WO2021067650A1 - Analyse de libération synchronisée de cellules incubées dans des nœuds de microenvironnement - Google Patents

Analyse de libération synchronisée de cellules incubées dans des nœuds de microenvironnement Download PDF

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Publication number
WO2021067650A1
WO2021067650A1 PCT/US2020/053878 US2020053878W WO2021067650A1 WO 2021067650 A1 WO2021067650 A1 WO 2021067650A1 US 2020053878 W US2020053878 W US 2020053878W WO 2021067650 A1 WO2021067650 A1 WO 2021067650A1
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WIPO (PCT)
Prior art keywords
node
cells
cell
nodes
medium
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PCT/US2020/053878
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English (en)
Inventor
Lydia L. Sohn
Molly KOZMINSKY
Roberto FALCÓN-BANCHS
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The Regents Of The University Of California
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Publication of WO2021067650A1 publication Critical patent/WO2021067650A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the invention provides devices and methods for Timed Release Analysis of Cells Incubated in Microenvironment Nodes (TRAIN).
  • the device comprises a microfluidic channel containing multiple nodes.
  • the device provides for specific cells, once tagged with a specific oligonucleotide sequence, to then be localized within a node via the complimentary oligonucleotide sequence.
  • These nodes can also contain patterned chemical and/or biological cues. Localized cells can also be stimulated by soluble cues flowed through the channel. Following exposure, cells can then be released in sequence, allowing different exposures to be linked with downstream analysis (immunophenotyping, mechano-phenotyping, protein analysis, etc.).
  • Features include the specific positioning of cells, allowing exposure of cells to several different conditions, and the ability to release the cells sequentially (or specific timepoints) for further analysis (immunophenotyping, mechano- phenotyping, protein analysis, etc.) once they have undergone treatment (e.g. drugs).
  • Applications include: drug testing, screening and/or discovery; multiplexed investigation of components of complex systems such as the tumor microenvironment; study of cell signaling or testing of technologies and interventions involving cell signaling; testing interactions between cells and tethered factors; testing interaction between different cell types; single cell analysis of different interventions; integration of interventions with different downstream analysis technologies such as RNA-seq or node-pore sensing or PCR, qRT-PCR, immunophenotyping, etc; analysis of microbial-based cancer therapies [007]
  • the invention provides a microfluidic device comprising a microfluidic channel containing a cell medium, and configured with multiple nodes located in series along the fluidic flowpath of the channel, wherein each node comprises a surface patterned with a corresponding oligonucleotide probe, and at each node is a cell labeled with a complementary oligonucleotide tag and immobilized at the node by hybridization of the tag with the oligonucleotide probe
  • each probe at each node is selective for a different cell type and immobilized at each node is a cell of the corresponding cell type;
  • each probe at each node is one of a plurality of probes at each node, wherein the probes are optionally selective for a different cell type or plurality of different cell types, and immobilized at each node is a cell/cells of the corresponding cell type/types;
  • the surface is a substrate surface, and the substrate comprises glass or quartz, preferably functionalized for attaching the probes, such as with aldehyde groups, or a plastic, such as cyclic olefin copolymer, polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), or thermal plastic, or a composite of a glass or plastic surface layer bonded to a glass or plastic substrate;
  • the nodes are in a size range to accommodate 1, 2, 5 or up to 10 or 100 or 1,000 or 10,000 cells, such as equivalent area to a circle of diameter of about 10 or 20 or 50 or 100 or 200 or 500 or 1000 or 2000 or 5000 um and/or the channels have a cross-sectional diameter to accommodate 1, 2, 5 or up to 10 or 100 cells, such as about 5 or 10 or 20 or 50 or 100 um;
  • the device further comprises at each node a different bioactive agent, such as a bioactive ligand, cell, receptor, drug, candidate agent, etc.;
  • a bioactive agent such as a bioactive ligand, cell, receptor, drug, candidate agent, etc.
  • the surface of one or more of the nodes is further patterned with a bioactive agent
  • each node is further patterned with a bioactive agent
  • the medium comprises a releasing reagent such as DNAse or a protease like trypsin or a trypsin analog operative to release the cells in series according to their respective node; and/or [018] the device is operably connected to a microfluidic pump and/or a downstream fluid analyzer.
  • a releasing reagent such as DNAse or a protease like trypsin or a trypsin analog operative to release the cells in series according to their respective node
  • the device is operably connected to a microfluidic pump and/or a downstream fluid analyzer.
  • the invention provides a method of using a disclosed device or embodiment thereof comprising: flowing the medium through the channel and analyzing over time medium exiting the device with a downstream fluid analyzer.
  • the method further comprises timed-release release of cells according to the node with the releasing agent.
  • the invention encompasses all combination of the particular embodiments recited herein, as if each combination had been laboriously recited.
  • Figs. la-b Two iterations of the photomask used for TRAIN.
  • Fig. la a composite of the masks showing the patterned regions for cells (red and green), alignment markers (yellow), and footprint of polydimethylsiloxane (PDMS) chamber (purple);
  • Fig. lb outline of channel used in second generation of the design.
  • Figs 2a-c TRAIN experimental schematic.
  • Fig. 2a Cells prelabeled with oligonucleotides of a specific sequence are introduced into the device.
  • Fig. 2b Cells are immobilized based on the nucleotide sequence and are cultured for the desired amount of time. During this phase they may be exposed to other stimuli, which can also be patterned.
  • Fig. 2c Cells are released using DNAse, trypsin and/or a trypsin analogue. The cells flow out of the device in series such that any stimuli can be coupled with the results of the downstream analysis.
  • Fig. 3. Demonstration of cell localization within device. Cells were patterned such that one green cell should be present in each of Chambers 1 and 3 and that one red cell should be present in each of Chambers 2 and 4.
  • TRAIN has broad general utility. Using a highly generalized protocol 1 , this platform has the capability to introduce cells of interest, recreate their microenvironment (surrounding cells), and present chemical cues this device can be used to study a wide variety of cell-cell or cell- ligand interaction. Applications of this device include to study disease where the contributions of the microenvironment are implicated in disease progression, e.g. cancer, or in situations that require the high-throughput exposure followed by specific analysis, e.g. drug testing/screening/discovery. [029] Advantages of this device include the high generalizability, in that the same design can be used to study any type of cell-cell or cell-ligand interaction.
  • the device can be extended to contain high numbers of nodes for multiplexing.
  • a significant advantage of this platform is the ability to sequentially release the cells and couple their specific cue with results from downstream analysis techniques such as RNA-seq or node pore sensing 2-4 or PCR, qRT-PCR, immunophenotyping, etc.
  • downstream analysis techniques such as RNA-seq or node pore sensing 2-4 or PCR, qRT-PCR, immunophenotyping, etc.
  • the ability to know what cues were presented to each cell makes this platform suitable for upstream integration of any number of downstream analysis techniques. Due to the microfluidic nature of the channel, relatively low reagent volumes are be used to perform experiments.
  • TRAIN provides many advantages over existing alternative technologies.
  • a microfluidic device integrated with complicated valve structures can be designed and fabricated to handle single-cells during the patterning and release steps. This is far from ideal since it involves difficult fabrication procedures and requires an elaborate actuation hardware, which would greatly increase the footprint of the device while also negatively affecting throughput.
  • Alternative assays could be performed in 96 well plate, but that would likely preclude single cell analysis. Additionally, cell loss during trypsinization/transfer to downstream analysis following exposure could be substantial.
  • One way to use the invention is to first identify a problem or question in which cell-cell interaction or cell-ligand interaction are highly relevant. Then obtain the cells and reagents of interest and label them with oligonucleotides.
  • Pattern and enclose the complimentary oligonucleotides within the TRAIN device Introduce cells and cues (such as additional cell types, ligands, etc.). Consider testing multiple cell types and multiple cues. Culture cells and cues for a pre-determined period of time, potentially with exposure to soluble cues, then release the cells directly to follow-up device or technology to allow downstream analysis. The results from this analysis can then be directly tied back to the cue to which the cell was exposed.
  • the design provides for multiple variations.
  • the number of nodes or conditions of interest can vary.
  • Cell types can vary.
  • the cue, whether it is another cell type, a ligand, or a component of the extracellular matrix, can be varied to explore different responses.
  • the amount of time the cells are maintained on the device can be adjusted. Soluble factors may be introduced. Different down-stream analysis techniques paired with TRAIN can also be selected.
  • Another available variation is provided when larger nodes are integrated in which cell sub populations can be grown instead of single-cells. Having multiple cells in the nodes facilitates downstream analysis since some technologies need more than one cell to produce conclusive results. This ability to culture multiple cells can also be used to study the effect of the cue of interest in cells and their lineage, allowing the use to determine if effects are acute or are maintained in later generations.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Sustainable Development (AREA)
  • Clinical Laboratory Science (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Fluid Mechanics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Dispositif microfluidique destiné à l'analyse de libération prolongée de cellules incubées dans des nœuds de microenvironnement (TRAIN), comprenant un canal microfluidique contenant un milieu de culture cellulaire, et configuré avec de multiples nœuds situés en série le long du trajet d'écoulement fluidique du canal. Chaque nœud comprend une surface ayant un motif avec une sonde oligonucléotidique correspondante et au niveau de chaque nœud, il y a une cellule marquée avec une étiquette oligonucléotidique complémentaire et immobilisée au niveau du nœud par hybridation de l'étiquette avec la sonde.
PCT/US2020/053878 2019-10-02 2020-10-02 Analyse de libération synchronisée de cellules incubées dans des nœuds de microenvironnement WO2021067650A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962909719P 2019-10-02 2019-10-02
US62/909,719 2019-10-02

