Liquid delivery system

Abstract

The methods and systems described herein relate to a reagent delivery system for delivering a plurality of reagents from a microwell array to a reaction channel and reducing air gaps or bubbles, or for removing cells that are not wrapped in a hydrogel cage. In some embodiments, the reagent is moved through the channel by a pump attached to the channel outlet and a pressure column applied to the channel inlet.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 419,655, filed on October 26, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporated by reference

[0004] All publications, patents, and patent applications mentioned in this specification are hereby 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 incorporated publications and patents or patent applications conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] Microfluidics has played a crucial role in single-cell analysis techniques, e.g., Zare et al., Annu. Rev. Biomed. Eng., 12:187-201 (2010); Valihrach et al., Int. J. Mol. Sci., 19:807 (2018); Murphy et al., The Analyst, 18:60-80 (2017); Shinde et al., Int. J. Mol. Sci., 19:3143 (2018); etc. However, there remain challenges in designing fluid and microfluidic systems that avoid common problems such as bubble formation or the persistence of bubbles once formed. Bubbles can have a negative impact on performance by obstructing flow, blocking reaction regions, and damaging delicate microstructures, e.g., Pereiro et al., LabChip, 19:2296 (2019). Especially in systems where reagent storage, flow, or transfer is in contact with the ambient atmosphere, such reagents may accumulate dissolved gases that can exceed saturation levels and lead to bubble formation within the microfluidic components. Summary of the Invention

[0006] In view of the above, the availability of new methods and devices for minimizing bubble formation in microfluidic systems that handle fluids exposed to the atmosphere would advance the field of microfluidics, especially in the area of single-cell analysis.

[0007] The methods and systems described herein relate to transferring liquid reagents from a reservoir exposed to the atmosphere to a fluid system where dissolved gases may rise above saturation levels, thereby risking the formation of destructive bubbles.

[0008] The present disclosure provides a method for moving a liquid in contact with the atmosphere through a channel, comprising: (a) providing a channel having an outlet and an inlet, the inlet being provided with an inlet reservoir, the channel containing a first liquid and being in fluid communication with a pump through its outlet, the pump moving the liquid through the channel at a predetermined rate; (b) transferring a second liquid in contact with the atmosphere from a reservoir to the inlet reservoir of 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 at the predetermined pressure. In some embodiments, the moving of the first and second liquids occurs over a predetermined duration such that the inlet reservoir does not empty of the first and second liquids. In some embodiments, the moving of the first and second liquids occurs over a predetermined duration such that a predetermined volume of the first and second liquids passes through the channel.

[0009] The present disclosure also provides a fluid delivery system, comprising: (a) one or more reaction channels, each of the one or more reaction channels 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 the liquid through the channel at a predetermined rate; (c) one or more supply reservoirs containing a liquid in contact with the atmosphere; (d) a pipette for transferring a second liquid from the one or more supply reservoirs to the inlet reservoir; and (e) a pressure manifold sealingly attached to the inlet reservoir, the pressure manifold applying a predetermined pressure above atmospheric pressure to the first and second liquids in the inlet reservoir.

[0010] The present disclosure also provides a method for delivering reagents to a cell analysis system, comprising: (a) providing a fluid device, the fluid device comprising (i) a channel comprising an inlet, an outlet, and a first surface having one or more cells disposed thereon, and 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 draws or moves a predetermined volume of liquid from the inlet reservoir into the channel under programmed control; (b) loading the assay reagent in contact with the atmosphere into the channel by transferring a volume of the assay reagent to the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir to pressurize the assay reagent at a predetermined pressure; and (d) pumping the assay reagent through the channel such that the assay reagent binds to the one or more cells disposed on the first surface of the channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A - 1EIllustrated is an embodiment for transferring a liquid exposed to the atmosphere into a closed reaction channel of a fluid system.

[0012] Figures 2A - 2B More particularly illustrated is an apparatus that can employ the systems and methods described herein to detect cells and synthetic hydrogel chambers.

[0013] Figure 3A Are photographs of various cells, some of which are trapped in the hydrogel structure and others are not and reside in the interstitial space between the hydrogel structures.

[0014] Figure 3B Are photographs of the various cells shown in Figure 3A after liquid has flowed through the flow cell without an applied overpressure pressure manifold while the pump removes fluid from the channel and leaves multiple cells in the interstitial space.

[0015] Figure 4A Are photographs of various cells, some of which are trapped in the hydrogel structure and others are not and reside in the interstitial space between the hydrogel structures.

[0016] Figure 4B Are photographs of the various cells shown in Figure 4A after liquid has flowed through the flow cell with an applied overpressure pressure manifold while the pump removes fluid from the channel. Compared with Figure 3B (without a pressure manifold), Figure 4B the proportion of cells left in the interstitial space in

[0017] Figure 5A Is a photograph of a flow cell that has channels E and F, both of which contain liquid and are relatively bubble-free.

[0018] Figure 5B Is Figure 5A a photograph of a flow cell that contains liquid incubated at 42 °C for 90 minutes, where channel F is pressurized at 5 PSI with a pressure manifold and channel E is at atmospheric pressure. Detailed Description

[0019] Unless otherwise indicated, the practice of the systems and methods described herein may employ conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques include, but are not limited to, the preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. Specific illustrations of suitable techniques can be obtained by reference to the examples below. However, other equivalent conventional procedures may of course be used. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Volumes I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, eds., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, eds., Protein Engineering Handbook (Wiley-VCH, 2009); etc. Guidance for selecting materials and components for performing specific functions can be found in available treatises and references on scientific instrumentation, including but not limited to Moore et al., Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013); etc.

[0020] The method and system involve delivering a fluid exposed to the atmosphere (e.g., from an open reservoir such as a microwell) to a closed reaction channel while minimizing the risk of bubble formation in the channel. In one aspect, the method includes transferring a liquid from a reservoir open to the atmosphere to an inlet reservoir at the inlet end of the reaction channel, and then sealingly attaching a pressure manifold to the inlet reservoir to deliver pressure to the liquid in the inlet channel and the main channel. In some cases, at the outlet end of the main channel, a pump or vacuum source draws or moves the liquid through the reaction channel at a predetermined rate while the pressure manifold delivers a pressure greater than ambient atmospheric pressure to the inlet reservoir. In some embodiments, the method includes a series of steps, which include: (a) depressurizing a first liquid in the channel; (b) delivering a second liquid to an inlet reservoir in fluid communication with the channel such that the first liquid and the second liquid form a single liquid body; (c) pressurizing the first liquid and the second liquid; and (d) drawing the first liquid and the second liquid through the channel at a predetermined rate. In some embodiments, such liquid delivery is part of a process that requires multiple such transfers, in which case steps (a) through (d) can be repeated multiple times. In some embodiments, "depressurizing" means equilibrating the pressure of the first liquid with atmospheric pressure. In some embodiments, prior to depressurizing, the first liquid is at a pressure above atmospheric pressure. In some embodiments, the step (c) of pressurizing the first liquid and the second liquid includes pressurizing the first liquid and the second liquid to a predetermined pressure. It should be noted that the ambient pressure or ambient atmospheric pressure refers to the pressure at the measurement location and typically ranges from about 14.5 inches of mercury to about 14.9 inches of mercury (or 14.5 pounds per square inch (psi) to 14.9 pounds per square inch and is typically about 14.7 psi).

