangular ultrathin membranes aligned parallel to each other. In some embodiments, the flow inserts are designed to fit over the whole area of the membrane-bearing chip. Inserts that cover the entire bottom of the well are sometimes referred to herein as “whole flow inserts” or “full-area flow inserts”.

[0035] In some embodiments, especially those in which there are two ultrathin membranes on the chip, the flow insert is sized to cover just one of the two ultrathin membranes. Cells cultured on the membrane not covered by the flow insert grow in a static medium, while those within the area covered by the flow channel of the flow insert grow in a flowing medium (culturing cells in flowing medium is sometimes referred to herein as growing the cells under “fluidic conditions”), permitting studying how the difference in static vs. fluidic conditions affects cell growth and cell interactions. Flow inserts sized to cover just a portion of a well are typically sized to cover approximately half the surface area of the well and are sometimes referred to herein as “half flow inserts. As noted, “half flow” inserts” are similar to full-area flow inserts, but with a narrower body. Fig. 4 is a drawing depicting a “half flow” insert. When paired with a membrane chip bearing a pair of ultrathin membranes, such inserts allow microphysiological system in which a half flow insert is used to provide a fluidflow (fluidic conditions) to cells on one of the ultrathin membrane, while cells growing on the second ultrathin membrane are in a non-flowing (static) fluid environment. Cells in the fluidic conditions are exposed to shear forces from the fluid flow, simulating the shear forces cells that are exposed to blood flow experience. Cells in the static conditions simulate cells, such as neurons, that are not directly exposed to shear forces from blood flow. In some embodiments, the cells grown in the static medium and in the fluidic conditions are of the same cell type. In some embodiments, the cells in the two conditions are of two different cell types.

[0036] The “whole flow” and “half flow” inserts of the invention therefore allow a MPS designed to provide only static conditions to cells grown on an ultrathin membrane in a well to be readily converted into a MPS that can provide fluidic conditions, or that can provide both static and fluidic conditions to cells grown on the ultrathin membrane or membranes (depending on whether the MPS has a single ultrathin membrane or paired ultrathin membranes).

[0037] In the course of studies underlying the present disclosure, it was realized that the inventive flow inserts had uses beyond simply converting a static well of a MPS to a fluidic well. It was realized that the inventive flow inserts also provide a means for providing a flow channel to any optically-clear lab equipment having with a flat surface impermeable to the fluid to be used, on which cells or microorganisms might be studied, such as a microscope slide, a cover slip, or a well in a tissue culture plate. Adhering one of the inventive devices to the microscope slide or other equipment is as simple as removing the protective coating on the flow insert and pressing the side of the insert bearing the now-exposed adhesive to the lab equipment of choice in the desired position. Further, not all MPS have a well containing an ultrathin membrane, and there is therefore not a slot or well into which a “flow insert” can be inserted. Such an MPS may have an area that is not configured to provide fluidic flow to cells grown in that area, but which is accessible from outside the MPS. Such an MPS can be afforded the ability to provide fluidic flow conditions to over cells in that area of the MPS by securing to that area of the MPS one of the inventive flow inserts.

[0038] In such uses, the inventive devices may not be considered to be called “flow inserts,” as the term “flow insert” implies that the device is to be inserted into the well of an MPS, and there is no slot or recess on lab equipment such as a microscope slide or a cover slip plate into which the inventive device can be inserted. For example, with respect to a microscopeslide or a cover slip, the “flow insert” is actually secured to the top surface of the slide or slip. In these embodiments, the inventive devices are more accurately referred to as “flow devices.” It will be understood, however, that as used herein, the terms “flow insert” and “flow device” refer to the same thing, and the difference in terminology is only intended to only by the use to which the practitioner decides to use it for a particular study. For convenience of reference, this disclosure will generally refer to the inventive devices as “flow inserts” regardless of whether the reference is to using the device as an insert into the well of a MPS or to provide a flow channel to a microscope slide, tissue culture plate, or similar lab equipment, but references to “flow devices” will refer to the same devices as do references to “flow inserts” unless otherwise specified or required by context.

