Liquid Delivery System

Abstract

The methods and systems described herein are directed to a reagent delivery system for delivering multiple reagents from a microwell array to a reaction channel with reduced voids or bubbles, or for removing cells not trapped in a hydrogel cage. In some embodiments, the reagents are moved through the channel by a pump attached to the channel outlet and a pressure column applied to the channel inlet.

Description

[Technical Field]

[0001] (cross reference) This application claims the benefit of U.S. Provisional Application No. 63 / 419,655, filed October 26, 2022, which is incorporated herein by reference in its entirety.

[0002] (Incorporated by reference) All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification supersede and / or supersede any such conflicting material. [Background technology]

[0003] Microfluidics plays an important role in techniques for single cell analysis, such as those described in Zare et al., Annu. Rev. Biomed. Eng., 12:187-201 (2010) (Non-Patent Document 1), Valihrach et al., Int. J. Mol. Sci., 19:807 (2018) (Non-Patent Document 2), Murphy et al., The Analyst, 18:60-80 (2017) (Non-Patent Document 3), Shinde et al., Int. J. Mol. Sci., 19:3143 (2018) (Non-Patent Document 4), etc. However, there remains a challenge in designing fluids and microfluidic systems that avoid common problems such as bubble formation or the persistence of formed bubbles. Bubbles can adversely affect performance by impeding flow, blocking reaction areas, and damaging fragile microstructures, as described, for example, in Pereiro et al., LabChip, 19:2296 (2019). Particularly in systems where reagent storage, flow, or transport is in contact with the ambient atmosphere, such reagents can accumulate dissolved gases that can exceed saturation levels and lead to bubble formation within microfluidic components. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Zare et al, Annu.Rev.Biomed.Eng.,12:187-201(2010) [Non-patent document 2] Valihrach et al,Int.J.Mol.Sci.,19:807(2018) [Non-patent document 3] Murphy et al,The Analyst,18:60-80(2017) [Non-patent document 4] Shinde et al,Int.J.Mol.Sci.,19:3143(2018) [Non-patent document 5] Pereiro et al,LabChip,19:2296(2019) Summary of the Invention [Means for solving the problem]

[0005] In light of the above, the availability of new methods and devices to minimize bubble formation in microfluidic systems processing fluids exposed to the atmosphere would advance the field of microfluidics, particularly in the area of ​​single cell analysis.

[0006] The methods and systems described herein are directed to transferring liquid reagents into fluidics systems from reservoirs exposed to the atmosphere, where dissolved gases may be elevated above saturation levels, thereby risking the formation of destructive bubbles.

[0007]

[0009] A method for moving a liquid in contact with the atmosphere through a channel is provided herein, the method including: (a) providing a channel having an outlet and an inlet with an inlet reservoir, the channel containing a first liquid and fluidly communicating with a pump through the outlet, the pump moving the liquid through the channel at a predetermined rate; (b) transferring a second liquid in contact with the atmosphere from the reservoir to an inlet reservoir at the inlet of the channel such that the transferred second liquid combines with the first liquid in the inlet reservoir; (c) attaching a pressure manifold to the inlet reservoir, the pressure manifold providing a predetermined pressure above atmospheric pressure to the first and second liquids in the channel; and (d) moving the first and second liquids through the channel at a predetermined rate by the pump under the predetermined pressure. In some embodiments, moving the first and second liquids occurs for a predetermined duration such that the inlet reservoir is not emptied of the first and second liquids. In some embodiments, the movement of the first and second liquids occurs for a predetermined duration such that a predetermined volume of the first and second liquids passes through the channel.

[0008] Also provided herein is a fluid delivery system comprising: (a) one or more reaction channels, each containing a first liquid and having an outlet and an inlet with an inlet reservoir; (b) a pump for each of the one or more reaction channels, the pump being in fluid communication with the outlet of the reaction channel and capable of moving liquid through the channel at a predetermined rate; (c) one or more supply reservoirs containing a liquid in contact with the atmosphere; (d) a pipettor for transferring a second liquid from the one or more supply reservoirs to the inlet reservoirs; and (e) a pressure manifold sealingly attached to the inlet reservoirs, the pressure manifold applying a predetermined pressure above atmosphere to the first and second liquids in the inlet reservoirs.

[0009] Also provided herein is a method for delivering reagents to a cell analysis system, the method including: (a) providing a fluidic device, the fluidic device including: (i) a channel, the channel having an inlet, an outlet, and a first surface, the surface having one or more cells disposed thereon, the inlet of the channel being fluidly connected to an inlet reservoir, and the outlet of the channel being fluidly connected to a pump, the pump under programmed control drawing or moving a predetermined volume of liquid in the inlet reservoir into the channel; (b) loading the channel with an assay reagent in contact with the atmosphere by transferring a volume containing the assay reagent to the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagent at a predetermined pressure; and (d) using the pump to draw the assay reagent through the channel such that the assay reagent combines with one or more cells disposed on the first surface of the channel. [Brief explanation of the drawings]

[0010] [Figure 1A] 1A-1E illustrate an embodiment for transferring an atmosphere-exposed liquid to an enclosed reaction channel of a fluidics system. [Figure 1B] 1A-1E illustrate an embodiment for transferring an atmosphere-exposed liquid to an enclosed reaction channel of a fluidics system. [Figure 1C] 1A-1E illustrate an embodiment for transferring an atmosphere-exposed liquid to an enclosed reaction channel of a fluidics system. [Figure 1D] 1A-1E illustrate an embodiment for transferring an atmosphere-exposed liquid to an enclosed reaction channel of a fluidics system. [Figure 1E] 1A-1E illustrate an embodiment for transferring an atmosphere-exposed liquid to an enclosed reaction channel of a fluidics system.

[0011] [Figure 2A] 2A-2B illustrate in more detail an apparatus that may employ the systems and methods described herein for detecting cells and synthesizing hydrogel chambers. [Figure 2B] 2A-2B illustrate in more detail an apparatus that may employ the systems and methods described herein for detecting cells and synthesizing hydrogel chambers.

[0012] [Figure 3A] FIG. 3A is a photograph of various cells, some of which are caged within the hydrogel structures and other cells are uncaged and reside in the interstitial spaces between the hydrogel structures.

[0013] [Figure 3B] FIG. 3B is a photograph of the various cells shown in FIG. 3A after flowing liquid through the row cell without the use of a pressure manifold that applies excess pressure while a pump moves the fluid out of the channel, leaving multiple cells in the interstitial spaces.

[0014] [Figure 4A] FIG. 4A is a photograph of various cells, some of which are caged within the hydrogel structures and other cells are uncaged and reside in the interstitial spaces between the hydrogel structures.

[0015] [Figure 4B]Figure 4B is a photograph of the various cells shown in Figure 4A after flowing liquid through the flow cell using a pressure manifold that applies overpressure while a pump moves the fluid out of the channel. The proportion of cells remaining in the interstitial space was significantly lower in Figure 4B (using the pressure manifold) compared to Figure 3B (without the pressure manifold).

[0016] [Figure 5A] FIG. 5A is a photograph of a flow cell with channels E and F, both of which contain liquid and are relatively bubble-free.

[0017] [Figure 5B] FIG. 5B is a photograph of the flow cell of FIG. 5A containing liquid incubated at 42° C. for 90 minutes with channel F pressurized at 5 PSI using a pressure manifold and channel E at atmospheric pressure. DETAILED DESCRIPTION OF THE INVENTION

[0018] The practice of the systems and methods described herein may employ, unless otherwise indicated, conventional techniques and procedures of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry that are within the skill of the art. Such conventional techniques include, but are not limited to, preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques may be had by reference to the examples herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and explanations can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: A Laboratory Manual; Retroviruses, and Molecular Cloning: A Laboratory Manual (all published by Cold Spring Harbor Laboratory Press), Site-directed Insertion of Transgenes by Renault and Duchateau (editors) (Springer, Heidelberg, 2013), and Protein Engineering Handbook by Lutz and Bornscheuer (editors) (Wiley-VCH, 2009). Guidance for selecting materials and components to perform specific functions can be found in available articles and references on scientific instrumentation, including, but not limited to, references such as Moore et al., Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge, MA), and Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013).