[0021] Embodiments of the method are as Figures 1A - 1E shown. The flow cell (109) includes four channels (116, 118, 120, and 122) (sometimes referred to as "main channels") and a body (108) that includes four inlet reservoirs (112a, 112b, 112c, and 112d), one inlet reservoir corresponding to each channel (116, 118, 120, and 122, respectively). In some embodiments, the inlet reservoir can include an inverted conical shape (or a partially conical shape) as Figure 1C or Figure 1E shown, and these inlet reservoirs are connected through the narrow portion of the inverted cone (sometimes referred to herein as the "throat" of the inlet reservoir), e.g., Figure 1A114a) and the inlets of the respective channels are in fluid communication with channels (116, 118, 120, and 122) respectively. The inlet reservoir can be formed in a body (such as (108)), which body (108) is combined with or otherwise sealingly attached to the flow cell (109). The enlarged view (110) gives a three-dimensional view of these components. The flow cell (109) can also include a first surface and a second surface to form the top and bottom boundaries of the channels. Referring to Figure 1C , the first surface can be in the form of a glass plate (105), which has a plurality of inlets (107a, 107b, 107c, and 107d) for the respective channels (116, 118, 120, and 122). The plurality of inlets (107a, 107b, 107c, and 107d) can be a series of through-holes in the glass plate (105), which through-holes are 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 plate (105) and the respective 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 an end opposite to one end of the inlet reservoir. In some embodiments, each outlet reservoir (113a, 113b, 113c, and 113d) is separately connected to a pump (115a, 115b, 115c, and 115d respectively) (and is in fluid communication with the pump). In some embodiments, such pumps are precision syringe pumps, which can be programmed to coordinate with the filling of the inlet reservoir (i.e., for example, in some embodiments, whenever the inlet reservoir is being filled, the syringe pump does not operate), and draw or move the liquid through its respective channel at a predetermined flow rate at predetermined intervals. The reagent reservoir can be a well 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., "pipettor")) transfers the liquid to all the inlet reservoirs of the flow cell simultaneously. For example, in Figure 1A a four-channel flow cell, the liquid in a subset of four wells (104) can be transferred (106) simultaneously to four inlet reservoirs (112a, 112b, 112c, and 112d). As Figure 1BAs shown, after delivering the liquid to the inlet reservoir, the pressure manifold (122) is sealingly attached to the inlet reservoir such that pressure can be applied to the delivered liquid and the liquid already present in the channels. The pressure manifold (122) can be a body that is 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 the inlet reservoirs. In some embodiments where each inlet reservoir is pressurized using a separate pressure source, such pressure sources can be adjusted to a pressure specific to its associated channel. In some embodiments, such specific pressure can depend on the amount of fluid resistance in its associated channel. Such differences in fluid resistance can be caused by different amounts of material (such as cells, gel microstructures, debris, etc.) in the channels. The latter embodiment is as shown in Figure 1B As shown, after the pressure manifold (122) is sealingly attached to the inlet reservoir, the pressure source (126) can generate a pressure (or a predetermined pressure) that is transferred to the inlet reservoir through the conduit (124) and the pressure manifold itself. In some embodiments, the predetermined pressure exceeds atmospheric pressure. In some embodiments, for example, in the case of causing the liquid to flow from the channels to the inlet reservoir, the predetermined pressure can be lower than atmospheric pressure.

[0022] For the above embodiments, Figure 1C and Figure 1DFurther illustrated is the movement of liquid through the inlet reservoir and channels during operation of the fluid transfer system. Cross-sectional view (111) (or FIG. (1)) shows the interior of the inlet reservoir and channels along plane (130), which plane (130) transverses a three-dimensional representation (110) of the body (108) forming the inlet reservoir. FIGS. (2)-(6) present the same cross-sectional view at different steps during the fluid transfer process. Returning to FIG. (1), the inlet reservoirs (112a, 112b, 112c, and 112d) can be in internal fluid communication with the interior of the channels (116, 118, 120, and 122), respectively, through the throat regions (114a, 114b, 114c, and 114d, respectively) of the inlet reservoir and the inlets of the channels, each of these throat regions including a narrow region of the inlet reservoir (the throat region 114a of the inlet reservoir is specifically shown). The shaded regions of the channels and the channel inlets in cross-section (111) represent the presence of a first liquid. According to the configuration of cross-section (111) (i.e., the first liquid in the channel and the inlet reservoir being empty (or nearly empty)), as shown in FIG. (2), a second fluid can be transferred (131) from one (or more) open reservoirs to the open inlet reservoir, for example, by using a pipette (132). After removing the pipette (132) (FIG. (3)), as shown, the first liquid (134) and the second liquid (136) can combine or coalesce with each other (at least partially) such that they form a single body of liquid for each channel. The pressure manifold (122) can be sealingly attached (140) to the inlet reservoirs (112a, 112b, 112c, and 112d); a predetermined pressure can be applied to the inlet reservoirs; and the pumps (115a, 115b, 115c, and 115d) can be actuated to begin pumping the liquid through their respective channels at a predetermined rate (FIGS. (4) and (5)). In some embodiments, the pressure can be adjusted to maintain a predetermined pressure if needed when the first and second liquids are being moved through the channels by the pumps. In some embodiments, the amount of liquid pumped through the channels is greater than the volume of a single channel. In some embodiments, the amount of liquid pumped or moved through the channels is a multiple of the channel volume. For example, the amount of liquid pumped or moved through the channels can be 2 times, 3 times, 4 times, 5 times, or 10 times the channel volume. In some embodiments, the amount of liquid pumped or moved through the channels can be less than the volume of a single channel. The volume of the inlet reservoirs (and the volume of the liquid delivered to these inlet reservoirs) can be adjusted according to the predetermined volume of liquid to be pumped or moved through the channels as needed. In some embodiments, the volume of the liquid delivered and the volume of the liquid pumped or moved in each cycle of the steps represented in FIGS. (1)-(6) are selected such that after pumping or moving the liquid, the resulting liquid level in the inlet reservoir is within or above the throat region of the inlet reservoir (e.g., Figure 1D of (138) in FIG. (5) ofFigure 1E as shown in (152) or (166) in []. Finally, as shown in FIG. (6), the original first liquid (as shown in the shaded area (134) in FIG. (3)) can be replaced by the new first liquid (as shown in the cross-hatched area (136) in FIG. (6)), so that the inlet reservoir is ready for the next cycle of the liquid transfer step.

[0023] Figure 1E Also illustrated is a cycle of steps for transferring a volume of liquid by the method described herein. A flow cell (150) is shown having an inlet reservoir (156) that is in fluid communication with a channel (154) and a pump (160) through a conduit (155) that connects the channel outlet to the pump (160). At the start of the transfer cycle, the level (152) of the first liquid can be in or near the throat region of the inlet reservoir, or in 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)) can be sealingly attached (170) to the inlet reservoir, and a predetermined pressure can be applied to the first and second liquids, after which the pump (160) is actuated (170) to draw or move a predetermined volume of the first and second liquids through the channel (154), thereby bringing the first liquid level and the second liquid level to (166), which readies the system for the next liquid transfer cycle (172).

[0024] Application

[0025] The reagent delivery methods and systems described herein can be used with a variety of analytical devices, particularly those used to analyze living biological materials such as single cells, because the formation and movement of bubbles can easily interfere with and / or distort measurements or operations. The reagent delivery methods and systems described herein can be particularly applicable to cell analysis systems that employ gel microstructures in flow cell channels, such as those described in International Patent Publication WO2022 / 150659 of Khurana et al. (which is incorporated herein by reference).

