WO2017210494A1 - Dosage de cellules multiplex à base microfluidique pour test de composés de médicaments - Google Patents

Dosage de cellules multiplex à base microfluidique pour test de composés de médicaments Download PDF

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Publication number
WO2017210494A1
WO2017210494A1 PCT/US2017/035560 US2017035560W WO2017210494A1 WO 2017210494 A1 WO2017210494 A1 WO 2017210494A1 US 2017035560 W US2017035560 W US 2017035560W WO 2017210494 A1 WO2017210494 A1 WO 2017210494A1
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Prior art keywords
wall
cells
gas
cell
microfluidic device
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PCT/US2017/035560
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English (en)
Inventor
Sabia ABIDI
Dimitrios P. PAPAGEORGIOU
Ming Dao
Subra Suresh
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Carnegie Mellon University
Massachusetts Institute Of Technology
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Publication of WO2017210494A1 publication Critical patent/WO2017210494A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • 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—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
    • G01N2015/012—
    • 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—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1497—Particle shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2203/00—Investigating strength properties of solid materials by application of mechanical stress
    • G01N2203/0058—Kind of property studied
    • G01N2203/0089—Biorheological properties

Definitions

  • a major challenge with in vitro investigations of the pathophysiological processes has been the lack of a well-controlled microenvironment to mimic in vivo circulating conditions.
  • painful vasoocclusions one of the hallmarks of sickle cell disease, are associated with rheological changes in blood flow resulting in blockage of microvasculature.
  • the polymerization of hemoglobin S in deoxygenated environments and its resultant sickling and decrease in deformability leads to this increase in blood viscosity.
  • Other factors, however, such as cell adhesion also contribute to blockage of blood flow, for example, by increasing the sickle RBC transit times in vessels, increasing the chances of sickling and reduction of deformability.
  • Cell adhesion is a form of communication and regulation between cells and their in vivo microenvironment. Often times dysfunction in cell specific adhesion is a sign of disease e.g. cancer, inflammatory diseases such as arthritis, sickle cell disease, etc. Detection and quantification of these adhesion specific changes would greatly aid in disease management. In addition, adhesion studies of cell populations can aid in the development of better tissue engineering alternatives and better drug treatment. In parallel, cell rigidity, stiffness, modulus or deformability is an indication of disease states, in many diseases such as cancer, malaria, sickle cell disease, leukemia, etc.
  • Controlled conditions may involve gas partial pressure modulation (diffusion rate control) using one or more gas mixtures, observing cell property (e.g. , adhesion, deformability) changes with different microenvironment conditions, such as precise shear stress modulation imposed by liquid flow profile, using different adhesive proteins (biophysical interaction between the cell/cell and/or cell/protein coated microchannel walls), under different temperatures, and osmolality control (constant or modulated osmolality).
  • the disclosure provides potential applications in patient specific disease diagnostics, screening drugs, studying drug efficacy, tissue engineering, etc.
  • Two pressure-driven flow systems were developed that can be paired with a two channel microfluidic -based platform to noninvasively study attachment and detachment of sickle cells, as one disease application, in a controlled microenvironment.
  • One system utilizes changes in gravitational potential while the other uses pressurized liquid or air to precisely control flow velocity or pressure experienced by cells.
  • the other uses pressurized liquid or air to precisely control flow velocity or pressure experienced by cells.
  • microfluidic platform provided herein also has gas control to enable the study of normoxic and hypoxic microenvironments, important for diseases such as sickle cell disease and cancer. Temperature and fluid and gas flow sensing capabilities also contribute to the controlled microenvironment.
  • the cell/fluidic channel in the device has been designed to resemble the size scale of post-capillary venules, sites associated with vasoocclusions. A protein functionalization scheme was used for the cell/fluidic channel to ensure resilient spatially homogeneous binding of protein to the channel that can be easily imaged throughout the assay.
  • the devices and systems provided herein offer the ability to study adhesion noninvasively in a well-controlled microenvironment that can be easily customized in terms of gas, temperature, protein, protein amount and flow rate experienced by cells to resemble the desired in vivo environment.