[0019] Methods and systems are directed to delivering fluids exposed to the atmosphere from open reservoirs (e.g., microwells, etc.) to closed reaction channels with minimal risk of inducing bubble formation within the channels. In one aspect, the methods involve transferring liquid from a reservoir open to the atmosphere to an inlet reservoir at the inlet end of the reaction channel, after which a pressure manifold is sealingly attached to the inlet reservoir and delivers pressure to the liquid in the inlet channel and the main channel. In some cases, a pump or vacuum source at the outlet end of the main channel draws or moves liquid through the reaction channel at a predetermined rate, while simultaneously delivering pressure in excess of ambient atmospheric pressure to the inlet reservoir. In some embodiments, the method includes a cycle of steps including: (a) depressurizing a first liquid in a channel; (b) delivering a second liquid to an inlet reservoir in fluid communication with the channel such that the first and second liquids form a single liquid body; (c) pressurizing the first and second liquids; and (d) drawing the first and second liquids through the channel at a predetermined rate. In some embodiments, such liquid delivery is part of a process requiring multiple such transfers, in which case steps (a)-(d) may be repeated multiple times. In some embodiments, "de-pressurizing" refers to equilibrating the first liquid pressure with atmospheric pressure. In some embodiments, prior to the de-pressurizing step, the first liquid is at a pressure higher than atmospheric pressure. In some embodiments, step (c) of pressurizing the first and second liquids includes pressurizing the first and second liquids to a predetermined pressure. It should be noted that ambient pressure or ambient atmospheric pressure refers to the pressure at the location of measurement and typically ranges from about 14.5 inches Hg to about 14.9 inches Hg (i.e., ranges from 14.5 pounds per square inch (psi) to 14.9 pounds per square inch, typically about 14.7 psi).

[0020] One embodiment of the method is illustrated in Figures 1A-1E. The flow cell (109) includes four channels (116, 118, 120, and 122) (sometimes referred to as "main channels"), and the body (108) includes four inlet reservoirs (112a, 112b, 112c, and 112d), one for each channel (116, 118, 120, and 122, respectively). In some embodiments, the inlet reservoirs may have an inverted cone shape (or partial cone shape) as shown in Figure 1C or 1E, and the inlet reservoirs are in fluid communication with the channels (116, 118, 120, and 122, respectively) through the narrow portion of the inverted cone (sometimes referred to herein as the "throat" of the inlet reservoir, e.g., 114a in Figure 1A) and the inlet of each channel. The inlet reservoirs may be formed within a body such as (108), which is bonded or otherwise sealingly attached to the flow cell (109). Detail view (110) provides a three-dimensional view of these components. The flow cell (109) may further include a first surface and a second surface for forming the upper and lower boundaries of the channels. Referring to FIG. 1C, the first surface may be in the form of a glass sheet (105) having multiple inlets (107a, 107b, 107c, and 107d) for respective channels (116, 118, 120, and 122). The multiple inlets (107a, 107b, 107c, and 107d) can be a series of through-holes in the glass sheet (105) aligned with the throat regions (114a, 114b, 114c, and 114d) of the inlet reservoirs (112a, 112b, 112c, and 112d). In various embodiments, a double-sided pressure-sensitive adhesive with corresponding through-holes can be used to bond the body (108) to the glass sheet (105) and the corresponding inlets (107a, 107b, 107c, and 107d). Each of the channels (116, 118, 120, and 122) can have an outlet (113a, 113b, 113c, and 113d, respectively) at the opposite end of the inlet reservoir.In some embodiments, each outlet reservoir (113a, 113b, 113c, and 113d) is separately connected to (fluidically communicates with) a pump (115a, 115b, 115c, and 115d, respectively). In some embodiments, such pumps are precision syringe pumps that can be programmed to draw or move liquid through their respective channels at predetermined flow rates at predetermined intervals in coordination with the filling of the inlet reservoirs (i.e., in some embodiments, the syringe pumps are deactivated each time an inlet reservoir is filled). The reagent reservoirs can be wells of a microtiter plate, such as a 96-well plate (100), where each well (e.g., 102) contains a reagent, and a liquid transfer system, such as an automated pipetting system (i.e., a "pipettor"), simultaneously transfers liquid to all of the inlet reservoirs of the flow cell. For example, in the four-channel flow cell of FIG. 1A, liquid in some four wells (104) can be simultaneously transferred (106) to four inlet reservoirs (112a, 112b, 112c, and 112d). As illustrated in FIG. 1B, after liquid is delivered to the inlet reservoirs, a pressure manifold (122) can be sealingly attached to the inlet reservoirs, thereby applying pressure to the delivered liquid and to the liquid already in the channels. The pressure manifold (122) can be a body sealingly attached to the body (108) containing the inlet reservoirs (112a, 112b, 112c, and 112d). In some embodiments, each inlet reservoir can be pressurized using a separate pressure source, or in other embodiments, a single pressure source can pressurize all of the inlet reservoirs. In some embodiments in which each inlet reservoir is pressurized using a separate pressure source, such pressure source can be adjusted to a pressure that is specific for its associated channel. In some embodiments, such characteristic pressure may depend on the amount of fluidic resistance within its associated channel. Such differences in fluidic resistance may result from different amounts of material (e.g., cells, gel microstructures, debris, etc.) within the channel. The latter embodiment is illustrated in FIG. 1B.After pressure manifold (122) is sealingly attached to the inlet reservoir, pressure source (126) can generate a pressure (or predetermined pressure) that is transferred to the inlet reservoir through conduit (124) and the pressure manifold itself. In some embodiments, the predetermined pressure is greater than atmospheric pressure. In some embodiments, the predetermined pressure can be less than atmospheric pressure, for example, under conditions where liquid is being forced to flow from the channel to the inlet reservoir.

[0021] 1C and 1D further illustrate the above embodiment, i.e., the movement of liquid through the inlet reservoir and channel during operation of the fluid transfer system. Cross-section (111) (or panel (1)) shows the inlet reservoir and channel interior along a plane (130) that intersects a three-dimensional representation (110) of the body (108) in which the inlet reservoir is formed. Panels (2)-(6) present the same cross-section at different steps in the fluid transfer process. Returning to panel (1), inlet reservoirs (112a, 112b, 112c, and 112d) can be in fluid communication with the interiors of the channels (116, 118, 120, and 122, respectively) through inlet reservoir throat regions (114a, 114b, 114c, and 114d, respectively), each comprising a narrow region of the inlet reservoir and a channel inlet (specifically shown with respect to inlet reservoir 114a). The shaded regions of the channels and channel inlets in cross section (111) indicate the presence of a first liquid. As shown in panel (2), a second fluid can be transferred (131) from the open reservoir (or reservoirs) to the open inlet reservoir, e.g., by use of a pipette (132), from the configuration of cross section (111) (i.e., with the first liquid in the channels and the inlet reservoirs empty (or nearly empty)). After removal of pipette (132) (panel (3)), the first liquid (134) and second liquid (136) shown may coalesce or merge (at least locally) with one another, thereby forming a single body of liquid for each channel. Pressure manifold (122) may be sealingly attached (140) to inlet reservoirs (112a, 112b, 112c, and 112d), a predetermined pressure may be applied to the inlet reservoirs, and pumps (115a, 115b, 115c, and 115d) may be activated and begin drawing liquid through their respective channels at a predetermined rate (panels (4) and (5)). In some embodiments, the pressure is adjusted as needed to maintain the predetermined pressure as the first and second liquids are moved through the channels by the pumps. In some embodiments, the amount of liquid drawn through the channels is greater than a single channel volume.In some embodiments, the amount of liquid drawn or displaced through a channel is a multiple of the channel volume. For example, the amount of liquid drawn or displaced through a channel can be two, three, four, five, or ten times the channel volume. In some embodiments, the amount of liquid drawn or displaced through a channel can be smaller than a single channel volume. The volumes of the inlet reservoirs (the volume of liquid delivered to them) can be adjusted depending on the predetermined volume of liquid desired to be drawn or displaced through the channel. In some embodiments, the volume of liquid delivered and the volume of liquid drawn or displaced in each cycle of the steps represented in panels (1)-(6) are selected so that after the liquid is drawn or displaced, the resulting level of liquid in the inlet reservoir is within or above the throat region of the inlet reservoir (e.g., as illustrated by (138) in panel (5) of Figure 1D or by (152) or (166) in Figure 1E). Finally, as shown in panel (6), the original first liquid (shown as the shaded area (134) in panel (3)) can be replaced by new first liquid (shown as the crosshatched area (136) in panel (6)), thereby making the inlet reservoir ready for the next cycle of the liquid transfer step.

[0022] 1E also illustrates a cycle of steps for transferring a volume of liquid according to the methods described herein. A flow cell (150) is shown, with an inlet reservoir (156) in fluid communication with a channel (154) and a pump (160) via a conduit (155) connecting the channel outlet to the pump (160). At the start of the transfer cycle, the level of the first liquid (152) can be at or near the throat region of the inlet reservoir or at or near the inlet of the channel. A second liquid can be transferred (168) to the inlet reservoir, thereby raising the level (158) of the combined first and second liquids in the inlet reservoir. A pressure manifold (162) (connected to a pressure source (164)) may be sealingly attached (170) to the inlet reservoirs, and a predetermined pressure may be applied to the first and second liquids, after which the pump (160) may be activated (170) to draw or move predetermined volumes of the first and second liquids through the channels (154), thereby bringing the first and second liquid levels (166) to ready the system for the next liquid transfer cycle (172).