[0026] Figures 2A - 2B Illustrated is a channel 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 that are programmably controlled for delivering beads and reagents to the channels. In this illustration, four channels (202, 204, 206, and 208) are shown, and an enlarged view (212) of a section (210) of channel 2 (204) is shown below. In Figure 2AIn the abstract view of the flow cell (200), the inlets, outlets, and other features of the channels 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 the channel 2 (204), a plurality of cells (e.g., (218)) are each encapsulated in a hydrogel chamber (e.g., (216)). In some cases, a portion of the plurality of cells are each encapsulated in a 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 used to form spatial barcodes. Thus, the reagent can be introduced into and removed from the interior of the hydrogel chamber by flowing the reagent (220) through the channel, while retaining the beads inside. Below the enlarged view (212) of the channel section (210) is shown an optical system (221) for photosynthesizing the hydrogel chambers at the location of the cells in the channel, e.g., as disclosed by Khurana et al. (cited above). An optical system having a configuration different from that of Figure 2A and Figure 2B can be employed to perform these functions. In some embodiments, one or more digital micromirror device (DMD)-objective lens systems for synthesizing the hydrogel structure can be employed to increase the synthesis speed by simultaneously synthesizing multiple structures.

[0027] Returning to Figure 2A, To enable photosynthesis in the hydrogel chamber, a light source (222) can generate a beam (223) of light of an appropriate wavelength (e.g., UV light), which beam (223) passes through an appropriate light mask or a beam shaping or beam steering (Galvo) system to shape the beam, thereby synthesizing the desired one or more structures in the channels. In some embodiments, a digital micromirror device (DMD) (224) is employed. In some embodiments, a physical light mask can be used. The chamber position, shape, and polymer matrix wall thickness can be determined at least in part based on the position information determined from the images collected by the detector (232). The position information can be the position of cells, nucleic acids, or any analyte of interest. The reflected light from the DMD (224) can be shaped using optics (e.g., collimating optics (228)) and directed through an objective lens system (234) into the channel 2 section (210). The objective lens (234) and the flow cell (200) can be moved relative to each other in the xy direction (236) to enable photosynthesis in the chamber at any position in any channel. In some embodiments, the flow cell (200) moves while the optical system (221) is stationary. In some embodiments, the objective lens (234) can also direct a beam (227) from a light source (229) to a target (such as a cell) on the first surface (214) and collect the optical signal (such as a fluorescence signal) from the assay performed on the first surface (214). Alternatively, optical signal collection can be performed with a separate objective lens as shown in Figure 2B . The information collected by the detector (232) or its counterpart in the Figure 2B embodiment, particularly the cell positions in their respective channels, can be utilized by a computer (238) and / or an auxiliary controller to direct the DMD (224) and control the translation device that controls the relative positions of the objective lens (234) and the flow cell (200) to synthesize hydrogel chambers of appropriate shape and size at appropriate positions.

[0028] Figure 2B An alternative optical system is illustrated, where the detection portion (250) of the optical system moves (272) independently of the movement (268) of the synthesis 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 optics, such as a dichroic mirror (262). As with Figure 2AAs in the embodiments, the detector (256) is operatively associated with the computer (264) and the integrated portion (252) of the optical system to provide position information to the integrated portion (252). The computer (264) and (238) are also operatively associated with a platform and / or motor that controls the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the integrated portion (252) of the optical system is located on the side of the first surface (264) opposite the detection portion (250). As with Figure 2A the embodiments, the integrated portion (252) of the optical system includes a component objective lens (274), a mirror (276), collimating optics (280), a DMD (282), and a light source (278).

[0029] In some embodiments, methods and systems for single cell analysis using a reagent delivery subsystem include: (a) providing a fluid device that includes (i) a channel that includes an inlet, an outlet, and a first surface having one or more cells disposed on the first surface, and 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 draws or moves a predetermined volume of liquid from the inlet reservoir into the channel under programmed control; (b) loading the assay reagent in contact with the atmosphere into the channel by transferring a volume of the assay reagent that includes the assay reagent into the inlet reservoir; (c) sealingly attaching a pressure manifold to the inlet reservoir to pressurize the assay reagent at a predetermined pressure; and (d) pumping the assay reagent through the channel such that the assay reagent binds to 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 with the assay reagent for a predetermined time. In some embodiments, such assay reagents are cell lysis reagents, transcription reagents, polynucleotide amplification reagents.

[0030] In some embodiments, methods and systems for single cell analysis using a reagent delivery subsystem include: (a) providing a fluidic device that includes: (i) a channel including 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 in the channel based on one or more optical signals from the one or more cells in the channel, 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 from the inlet reservoir through the channel; (b) loading one or more cells into the channel by transferring a volume of a mixture of one or more cells and one or more polymer precursors to the inlet reservoir, sealingly attaching a pressure manifold to the inlet reservoir to pressurize the mixture at a predetermined pressure, and pumping the mixture through the channel at a predetermined rate such that the cells of the mixture are disposed on the first surface of the channel; (c) synthesizing the one or more chambers in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form a polymeric matrix wall of the chambers, such that each chamber encapsulates a single cell of the one or more cells, wherein the location of each of the synthesized chambers on the first surface is determined by the location of the cell encapsulated by each of the synthesized chambers as identified by the detector.

[0031] In some embodiments, methods and systems for single cell analysis using a reagent delivery subsystem include: (a) providing a fluid device that includes: (i) a channel that includes an inlet, an outlet, and a first surface having one or more cells disposed on the first surface, (ii) a spatial energy modulation element in optical communication with the first surface, and (iii) a detector that identifies the location of the one or more cells in the channel based on one or more optical signals from the one or more cells in the channel, 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 draws a predetermined volume of liquid from the inlet reservoir into the channel under programmed control; (b) loading the one or more polymer precursors into the channel by transferring a volume of liquid that includes one or more polymer precursors to the inlet reservoir, sealingly attaching a pressure manifold to the inlet reservoir to pressurize the liquid at a predetermined pressure, and pumping the liquid through the channel such that the one or more polymer precursors bind to the one or more cells disposed on the first surface of the channel; (c) synthesizing one or more chambers in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form a polymeric matrix wall of the chamber, such that each chamber encapsulates a single cell of the one or more cells, wherein the location of each of the synthesized chambers on the first surface is determined by the location of the cell encapsulated by each of the synthesized chambers as identified by the detector.

[0032] It should be understood that the term "detector" as used herein can include, but is not limited to: a microscope element that collects and optionally magnifies an image of a portion of the channel; and an image analysis element that includes software for identifying cells and associated location information. A computer element uses such information generated by the detector and user input to generate commands for other elements, such as the spatial energy modulation element, to perform various functions, including but not limited to synthesizing chambers, "as needed" degradation of chambers, selective photodegradation of chambers, etc. Configurations of such embodiments are illustrated in the Figures 2A - 2B above. In some embodiments, the channel of the fluid device further includes a second surface, wherein the first surface and the second surface are disposed opposite each other across the channel, and wherein the polymeric matrix wall of the chamber extends from the first surface to the second surface to form chambers each having an interior. In some embodiments, the chambers in the channel each encapsulate a single cell. In some embodiments, both the first wall and the second wall are made of a light-transmissive material, such as glass, plastic, etc., and are positioned such that the first surface and the second surface are substantially parallel to each other. The perpendicular distance between the first surface and the second surface can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm.

[0033] As described above, any one of the first surface, the second surface, or the polymeric matrix wall of the chamber can include 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 assay reagents (such as oligonucleotide labels from antibodies), and the like. Derivatized surfaces for such purposes are well known to those skilled in the art, as evidenced by the following references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above); etc.

[0034] As described above, in some embodiments, the fluidic device of the method includes a detector or is operatively associated with a detector, which can share the optical path of the spatial energy modulation element, or can be disposed near a second wall or on the opposite side of the first wall from the spatial energy modulation element in an embodiment having only a first wall and a first surface (such as a well). The detector is positioned such that it can detect an optical signal from a cell in the channel or detect an optical signal near a cell in the channel, e.g., the cell is distributed on the first surface in the chamber. In some embodiments, the first wall and the second wall each comprise a light-transmissive material, e.g., such that the spatial energy modulation element can project light energy into the interior of the channel and such that the detector can detect optical signals (such as fluorescence emission or reflected light from a biological component). In some embodiments, the projected energy from the spatial energy modulation element is light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element can have a complex cross-section, thereby allowing (in various embodiments) the simultaneous synthesis of multiple chambers. Light-transmissive materials include but are not limited to materials such as glass, quartz, plastic, etc.