  • microfluidic platforms make them an ideal platform to study sickle cell disease and hematologic pathophysiology.
  • Several platforms have specifically focused on quantification of red blood cell deformability and adhesion (e.g., to a protein coated substratum) for diseases such as sickle cell disease.
  • These platforms however have not accounted for hypoxia, which is central to recapitulating the sickle cell vasoocclusive microenvironment, and its role in deformability and adhesion.
  • microfluidic platform that has improved on current microfluidic devices in its ability to control the gas mixture and its diffusion rate into the cellular microenvironment using a deformable PDMS membrane. This improvement enables the study of hypoxic microenvironments relevant for sickle cell disease and other diseases such as cancer.
  • Other advantageous features of the platform include the ability to customize the flow profile to pulsatile or continuous flow to simulate the precise shear rate experienced by sickle cells at sites of vasoocclusion and ability to modulate temperature and geometry of a microfluidic channel.
  • a protein functionalization scheme that can be quantified and imaged and is robust enough to stay intact during flow rate modulation has also been utilized.
  • Uses of the methods, devices and systems of the present disclosure include recapitulating complex in vivo microenvironments for tissue engineering and drug testing applications though systematic study of individual parameters such as gas, geometry, shear stress, protein and how they contribute to diseases such as cancer or blood-related diseases.
  • image analysis can be time-intensive and may be automated to facilitate analysis. This can be done by using available high-end commercial software packages, or by developing customized image analysis packages.
  • Red blood cell Red blood cell
  • RBC RBC diseases, especially those that involve hemolysis, are often closely linked to RBCs' fragility or susceptibility to hemolysis.
  • Commonly used fragility testing methods including the osmotic fragility test and various forms of mechanical fragility tests, are not single-cell assays; they cannot be used to obtain the exact pressure or stress under which individual RBCs lyse, and they all require relatively large blood volume (-3-20 mL).
  • microfluidic based assays for testing mechanical fragility at single cell level requiring a small blood volume.
  • the assay can be designed to handle 20-100 cells per test; however due to the inherent advantages of microfluidic processability utilizing automated RBC loading and fluidic multiplexing, it can be easily extended with further engineering development for hemolytic screening of 2,000-10,000 cells in less than an hour.
  • the setup can also be combined with a precise control of the varying gas microenvironment (including oxygen, nitric oxide etc.) for RBCs being tested.
  • This setup is expected to have high impact in providing a fundamentally new microfluidic -based assay for RBC mechanical fragility profiling at single-cell level requiring only a small blood volume (-100 ⁇ ), which can be used for being part of the assessment of the damage in stored blood (blood storage lesion), for the diagnosis of hemolytic diseases/disorders such as hereditary spherocytosis, and for providing new fundamental insights for various hemolytic diseases such as sickle cell anemia.
  • the invention is a high throughput method of measuring a
  • morphological and/or mechanical property of an individual cell under controlled gas conditions comprising: flowing a fluid comprising a plurality of cells through a channel comprising a wall, wherein at least a portion of the wall is coated with at least one protein, obtaining at least one measurement of a cell in the fluid; and regulating a level of gas in the fluid.
  • the property is a morphological property.
  • the morphological property is cell shape.
  • the cell shape is abnormal.
  • the cell shape is round, disk shaped, biconcave, oblong, or sickle shaped.
  • the morphological property is cell texture.
  • the cell texture is abnormal.
  • the cell texture is smooth, coarse, or spiky.
  • the measurement is a fraction of cells with an abnormal shape or texture. In another embodiment the measurement is a delay time of an abnormal cell shape change. In another embodiment the measurement is a delay time of recovering from an abnormal shape change. In yet another embodiment the cell shape change is sickling or unsickling.
  • the cells are bound to the portion of the wall that is coated with at least one protein. In other embodiments the cells are not bound to the portion of the wall that is coated with at least one protein. In some embodiments the measurement is used to determine a proportion of the cells that have an abnormal shape or texture at a certain temperature, flow rate and/or gas concentration.
  • the property is a mechanical property.
  • the mechanical property is adhesiveness.