[0023] (applicable) The reagent delivery methods and systems described herein may be used with a wide range of analytical devices, particularly devices for analyzing live biological material such as single cells due to their sensitivity to measurement or operation interruption and / or distortion due to bubble formation and movement. The reagent delivery methods and systems described herein may be particularly applicable to cellular analysis systems employing gel microstructures in flow cell channels, such as those described in International Patent Publication No. WO 2022 / 150659 to Khurana et al. (incorporated herein by reference).

[0024] 2A-2B illustrate the channels of a flow cell fabricated and operated according to Khurana et al. (cited above). The flow cell (200) can be a component of a fluidic device that provides one or more channels and liquid handling components for delivering beads and reagents to the channels under programmable control. In this figure, four channels (202, 204, 206, and 208) are shown, with a detailed view (212) of a segment (210) of channel 2 (204) shown below. In the summarized view of the flow cell (200) in FIG. 2A, the channel inlets, outlets, and other features are not shown. In some embodiments, the distance between the lower surface ("first" surface 214) and the upper surface ("second" surface 215) (i.e., the internal height of the channel) can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm. On the first surface (214) of channel 2 (204), a plurality of cells, e.g., (218), are each encapsulated by a hydrogel chamber, e.g., (216). In some cases, only a portion of the plurality of cells is encapsulated by the hydrogel chamber. In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to be impermeable to cells but permeable to reagents for forming spatial barcodes. Thus, reagents can be introduced into (or removed from) the interior of the hydrogel chamber by flowing the reagents through the channel (220), while the beads are retained inside. Shown below the detailed view (212) of channel segment (210) is an optical system (221) for photosynthesizing the hydrogel chamber at the location of cells within the channel, as disclosed, for example, in Khurana et al. (cited above). Optical systems with configurations different from those of FIGS. 2A and 2B can also be employed to perform these functions. In some embodiments, one or more digital micromirror device (DMD) / objective lens subsystems for synthesizing hydrogel structures can be employed to increase the speed of synthesis by synthesizing multiple structures simultaneously.

[0025] Returning to FIG. 2A, to photosynthesize the hydrogel chamber, a light source (222) can generate a light beam (223) of an appropriate wavelength of light (e.g., UV light), which passes through an appropriate optical mask or beam-shaping or beam-steering (Galvo) system to shape the beam to synthesize the desired structure or structures within the channel. In some embodiments, a digital micromirror device (DMD) (224) is employed. In some embodiments, a physical optical mask can be employed. Chamber location, shape, and polymer matrix wall thickness can be determined from positional information determined, at least in part, from images collected by detector (232). The positional information can be the location of cells, nucleic acids, or any analyte of interest. Reflected light from the DMD (224) can be shaped using optics, such as collimating optics (228), and directed into the channel 2 segment (210) through an objective lens system (234). The objective lens (234) and flow cell (200) can move relative to one another in the XY direction (236) to capture chambers anywhere within any of the channels. In some embodiments, the flow cell (200) moves and the optical system (221) is stationary. In some embodiments, the objective lens (234) can also direct a light beam (227) from the light source (229) to a target, such as a cell, on the first surface (214) and collect optical signals, such as fluorescent signals, from an assay performed on the first surface (214). Alternatively, optical signal collection can be performed using a separate objective lens, as shown in FIG. 2B. Information collected by detector (232) or its counterpart in the embodiment of FIG. 2B, particularly the cell positions within their respective channels, can be employed by computer (238) and / or an auxiliary controller to instruct DMD (224) and the translation device, which controls the relative position of objective lens (234) and flow cell (200), to synthesize hydrogel chambers of appropriate shape and size at appropriate locations.

[0026] FIG. 2B illustrates an alternative optical system in which the detection portion (250) of the optical system moves (272) independently of the movement (268) of the combining portion (252) of the optical system. The detection portion (250) of the optical system includes a detector (256), an objective lens (258), a light source (260), and interconnecting optical elements (such as a dichroic mirror (262)). Similar to the embodiment of FIG. 2A, the detector (256) is operatively associated with a computer (264) and the combining portion (252) of the optical system, providing positional information to the combining portion (252). The computers (264) and (238) are also operatively associated with a stage and / or motors that control the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the combining portion (252) of the optical system is located on the other side of the first surface (264) from the detection portion (250). Similar to the embodiment of FIG. 2A, the combining section (252) includes components such as an objective lens (274), a mirror (276), a collimating optical system (280), a DMD (282), and a light source (278).

[0027] In some embodiments, methods and systems for single cell analysis employing a reagent delivery subsystem include: (a) providing a fluidic device comprising: (i) a channel, the channel comprising an inlet, an outlet, and a first surface, the first surface having one or more cells disposed thereon; the inlet of the channel in fluid communication with an inlet reservoir; and the outlet of the channel in fluid communication with a pump, the pump under programmed control drawing or moving a predetermined volume of liquid in the inlet reservoir into the channel; (b) loading the channel with an assay reagent in contact with the atmosphere by transferring a volume containing the assay reagent to the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagent at a predetermined pressure; and (d) drawing the assay reagent through the channel using the pump such that the assay reagent combines with the one or more cells disposed on the first surface of the channel. In some embodiments, such methods further include incubating the one or more cells and the assay reagent for a predetermined period of time. In some embodiments, such assay reagents are cell lysis reagents, transcription reagents, polynucleotide amplification reagents.

[0028] In some embodiments, methods and systems for single cell analysis employing a reagent delivery subsystem include: (a) providing a fluidic device comprising: (i) a channel having an inlet, an outlet, and a first surface; (ii) a spatial energy modulation element in optical communication with the first surface; and (iii) a detector that identifies the location of one or more cells within the channel based on one or more optical signals from the one or more cells, wherein the inlet of the channel is in fluid communication with an inlet reservoir and the outlet of the channel is in fluid communication with a pump that, under programmed control, draws a predetermined volume of liquid through the channel and into the inlet reservoir; and (b) transferring a volume of a mixture of one or more cells and one or more polymer precursors into the inlet reservoir. Thus, the method includes loading one or more cells into the channel, sealingly attaching a pressure manifold to the inlet reservoir, pressurizing the mixture at a predetermined pressure, and using a pump to draw the mixture through the channel at a predetermined rate so that the cells of the mixture are disposed on a first surface of the channel; and (c) synthesizing one or more chambers within the channel by projecting light into the channel using a spatial energy modulation element, such that each chamber encapsulates a single cell of the one or more cells, wherein the projected light causes crosslinking of one or more polymer precursors to form a polymer matrix wall of the chamber, and the position of each synthesized chamber on the first surface is determined by the position of the encapsulated cell, which is thereby identified by a detector.

[0029] In some embodiments, methods and systems for single cell analysis employing a reagent delivery subsystem include: (a) providing a fluidic device comprising: (i) a channel having an inlet, an outlet, and a first surface, the channel having one or more cells disposed thereon; (ii) a spatial energy modulation element in optical communication with the first surface; and (iii) a detector that identifies the location of one or more cells within the channel based on one or more optical signals from the one or more cells, the inlet of the channel being in fluid communication with an inlet reservoir and the outlet of the channel being in fluid communication with a pump that, under programmed control, draws a predetermined volume of liquid in the inlet reservoir into the channel; and (b) providing a volume of a fluid containing one or more polymer precursors in the inlet reservoir. (c) loading one or more polymer precursors into the channel by transferring a liquid, sealingly attaching a pressure manifold to the inlet reservoir, and drawing the liquid through the channel using a pump so that the one or more polymer precursors pressurize the liquid at a predetermined pressure and combine with one or more cells disposed on a first surface of the channel; and (d) synthesizing one or more chambers within the channel, each chamber encapsulating one or more single cells of the one or more cells, by projecting light into the channel using a spatial energy modulation element, such that the projected light causes cross-linking of the one or more polymer precursors and forms a polymer matrix wall of the chamber, wherein the position of each synthesized chamber on the first surface is determined by the position of the encapsulated cell, thereby identified by a detector.

[0030] It should be understood that the term "detector" as used herein can include, but is not limited to, a microscopy component that collects and optionally magnifies an image of a portion of the channel, and an image analysis component equipped with software for identifying cells and associated location information. The computer component uses such information generated by the detector, along with user input, to generate commands for other components, such as spatial energy modulation components, which perform various functions, including, but not limited to, synthesizing chambers, disassembling chambers "on-demand," selectively photolyzing chambers, and the like. The configuration of such an embodiment is illustrated in FIGS. 2A-2B, described above. In some embodiments, the channel of the fluidic device further comprises a second surface, the first and second surfaces being disposed opposite each other across the channel, and the polymer matrix walls of the chambers extending from the first surface to the second surface to form a chamber, each having an interior. In some embodiments, each of the chambers in the channel encapsulates a single cell. In some embodiments, both the first wall and the second wall are made of an optically transparent material such as glass, plastic, etc., and are positioned such that the first and second surfaces are substantially parallel to one another. The perpendicular distance between the first and second surfaces can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm.