[0035] The spatial energy modulation element that uses light energy to achieve polymerization can include a physical light mask or a virtual light mask, such as a digital micromirror device (DMD). The following references, which are hereby incorporated by reference, provide guidance in the selection and operation of a DMD for achieving photopolymerized gels: Chung et al., U.S. Patent 10464307; Hribar et al., U.S. Patent 10351819; Das et al., U.S. Patent 9561622; Huang et al., Biomicrofluidics, 5:034109 (2011); etc.

[0036] Gel chamber

[0037] The methods and devices of Khurana et al. can employ a variety of photosynthetic gels and degradable gels for cell analysis. The following references, which are hereby incorporated by reference, provide guidance for selecting such gels to obtain desired properties (including but not limited to properties such as biocompatibility, porosity, gelation rate, degradation rate, etc.): 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); Bowman et al., U.S. Patent 9631092; LeValley et al., ACS Appl. Bio. Mater., 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 US2016 / 0177030; Shih et al., Biomacromolecules, 13(7):2003-2012 (2012); etc. In some embodiments, free radical polymerization is carried out using a photoinitiator to form a photosynthetic gel. In some embodiments, the photoinitiator includes Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), or eosin-Y (see, for example, Choi et al., Biotechniques, 66(1):40-53 (2019)). In some embodiments, the hydrogel precursor includes 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 includes PEG or multi-arm PEG. In some embodiments, the polymer precursor includes an enzymatically degradable crosslinker. In some embodiments, such enzymatically degradable crosslinkers can be degraded by esterase or peptidase. In some embodiments, the polymer precursor includes a photocleavable crosslinker. In some embodiments, such photocleavable crosslinkers include nitrobenzyl.In some embodiments, such photocleavable crosslinkers include coumarin moieties. In some embodiments, the photocleavable hydrogels are used in conjunction with the methods described herein, e.g., because the photocleavage of the hydrogel chambers can be selective and as needed, such that a designated hydrogel chamber can be degraded without affecting unselected hydrogel chambers. In some embodiments, the hydrogel chambers are degraded non-selectively such that all hydrogel chambers in a given channel (or other vessel) are degraded simultaneously. In some embodiments, such non-selective degradation is carried out with a cleavage reagent that specifically cleaves labile bonds in the hydrogel. For example, such cleavage agents include reducing agents. In some embodiments, such non-specific degradation is carried out with an enzyme that cleaves bonds or chemical elements in the hydrogel. Chemical elements include, but are not limited to, peptides, polysaccharides, and oligonucleotides.

[0038] In the figures, for convenience, the hydrogel chambers are illustrated as being placed in isolation and not connected to adjacent chambers, and are shown as having a cylindrical or toroid-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 the proliferation assay, each synthesized hydrogel chamber has the same shape and area, e.g., a toroid-like shape having an internal area in the range selected from 0.001 to 0.01 mm 2

[0039] Porosity ​。In some embodiments, the hydrogel porosity is selected to allow selected reagents to pass through while preventing other reagents or objects (such as proteins of cells or lysed cells) from passing through. In some embodiments, the polymer chains crosslinking the hydrogel structure form a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some embodiments, the average diameter of the pores is from about 2 nm to about 25 nm, or from about 5 nm to about 20 nm. In some embodiments, the average pore diameter is selected to prevent cell proteins from passing through. In some embodiments, the average pore diameter is selected to prevent cell proteins with a molecular weight of 1 kilodalton or greater from passing through. In some embodiments, the average pore diameter is selected to prevent cell proteins with a molecular weight of 5 kilodaltons or greater from passing through. In some embodiments, the pore size of the hydrogel structure is tuned by changing the ratio of the concentration of the polymer precursor to the concentration of the crosslinker, changing the pH, salt concentration, temperature, light intensity, etc. Guidance for selecting materials and conditions to control 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.3013.03.03 (2014); Annabi et al., Tissue Engineering, part B, 16(4):371-383 (2010); etc.

[0040] Size and shape of the hydrogel chamber。In some embodiments, the polymeric matrix walls of the chamber inhibit the passage of a predetermined component, such as mammalian cells, bacterial cells, or proteins from lysed cells. In some embodiments, the polymeric matrix walls extend from a first surface to a second surface (parallel to the first surface) to form a chamber within the channel. In some embodiments, the chamber has a polymeric matrix wall and an interior. In some embodiments, the size of the interior of the chamber is designed to encapsulate cells, such as mammalian cells. For example, such a chamber can include a cylindrical shell or a polygonal shell that includes an interior space or an interior and a polymeric matrix wall. In some embodiments, such a chamber can have an annulus-like cross-section. As used herein, the term "annulus-like cross-section" means a cross-section that is topologically equivalent to an annulus. In some embodiments, the interior space or interior of the chamber has an inner diameter of 1 μm to 500 μm and a volume ranging from 1 picoliter to 200 nanoliters or 100 picoliters to 100 nanoliters or 100 picoliters to 10 nanoliters. In some embodiments, the thickness of the polymeric matrix wall is at least 1 μm (micrometers). In some embodiments, the value of the height of the chamber having an annulus-like cross-section ranges from 10 μm to 500 μm or ranges from 50 μm to 250 μm. In some embodiments, the aspect ratio (i.e., height / width) of the polymeric matrix wall having an annulus-like cross-section is 1 or less. In some embodiments, the aspect ratio and the polymeric matrix wall thickness are selected to maximize the chamber stability against forces (such as the flow of reagents through the channel), washing, etc. In some embodiments, at least one polymeric matrix wall is a hydrogel wall. In some embodiments, at least one polymeric matrix is degradable. In some embodiments, the degradation of at least one polymeric matrix is "on demand". In some embodiments, the chambers in the channel are discontinuous. In some embodiments, the chambers in the channel can be adjacent to adjacent chambers. In some embodiments, the chambers can share a polymeric matrix wall with each other. In some embodiments, the chamber can be synthetic, having slits or other orifices large enough to allow certain components (e.g., beads) to pass through, but small enough to prevent other components (e.g., cells) from passing through.

[0041] Hydrogel compositions. As mentioned above, hydrogel compositions can vary widely, and hydrogels can be formed by various 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.

[0042] Hydrogel degradation 。In some embodiments, the hydrogel chambers are generally degradable or depolymerizable within the channels or "as needed" within the channels. Commonly degradable hydrogel chambers are degraded by treatment with a degradation agent or (equivalently) a depolymerization agent that is exposed to all the 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 reagents or degradation reagents). In some embodiments, degradation as needed can be implemented using a polymer precursor that permits photocrosslinking and photodegradation, for example, using different wavelengths for crosslinking and degradation. For example, eosin Y can be used for free radical polymerization at defined regions using a 500 nm wavelength, and thereafter irradiation at 380 nm can be used to cleave the crosslinker. In other embodiments, a photocaged hydrogel cleavage reagent can be included in the formation of the polymer matrix walls. For example, an acid-labile crosslinker (such as an ester, etc.) can be used to produce the hydrogel, and then UV light can be used to generate local acidic conditions, which in turn degrade the hydrogel. In some embodiments, at least one polymer matrix can be degraded by at least one of the following: (i) contacting at least one polymer matrix with a cleavage reagent; (ii) heating at least one polymer matrix to at least 90 °C; or (iii) exposing at least one polymer matrix to light of a wavelength that cleaves a photocleavable crosslinker that crosslinks the polymers of at least one polymer matrix. In some embodiments, 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 the polymer matrix or hydrogel can be functionalized by coupling a functional group to the polymer matrix or hydrogel.