  • the mechanical property is adhesiveness to the portion of the wall that is coated with at least one protein.
  • the cell is bound to a fixed position on the portion of the wall that is coated with at least one protein. In other embodiments the cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein.
  • the measurement is a number of and/or fraction of cells that bind to the portion of the wall that is coated with at least one protein. In other embodiments the measurement is a rate at which cells bind to the portion of the wall that is coated with at least one protein. In another embodiment the measurement is a speed that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein. In yet another embodiment the measurement is a distance that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein.
  • the measurement is a number of and/or a fraction of cells that detach from the portion of the wall that is coated with at least one protein. In another embodiment the measurement is used to determine a number of and/or fraction of cells that bind to the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In other embodiments the measurement is used to determine a rate at which cells bind to the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration.
  • the measurement is used to determine an average speed that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In other embodiments the measurement is used to determine an average distance that a cell slides, rolls, or tumbles along the portion of the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration. In another embodiment the measurement is used to determine a number of and/or a fraction of cells that detach from the wall that is coated with at least one protein at a certain temperature, flow rate and/or gas concentration.
  • the method comprises contacting the cell with the wall that is coated with at least one protein while the fluid is flowing through the channel. In other embodiments the method further comprises stopping the flow of the fluid through the channel.
  • the method comprises contacting the cell with the wall that is coated with at least one protein while the fluid is not flowing through the channel. In other embodiments the method further comprises starting the flow of the fluid through the channel.
  • the mechanical property is deformability.
  • the cell is bound to a fixed position on the portion of the wall that is coated with at least one protein.
  • the measurement is an amount that the cell deforms. In other embodiments the measurement is the distance that a cell stretches. In another embodiment the measurement is a ratio of the length versus the width of the cell. In another embodiment the measurement is used to determine an amount a cell deforms at a certain temperature, flow rate and/or gas concentration. In yet another embodiment the measurement is used to determine an average amount that cells deform at a certain temperature, flow rate and/or gas concentration.
  • the cells are from a subject. In another embodiment the cells are from a blood sample. In other embodiments the cells comprise red blood cells, white blood cells, stem cells or epithelial cells. In another embodiment the cells are red blood cells. In yet another embodiment the cells comprise one or more tumor cells.
  • the gas is selected from the group consisting of oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide and/or methane.
  • the gas is oxygen.
  • the level of the gas in the fluid is regulated to be at a concentration of less than 5%.
  • the level of the gas in the fluid is regulated to be at a concentration from 5% to 20%.
  • the level of the gas in the fluid is regulated to be at a
  • the level of the gas in the fluid is regulated to be at a concentration from 40% to 60%. In other embodiments the level of the gas in the fluid is regulated to be greater than 60%. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 20%. In another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 5%. In other embodiments the level of the gas in the fluid is regulated to be at a concentration of about 2%. In other embodiments the level of the gas in the fluid is regulated to be at a concentration of about 20% oxygen, 5% carbon dioxide and about 75% nitrogen.
  • the level of the gas in the fluid is regulated to be at a concentration of about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet another embodiment the level of the gas in the fluid is regulated to be at a concentration of about 2% oxygen, 5% carbon dioxide and about 93% nitrogen.
  • the property is measured at two or more different gas concentrations. In other embodiments the gas concentration is increased. In another embodiment the gas concentration is decreased. In yet another embodiment the property is measured as a function of time and as a function of gas concentration.
  • the cells are from a subject having or suspected of having a condition or disease selected from the group consisting of sickle cell disease (SCD), sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes and leukemia.
  • SCD sickle cell disease
  • SCT sickle cell trait
  • spherocytosis ovalocytosis
  • alpha thalassemia beta thalassemia
  • delta thalassemia malaria
  • anemia diabetes and leukemia
  • the cells are from a subject having or suspected of having sickle cell disease.
  • the fluid comprising the cells is flowed at a predetermined flow rate.
  • the flow rate is in a range of about 0.01 ⁇ /min to about 1000 ⁇ /min.
  • the flow rate is in a range of about 0.1 ⁇ /min to about 50 ⁇ /min.