[0039] The inventive devices have a top surface, bottom surface, height between the top surface and the bottom surface, a width defined by exterior sides of the device, a first port and a second port. Each port has an opening at the top of the device, which opening extends vertically from the top surface of the device to the bottom surface of the device, with the first port and the second port fluidly connected across at least a portion of the width of the device by an open channel defined along the bottom surface of the device by interior sides. When the device is secured to a substrate, such as the apical surface of the well of a MPS, the open channel forms a flow channel. Fluid introduced into the first port flows down the length of the first port to the flow channel, through the flow channel, and up the length of the second port, where it can exit the device. To permit the fluid to flow through the device from the first port, across the flow channel, and out through the the second port, a pressure is provided, which pressure is regulated to cause the fluid to flow across the flow channel at a rate desired by the practitioner. For convenience of reference, the first port, through which a fluid is introduced into the device is sometimes referred to herein as the “input port,” while the second port, through which the medium or other fluid exits the flow insert or other device of the invention is sometimes referred to herein as the “exit port.” The practitioner can, of course, choose to reverse the direction of the fluidic flow at any time simply by reversing which of the two ports is used as the input port, with the port originally serving as the input port becoming the exit port.

[0040] Both the full-area flow insert and the half-area flow insert can be inserted into a MPS in minutes, change the device from one that cultures cells on the membrane from static culture conditions to fluidic flow culture conditions, and provide sufficient gas exchange for cell culture. Testing of the flow inserts demonstrated leak-proof operation, and appropriateshear stress conditions on the cells in the flow channel, as evidenced by, for example, morphological alignment of cells along the flow axis. In preferred embodiments, the flow insert is made of a biocompatible material.Studies underlying this disclosure using cells cultured on ultrathin membranes under static conditions without the flow insert showed that the cells did not do well when the flow insert was placed over the existing culture and then subjected to fluid flow. Users of the inventive flow inserts choosing to provide fluidic conditions to cells on the ultrathin membrane of a MPS or on a piece of lab equipment, such as a microscope slide, should therefore first secure the flow insert to the MPS or other piece of lab equipment and then flow cells into the flow channel of the flow insert to seed the cells in the flow channel. This is conveniently done by placing the cells in a selected medium, such as a compatible cell culture medium, and flowing the medium containing the cells into the input port of the flow insert until the desired cell density is reached in the flow channelSizes and Materials

[0041] The flow insert is conveniently made of several components. A typical configuration is shown in an exploded view in Fig. 2. In Fig. 2, the largest component is labeled as “LI,” which in the exemplar embodiment shown is made of PDMS. In this example, LI is a piece of PDMS that is 3 mm thick and that has cylindrical openings extending through the otherwise solid PDMS on the left and right that will become the ports when the device is completed. A second layer, L2, made of silicone coated with a pressure sensitive adhesive (“PSA”) on both sides attaches LI to a layer of a harder material (labeled L3), in this case, polyethylene terephthalate, or “PET.” L3 is in turn connected to a second layer of silicone coated with PSA, L4. Once assembled, the PSA on the bottom surface of L4 is covered with a peelable material (not shown), which protects the PSA until the practitioner wishes to secure the device to the well of a MPS or other piece of equipment. As can be seen, each of layers L2, L3, and L4 has a central channel which, when the four layers are assmbled, aligns to form a flow channel fluidly connecting the port on the left side of LI to the port on the right side of LI. When the assembled device is secured to the bottom of the well of a MPS or another piece of equipment, the flow channel is closed off on the bottom.

[0042] For embodiments in which a whole-area flow insert is to be inserted into the well of a MPS device intended to hold an ultrathin membrane chip, and for whole-area flow insertsused as flow devices, the inserts are sized to fit over a 5.4 mm x 5.4 mm membrane chip (references below to “chips” refer to chips bearing an ultrathin membrane).