[0043] While the methods and systems have been described herein with reference to several specific example embodiments, those skilled in the art will recognize that many changes can be made thereto without departing from the spirit and scope of the disclosure. In addition to those discussed above, the methods and systems described herein can also be applicable to various sensor embodiments and other subjects.

[0044] Additional embodiments

[0045] Aspect 1. A method for moving a liquid through a channel, comprising:

[0046] (a) Provide the channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel contains a first liquid and is in fluid communication with the outlet, wherein a pump is fluidly coupled to the outlet, and the pump is configured to move the liquid through the channel;

[0047] (b) Transfer a second liquid to the inlet reservoir, wherein the transferred second liquid is in contact with the atmosphere;

[0048] (c) Attach a pressure manifold to the inlet reservoir, the pressure manifold providing a pressure above atmospheric pressure to the first liquid and the second liquid; and

[0049] (d) Actuate the pump to withdraw at least a portion of the first liquid from the channel via the outlet and move at least a portion of the second liquid into the channel, wherein the pump is programmed to move the fluid at a predetermined rate while under the provided pressure.

[0050] In various aspects described herein, the first liquid may include a buffer (e.g., phosphate buffered saline), various types of cells, and / or a polymer precursor configured to polymerize into a cell cage.

[0051] In various aspects described herein, the second liquid may include a buffer (e.g., phosphate buffered saline), a lysis agent, a fluorescently labeled antibody, cell culture medium, and / or a hydrogel degrading agent.

[0052] In various aspects described herein, the pressure may be a constant value, a predetermined value, a range of pressure values, or an overpressure value greater than ambient atmospheric pressure.

[0053] Aspect 2. The method according to aspect 1, wherein the pressure manifold provides the pressure above atmospheric pressure before the pump is actuated to withdraw the first liquid and the second liquid through the channel.

[0054] Aspect 3. The method according to aspect 1, wherein the pressure manifold provides the pressure above atmospheric pressure before the pump is actuated to withdraw the first liquid and the second liquid through the channel, and wherein the pressure manifold continues to provide the pressure above atmospheric pressure during the actuation of the pump to withdraw the first liquid and the second liquid through the channel.

[0055] Aspect 4. The method according to aspect 1, wherein the pressure manifold provides the pressure above atmospheric pressure while the pump is actuated to withdraw the first liquid and the second liquid through the channel.

[0056] Aspect 5. The method according to any one of aspects 1 - 4, wherein attaching the pressure manifold to the inlet reservoir forms a sealed attachment between the inlet reservoir and the pressure manifold.

[0057] Aspect 6. The method according to any one of aspects 1 - 5, further comprising moving at least 90% or more of the second liquid out of 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 moved out of the channel such that the channel contains gas from the pressure manifold.

[0058] Aspect 7. The method according to any one of aspects 1 - 6, wherein the first liquid forms a plurality of bubbles in the channel, and actuating the pump to draw at least a portion of the first liquid out of the channel causes a portion of the plurality of bubbles in the channel to flow out of the outlet.

[0059] Aspect 8. The method according to aspect 7, wherein the portion of the plurality of bubbles ranges from about 50% to 100%. The combination of using an actuated pump to draw out the liquid and a pressure manifold providing a pressure above atmospheric pressure increases the yield of removing bubbles in an effective manner. For various reasons, it may be difficult to remove bubbles from the channel by flushing the liquid through the channel using only the pressure manifold or only the pump.

[0060] Aspect 9. The method according to any one of aspects 1 - 8, wherein the first liquid includes a plurality of cells in the channel, and actuating the pump to draw at least a portion of the first liquid out of the channel causes a portion of the plurality of cells in the channel to flow out of the outlet.

[0061] Aspect 10. The method according to aspect 9, wherein the portion of the plurality of cells ranges from about 50% to 100%. The combination of using an actuated pump to draw out the liquid and a pressure manifold providing a pressure above atmospheric pressure increases the yield of removing cells in an effective manner, which is an unexpected result. For various reasons, it may be difficult to remove cells from the channel by flushing the liquid through the channel using only the pressure manifold or only the pump.

[0062] Aspect 11. The method according to any one of aspects 1 - 10, wherein the pump generates a vacuum during actuating the pump to draw at least a portion of the first liquid out of the channel.

[0063] Aspect 12. The method according to any one of aspects 1 - 11, wherein the pump includes an injection pump.

[0064] Aspect 13. The method according to any one of aspects 1-12, wherein at least a portion of the second liquid moves within a predetermined duration such that the second liquid in the inlet reservoir is not emptied.

[0065] Aspect 14. The method according to any one of aspects 1-12, wherein at least a portion of the second liquid moves over a period of time such that the first liquid and the second liquid pass through the channel, thereby filling the channel with air.

[0066] Aspect 15. The method according to any one of aspects 1-14, wherein the pressure provided by the pressure manifold is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

[0067] Aspect 16. The method according to any one of aspects 1-14, wherein the pressure provided by the pressure manifold has a difference from the atmospheric pressure in the range of about 2 pounds per square inch to about 5 pounds per square inch.

[0068] Aspect 17. A method of delivering a reagent to a cell analysis system, comprising:

[0069] Providing a fluid device, the fluid device comprising:

[0070] (i) A channel including an inlet and an outlet;

[0071] (ii) A spatial energy modulation element optically communicating with the channel;

[0072] (iii) A detector that identifies the location of one or more cells in the channel based on one or more optical signals from the one or more cells in the channel,

[0073] wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein the 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;

[0074] Loading a mixture of the one or more cells and one or more polymer precursors into the inlet reservoir;

[0075] Sealingly attaching a pressure manifold to the inlet reservoir to pressurize the mixture to an elevated pressure greater than ambient pressure; and

[0076] Moving the mixture into the channel with the pump such that the one or more cells of the mixture are disposed in the channel;

[0077] One or more chambers are synthesized in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form a polymeric matrix wall of the chamber, such that each chamber encapsulates a single cell of the one or more cells, wherein the position of each of the synthesized chambers is determined by the position identified by the detector.

[0078] Aspect 18. The method according to aspect 17, wherein the mixture comprises a first liquid, wherein after synthesizing the one or more chambers in the channel, a portion of the one or more cells is disposed in an interstitial space outside the chambers, the method further comprising:

[0079] Loading a second liquid into the inlet reservoir;

[0080] Sealingly attaching the pressure manifold to the inlet reservoir to pressurize the second liquid to the elevated pressure greater than the ambient pressure; and

[0081] Moving the second liquid into the channel with the pump such that a small portion of the cells disposed in the interstitial space flow out of the channel via the outlet.

[0082] Aspect 19. The method according to aspect 18, wherein the small portion of the cells in the interstitial space ranges from about 50% to 100%. Figure 3A Various cells are shown, where some cells are trapped in the hydrogel structure (216) and other cells (such as 218) are not trapped and reside in the interstitial space between the hydrogel structures. Figure 3B The channel after flushing with the second liquid is shown, where the second liquid is not pressurized with a pressure manifold and only a pump is used to move the liquid by sucking the liquid from the outlet. Figure 3B It is shown that, compared with before Figure 3A washing the channel, a large number of remaining cells (such as 218) are located in the interstitial space. Figure 4A Various cells are shown, where some cells are trapped in the hydrogel structure (216) and other cells (such as 218) are not trapped and reside in the interstitial space between the hydrogel structures. Figure 4B The channel after flushing with the second liquid is shown, where the second liquid is pressurized with a pressure manifold while a pump is used to move the liquid by sucking the liquid from the outlet. Figure 4B It is shown that the amount of remaining cells in the interstitial space is relatively small, which is compared with Figure 3BThere is a stark contrast in the relatively large amount of remaining cells when the pressure manifold is not used as shown. It should be noted that under certain conditions, the cell and hydrogel structures may be fragile with respect to the flowing liquid, and the flushing with the pressure manifold and the pump that sucks the liquid from the outlet results in benign conditions that do not disrupt the hydrogel and also results in effective removal of the interstitial cells.