  • the flow rate is in a range of about 0.1 ⁇ /min to about 10 ⁇ /min.
  • the flow rate is in a range of about 0.1 ⁇ /min to about 1 ⁇ /min.
  • the fluid comprising the cells is flowed at a predetermined pressure gradient.
  • the pressure gradient is in a range of about 0.01 Pa/ ⁇ to 10 Pa/ ⁇ .
  • the pressure gradient is in a range of about 0.1 Pa/ ⁇ to 5 Pa/ ⁇ .
  • the pressure gradient is in a range of about 0.1 Pa/ ⁇ to 2 Pa/ ⁇ .
  • the flow rate or pressure gradient is increased relative to the predetermined flow rate or predetermined pressure gradient. In other embodiments the flow rate or pressure gradient is decreased relative to the predetermined flow rate or predetermined pressure gradient. In another embodiment the flow rate or pressure gradient is ceased. In other embodiments the flow rate or pressure gradient is continuous. In another embodiment the flow rate or pressure gradient is not continuous. In yet another embodiment the fluid is pulsed through the channel.
  • the property is measured after one or more reoxygenation (ReOxy) cycles. In other embodiments the property is measured after at least 5, 10, 20, 50, or 100 reoxygenation (ReOxy) cycles. In another embodiment the property is measured after one or more deoxygenation (DeOxy) cycles. In yet another embodiment the property is measured after at least 5, 10, 20, 50, or 100 deoxygenation (DeOxy) cycles.
  • the fluid comprising the cells is flowed at a predetermined temperature. In other embodiments the temperature is a physiological temperature.
  • the protein comprises a cell surface protein or extracellular matrix (ECM) protein.
  • the cell surface protein is a cell adhesion molecule.
  • the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor.
  • the ECM protein is collagen, laminin, or fibronectin.
  • the protein comprises an antibody.
  • the fluid comprising a plurality of cells is flowed through the device described herein.
  • the invention is a microfluidic device comprising: a structure defining a microfluidic channel that comprises a first wall adjacent to the microfluidic channel, wherein at least a portion of the first wall is coated with at least one protein; and a second wall adjacent to the microfluidic channel, wherein at least a portion of the second wall comprises a gas permeable membrane or film.
  • the first wall is a top wall, a bottom wall, and/or a side wall.
  • the second wall is a top wall, a bottom wall, and/or a side wall.
  • the first wall and the second wall are different walls of the microfluidic channel.
  • the first wall and the second wall are the same wall of the microfluidic channel.
  • at least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or at least 90% of the second wall comprises a gas permeable membrane or film.
  • the second wall is a top wall. In other embodiments the second wall is a bottom wall or a side wall. In other embodiments the entire second wall comprises a gas permeable membrane or film. In another embodiment the second wall is a top wall. In yet another embodiment the second wall is a bottom wall or a side wall.
  • the first wall is a bottom wall. In other embodiments the first wall is a top wall, or a side wall. In another embodiment at least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or at least 90% of the first wall is coated with at least one protein. In other embodiments the first wall is a bottom wall. In another embodiment the first wall is a top wall or a side wall. In some embodiments the entire wall is coated with at least one protein.
  • the first wall is a bottom wall. In other embodiments the first wall is a top wall or a side wall.
  • the channel comprises a substantially planar transparent first wall and/or second wall.
  • the first wall is a substantially planar transparent wall.
  • the second wall is a substantially planar transparent wall.
  • the substantially planar transparent wall is glass or plastic.
  • the substantially planar transparent wall has a thickness in a range of 0.05 mm to 0.2 mm. In another embodiment the substantially planar transparent wall permits observation into the microfluidic channel by microscopy.
  • the protein comprises a cell surface protein or extracellular matrix (ECM) protein.
  • the cell surface protein is a cell adhesion molecule.
  • the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor.
  • the ECM protein is collagen, laminin, or fibronectin.
  • the protein comprises an antibody.