[0031] As described above, any of the first surface, second surface, or polymer matrix walls of the chamber may comprise capture elements and other functional groups for performing various operations, including, but not limited to, capturing beads, capturing cells, capturing analytes (such as mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing components of analytical reagents (such as oligonucleotide labels from antibodies), etc. Derivatizing surfaces for such purposes is well known to those of skill in the art, as evidenced by the following references, such as the Integrated DNA Technologies brochure by Hermanson (cited above).

[0032] As described above, in some embodiments, the fluidic device of the method comprises or is operably associated with a detector that may share the optical path of the spatial energy modulation element, or may be positioned adjacent to the second wall, or in some embodiments, opposite the first wall from the spatial energy modulation element, such as a well having only a first wall and a first surface. The detector is positioned so that it can detect optical signals from, for example, cells in a channel distributed on the first surface within the chamber, or adjacent thereto. In some embodiments, each of the first and second walls comprises an optically transparent material, for example, so that the spatial energy modulation element can project optical energy into the interior of the channel, and the detector can detect optical signals, such as fluorescent emission or reflected light from the biological component. In some embodiments, the energy projected from the spatial energy modulation element is optical energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element can have a complex cross-section, which (in various embodiments) allows for simultaneous synthesis of multiple chambers. Optically transparent materials include, but are not limited to, materials such as glass, quartz, plastic, etc.

[0033] The spatial energy modulation element that uses light energy for polymerization can comprise a physical light mask or a virtual light mask, such as a digital micromirror device (DMD). The following references (hereby incorporated by reference): Chung et al., U.S. Pat. No. 10,464,307; Hribar et al., U.S. Pat. No. 10,351,819; Das et al., U.S. Pat. No. 9,561,622; Huang et al., Biomicrofluidics, 5:034109 (2011), etc., provide guidance in selecting and operating a DMD for photopolymerizing gels.

[0034] (Gel chamber) The method and apparatus of Khurana et al. can employ a wide variety of photosynthesizable and degradable gels for cell analysis. Guidance for selecting such gels for desired properties, including but not limited to properties such as biocompatibility, porosity, gelation rate, degradation rate, etc., is provided in the following references (incorporated by reference): Kharkar et al., Chem. Soc. Rev., 42:7335-7372 (2013); Kharkar et al., Polymer Chem., 6(31):5565-5574 (2015); Neumann et al., Acta Biomater., 39:1-11 (2016); DeForest et al., Nature Chemistry, 3(12):925-931 (2012); U.S. Pat. No. 9,631,092 to Bowman et al.; ACS Appl. Biomater., 3(10):6944-6958 (2020); Kabb et al., ACS Appl. Mater. Interfaces, 10:16793-16801 (2018), Fairbanks et al., Macromolecules, 44:2444-2450 (2011), Fairbanks et al., Adv. Mater., 21(48):5005-5010 (2009), Sugiura et al., U.S. Patent Publication No. US2016 / 0177030, Shih et al., Biomacromolecules, 13(7):2003-2012 (2012), etc. In some embodiments, photosynthetic gels are formed using a photoinitiator for radical polymerization. In some embodiments, the photoinitiator comprises Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), or Eosin-Y (see, e.g., Choi et al., Biotechniques, 66(1):40-53 (2019)).In some embodiments, the hydrogel precursor comprises hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly(ethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), and poly(vinyl alcohol). In some embodiments, the polymer precursor comprises PEG or multi-arm PEG. In some embodiments, the polymer precursor comprises an enzymatically degradable cross-linking agent. In some embodiments, such enzymatically degradable cross-linking agents are degradable by esterases or peptidases. In some embodiments, the polymer precursor comprises a photodegradable cross-linking agent. In some embodiments, such a photodegradable cross-linking agent comprises a nitrobenzyl group. In some embodiments, such a photodegradable cross-linking agent comprises a coumarin moiety. In some embodiments, photodegradable hydrogels are used with the methods described herein, for example, because photolysis of hydrogel chambers can be performed selectively and on demand, such that defined hydrogel chambers can be degraded without affecting non-selective hydrogel chambers. In some embodiments, hydrogel chambers are non-selectively degraded, such that all hydrogel chambers in a given channel (or other container) are simultaneously degraded. In some embodiments, such non-selective degradation is performed using a cleavage reagent that specifically cleaves labile bonds in the hydrogel. For example, such a cleavage agent includes a reducing agent. In some embodiments, such non-specific degradation is performed using an enzyme that cleaves bonds or chemical entities in the hydrogel. Chemical entities include, but are not limited to, peptides, polysaccharides, and oligonucleotides.

[0035] For convenience, the hydrogel chambers are illustrated in the figures as being isolated, without connection to adjacent chambers, and as having a cylindrical or ring-like shape; however, the spatial energy modulation element can synthesize chambers of different shapes and sizes as may be useful for a particular application. In some embodiments of proliferation assays, each hydrogel chamber synthesized has the same shape and area, e.g., 0.001-0.01 mm. 2 The inner surface of the tubular member has an inner surface area selected from the range of 0.1 to 0.5 mm.

[0036] Porosity: In some embodiments, the hydrogel porosity is selected to allow the passage of a selected reagent while simultaneously preventing the passage of other reagents or entities, such as cells or lysed cellular proteins. In some embodiments, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some embodiments, the pores have an average diameter of about 2 nm to about 25 nm, or about 5 nm to about 20 nm. In some embodiments, the average pore diameter is selected to prevent the passage of cellular proteins. In some embodiments, the average pore diameter is selected to prevent the passage of cellular proteins having a molecular weight of 1 kilodalton or greater. In some embodiments, the average pore diameter is selected to prevent the passage of cellular proteins having a molecular weight of 5 kilodaltons or greater. In some embodiments, the pore size of the hydrogel structure is adjusted by varying the ratio of the concentration of polymer precursor to the concentration of crosslinking agent, by varying pH, salt concentration, temperature, light intensity, etc. Guidance for selecting materials and conditions for controlling hydrogel porosity can be found in the following references: Jung et al., Biochem. Eng. J., 135:123-132 (2018); Winther et al., Biochim. Biophys. Acta, 1840(2):doi:10.1016 / j.bbagen.3013.03.031 (2014); Annabi et al., Tissue Engineering, part B, 16(4):371-383 (2010), etc.

[0037] Size and Shape of the Hydrogel Chamber: In some embodiments, the polymer matrix wall of the chamber inhibits passage of predetermined components, such as proteins from mammalian cells, bacterial cells, or lysed cells. In some embodiments, the polymer matrix wall extends from a first surface to a second surface (parallel to the first surface), forming a chamber within the channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the interior of the chamber is sized to encapsulate cells, such as mammalian cells. For example, such a chamber may comprise a cylindrical or polygonal shell, with an interior space or with an interior and a polymer matrix wall. In some embodiments, such a chamber may have an annular-like cross-section. As used herein, the term "annular-like cross-section" means a cross-section that is topologically equivalent to a ring. In some embodiments, the chamber's interior space, i.e., its interior, has an inner diameter of 1 μm to 500 μm and a volume in the range of 1 picoliter to 200 nanoliters, or 100 picoliters to 100 nanoliters, or 100 picoliters to 10 nanoliters. In some embodiments, the polymer matrix wall has a thickness of at least 1 μm (micrometer). In some embodiments, the height of the chamber with a ring-like cross-section has a value in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm. In some embodiments, the polymer matrix wall with a ring-like cross-section has an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, the aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability against forces such as reagent flow through the channel, washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, degradation of at least one polymer matrix is ​​"on demand." In some embodiments, the chambers within a channel are non-contiguous.In some embodiments, chambers within a channel may be adjacent to adjacent chambers. In some embodiments, chambers may share polymer matrix walls with one another. In some embodiments, chambers may be synthesized with slits or other orifices large enough to allow passage of some components, e.g., beads, but small enough to prevent passage of other components, e.g., cells.

[0038] Hydrogel Composition: As noted above, hydrogel compositions can vary widely, and hydrogels can be formed by a variety of methods. Biocompatible hydrogel precursors include, but are not limited to, hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly(ethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), and poly(vinyl alcohol). In some embodiments, the hydrogel is formed by photoinitiated free radical crosslinking. In some embodiments, the hydrogel is formed by photoinitiated thiol-ene reaction.