[0043] Fig. 5 shows an exemplar full-area insert which has been secured to a standard 5.4 mm x 5.4 mm chip with the membrane in the chip visible under the flow insert. In the exemplar insert shown, the two ports each have a diameter of 0.8 mm, with the centers of the ports 3.5 mm apart. The two ports are within the overall length of the flow channel, which in the insert shown is 4.4 mm, leaving 0.5 mm of the insert on each end of the insert (that is, to the left of the left-hand port and to the right of the port on the right side). That 0.5 mm of the insert on each side (and the rest of the perimeter of the insert) is covered by a PSA that is bonding the insert to the chip below it. In this exemplar chip, there is 0.4 mm between the interior edge of the port (the edge of the port closest to the center) and the short edge of the ultrathin membrane in the chip. The size of the ports can be varied by ± 20%, ± 15%, ± 10%, or by ± 5%, with each term to the right being preferred to any term to its left. The flow channel can be shorter, but should not be longer when used with a 5.4 mm x 5.4 mm chip, as a minimum of 0.5 mm of bonding space needs to be available on the insert to the left and right of the flow channel so that the PSA can secure the insert to the material forming the chip without leakage. The channel height in this example is 0.2 mm, with a total channel volume of 4.23 pl.

[0044] The flow inserts are preferably made of a biocompatible material. In some preferred embodiments, the biocompatible material is polydimethylsiloxane, or “PDMS.” In some embodiments, the biocompatible material for LI is acrylic. In some embodiments, the biocompatible material for LI is cyclic olefin copolymer (“COC”). In some embodiments, the biocompatible material for LI is acetal. In some embodiments, the biocompatible material for LI is polycarbonate. In some embodiments, the biocompatible material for LI is glycol- modified PET, or “PETG”. In some embodiments, the biocompatible material for LI is polyimide. In some embodiments, the biocompatible material for LI is fluorinated ethylene propylene (“FEP”). In some embodiments, the biocompatible material for LI is polytetrafluoroethylene (“PTF”). In some embodiments, the biocompatible material for LI is polystyrene. In some embodiments, the biocompatible material for LI is polypropylene. In some embodiments, the biocompatible material for LI is silicone. Each of Layers 2-4 can be independently selected from one of these materials.

[0045] The flow devices are conveniently used with standard laboratory equipment used to culture or to observe cells, such as glass slides, cover slips, and wells of tissue culture plates (“TCPs”). Glass slides typically are rectangles with a length of 75 mm (2.95 inches) x 25 mm (0.984 inches), while cover slips are typically 25 mm x 25 mm squares. In some embodiments, the flow devices used on glass slides or cover slips are the same size as those designed for the wells of a MPS. In some embodiments, larger flow inserts can be used with glass slides or cover slips, as their larger size permits. It is contemplated that the maximum size for a flow device will be 25 mm so that the flow device can be used on both glass slides and cover slips. As the pressures used to push culture medium through a longer flow channel may need to be somewhat higher than the pressure needed to push culture medium through a 4.4 mm flow channel, the flow channel of a 25 mm flow device should be no more than 23 mm so that there can be 1 mm of material on which PSA can be positioned on either side of the flow channel.

[0046] TCPs are typically configured with a plurality of flat bottom cylindrical wells, with the wells in TCPs having more wells typically having a smaller diameter than the wells of TCPs with fewer wells. It is contemplated that the inventive flow devices can be used with TCPs having six or fewer wells. The wells of TCPs with six wells, for example, typically have a diameter of 6.5 mm, which is large enough to accept a flow insert of dimensions that fit into the well of a MPS device that accepts a 5.4 mm x 5.4 mm ultrathin membrane chip. The flow devices are contemplated for use with TCPs that are optically clear, as opposed to TCPs that are white or black used in studies involving fluorescence.Pressure Sensitive Adhesives and Protective Covers

[0047] The flow inserts or flow devices attach to the MPS or other lab equipment using pressure sensitive adhesives, or “PSAs,” which are covered until the time of use by a protective, peelable material, such as a film.