  • the microfluidic channel has a height from a top wall to a bottom wall in a range of 1 ⁇ to 50 ⁇ . In another embodiment the microfluidic channel has a height from a top wall to a bottom wall in a range of 1 ⁇ to 20 ⁇ . In other embodiments the microfluidic channel has a height from a top wall to a bottom wall in a range of 5 ⁇ to 20 ⁇ . In another embodiment the microfluidic channel has a height from a top wall to a bottom wall in a range of 10 ⁇ to 20 ⁇ . In another embodiment the microfluidic channel has a height from a top wall to a bottom wall of 15 ⁇ .
  • the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel in a range of 0.1 mm to 3 mm. In other embodiments the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel in a range of 1 mm to 2 mm. In another embodiment the microfluidic channel has a width from a first side wall of the microfluidic channel to a second side wall of the microfluidic channel of 1.3 mm.
  • the microfluidic channel has a length in a range of 1 mm to 10 mm. In other embodiments the microfluidic channel has a length in a range of 2 mm to 5 mm. In another embodiment the microfluidic channel has a length of 3 mm. In some embodiments the microfluidic channel comprises at least one inlet at a first end of the microfluidic channel and/or at least one outlet at a second end of the microfluidic channel. In other embodiments the microfluidic channel comprises at least one inlet at a first end of the microfluidic channel. In another embodiment the microfluidic channel comprises at least one outlet at a second end of the microfluidic channel.
  • the microfluidic device further comprises a reservoir fluidically connected with the microfluidic channel.
  • the microfluidic device further comprises a pump that is connected to the reservoir, and is configured to perfuse fluid from the reservoir to the microfluidic channel.
  • the reservoir comprises an inlet at a first end of the reservoir and an outlet at a second end of the reservoir.
  • the microfluidic device further comprises a microscope configured to permit observation within the microfluidic channel through the first wall of the microfluidic device. In another embodiment at least one measurement of a cell that passes through one of the microfluidic channels can be obtained.
  • the microfluidic device further comprises a heat transfer element that directly or indirectly contacts the microfluidic device.
  • the heat transfer element is configured to maintain a fluid in the microfluidic channel at a predetermined temperature.
  • the predetermined temperature is a physiologically relevant temperature.
  • the physiologically relevant temperature is in a range of 30 °C to 45 °C.
  • the physiologically relevant temperature is 37 °C.
  • the physiologically relevant temperature is 41 °C.
  • the microfluidic device further comprises a gas channel, wherein the gas channel comprises a wall that contacts the second wall of the microfluidic device, and wherein the gas channel contacts at least a portion of the gas permeable membrane or film of the microfluidic device. In other embodiments the gas channel contacts the entire portion of the gas permeable membrane or film. In another embodiment the gas channel comprises an inlet at first end of the gas channel. In other embodiments the gas channel comprises an outlet at a second end of the gas channel.
  • the gas permeable membrane or film comprises polydimethylsiloxane (PDMS), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose triacetate (CTA), or poly(methyl methacrylate) (PMMA).
  • PDMS polydimethylsiloxane
  • HPC hydroxypropyl cellulose
  • HPMC hydroxypropyl methylcellulose
  • CTA cellulose triacetate
  • PMMA poly(methyl methacrylate)
  • PMMA poly(methyl methacrylate)
  • the gas permeable membrane or film comprises polydimethylsiloxane (PDMS).
  • the invention is a method for identifying a therapeutic agent, the method comprising: perfusing a fluid comprising one or more cells through the device described herein; administering one or more compounds to the fluid, or wherein the fluid comprises the one or more compounds; determining a property of one or more of the cells; and comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the therapeutic effectiveness of the compound.
  • two or more of the compounds are administered to the fluid sequentially. In other embodiments two or more of the compounds are administered simultaneously. In another embodiment the method further comprises identifying an effective therapeutic agent based on the comparison above.
  • the cells are from a subject. In other embodiments the method further comprises administering the effective therapeutic agent to the subject.
  • the compounds are from a library of compounds. In other embodiments the compounds are candidate therapeutic agents.
  • the invention is a method for analyzing a condition or disease in a subject, the method comprising: perfusing a fluid comprising one or more cells from the subject through the device described herein; determining a property of one or more of the cells; and comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject.