[0039] Hydrogel Degradation: In some embodiments, hydrogel chambers are degradable or depolymerizable either globally within the channel or "on demand" within the channel. Generally, degradable hydrogel chambers are degraded by treatment with a degradation agent, or equivalently, a depolymerization agent, exposed to all chambers within the channel. Depolymerization agents include, but are not limited to, heat, light, and / or chemical depolymerization reagents (sometimes also referred to as cleavage or degradation reagents). In some embodiments, on-demand degradation can be implemented using polymer precursors that allow for photocrosslinking and photodegradation, e.g., using different wavelengths for crosslinking and degradation. For example, eosin Y can be used for radical polymerization in a defined region using a 500 nm wavelength, followed by illumination at 380 nm to cleave the crosslinking agent. In other embodiments, a photocaged hydrogel cleavage reagent can be included within the polymer matrix wall former. For example, an acid-labile crosslinking agent (such as an ester) can be used to generate a hydrogel, and then UV light can be used to generate localized acidic conditions that subsequently degrade the hydrogel. In some embodiments, the at least one polymer matrix is ​​degradable by at least one of (i) contacting the at least one polymer matrix with a cleavage reagent, (ii) heating the at least one polymer matrix to at least 90°C, or (iii) exposing the at least one polymer matrix to light of a wavelength that cleaves a photocleavable crosslinking agent that crosslinks the polymers of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage reagent degrades the hydrogel. In some embodiments, the cleavage reagent comprises a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage reagent, or a combination thereof. In some embodiments, the cleavage reagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof.In some embodiments, the surface of a polymer matrix or hydrogel can be functionalized by attaching functional groups to the polymer matrix or hydrogel.

[0040] Although the methods and systems are described herein with reference to certain specific exemplary embodiments, those skilled in the art will recognize that many modifications may be made thereto without departing from the spirit and scope of the present disclosure. The methods and systems described herein may be applicable to a variety of sensor implementations and other subject matter in addition to those discussed above.

[0041] Additional Embodiments Aspect 1. A method of moving a liquid through a channel, the method comprising: (a) providing a channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel containing a first liquid and in fluid communication with the outlet, and a pump is fluidly coupled to the outlet, the pump configured to move the liquid through the channel; (b) transferring a second liquid to an inlet reservoir, the transferred second liquid being in contact with the atmosphere; (c) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first and second liquids; (d) operating a pump to draw at least a portion of the first liquid from the channel through the outlet and to move at least a portion of the second liquid into the channel, the pump being programmed to move the fluids at a predetermined rate while under the provided pressure.

[0042] In various aspects described herein, the first liquid can include a buffer solution (e.g., phosphate buffered saline), various types of cells, and / or polymer precursors configured to polymerize into cell cages.

[0043] In various aspects described herein, the second liquid can include a buffer (e.g., phosphate buffered saline), a lysis agent, a fluorescently labeled antibody, cell culture medium, and / or a hydrogel degradation agent.

[0044] In various aspects described herein, the pressure can be a constant value, a predetermined value, a range of pressure values, or an overpressure value above ambient atmospheric pressure.

[0045] Aspect 2. The method of aspect 1, wherein the pressure manifold provides a pressure above atmospheric pressure before actuating the pumps to draw the first and second liquids through the channels.

[0046] Aspect 3. The method of aspect 1, wherein the pressure manifold provides a pressure above atmospheric pressure before operating the pump to draw the first and second liquids through the channel, and the pressure manifold continues to provide a pressure above atmospheric pressure throughout operating the pump to draw the first and second liquids through the channel.

[0047] Aspect 4. The method of aspect 1, wherein the pressure manifold provides a pressure above atmospheric pressure simultaneously with actuating the pump to draw the first and second liquids through the channel.

[0048] Aspect 5. The method of any one of aspects 1-4, wherein attaching the pressure manifold to the inlet reservoir forms an attached seal between the inlet reservoir and the pressure manifold.

[0049] Aspect 6. The method of any one of aspects 1-5, further comprising removing at least 90% or more of the second liquid from the channel such that the channel contains gas from the pressure manifold. In other aspects, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the second liquid can be displaced from the channel such that the channel contains gas from the pressure manifold.

[0050] Aspect 7. A method according to any one of aspects 1-6, wherein the first liquid forms a plurality of bubbles in the channel, and operating the pump to draw at least a portion of the first liquid from the channel causes a portion of the plurality of bubbles in the channel to flow out through the outlet.

[0051] Aspect 8. The method of aspect 7, wherein a portion of the plurality of bubbles ranges from about 50% to 100%. Using a combination of operating a pump to draw liquid and a pressure manifold that provides pressure above atmospheric pressure has improved the yield for removing bubbles in an efficient manner. For various reasons, it can be difficult to remove bubbles from a channel by pumping liquid through the channel using only a pressure manifold or only a pump.

[0052] Aspect 9. The method of any one of aspects 1-8, wherein the first liquid comprises a plurality of cells in a channel, and actuating the pump to draw at least a portion of the first liquid from the channel causes a portion of the plurality of cells in the channel to flow out through the outlet.

[0053] Aspect 10. The method of Aspect 9, wherein the portion of the plurality of cells ranges from about 50% to 100%. Using a combination of actuating a pump to draw in liquid and a pressure manifold to provide pressure above atmospheric pressure improved the yield of cell removal in an efficient manner, which was an unexpected result. For various reasons, it can be difficult to remove cells from a channel by pumping liquid through the channel using only a pressure manifold or only a pump.

[0054] Aspect 11. The method of any one of aspects 1-10, wherein the pump creates a vacuum while actuating the pump to draw at least a portion of the first liquid from the channel.

[0055] Aspect 12. The method of any one of aspects 1-11, wherein the pump comprises a syringe pump.

[0056] Aspect 13. The method of any one of aspects 1-12, wherein the movement of at least a portion of the second liquid occurs for a predetermined duration such that the second liquid in the inlet reservoir is not emptied.

[0057] Aspect 14. The method of any one of aspects 1-12, wherein the movement of at least a portion of the second liquid occurs for a duration such that the first and second liquids pass through the channel and leave the channel filled with air.

[0058] Aspect 15. The method of any one of Aspects 1-14, wherein the pressure provided from the pressure manifold is greater than 1 time atmospheric pressure but less than 5 times atmospheric pressure.

[0059] Aspect 16. The method of any one of Aspects 1-14, wherein the pressure provided from the pressure manifold and atmospheric pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

[0060] Aspect 17. A method of delivering a reagent to a cellular analysis system, the method comprising: A fluidic device is provided, the fluidic device comprising: (i) a channel having an inlet and an outlet; (ii) a spatial energy modulation element in optical communication with the channel; (iii) a detector that identifies the location of one or more cells within the channel based on one or more optical signals from the one or more cells, wherein an inlet of the channel is in fluid communication with an inlet reservoir and an outlet of the channel is in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir through the channel; and and loading a mixture of one or more cells and one or more polymer precursors into an inlet reservoir; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the mixture to an elevated pressure above ambient pressure; moving the mixture into the channel with a pump such that one or more cells of the mixture are disposed within the channel; synthesizing one or more chambers within the channel, each chamber encapsulating a single cell of the one or more cells, by projecting light into the channel using a spatial energy modulation element, such that the projected light causes cross-linking of one or more polymer precursors to form a polymer matrix wall of the chamber; Including, A method wherein the location for each of the combined chambers is determined by a position identified by a detector.

[0061] Aspect 18. The mixture comprises a first liquid, and after forming one or more chambers in the channel, a portion of the one or more cells is disposed in an interstitial space outside the chamber, and the method further comprises: loading the inlet reservoir with a second liquid; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the second liquid to an elevated pressure above ambient pressure; using a pump to move a second liquid into the channel such that a small portion of the cells disposed in the interstitial space exit the channel through the outlet; 18. The method of aspect 17, further comprising:

[0062] Aspect 19. The method of aspect 18, wherein the fraction of cells in the interstitial spaces ranges from about 50% to 100%. Figure 3A shows various cells, some of which are caged within the hydrogel structures (216) and other cells (e.g., 218) are uncaged and reside within the interstitial spaces between the hydrogel structures. Figure 3B shows the channel after flowing with a second liquid, in which a pressure manifold was not used to pressurize the second liquid and only a pump was used to move the liquid by drawing liquid from the outlet. Figure 3B shows a significant amount of remaining cells (e.g., 218) present within the interstitial spaces, compared to Figure 3A before cleaning of the channel. Figure 4A shows various cells, some of which are caged within the hydrogel structures (216) and other cells (e.g., 218) are uncaged and reside within the interstitial spaces between the hydrogel structures. Figure 4B shows the channel after flowing with a second liquid, where a pressure manifold was used to pressurize the second liquid while a pump was used to move the liquid by drawing it from the outlet. Figure 4B shows that there are relatively few remaining cells in the interstitial spaces, in contrast to the larger amount of remaining cells when no pressure manifold was used as shown in Figure 3B. Note that under certain conditions, cells and hydrogel structures can be fragile to flowing liquid, and flowing with a pressure manifold and pump drawing liquid from the outlet resulted in a benign condition in which the hydrogel was not perturbed, while at the same time resulting in efficient removal of interstitial cells.