[0083] Aspect 20. The method according to any one of aspects 18 - 19, wherein the first liquid forms a plurality of bubbles in the interstitial space in the channel, and wherein moving the second liquid into the channel with the pump causes a portion of the plurality of bubbles disposed in the interstitial space to flow out of the channel via the outlet.

[0084] Aspect 21. The method according to aspect 20, wherein the portion of the plurality of bubbles ranges from about 50% to 100%.

[0085] Aspect 22. The method according to any one of aspects 17 - 21, wherein the pump creates a vacuum at the outlet during the moving of the mixture into the channel.

[0086] Aspect 23. The method according to any one of aspects 18 - 21, wherein the pump creates a vacuum at the outlet during the moving of the second liquid into the channel.

[0087] Aspect 24. The method according to any one of aspects 17 - 23, wherein the pump comprises an injection pump.

[0088] Aspect 25. The method according to any one of aspects 18 - 24, wherein the moving of the second liquid occurs over a period of time such that the first liquid and the second liquid pass through the channel, thereby filling the channel with air.

[0089] Aspect 26. The method according to any one of aspects 17 - 25, wherein the elevated pressure is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

[0090] Aspect 27. The method according to any one of aspects 17 - 25, wherein the difference between the elevated pressure provided by the pressure manifold and the ambient pressure ranges from about 2 pounds per square inch to about 5 pounds per square inch.

[0091] Aspect 28. The method according to any one of aspects 17 - 27, wherein the sealing attachment of the pressure manifold to the inlet reservoir to pressurize the mixture to the elevated pressure greater than the ambient pressure occurs before the moving of the mixture into the channel with the pump.

[0092] Aspect 29. The method according to any one of aspects 17 - 27, wherein the pressure manifold is sealingly attached to the inlet reservoir to pressurize the mixture to the elevated pressure greater than the ambient pressure occurs before the mixture is moved into the channel by the pump, and wherein during the moving of the mixture into the channel by the pump, the pressure manifold continues to provide the elevated pressure.

[0093] Aspect 30. The method according to 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.

[0094] Aspect 31. A fluid delivery system, comprising:

[0095] One or more reaction channels, each of the one or more reaction channels containing a first liquid and having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet;

[0096] A pump, the pump being in fluid communication with the outlet of the one or more reaction channels, wherein the pump is configured to move liquid through the channel at a predetermined rate;

[0097] One or more supply reservoirs, the one or more supply reservoirs containing liquid in contact with the atmosphere;

[0098] A pipette for transferring a second liquid from the one or more supply reservoirs to the inlet reservoir; and

[0099] A pressure manifold, the pressure manifold being sealingly attached to the inlet reservoir, wherein the pressure manifold is configured to apply a predetermined pressure above atmospheric pressure to the first liquid and the second liquid.

[0100] Aspect 32. A method of delivering a reagent to a cell analysis system, comprising:

[0101] Providing a fluid device, the fluid device comprising a channel, the channel including an inlet, an outlet, and a surface having one or more cells disposed on the surface, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein 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;

[0102] Loading an assay reagent in contact with the atmosphere into the inlet reservoir;

[0103] Sealingly attaching a pressure manifold to the inlet reservoir so as to pressurize the assay reagent to an elevated pressure greater than the ambient pressure;

[0104] Use the pump to move the assay reagent through the channel such that the assay reagent contacts the one or more cells disposed on the surface of the channel.

[0105] Aspect 33. A method of delivering a reagent to a cell analysis system, comprising:

[0106] Providing a fluid device, the fluid device comprising:

[0107] (i) a channel comprising an inlet, an outlet, and a surface, the surface having one or more cells disposed on the surface,

[0108] (ii) a spatial energy modulation element optically communicating with the surface,

[0109] (iii) a detector that identifies the position of the one or more cells in the channel based on one or more optical signals from the one or more cells in the channel,

[0110] wherein the inlet of the channel is in fluid communication with an inlet reservoir, and

[0111] wherein the outlet of the channel is in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir to the channel;

[0112] Loading a liquid comprising one or more polymer precursors into the inlet reservoir; sealingly attaching a pressure manifold to the inlet reservoir to pressurize the liquid at an elevated pressure greater than ambient pressure; and using the pump to move the liquid through the channel;

[0113] Synthesizing one or more chambers in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, such that each chamber encapsulates a single cell of the one or more cells, wherein the position of each of the synthesized chambers on the surface is determined by the position identified by the detector.

[0114] Aspect 34. A method of delivering a reagent to a cell analysis system, comprising:

[0115] Providing a fluid device, the fluid device comprising a channel that comprises an inlet, an outlet, and a surface, the surface having one or more cells disposed on the surface, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein the outlet of the channel is in fluid communication with a pump configured to move a predetermined volume of liquid from the inlet reservoir to the channel;

[0116] Load a measurement reagent in contact with the atmosphere into the inlet reservoir;

[0117] Sealably attach a pressure manifold to the inlet reservoir to pressurize the measurement reagent at an elevated pressure greater than ambient pressure; and

[0118] Use the pump to move the measurement reagent through the channel such that the measurement reagent contacts the one or more cells disposed on the surface of the channel.

[0119] Aspect 35. The method according to aspect 34, further comprising incubating the one or more cells in the measurement reagent for a predetermined time.

[0120] Aspect 36. The method according to aspect 34, wherein each of the one or more cells is encapsulated in a hydrogel chamber.

[0121] Aspect 37. The method according to any one of the preceding aspects, wherein the pressure manifold includes a conduit that includes a single gas input port and a plurality of gas output ports, wherein the plurality of gas output ports are in fluid communication with the single gas input port.

[0122] Aspect 38. The method according to any one of the preceding aspects, wherein the pressure manifold includes a conduit that includes a single gas input port and a plurality of gas output ports.

[0123] Aspect 39. A method for moving a liquid through a channel, the method comprising:

[0124] (a) Provide the channel having an outlet and an inlet,

[0125] wherein an inlet reservoir is fluidly coupled to the inlet, the channel contains a first liquid and is in fluid communication with the outlet, wherein a pump is fluidly coupled to the outlet and is configured to move the liquid through the channel;

[0126] (b) Attach a pressure manifold to the inlet reservoir, the pressure manifold providing a pressure above atmospheric pressure to the first liquid;

[0127] (c) Actuate the pump to withdraw at least 90% or more of the first liquid from the channel via the outlet and move gas from the pressure manifold into the channel, wherein the pump is programmed to move the fluid at a predetermined rate while at the provided pressure;

[0128] (d) Transfer a second liquid to the inlet reservoir, wherein the transferred second liquid is in contact with the atmosphere;

[0129] (e) Attach the pressure manifold to the inlet reservoir, the pressure manifold providing the pressure above atmospheric pressure to the second liquid; and

[0130] (f) Actuate the pump to withdraw at least a portion of the gas from the passage via the outlet and move at least a portion of the second liquid into the passage, wherein the pump is programmed to move the fluid at the predetermined rate while at the provided pressure.

[0131] Aspect 40. The method according to aspect 39, wherein before actuating the pump to withdraw the gas from the passage, the pressure manifold provides the pressure above atmospheric pressure.