  • the method of analyzing the condition or disease is a method for detecting the presence or absence of the condition or disease in the subject, and wherein the property is indicative of the presence of the condition or disease in the subject. In other embodiments the method of analyzing the condition or disease is a method for detecting the presence or absence of the condition or disease in the subject, and wherein the property is indicative of the absence of the condition or disease in the subject. In another embodiment the method of analyzing the condition or disease is a method for determining the severity of a condition or disease in the subject, and wherein the property is indicative of the severity of the condition or disease in the subject. In other embodiments the method of analyzing the condition or disease is a method for predicting vaso-occlusion crises in a subject, and wherein the property is indicative of a likelihood that the subject will undergo vaso-occlusion crisis.
  • the cells comprise blood cells.
  • the condition or disease is selected from the group consisting of sickle cell disease (SCD), sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes and leukemia.
  • the condition or disease is sickle cell disease.
  • the property is a mechanical property. In another embodiment the property is deformability, or adhesiveness.
  • the property is deformability. In other embodiments the property is adhesiveness.
  • the invention is a method for monitoring the effectiveness of a therapeutic agent for treating a disease or condition in a subject comprising: (a) perfusing a fluid comprising one or more cells from the subject through the device described herein; (b) determining a property of one or more of the cells; (c) treating the subject with the therapeutic agent; and (d) repeating steps (a) and (b) at least once wherein a difference in the property of one or more cells is indicative of the effectiveness of the therapeutic agent.
  • the invention is a method for determining the effectiveness of a therapeutic comprising: (a) obtaining a biological sample from a subject comprising a cell; (b) perfusing a fluid comprising one or more cells from the subject through the device described herein; (c) determining a property of one or more of the cells; (d) contacting the biological sample comprising a cell with the therapeutic; (e) perfusing a fluid comprising the product of (d) through the device described hereon; (f) determining a property of one or more of the cells from (e); and (g) comparing the property of one or more cells from (c) with the property of one or more cells from (f), wherein the results of the comparison are indicative of the effectiveness of the therapeutic.
  • the therapeutic is for treating sickle cell disease.
  • the therapeutic is hydroxyurea (HU) or 5- hydroxymethylfurfural (Aes-103).
  • the invention is a real-time method for quantifying cell
  • morphological kinetics in response to varying levels of gas comprising: (a) perfusing a fluid comprising one or more blood cells through the device described herein, wherein the fluid has a first level of gas; (b) determining a property of one or more of the cells from (a); (c) perfusing a fluid comprising one or more cells through the device described herein; wherein the fluid has a second level of gas that is different from the first level; (d) determining a property of one or more of the cells from (c); and (e) quantifying the cell morphological kinetics of the cells from (b) and (d).
  • the cell morphological kinetics are cell sickling and/or unsickling kinetics.
  • the invention is a system comprising any of the microfluidic devices provided herein and a gas source, wherein the gas source is connected to the gas channel of the microfluidic device via one or more supply lines.
  • the gas source is configured to flow gas from the gas source into the gas channel of the microfluidic device.
  • the gas source comprises oxygen, nitrogen, and/or carbon dioxide.
  • the gas source is connected to the gas channel of the microfluidic device via a gas flow sensor and/or a gas flow regulator.
  • system further comprises a microscope configured to observe one or more cells within the microfluidic device.
  • system further comprises a reservoir that is fluidically connected to the microfluidic channel of the microfluidic device via one or more supply lines.
  • the reservoir comprises a fluid, and wherein the fluid comprises a plurality of cells.
  • the reservoir is connected to the microfluidic channel via a flow sensor.
  • the reservoir is connected to the microfluidic channel via a pressure regulator and or a fluid flow sensor.
  • the pressure regulator is configured to pulsate the fluid from the reservoir into the microfluidic device.
  • system further comprises a temperature regulator that is configured to regulate the temperature of a fluid within the microfluidic device.
  • the system further comprises a computer in electronic communication with the gas flow sensor, the gas flow regulator, the pressure regulator, and/or the fluid flow sensor, wherein the computer comprises an input interface configured to receive information from the fluid flow sensor and/or the gas flow sensor, and an output interface configured to provide an output signal to the pressure regulator and/or the gas flow regulator.