[0063] Aspect 20. A method according to any one of aspects 18-19, wherein the first liquid forms a plurality of bubbles in the gap space within the channel, and a pump is used to move the second liquid into the channel so that some of the plurality of bubbles disposed in the gap space flow out of the channel through the outlet.

[0064] Aspect 21. The method of aspect 20, wherein a portion of the plurality of bubbles ranges from about 50% to 100%.

[0065] Aspect 22. The method of any one of aspects 17-21, wherein the pump creates a vacuum at the outlet while moving the mixture into the channel.

[0066] Aspect 23. The method of any one of aspects 18-21, wherein the pump creates a vacuum at the outlet while moving the second liquid into the channel.

[0067] Aspect 24. The method of any one of aspects 17-23, wherein the pump comprises a syringe pump.

[0068] Aspect 25. The method of any one of aspects 18-24, wherein the movement of the second liquid occurs for a duration such that the first and second liquids pass through the channel, leaving the channel filled with air.

[0069] Aspect 26. The method of any one of Aspects 17-25, wherein the elevated pressure is greater than 1 time atmospheric pressure but less than 5 times atmospheric pressure.

[0070] Aspect 27. The method of any one of aspects 17-25, wherein the elevated pressure from the pressure manifold and the ambient pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

[0071] Aspect 28. The method of any one of aspects 17-27, wherein the pressure manifold is sealed and attached to the inlet reservoir, and pressurizing the mixture to an elevated pressure above ambient pressure occurs before using a pump to move the mixture into the channel.

[0072] Aspect 29. The method of any one of aspects 17-27, wherein the pressure manifold is sealed and attached to the inlet reservoir, and pressurizing the mixture to an elevated pressure above ambient pressure occurs before using the pump to move the mixture into the channel, and the pressure manifold continues to provide the elevated pressure throughout using the pump to move the mixture into the channel.

[0073] Aspect 30. The method of any one of aspects 18-29, further comprising removing at least 90% or more of the second liquid from the channel such that the channel contains gas from the pressure manifold.

[0074] Aspect 31. A fluid delivery system, comprising: one or more reaction channels each containing a first liquid and having an outlet and an inlet, the inlet reservoir fluidly coupled to the inlet; a pump in fluid communication with the outlet of one or more reaction channels, the pump configured to move liquid through the channel at a predetermined rate; one or more supply reservoirs containing a liquid in contact with the atmosphere; a pipettor for transferring a second liquid from one or more supply reservoirs to the inlet reservoir; a pressure manifold sealingly attached to the inlet reservoir; Equipped with The fluid delivery system, wherein the pressure manifold is configured to apply a predetermined pressure above atmospheric pressure to the first and second liquids.

[0075] Aspect 32. A method for delivering a reagent to a cellular analysis system, the method comprising: providing a fluidic device comprising a channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon, the inlet of the channel in fluid communication with an inlet reservoir, and the outlet of the channel in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; loading an inlet reservoir with an assay reagent in contact with the atmosphere; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagents at an elevated pressure above ambient pressure; moving an assay reagent through the channel using a pump so that the assay reagent contacts one or more cells disposed on a surface of the channel; A method comprising:

[0076] Aspect 33. A method for delivering a reagent to a cellular analysis system, the method comprising: A fluidic device is provided, the fluidic device comprising: (i) a channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon; (ii) a spatial energy modulating element in optical communication with the surface; (iii) a detector that identifies the location of one or more cells within the channel based on one or more optical signals from the one or more cells, wherein an inlet of the channel is in fluid communication with an inlet reservoir and an outlet of the channel is in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; and and loading a liquid containing one or more polymer precursors into an inlet reservoir; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the liquid at an elevated pressure above ambient pressure; moving a liquid through the channel using a pump; synthesizing one or more chambers within the channel, each chamber encapsulating a single cell of the one or more cells, by projecting light into the channel using a spatial energy modulation element, such that the projected light causes cross-linking of one or more polymer precursors to form a polymer matrix wall of the chamber; Including, A method wherein the location for each of the synthesized chambers on the surface is determined by the position identified by the detector.

[0077] Aspect 34. A method for delivering a reagent to a cellular analysis system, the method comprising: A fluidic device is provided, the fluidic device comprising: a channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon, the inlet of the channel in fluid communication with an inlet reservoir, and the outlet of the channel in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; loading an inlet reservoir with an assay reagent in contact with the atmosphere; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagents at an elevated pressure above ambient pressure; moving an assay reagent through the channel using a pump so that the assay reagent contacts one or more cells disposed on a surface of the channel; A method comprising:

[0078] Aspect 35. The method of aspect 34, further comprising incubating one or more cells in the assay reagent for a predetermined period of time.

[0079] Aspect 36. The method of aspect 34, wherein each of the one or more cells is encapsulated by a hydrogel chamber.

[0080] Aspect 37. The method of any one of the preceding aspects, wherein the pressure manifold comprises a conduit including a single gas input port and a plurality of gas output ports, the plurality of gas output ports being in fluid communication with the single gas input port.

[0081] Aspect 38. The method of any one of the preceding aspects, wherein the pressure manifold comprises a conduit, the conduit including a single gas input port and a plurality of gas output ports.

[0082] Aspect 39. A method for moving a liquid through a channel, the method comprising: (a) providing a channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel containing a first liquid and in fluid communication with the outlet, and a pump is fluidly coupled to the outlet, the pump configured to move the liquid through the channel; (b) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first liquid; (c) operating a pump to draw at least 90% or more of the first liquid from the channel through the outlet and to move gas from the pressure manifold into the channel, the pump being programmed to move fluid at a predetermined rate while under the provided pressure; (d) transferring a second liquid to the inlet reservoir, the transferred second liquid being in contact with the atmosphere; (e) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the second liquid; (f) operating a pump to draw at least a portion of the gas from the channel through the outlet and to move at least a portion of the second liquid into the channel, the pump being programmed to move the fluid at a predetermined rate while under the provided pressure; A method comprising:

[0083] Aspect 40. The method of aspect 39, wherein the pressure manifold provides a pressure above atmospheric pressure before actuating the pump to draw gas from the channel.

[0084] Aspect 41. The method of aspect 39, wherein the pressure manifold provides a pressure above atmospheric pressure before operating the pump to draw gas from the channel, and the pressure manifold continues to provide a pressure above atmospheric pressure throughout operating the pump to draw at least a portion of the gas from the channel.

[0085] Aspect 42. The method of any one of aspects 39-41, wherein attaching the pressure manifold to the inlet reservoir forms an attached seal between the inlet reservoir and the pressure manifold.

[0086] Aspect 43. The method of any one of aspects 39-42, wherein the pump creates a vacuum while operating the pump to draw at least a portion of the gas or first liquid from the channel.

[0087] Aspect 44. The method of any one of aspects 39-43, wherein the pump comprises a syringe pump.

[0088] Aspect 45. The method of any one of Aspects 39-44, wherein the pressure provided from the pressure manifold is greater than 1 time atmospheric pressure but less than 5 times atmospheric pressure.

[0089] Aspect 46. The method of any one of aspects 39-44, wherein the pressure provided from the pressure manifold and atmospheric pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

[0090] Aspect 47. The method of any one of the preceding aspects, wherein the pressure manifold outputs a gas for providing the pressure, and the gas is selected from the group consisting of air, carbon dioxide, nitrogen, argon, and combinations thereof.

[0091] Aspect 48. The method of any one of the preceding aspects, wherein the pressure manifold outputs a gas for providing pressure, the gas comprising 5% carbon dioxide, and the second liquid comprises a cell culture medium that is equilibrated with 5% carbon dioxide such that the pH of the cell culture medium does not change by more than 10%.

[0092] Aspect 49. The method of any one of the preceding aspects, wherein the pressure manifold outputs a gas for providing pressure, the gas comprising nitrogen or argon, and the second liquid comprises anaerobic cells or anaerobic organisms.

[0093] Aspect 50. The method of any one of the preceding aspects, wherein the pressure manifold provides a pressure above atmospheric pressure for a predetermined duration before actuating the pump to draw at least a portion of the first or second liquid from the channel. The predetermined duration can range from 5 minutes to 1 day. The pressure manifold providing the pressure for the predetermined duration causes a reduction in bubble formation for the predetermined duration compared to a situation in which no pressure manifold was used.

[0094] Aspect 51. A method of incubating a liquid in a channel, the method including: (a) providing a channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel containing a first liquid and in fluid communication with the outlet, a pump fluidly coupled to the outlet, the pump configured to move the liquid through the channel; and (b) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first liquid, wherein the pump is not activated, causing the outlet to be sealed and the liquid to be quiescent. FIG. 5A shows a flow cell having channels E and F in which the liquid in the channels was relatively bubble-free. Channel F was pressurized using the pressure manifold at 5 PSI in the inlet reservoir and the outlet was closed for 90 minutes at 42° C. Channel E was left at atmospheric pressure for 90 minutes at 42° C. FIG. 5B shows that after 90 minutes, channel F remained relatively bubble-free and channel E had multiple bubbles 502. Thus, the channel with applied pressure from the manifold served to reduce bubble formation over a period of time at elevated temperatures.