[0048] PSAs were developed over 50 years ago and their characteristics are well known in the art. See, generally, Czech, Z., “Pressure-sensitive acrylic adhesives (PSAs): how it began and the present state of art,” ChemTexts 2024, v. 10, article 6; doi.org / 10.1007 / s40828-024- 00189-w; Yarusso, “Effect of rheology on PSA Performance,” in Dillard, et al, eds. THE MECHANICS OF ADHESION, 2002, 1:499-533; doi.org / 10.1016 / B978-0-444-51140-9.50040-8; Cognard, P., ed. HANDBOOK OF ADHESIVES AND SEALANTS, VOLS. 1 AND 2, Elsevier Science, 2006; and Petrie, E., HANDBOOK OF ADHESIVES & SEALANTS, McGraw Hill, 3rdEd., 2021. Anumber of PSAs are known in the art and commercially available. There are four main types of PSAa: natural rubber PSAs, synthetic rubber PSAs, acrylic PSAs, and silicone PSAs. Biocompatible PSAs of acrylic, silicone, or polyurethane have been developed for topical use in closing wounds and for surgical repairs, as discussed in, e.g., Fitzgerald, D., et al., Progress in Polymer Sci, July 2023, 142:101692; doi.org / 10.1016 / j.progpolymsci.2023. 101692; Pradeep, S., et al., J Adhesion, 2023, 99(14): 2145-2166; doi.org / 10.1080 / 00218464.2023.2176761; Fitzgerald, D„ et al., Nat Rev Mater, 2023, 8(l):3-5, doi: 10.1038 / s41578-022-00516-y. Biocompatibility of adhesives can be validated by United States Pharmacopeia (USP) VI Standardization, International Standards Organization (ISO) 10993 Certification, or both.

[0049] Commercially available silicone PSAs and acrylic PSAs were tested in studies underlying the present disclosure. Several PSAs sold by 3M™ using different acrylic adhesives were compared. The PSAs are provided as double coated tapes with a protective, peelable coating. One, using an acrylic adhesive which 3M™ refers to as 200MP, which is used for bonding high surface energy substrates such as metal and glass, developed fluid leaks under pressure. A second product, using an exemplar acrylic adhesive, called “300LSE” that bond to low surface energy substrates such as plastics, did not develop fluid leaks under pressure and was thus deemed suitable for use in the inventive flow inserts. The letters “LSE” in the adhesive’s name is apparently an initialism for “low surface energy.” Accordingly, adhesives that adhere to low surface energy materials are preferred. Adhesives such as 300LSE are typically provided in several formats, one of which is double sided tape covered on each side by a protective coating that peeled off when the tape is to be placed on the item to be bonded to a second item. The present invention contemplates the use of such double-sided tapes. The manufacturers of such double-sided tapes have selected peelable protective coatings suitable for the various tapes they sell, including tapes bearing adhesives such as 300LSE that bond to low surface energy surfaces, and have made tapes with such protective coatings commercially available to all, including those of skill in this art. 3M™ alone lists no fewer than six double coated tapes (product numbers 93005LE, 93005LEB, 93015LE, 9495LE, and 93020LE, and four adhesive transfer tapes (9471LE, 9671LE, 9472LE, and 9672LE), all of which use 300LSE as the adhesive and all of which are coated with a peelable, protective coating. It is believed that PSAs of any type can be used in embodiments of the inventive flow devices, including natural rubber and synthetic rubber PSAs, so long as the PSA of that material can bond to low surface energy materials.

[0050] Surface energy is a well understood property of all materials. As stated on an“Introduction to Surface Energy” webpage provided by 3M™adhesion / introduction-surface-eoergy / ), surface energy is “a measure of a how attracted a material’s molecules are to each other and to other materials’ molecules. Surface energy is a good measure of how easy or hard the surface may be to adhere.” Materials with a surface energy below 36 dynes / cm are considered low energy. The surface energy of most common materials used for flow devices, MPSs, and lab equipment are known and readily available on the webpages of adhesive suppliers, such as the following 3M™ webpage:adhesion / categorizing-surihce-energy / . The surface energy of many common plastics and polymers used in lab equipment is either low energy or a little above the 36 dyn / cm point defining low energy from medium surface energy. As adhesives that bind low surface energy materials can also bind higher surface energy materials, adhesives that bond to low surface energy materials, such as polyethylmethacrylate (PEMA, surface energy 35.9) can also bond to the polystyrene used for some commercially available tissue culture plates (surface energy 40.7) or to the glass used for microscope slipes and cover slips. They are thus suitable for bonding the inventive flow devices to lab equipment to provide such lab equipment with fluidic flow capabilities. It is believed that PSAs of any type can be used in embodiments of the inventive flow devices, including natural rubber and synthetic rubber PSAs, so long as the PSA of that material can bond to low surface energy materials.