  • the computer is in electronic communication with the microscope, and wherein the input interface is configured to receive information from the microscope, and wherein the output interface is configured to provide an output signal to the microscope.
  • the computer is in electronic communication with the temperature regulator, and wherein the input interface is configured to receive information from the temperature regulator, and wherein the output interface is configured to provide an output signal to the temperature regulator.
  • the invention is a kit comprising a microfluidic device, wherein the microfluidic device comprises: a structure defining a microfluidic channel that comprises a first wall adjacent to the microfluidic channel; and a second wall adjacent to the microfluidic channel, wherein at least a portion of the second wall comprises a gas permeable membrane or film.
  • the invention is a kit comprising any of the systems described herein.
  • the protein is in a container.
  • the protein is lyophilized.
  • the protein is in solution.
  • the protein comprises a cell surface protein or extracellular matrix (ECM) protein.
  • the cell surface protein is a cell adhesion molecule.
  • the cell surface protein is an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G- protein coupled receptor.
  • the ECM protein is collagen, laminin, or fibronectin.
  • the protein comprises an antibody.
  • kit further comprises instructions for coating a wall of the microfluidic channel with the protein.
  • FIG. 1- shows a non-limiting schematic representation of a system configuration for a microfluidic device.
  • FIGs. 2A-2C - show an example of protein functionalization and quantification.
  • FITC-labeled fibronectin (FIG. 2A) and Rhodamine-labeled laminin (FIG. 2B) functionalized microfluidic channels at 100 ⁇ g/ml concentration imaged with a lOx objective lens.
  • Protein quantification of a fibronectin functionalized channel using a Coomassie Blue assay FIG. 2C). Mean +/- standard deviation.
  • FIGs. 3A-3B - are exemplary data showing that lighter density fraction sickle RBCs are more adherent to fibronectin. Density fractionation of sample showing four bands of increasing density (FIG. 3A). Adhesion percentage of individual density fractions under oxygenated and low flow conditions (FIG. 3B).
  • FIGs. 4A-4B - show exemplary detachment profiles for sickle cells adherent to fibronectin (FIG. 4A) and laminin (FIG. 4B) (50 ⁇ g/ml) after 15 minutes of static incubation. Times and shear rates corresponding to cells detached are provided. Percentage of cells detached are indicated by blue bars. Shear rate is indicated by a red dotted line.
  • FIG. 5 - shows examples of increased tethering of deformable sickle RBCs with increasing flow rates. Numbers listed to the right indicate time. Blue triangle represents increasing flow rate. Orange line represents length of tether as tethers cannot be observed at 63x objective magnification.
  • sickle RBCs were incubated statically with 40 ⁇ g/ml of laminin functionalized microfluidic device for 15 min. After incubation, flow rate was steadily increased to detach adherent cells.
  • FIGs. 6A-6B - show exemplarydetachment profiles for sickle cells adherent to fibronectin under oxygenated (FIG. 6A) and deoxygenated (FIG. 6B) conditions after 15 minutes of static incubation. Red dotted line corresponds to pressure (mbar) cells experience for detachment.
  • FIG. 7 - shows exemplary hypoxia and dose-specific adhesion of sickle RBCs.
  • Sickle RBCs were incubated for 15 min statically under oxygenated or deoxygenated conditions with fibronectin at the indicated concentrations.
  • FIGs. 8A-8B - show exemplary elongation ratio measurements in a deformable sickle cell at normoxia. Images of cell depicting (FIG. 8A) ratio of major to minor axes initially and (FIG. 8B) ratio of major to minor axes of right before detachment.
  • FIG. 9 - shows exemplary relaxation ratio measurements in deformable sickle cells. Time course images showing response of deformable sickle cell to perturbation (left; shaded in blue) and relaxation (right).
  • FIGs. 10A-10B - show an exemplary elongation response of same cell to 2000 mbar pressure perturbation under oxygenated (FIG. 10A) and deoxygenated (FIG. 10B) conditions.