[0095] (definition) Unless specifically defined otherwise herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those of standard treatises and texts in the field, such as Kornberg and Baker's DNA Replication, Second Edition (W.H. Freeman, New York, 1992), Lehninger's Biochemistry, Second Edition (Worth Publishers, New York, 1975), Strachan and Read's Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999), and Abbas et al.'s Cellular and Molecular Immuology, 6th edition (Saunders, 2007).

[0096] "Assay" refers to a process for detecting or measuring cellular characteristics or properties of a single cell or a population of cells. Typically, the process steps of an assay involve a chemical, biochemical, or molecular reaction (such as cleavage of a junction, specific conjugation of complementary components, an enzymatic reaction, or dissolution of complementary components) or a change in physical state (such as an increase or decrease in temperature or a change in energy level), resulting in the generation of a signal (or signals) from which the presence, absence, or magnitude of a cellular-related quantity can be inferred. The nature of the signal produced by an assay can vary widely and can include, but is not limited to, an electrical signal, an optical signal, a chemical signal, or a material signal. A material signal involves the production of a material containing information that can be extracted. For example, a material signal can be the amplification of a polynucleotide whose length, quantity, composition, or nucleotide sequence indicates a cellular characteristic. For example, a barcode oligonucleotide can be a material signal. The cellular characteristics and properties detected and measured can vary widely and include, but are not limited to, cytotoxicity, viability, growth capacity under selected conditions, size, shape, motility, cell surface i.e., membrane protein type and profile, secreted protein type and profile, metabolite production, transcriptome, gene copy number, gene or allele discrimination, chromatin accessibility profile, vector copy number for engineered or infected cells, etc. Assays of particular interest for cell-based therapies include, but are not limited to, cytotoxicity, viability, activation, growth capacity under selected conditions, chromatin accessibility profile, cell surface i.e., membrane protein type and profile, secreted protein type and profile, intracellular proteins, transcriptome, vector copy number, etc. As used herein, an "assay reagent" is a liquid used in an assay. Assay reagents can include, but are not limited to, pH buffered solutions, enzyme buffer solutions with or without enzymes, molecular or cellular stains or dyes, lysing agents, gel-resolving reagents, cell suspensions, salt solutions, wash solutions, cell growth media, etc.

[0097] A "cell" refers to a biological cell that can be assayed by the methods and systems described herein, including, but not limited to, a vertebrate, invertebrate, eukaryotic, mammalian, microbial, protozoan, prokaryotic, bacterial, worm, or fungal cell. In some embodiments, a mammalian cell is assayed by the methods and systems described herein. In particular, any mammalian cell that can be or has been genetically modified for use in medical, industrial, environmental, or therapeutic processes can be analyzed by the methods and systems described herein. In some embodiments, a "cell" as used herein includes a genetically modified mammalian cell. In some embodiments, a "cell" includes a stem cell. In some embodiments, a "cell" refers to a cell modified by CRISPR Cas9 technology. In some embodiments, a "cell" refers to a cell of the immune system, including, but not limited to, a cytotoxic T lymphocyte, a regulatory T cell, a CD4+ T cell, a CD8+ T cell, a natural killer cell, an antigen-presenting cell, or a dendritic cell. Of particular interest are cytotoxic T lymphocytes that are engineered for therapeutic applications such as cancer therapy.

[0098] "Hydrogel" refers to a gel comprising a cross-linked hydrophilic polymer network with the ability to absorb and retain large amounts of water (e.g., 60-90 percent or 70-80 percent water) without dissolution due to the establishment of physical or chemical bonds between polymer chains, which may be covalent, ionic, or hydrogen bonds. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels may comprise natural or synthetic polymers and may be reversible (i.e., degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers may include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Natural hydrogel polymers may include alginate, hyaluronic acid, and collagen. The following references describe hydrogels and their biomedical uses: Drury et al., Biomaterials, 24:4337-4351 (2003); Garagorri et al., Acta Biomatter, 4(5):1139-1147 (2008); Caliari et al., Nature Methods, 13(5):405-414 (2016); Bowman et al., U.S. Pat. No. 9,631,092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0099] "Polymer matrix" generally refers to a phase material (e.g., a continuous phase material) that includes at least one polymer. In some embodiments, the polymer matrix refers not only to at least one polymer but also to the interstitial spaces not occupied by the polymer. The polymer matrix may be composed of one or more types of polymer. The polymer matrix may include linear, branched, and cross-linked polymer units. The polymer matrix may also include non-polymeric species intercalated within the interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous. For example, the term "polymer matrix" may encompass dried hydrogels, hydrated hydrogels, and hydrogels including glass fibers. The polymer matrix may include polymer precursors, which generally refer to one or more molecules whose activation can trigger or initiate a polymer reaction. The polymer precursors can be activated by electrochemical energy, photochemical energy, photons, magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which are polymerized to produce a polymer matrix) and crosslinking compounds, which may include photoinitiators, and other compounds necessary or useful to generate a polymer matrix, particularly a polymer matrix that is a hydrogel.

[0100] While preferred embodiments of the systems and methods described herein have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the systems and methods described herein be limited by the specific examples provided herein. While the systems and methods described herein have been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the systems and methods described herein. Furthermore, it should be understood that all aspects of the systems and methods described herein are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the systems and methods described herein may be employed in practicing the systems and methods described herein. It is therefore contemplated that the systems and methods described herein also encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the systems and methods described herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method of moving a liquid through a channel, the method comprising: (a) providing said channel having an outlet and an inlet, an inlet reservoir fluidly coupled to the inlet; the channel contains a first liquid; a pump fluidly coupled to the outlet; and (b) transferring a second liquid to the inlet reservoir, the transferred second liquid being in contact with the atmosphere; and (c) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first and second liquids; (d) operating the pump to draw at least a portion of the first liquid from the channel through the outlet and to move at least a portion of the second liquid into the channel; Including, The method wherein the pump is programmed to move fluid at a predetermined rate while under the pressure provided by the pressure manifold.

2. The method of claim 1 , wherein the pressure manifold provides the pressure above atmospheric pressure prior to the actuation in (d).

3. 2. The method of claim 1, wherein the pressure manifold provides the pressure above atmospheric pressure before the actuation in (d), and the pressure manifold continues to provide the pressure above atmospheric pressure during the actuation in (d).

4. The method of claim 1 , wherein the pressure manifold provides the pressure above atmospheric pressure simultaneously with the actuation in (d).

5. The method of any one of claims 1-4, wherein in (c), an associated seal is formed between the inlet reservoir and the pressure manifold.

6. The method of any one of claims 1-5, further comprising removing at least 90% or more of the second liquid from the channel such that the channel contains gas from the pressure manifold.

7. 7. The method of claim 1, wherein the first liquid forms a plurality of bubbles in the channel, and in (d), at least some of the bubbles in the channel flow out through the outlet.

8. The method of claim 7 , wherein the portion of the plurality of bubbles ranges from about 50% to about 100% of the bubbles.

9. The method of any one of claims 1 to 8, wherein the first liquid contains a plurality of cells in the channel, and in (d), at least a portion of the plurality of cells in the channel flows out through the outlet.

10. 10. The method of claim 9, wherein the portion of the plurality of cells ranges from about 50% to about 100% of the cells.

11. The method of any one of claims 1 to 10, wherein in (d), the pump generates a vacuum.

12. The method of any one of claims 1-11, wherein the pump comprises a syringe pump.

13. A method according to any preceding claim, wherein the movement of at least the portion of the second liquid occurs for a predetermined duration such that the second liquid in the inlet reservoir is not emptied.

14. 13. The method of any one of claims 1-12, wherein the movement of at least the portion of the second liquid occurs for a duration such that the first and second liquids pass through the channel, thereby leaving the channel filled with air.

15. 15. The method of any one of claims 1-14, wherein the pressure provided by the pressure manifold is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

16. 15. The method of any one of claims 1-14, wherein the pressure provided by the pressure manifold and the atmospheric pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

17. 1. A method for delivering a liquid to a cellular analysis system, the method comprising: (a) providing a fluidic device, the fluidic device comprising: (i) a channel having an inlet and an outlet; (ii) a spatial energy modulating element in optical communication with the channel; (iii) a detector that identifies the location of one or more cells within the channel based on one or more optical signals from the one or more cells; Equipped with the inlet of the channel is in fluid communication with an inlet reservoir; the outlet of the channel is in fluid communication with a pump, the pump configured to move a predetermined volume of liquid from the inlet reservoir through the channel; (b) loading the mixture of the one or more cells and one or more polymer precursors into the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the mixture to an elevated pressure above ambient pressure; (d) using the pump to move the mixture into the channel such that the one or more cells of the mixture are disposed within the channel; (e) synthesizing one or more chambers within the channel, each chamber enclosing a single cell of the one or more cells, by projecting light into the channel using the spatial energy modulation element, such that the projected light causes the one or more polymer precursors to form polymer matrix walls of one or more chambers; Including, A method wherein a location for each of the combined chambers is determined by the position of the one or more cells identified by the detector.