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6790330B2 (en) * 2000-06-14 2004-09-14 Board Of Regents, The University Of Texas System Systems and methods for cell subpopulation analysis
US20050202504A1 (en) * 1995-06-29 2005-09-15 Affymetrix, Inc. Miniaturized genetic analysis systems and methods
US7807454B2 (en) * 2006-10-18 2010-10-05 The Regents Of The University Of California Microfluidic magnetophoretic device and methods for using the same
US8062856B2 (en) * 1999-08-05 2011-11-22 Cellomics, Inc. System for cell-based screening
US8900828B2 (en) * 2006-05-01 2014-12-02 Cepheid Methods and apparatus for sequential amplification reactions
US20190144936A1 (en) * 2016-01-15 2019-05-16 Massachusetts Institute Of Technology Semi-permeable arrays for analyzing biological systems and methods of using same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050202504A1 (en) * 1995-06-29 2005-09-15 Affymetrix, Inc. Miniaturized genetic analysis systems and methods
US8062856B2 (en) * 1999-08-05 2011-11-22 Cellomics, Inc. System for cell-based screening
US6790330B2 (en) * 2000-06-14 2004-09-14 Board Of Regents, The University Of Texas System Systems and methods for cell subpopulation analysis
US8900828B2 (en) * 2006-05-01 2014-12-02 Cepheid Methods and apparatus for sequential amplification reactions
US7807454B2 (en) * 2006-10-18 2010-10-05 The Regents Of The University Of California Microfluidic magnetophoretic device and methods for using the same
US20190144936A1 (en) * 2016-01-15 2019-05-16 Massachusetts Institute Of Technology Semi-permeable arrays for analyzing biological systems and methods of using same

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