[0132] Aspect 41. The method according to aspect 39, wherein before actuating the pump to withdraw the gas from the passage, the pressure manifold provides the pressure above atmospheric pressure, wherein during actuating the pump to withdraw at least a portion of the gas from the passage, the pressure manifold continues to provide the pressure above atmospheric pressure.

[0133] Aspect 42. The method according to any one of aspects 39 - 41, wherein attaching the pressure manifold to the inlet reservoir forms a sealed attachment between the inlet reservoir and the pressure manifold.

[0134] Aspect 43. The method according to any one of aspects 39 - 42, wherein during actuating the pump to withdraw at least the portion of the gas or the first liquid from the passage, the pump generates a vacuum.

[0135] Aspect 44. The method according to any one of aspects 39 - 43, wherein the pump comprises an injection pump.

[0136] Aspect 45. The method according to any one of aspects 39 - 44, wherein the pressure provided by the pressure manifold is greater than 1 times atmospheric pressure but less than 5 times atmospheric pressure.

[0137] Aspect 46. The method according to any one of aspects 39 - 44, wherein the difference between the pressure provided by the pressure manifold and the atmospheric pressure ranges from about 2 pounds per square inch to about 5 pounds per square inch.

[0138] Aspect 47. The method according to any one of the foregoing aspects, wherein the pressure manifold outputs a gas to provide the pressure, wherein the gas is selected from air, carbon dioxide, nitrogen, argon, and combinations thereof.

[0139] Aspect 48. The method according to any one of the foregoing aspects, wherein the pressure manifold outputs a gas to provide the pressure, wherein the gas contains 5% carbon dioxide, and the second liquid comprises a cell culture medium balanced with 5% carbon dioxide such that the pH change of the cell culture medium does not exceed 10%.

[0140] Aspect 49. The method according to any one of the foregoing aspects, wherein the pressure manifold outputs a gas to provide the pressure, wherein the gas contains nitrogen or argon and the second liquid comprises anaerobic cells or anaerobic organisms.

[0141] Aspect 50. The method according to any one of the foregoing aspects, wherein before actuating the pump to withdraw at least a portion of the first liquid or the second liquid from the channel, the pressure manifold provides the pressure above atmospheric pressure for a predetermined period of time. The predetermined period of time can range from 5 minutes to 1 day. The pressure manifold that provides the pressure for the predetermined period of time results in a reduction in the formation of bubbles during the predetermined period of time compared to a situation where no pressure manifold is used.

[0142] Aspect 51. A method for incubating a liquid in a channel, comprising: (a) providing the channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel contains a first liquid and is in fluid communication with the outlet, wherein a pump is fluidly coupled to the outlet, the pump being configured to move the liquid through the channel; (b) attaching a pressure manifold to the inlet reservoir, the pressure manifold providing a pressure above atmospheric pressure to the first liquid, wherein the pump is not actuated, thereby causing the outlet to be sealed and the liquid to be in a stationary state. Figure 5A A flow cell having channels E and F is shown, wherein the liquid in the channels is relatively bubble-free. Channel F is pressurized with a pressure manifold at 5 PSI at the inlet reservoir, and the outlet is closed at 42 °C for 90 minutes. Channel E is placed at atmospheric pressure at 42 °C for 90 minutes. Figure 5B It is shown that after 90 minutes, channel F remains relatively bubble-free, and channel E has a plurality of bubbles 502. Thus, the channel with the pressure applied by the manifold helps to reduce the formation of bubbles over a period of time and at an elevated temperature.

[0143] Definition

[0144] Unless otherwise clearly defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow standard treatises and texts in the art, e.g., Kornberg and Baker, DNA Replication, 2nd ed. (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immunology, 6th ed. (Saunders, 2007).

[0145] "Assay" refers to a process for detecting or measuring a cellular characteristic or property of a single cell or cell population. Generally, the process steps of an assay include chemical, biochemical, or molecular reactions (such as cleavage of bonds, specific binding of complementary components, enzyme reactions, dissolution of complementary components, etc.) or changes in physical state (such as increase or decrease in temperature, change in energy level, etc.), and result in the generation of one or more signals from which the presence, absence, or magnitude of a cell-related quantity can be inferred. The nature of the signals generated by an assay can vary widely and can include, but are not limited to, electrical signals, optical signals, chemical signals, or material signals. Material signals include the production of a substance that includes information that can be extracted. For example, a material signal can be the amplification of a polynucleotide, the length, number, composition, or nucleotide sequence of which indicates a cellular characteristic. For example, a barcoded oligonucleotide can be a material signal. The cellular characteristics or properties that are detected or measured can vary widely and include, but are not limited to, cytotoxicity, viability, proliferation ability under selected conditions, size, shape, motility, type and distribution pattern of cell surface or cell membrane proteins, type and distribution pattern of secreted proteins, production of metabolites, transcriptome, gene copy number, gene or allele identity, chromatin accessibility distribution pattern, vector copy number of engineered or infected cells, etc. Assays of particular interest for cell-based therapies include, but are not limited to, cytotoxicity, viability, activation, proliferation ability under selected conditions, chromatin accessibility distribution pattern, type and distribution pattern of cell surface or membrane proteins, type and distribution pattern of secreted proteins, intracellular proteins, transcriptome, vector copy number, etc. As used herein, "assay reagent" is a liquid used in an assay. Assay reagents can include, but are not limited to, pH buffer solutions, enzyme buffer solutions with or without enzymes, molecular or cellular stains or dyes, lysis agents, gel degradation reagents, cell suspensions, salt solutions, wash solutions, cell growth media, etc.

[0146] "Cell" refers to biological cells that can be measured by the methods and systems described herein, including but not limited to vertebrate cells, invertebrate cells, eukaryotic cells, mammalian cells, microbial cells, protozoan cells, prokaryotic cells, bacterial cells, insect cells, or fungal cells. In some embodiments, mammalian cells are measured by the methods and systems described herein. In particular, any mammalian cells that can or have been genetically altered for use in medical, industrial, environmental, or therapeutic processes can be analyzed by the methods and systems described herein. In some embodiments, "cell" as used herein includes genetically modified mammalian cells. In some embodiments, "cell" includes stem cells. In some embodiments, "cell" refers to cells modified by CRISPR Cas9 technology. In some embodiments, "cell" refers to cells of the immune system, including but not limited to cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen-presenting cells, or dendritic cells. Of particular interest are cytotoxic T lymphocytes engineered for therapeutic applications such as cancer therapy.

[0147] "Hydrogel" means a gel comprising a crosslinked hydrophilic polymer network that is capable of absorbing and retaining large amounts of water (e.g., 60 to 90 percent water, or 70 to 80 percent) without dissolving due to the establishment of physical or chemical bonds (which can be covalent, ionic, or hydrogen bonds) between the polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels can comprise natural or synthetic polymers and can be reversible (i.e., degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers can include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Natural hydrogel polymers can 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. Patent 9,631,092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0148] "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 to at least one polymer and the interstitial spaces not occupied by the polymer. The polymer matrix can be composed of one or more types of polymers. The polymer matrix can include linear, branched, and crosslinked polymer units. The polymer matrix can also include non-polymeric species inserted into the interstitial spaces not occupied by the polymer chains. The inserted species can be solid, liquid, or gaseous species. For example, the term "polymer matrix" can encompass dry hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. The polymer matrix can include polymer precursors, which generally refer to one or more molecules that can trigger or initiate a polymerization reaction upon activation. 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 the polymer matrix) and crosslinking compounds, which can include photoinitiators and other compounds necessary or useful for producing the polymer matrix (especially a polymer matrix in the form of a hydrogel).