18. the mixture comprises a first liquid, and after the combining in (e), a portion of the one or more cells are disposed in an interstitial space outside the chamber, the method further comprising: (f) loading the inlet reservoir with a second liquid; (g) sealingly attaching the pressure manifold to the inlet reservoir and pressurizing the second liquid to the elevated pressure above the ambient pressure; (h) using the pump to move the second liquid into the channel such that a small portion of the cells disposed in the interstitial space exit the channel through the outlet; 20. The method of claim 17, further comprising:

19. 20. The method of claim 18, wherein the portion of the one or more cells in the interstitial space ranges from about 50% to about 100.

20. 20. The method of claim 18 or 19, wherein the first liquid forms a plurality of bubbles in the interstitial spaces within the channel, and in (h), some of the bubbles disposed in the interstitial spaces exit the channel through the outlet.

21. The method of claim 20, wherein the portion of the plurality of bubbles ranges from about 50% to about 100%.

22. 22. The method of any one of claims 17-21, wherein in (d), the pump creates a vacuum at the outlet.

23. 22. The method of any one of claims 18-21, wherein in (h), the pump creates a vacuum at the outlet.

24. The method of any one of claims 17-23, wherein the pump comprises a syringe pump.

25. 25. A method according to any one of claims 18-24, wherein the movement of the second liquid occurs for a duration such that the first and second liquids pass through the channel, thereby leaving the channel filled with air.

26. 26. A method according to any one of claims 17 to 25, wherein the elevated pressure is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

27. 26. The method of any one of claims 17-25, wherein the elevated pressure provided by the pressure manifold and the ambient pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

28. 28. The method of any one of claims 17-27, wherein (c) occurs before (d).

29. 28. The method of any one of claims 17-27, wherein (c) occurs before (d), and the pressure manifold continues to provide the elevated pressure throughout (d).

30. 30. The method of any one of claims 18-29, further comprising removing at least 90% or more of the second liquid from the channel such that the channel contains gas from the pressure manifold.

31. 1. A fluid delivery system, comprising: one or more reaction channels each containing a first liquid, wherein each reaction channel of the one or more reaction channels comprises an outlet and an inlet, and an inlet reservoir is fluidly coupled to the inlet; a pump in fluid communication with the outlet, the pump configured to move liquid through the channel at a predetermined rate; one or more supply reservoirs containing a second liquid in contact with the atmosphere; a pipettor for transferring the second liquid from the one or more supply reservoirs to the inlet reservoir; a pressure manifold sealingly attached to the inlet reservoir; Equipped with The fluid delivery system, wherein the pressure manifold is configured to apply a pressure above atmospheric pressure to the first and second liquids.

32. 1. A method for delivering a reagent to a cellular analysis system, the method comprising: providing a fluidic device comprising a channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon, the inlet of the channel in fluid communication with an inlet reservoir and the outlet of the channel in fluid communication with a pump, the pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; loading the inlet reservoir with an assay reagent in contact with the atmosphere; sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagents at an elevated pressure above ambient pressure; moving the assay reagent through the channel using the pump so that the assay reagent contacts the one or more cells disposed on the surface of the channel; A method comprising:

33. 1. A method for delivering a reagent to a cellular analysis system, the method comprising: (a) providing a fluidic device, the fluidic device comprising: (i) a channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon; (ii) a spatial energy modulating element in optical communication with said surface; (iii) a detector that identifies a location of the one or more cells within the channel based on one or more optical signals from the one or more cells, wherein the inlet of the channel is in fluid communication with an inlet reservoir and the outlet of the channel is in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; and and (b) loading a liquid containing one or more polymer precursors into the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the liquid at an elevated pressure above ambient pressure; (d) moving the liquid through the channel using the pump; (e) synthesizing one or more chambers within the channel, each chamber enclosing a single cell of the one or more cells, by projecting light into the channel using the spatial energy modulation element, such that the projected light causes the one or more polymer precursors to form polymer matrix walls of one or more chambers; Including, A method wherein a location for each of the synthesized chambers on the surface is determined by the position of the one or more cells identified by the detector.

34. 1. A method for delivering a reagent to a cellular analysis system, the method comprising: (a) providing a fluidic device, the fluidic device comprising: (i) a channel, the channel having an inlet, an outlet, and a surface, the surface having one or more cells disposed thereon, the inlet of the channel in fluid communication with an inlet reservoir, and the outlet of the channel in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir into the channel; (b) loading the inlet reservoir with an assay reagent in contact with the atmosphere; (c) sealingly attaching a pressure manifold to the inlet reservoir and pressurizing the assay reagents at an elevated pressure above ambient pressure; (d) moving the assay reagent through the channel using the pump so that the assay reagent contacts the one or more cells disposed on the surface of the channel; A method comprising:

35. 35. The method of claim 34, further comprising incubating the one or more cells in the assay reagent for a predetermined period of time.

36. 35. The method of claim 34, wherein each of the one or more cells is encapsulated by a hydrogel chamber.

37. 10. The method of claim 1, wherein the pressure manifold comprises a conduit having a single gas input port and a plurality of gas output ports, the plurality of gas output ports being in fluid communication with the single gas input port.

38. 10. The method of claim 1, wherein the pressure manifold comprises a conduit having a single gas input port and a plurality of gas output ports.

39. 1. A method of moving a liquid through a channel, the method comprising: (a) providing said channel having an outlet and an inlet, an inlet reservoir fluidly coupled to the inlet; the channel contains a first liquid; a pump fluidly coupled to the outlet; and (b) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first liquid; (c) operating the pump to draw at least 90% or more of the first liquid from the channel through the outlet and to move gas from the pressure manifold into the channel, the pump being programmed to move fluid at a predetermined rate while under the pressure provided by the pressure manifold; (d) transferring a second liquid to the inlet reservoir, the transferred second liquid being in contact with the atmosphere; and (e) attaching the pressure manifold to the inlet reservoir, the pressure manifold providing the pressure above atmospheric pressure to the second liquid; (f) operating the pump to draw at least a portion of the gas from the channel through the outlet and to displace at least a portion of the second liquid into the channel; Including, The method wherein the pump is programmed to move fluid at the predetermined rate while under the pressure provided by the pressure manifold.

40. 40. The method of claim 39, wherein the pressure manifold provides the pressure above atmospheric pressure prior to the actuation in (f).

41. 40. The method of claim 39, wherein the pressure manifold provides the pressure above atmospheric pressure before the actuation in (f), and the pressure manifold continues to provide the pressure above atmospheric pressure during the actuation in (f).

42. 42. The method of any one of claims 39-41, wherein in (b) and (e) an associated seal is formed between the inlet reservoir and the pressure manifold.

43. 43. A method according to any one of claims 39 to 42, wherein the pump creates a vacuum during the actuation of the pump so as to draw at least a portion of the gas or first liquid from the channel.

44. The method of any one of claims 39-43, wherein the pump comprises a syringe pump.

45. 45. The method of any one of claims 39-44, wherein the pressure provided by the pressure manifold is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

46. 45. The method of any one of claims 39-44, wherein the pressure provided by the pressure manifold and the atmospheric pressure have a difference ranging from about 2 pounds per square inch to about 5 pounds per square inch.

47. 10. The method of claim 1, wherein the pressure manifold outputs a gas for providing the pressure, the gas being selected from the group consisting of air, carbon dioxide, nitrogen, argon, and combinations thereof.

48. 48. The method of any one of claims 1-16, 18-31, 39-47, wherein the pressure manifold outputs a gas to provide the pressure, the gas comprising 5% carbon dioxide, and the second liquid comprises a cell culture medium equilibrated with 5% carbon dioxide such that the pH of the cell culture medium in the channel does not change by more than 10%.

49. 49. The method of any one of claims 1-16, 18-31, 39-48, wherein the pressure manifold outputs a gas to provide the pressure, the gas comprising nitrogen or argon, and the second liquid comprises anaerobic cells or organisms.

50. 1. A method of incubating a liquid in a channel, the method comprising: (a) providing the channel having an outlet and an inlet, an inlet reservoir fluidly coupled to the inlet, the channel containing a first liquid, and a pump fluidly coupled to the outlet; (b) attaching a pressure manifold to the inlet reservoir that provides a pressure above atmospheric pressure to the first liquid; Including, A method wherein the pump is not activated, thereby causing the outlet to be sealed and the liquid to be stationary.

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