[0051] In some embodiments, the flow devices are made using PSAs with adhesives that bond low surface energy substrates such as plastics. (The silicone chip to which the flow insert bonds is not plastic, but is bonded successfully by PSAs that bond low surface energy materials.) A further flow insert that did not leak when applied to a membrane chip was made with a silicone PSA that bonds to plastic. In some embodiments, the PSA comprises an acrylic adhesive that bonds low surface energy substrates. In some embodiments, the PSA comprises a silicon adhesive that bonds low surface energy substrates. The PSA can be an adhesive transfer tape. The PSA can be a differential adhesive transfer tape, which by definition has a different adhesive on each side to two different surfaces to be bonded to each other. In some embodiments, the inventive device is a flow device intended to bond to a glass surface, such as a microscope slide, and the PSA is a differential adhesive transfer tape which has an adhesive that bonds to low energy surfaces on the side bonding to the bottom ofthe flow device, and the second adhesive is one that bonds to high surface energy materials, such as glass. If two adhesives are used, one facing towards the flow insert and the second towards the surface to which the flow insert is to be secured, the adhesives facing the flow insert has to bond the material of which the flow device is made of and the adhesive facing the surface to which the flow insert is to be secured can bond to material of the surface energy of that material. As noted, an adhesive capable of bonding to a low surface energy material will be able to bond to materials of higher surface energy. It is believed that PSAs of any type can be used in embodiments of the inventive flow devices, including natural rubber and synthetic rubber PSAs, so long as the PSA of that material can bond to low surface energy materials or to high energy material.

[0052] It is expected that the flow insert will be positioned and attached to the MPS or the surface of other lab equipment on which it is to be used before cells are introduced onto the device by seeding them into the flow channel. Thus, it is expected that the cells themselves will not be in direct contact with the PSA. Further, if the flow insert or device is pressed into place securely, there will be little space for culture medium to seep under the edge of the flow insert and come into contact with the PSA. Thus, it is not expected that whether the PSA is toxic or non-toxic will be important. For an extra measure of protection to reduce the chance that sensitivity of the cells used in an experiment to the PSA will create variability in the results of the studies to be undertaken, a biocompatible PSA can be used. It is anticipated that any PSA that is non-toxic, such as any of the biocompatible adhesives reported in the articles noted above, can be used to secure and seal the flow insert or device to the MPS or to another piece of lab equipment. The cells cultured on the ultrathin membrane are typically vascular endothelial cells, bronchial epithelial cells, or brain endothelial cells.

[0053] Any particular biocompatible PSA can be readily tested for potential toxicity to the cells to be used in a particular experiment (sometimes referred to as the “cells of interest”). A cell culture plate, such as a 24-well plate, is chosen, and a sample of the PSA to be tested is placed in the bottom of a first well. Equal amounts of cell culture medium known to be able to support the growth of the cells of interest are added to the first well containing the PSA, and to a second well, which does not. An equal number of the cells of interest are then added to the first and to the second well, and the cell culture plate is then incubated for 24 hours under the same conditions for both wells. The growth and viability of the cells in the two wells is then observed. If poor growth or obviously greater mortality is observed with respect to the cells in the well with the PSA as compared to the well without the PSA, then the PSAbeing tested is not suitable as an adhesive for use in the inventive devices. If the cells have grown approximately equally in both wells, and if there is no evident difference in mortality between the two wells, than the PSA is suitable for use on the inventive devices. In some embodiments the cells are human cells. In some embodiments the human cells are bronchial epithelial cells. In some embodiments the human cells are brain endothelial cells.