[0149] While the preferred embodiments of the systems and methods described herein have been shown and described, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The systems and methods described herein are not intended to be limited by the specific examples provided in this specification. 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 to be construed in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the systems and methods described herein. In addition, it should be understood that all aspects of the systems and methods described herein are not limited to the specific descriptions, configurations, or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the systems and methods described herein can be employed in practicing the systems and methods described herein. Accordingly, it is contemplated that the systems and methods described herein should also cover any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the systems and methods described herein and are intended to cover methods and structures within the scope of these claims and their equivalents.

Claims

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

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

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

4. The method according to claim 1, wherein during the actuation in (d), the pressure manifold provides the pressure above atmospheric pressure.

5. The method according to any one of claims 1-4, wherein in (c), a sealed attachment is formed between the inlet reservoir and the pressure manifold.

6. The method according to any one of claims 1-5 further comprises: Removing at least 90% or more of the second liquid from the channel such that the channel contains gas from the pressure manifold.

7. The method according to any one of claims 1-6, wherein the first liquid forms a plurality of bubbles in the channel, and wherein in (d), at least a portion of the plurality of bubbles in the channel flows out of the outlet.

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

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

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

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

12. The method according to any one of claims 1-11, wherein the pump includes an injection pump.

13. The method according to any one of claims 1-12, wherein the movement of at least the portion of the second liquid occurs over a predetermined duration such that the second liquid in the inlet reservoir is not emptied.

14. The method according to any one of claims 1-12, wherein at least a portion of the movement of the second liquid occurs over a period of time such that the first liquid and the second liquid pass through the channel, thereby filling the channel with air.

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

17. A method of delivering a liquid to a cell analysis system, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel including an inlet and an outlet; (ii) a spatial energy modulation element optically communicating with the channel; (iii) a detector that identifies the location of one or more cells in the channel based on one or more optical signals from the one or more cells in the channel, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein the 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; (b) loading a 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 to pressurize the mixture to an elevated pressure greater than ambient pressure; (d) moving the mixture into the channel with the pump such that the one or more cells of the mixture are disposed in the channel; and (e) synthesizing the one or more chambers in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes the one or more polymer precursors to form a polymer matrix wall of the one or more chambers, such that each chamber encapsulates a single cell of the one or more cells, wherein the location of each of the synthesized chambers is determined by the location of the one or more cells identified by the detector.

18. The method according to claim 17, wherein the mixture comprises a first liquid, wherein after the synthesis in (e), a portion of the one or more cells is disposed in an interstitial space outside the chamber, and wherein the method further comprises: (f) loading a second liquid into the inlet reservoir; (g) sealingly attaching the pressure manifold to the inlet reservoir to pressurize the second liquid to the elevated pressure greater than the ambient pressure; and (h) moving the second liquid into the channel with the pump such that a small portion of the cells disposed in the interstitial space flow out of the channel via the outlet.

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

20. The method according to claim 18 or 19, wherein the first liquid forms a plurality of bubbles in the interstitial space in the channel, and wherein in (h), a portion of the plurality of bubbles disposed in the interstitial space flows out of the channel via the outlet.

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

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

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

24. The method according to any one of claims 17-23, wherein the pump comprises an injection pump.

25. The method according to any one of claims 18-24, wherein the movement of the second liquid occurs over a period of time such that the first liquid and the second liquid pass through the channel, thereby filling the channel with air.

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

27. The method according to any one of claims 17-25, wherein the elevated pressure provided by the pressure manifold has a difference from the ambient pressure in the range of about 2 pounds per square inch to about 5 pounds per square inch.

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

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

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

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

32. A method of delivering a reagent to a cell analysis system, the method comprising: Provide a fluid device, the fluid device including a channel, the channel including an inlet, an outlet, and a surface having one or more cells disposed on the surface, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein 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; Load a measurement reagent in contact with the atmosphere into the inlet reservoir; Sealingly attach a pressure manifold to the inlet reservoir so as to pressurize the measurement reagent at an elevated pressure greater than ambient pressure; And Use the pump to move the measurement reagent through the channel such that the measurement reagent contacts the one or more cells disposed on the surface of the channel.

33. A method of delivering a reagent to a cell analysis system, the method comprising: (a) Provide a fluid device, the fluid device including: (i) A channel including an inlet, an outlet, and a surface having one or more cells disposed on the surface, (ii) A spatial energy modulation element optically in communication with the surface, (iii) A detector that identifies the position of the one or more cells in the channel based on one or more optical signals from the one or more cells in the channel, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein 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; (b) Load a liquid including one or more polymer precursors into the inlet reservoir; (c) Sealingly attach a pressure manifold to the inlet reservoir so as to pressurize the liquid at an elevated pressure greater than ambient pressure; (d) Use the pump to move the liquid through the channel; and (e) Synthesize the one or more chambers in the channel by projecting light into the channel with the spatial energy modulation element such that the projected light causes the one or more polymer precursors to form a polymer matrix wall of the one or more chambers, such that each chamber encapsulates a single cell of the one or more cells, wherein the position of each of the synthesized chambers on the surface is determined by the position of the one or more cells identified by the detector.

34. A method of delivering a reagent to a cell analysis system, the method comprising: (a) Provide a fluid device, the fluid device including: (i) A channel including an inlet, an outlet, and a surface having one or more cells disposed on the surface, wherein the inlet of the channel is in fluid communication with an inlet reservoir, and wherein 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; (b) Load a measurement reagent in contact with the atmosphere into the inlet reservoir; (c) Sealingly attach a pressure manifold to the inlet reservoir so as to pressurize the measurement reagent at an elevated pressure greater than ambient pressure; and (d) Use the pump to move the assay reagent through the channel such that the assay reagent contacts the one or more cells disposed on the surface of the channel.

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

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

37. The method according to any one of the preceding claims, wherein the pressure manifold comprises a conduit, wherein the conduit comprises a single gas input port and a plurality of gas output ports, and wherein the plurality of gas output ports are in fluid communication with the single gas input port.

38. The method according to any one of the preceding claims, wherein the pressure manifold comprises a conduit, wherein the conduit comprises a single gas input port and a plurality of gas output ports.

39. A method for moving a liquid through a channel, the method comprising: (a) Providing the channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, the channel containing a first liquid, wherein a pump is fluidly coupled to the outlet; (b) Attaching a pressure manifold to the inlet reservoir, the pressure manifold providing a pressure above atmospheric pressure to the first liquid; (c) Actuating the pump to withdraw at least 90% or more of the first liquid from the channel via the outlet and move gas from the pressure manifold into the channel, wherein the pump is 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, wherein the transferred second liquid is in contact with the atmosphere; (e) Attaching the pressure manifold to the inlet reservoir, the pressure manifold providing the pressure above atmospheric pressure to the second liquid; and (f) Actuating the pump to withdraw at least a portion of the gas from the channel via the outlet and move at least a portion of the second liquid into the channel, wherein the pump is programmed to move fluid at the predetermined rate while under the pressure provided by the pressure manifold.

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

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

42. The method according to any one of claims 39 - 41, wherein in (b) and (e), a sealed attachment is formed between the inlet reservoir and the pressure manifold.

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

44. The method according to any one of claims 39 - 43, wherein the pump comprises an injection pump.

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

47. The method according to any one of the preceding claims, wherein the pressure manifold outputs a gas to provide the pressure, and wherein the gas is selected from air, carbon dioxide, nitrogen, argon, and combinations thereof.

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

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

50. A method for incubating a liquid in a channel, comprising: (a) providing the channel having an outlet and an inlet, wherein an inlet reservoir is fluidly coupled to the inlet, wherein the channel contains a first liquid, and wherein a pump is fluidly coupled to the outlet; and (b) attaching a pressure manifold to the inlet reservoir, the pressure manifold providing a pressure above atmospheric pressure to the first liquid, wherein the pump is not actuated, thereby causing the outlet to be sealed and the liquid to be in a stationary state.

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