[0054] Peelable films and materials that protect a PSA until the user chooses to peel them off and expose the PSA are well known and commercially available, as evidenced by the “peel and seal” envelopes found in any office supply store. In the case of the inventive devices, the “peel and seal” PSA is disposed around the edge of the flow insert or flow device, and the protective coating is peeled off to expose the PSA, allowing the PSA to be pressed against the MPS or piece of lab equipment to which the user wishes to secure the flow insert or device. The protective coating is a flexible material which is selected for its ability to adhere to the PSA strongly enough that it can stay on the PSA until a force is exerted on the material to peel it off, but does not adhere so strongly to the PSA that the PSA comes off the flow insert or device when the protective coating is peeled off. The removable tape used on the flap of a “peel and seal” envelope, which has a plastic coating allowing it to adhere removably to the adhesive on the envelope flap, is illustrative of a suitable peelable material for use on the PSA. As combinations of PSAs and peelable protective films or coatings are not only well known in the art, but also commercially available, it is believed that selection of protective films or other coatings suitable for releasably coating the PSA is well within the competence of the person of skill and do not need to be taught in detail here.Ultrathin membranes and pore size ranges

[0055] As noted above, in some embodiments, the flow inserts are used to create the ability to provide fluidic flow over a porous, ultrathin membrane on which cells can be disposed. Ultrathin (< 400 nm thick) precision pore membranes have been made and their properties explored, as exemplified by Striemer, et al., Nature, 2007. 445(7129): p. 749-753; DesOrmeaux, et al., Nanoscale, 2014. 6(18): p. 10798-10805; and Winans, et al., J Memb Sci, 2016. 499: p. 282-289. Ultrathin membranes exhibit a unique combination of filtration properties. They are exceptionally permeable, enabling very low-pressure filtration in microfluidic devices. References to a “membrane” in this disclosure refers to an ultrathin membrane unless otherwise specified.

[0056] Ultrathin membranes have been made using pure silicon, silicon nitride, glass (SiCU), MgF2, gold, graphene, and various polymers. Because of their extreme thinness, ultrathin membranes are sometimes referred to as “2D membranes.” It is contemplated that ultrathin membranes made of any of the materials mentioned above can be used in the inventive devices and methods.

[0057] In preferred embodiments, the ultrathin membranes are made of silicon, silicon nitride, silicon oxide, or silicon dioxide, as ultrathin membranes made of these materials are particularly robust. In some preferred embodiments, the ultrathin membrane is a silicon nitride ultrathin membrane.

[0058] Ultrathin membranes are so thin as to be transparent. They therefore better facilitate use of microscopy to monitor the device, for example to observe cells on the ultrathin membrane.

[0059] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS1. A flow insert for a microphy siological system (MPS) having a well in a top compartment of said MPS sized to accept a 5.4 mm x 5.4 mm membrane chip bearing one ultrathin membranes or or a pair of ultrathin membranes, said flow insert comprising:(a) a body having a top, sides, and a bottom, which bottom has a perimeter,(b) a flow channel defined by a continuous open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel,(c) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and(d) a second port fluidly connected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert(i) is 5.4 mm x 5.4 mm or smaller, and(ii) has disposed around its perimeter a pressure-sensitive adhesive that bonds to low surface energy materials.

2. The flow insert of claim 1, wherein said pressure-sensitive adhesive is covered by a protective coating.

3. The flow insert of claim 2, wherein said protective coating is a film.

4. The flow insert of claim 1, wherein said pressure-sensitive adhesive is non-toxic to human cells.

5. The flow insert of claim 4, wherein said human cells are selected from bronchial epithelial cells and brain endothelial cells.

6. The flow insert of claim 1, wherein said pressure-sensitive adhesive is a silicone pressure-sensitive adhesive.

7. The flow insert of claim 1, wherein said pressure-sensitive adhesive is an acrylic rubber pressure-sensitive adhesive.

8. The flow insert of claim 1, wherein said pressure-sensitive adhesive is a natural rubber pressure-sensitive adhesive.

9. The flow insert of claim 1, wherein said pressure-sensitive adhesive is a synthetic rubber pressure-sensitive adhesive.

10. The flow insert of claim 1, wherein said membrane chip has a pair of ultrathin membranes and said flow insert is sized to cover only one of said ultrathin membranes.

11. The flow insert of claim 1, wherein said flow insert is sized to cover one of half of the area of said bottom of said well, ±20%.

12. The flow insert of claim 1, wherein said flow insert is sized to cover half of the area of said bottom of said well, ±10%.

13. The flow insert of claim 1, wherein said flow insert is sized to cover half of the area of said bottom of said well.

14. A flow device to introduce fluidic flow over a top surface of a piece of laboratory equipment, said top surface of said laboratory equipment being optically clear, said flow device comprising:(a) a body having a top, sides, and a bottom, which bottom has a perimeter,(b) a flow channel defined by an open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel,(c) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and(d) a second port fluidly connected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert has disposed around said perimeter a pressuresensitive adhesive that bonds to low surface energy material.

15. The flow device of claim 14, wherein said pressure-sensitive adhesive is covered by a protective coating.

16. The flow insert of claim 14, wherein said pressure-sensitive adhesive is non-toxic to human cells.

17. The flow insert of claim 14, wherein said human cells are selected from vascular endothelial cells and brain endothelial cells.

18. The flow insert of claim 14, wherein said pressure-sensitive adhesive is a natural rubber pressure-sensitive adhesive.

19. The flow insert of claim 14, wherein said pressure-sensitive adhesive is a synthetic rubber pressure-sensitive adhesive.

20. The flow insert of claim 14, wherein said pressure-sensitive adhesive is an acrylic pressure-sensitive adhesive.

21. The flow insert of claim 14, wherein said pressure-sensitive adhesive is a silicone pres sure- sensitive adhesive.

22. The flow insert of claim 14, wherein said laboratory equipment is a glass microscope slide.

23. The flow insert of claim 14, wherein said laboratory equipment is a cover slip.

24. The flow insert of claim 14, wherein said laboratory equipment is a well of a tissueculture plate.

25. A system for providing fluidic flow over a surface of a piece of laboratory equipment not having said fluidic flow, said systems comprising(a) a flow device for introducing said fluidic flow over a surface of a piece of laboratory equipment, said surface of said laboratory equipment being optically clear, said flow device comprising:(i) a body having a top, sides, and a bottom, which bottom has a perimeter,(ii) a flow channel defined by an open space within said bottom of said body, said flow channel having a length, said length having a first end and a second end, said ends being disposed opposite each other along said length of said flow channel,(iii) a first port fluidly connected to said first end of said flow channel and fluidly connected to an opening on said top of said flow insert, and(iv) a second port fluidly connected to said second end of said flow channel and fluidly connected to an opening on said top of said flow insert, which bottom of said flow insert has disposed around said perimeter a pressuresensitive adhesive that bonds to low surface energy material,(b) a piece of optically clear laboratory equipment to which said flow device is bonded by said pressure-sensitive adhesive,(c) a means of introducing fluid under a pressure into said first port of said flow device, and(d) a means of receiving fluid from said second port of said flow device.

26. The system of claim 25, wherein said pressure-sensitive adhesive is non-toxic to human cells.

27. The system of claim 25, wherein said human cells are selected from vascular endothelial cells and brain endothelial cells.

28. The system of claim 25, wherein said pressure-sensitive adhesive is an acrylic pressure-sensitive adhesive.

29. The system of claim 25, wherein said pres sure- sensitive adhesive is a silicone pres sure- sensitive adhesive.

30. The system of claim 25, wherein said piece of laboratory equipment is a glass microscope slide.

31. The system of claim 25, wherein said piece of laboratory equipment is a cover slip.

32. The system of claim 25, wherein said piece of said laboratory equipment is a well of a tissue-culture plate.

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