  • FIG. 11 - is an exemplary schematic representation of the device design.
  • FIG. 12 - is an exemplary schematic depicting a closed loop gravitational potential driven flow design setup.
  • FIG. 13 - is an exemplary schematic depicting open loop gravitational potential driven flow design setup.
  • FIG. 14 - is an exemplary schematic depicting pneumatic flow achieved through pressurized container or under vacuum reservoir.
  • FIG. 15 - is an exemplary schematic depicting pneumatic flow achieved through pressurized reservoir and single syringe as a source of back pressure.
  • FIG. 16 - is an exemplary schematic depicting pneumatic flow achieved through two pressurized reservoirs.
  • FIG. 17 - is an exemplary shape classification of oxygenated and deoxygenated sickle cells for fibronectin and laminin adhesion studies.
  • Cell shapes are traditional classifications in sickle cell literature with elongated and irreversibly sickled cells having increasing aspect ratios as compared to discocyte populations. At least 100 cells were counted for each condition. Mean +/- standard deviation.
  • FIG. 18 - shows gas pressure-induced hemolysis (gas mixture of 20% 0 2 , 5% C0 2 , and 75%N 2 ).
  • Panels show exemplary images of red blood cells within the flow channel using an exemplary microfluidic device. Characteristic instances of red blood cell (RBC) lysis events are shown with dotted ovals.
  • RBC red blood cell
  • FIG. 19 - shows a magnified view of gas pressure-induced hemolysis of Fig. 18.
  • Panels show exemplary images of red blood cells within the flow channel using an exemplary microfluidic device.
  • the red blood cell shown experiences initial pressure under normoxic conditions, Pini t n o r mox i a ; from that point onward the increase in the control channel pressure is ⁇ 6 psi/min.
  • FIG. 20 - shows a cross-sectional schematic representation of an exemplary micro- hemolytic device.
  • the dual-layer design comprises a flow channel, where one or more cells (e.g. a red blood cell in suspension) flow through the flow channel.
  • the design also comprises a control channel, where the pressure can be modulated (e.g., increased) to deform the membrane, thereby narrowing the flow channel.
  • the narrowing of the flow channel may exert a force onto one or more cells flowing through the flow channel.
  • the figure shows representative RBCs in the flow channel, which may be observed under a microscope objective lens. In the exemplary device shown, a PDMS membrane under pressure is deformed to narrow the flow channel.
  • FIG. 21 - shows gas pressure-induced hemolysis under hypoxic (DeOxy) gas conditions (gas mixture of 2% 0 2 , 5% C0 2 , 93% N 2 ) under the identical experimental conditions as Figs. 18 & 19; and control channel pressure rate ⁇ 10 psi/min.
  • FIG. 22 - shows morphological heterogeneity of adherent sickle cells under shear flow and steady state hypoxia.
  • the image shows adherent sickle cell heterogeneity after approximately 10 minutes of shear flow under hypoxic conditions. Walls of the flow channel are coated with fibronectin (FN-coated). Cells a-n are labelled.
  • FIG. 23 - shows the transition from single point (weak) adhesion to multiple point (firm) adhesion under shear flow and steady state hypoxia.
  • FIG. 24 - shows time-dependent adherent sickle shape change under shear flow and steady state hypoxia of cells "i", "f ' and "n” of Fig. 22.
  • These characteristic snapshots showcase cells with minimal shape change and non-protruded polymer fibers outwards of the cell.
  • cell “i” between 0 s ⁇ t ⁇ 4min there is noticeable reorganization of polymerized content; the cell tends to form a tri-lobe after ⁇ 9 min.
  • cells “f ' and "n” the changes in morphology are minimal indicating the stable morphological steady state under
  • FIG. 25 - shows time-dependent adherent sickle shape change under shear flow and steady state hypoxia of an adhered reticulocyte.
  • Panels show successive images of the cell shape change of "cell b," as indicated in FIG. 22.
  • Described herein are devices, methods, and systems for assessing cell properties, such as adhesion (e.g., to a protein coated on a substrate), morphology, and deformability under controlled gas environments. Accordingly, a microfluidics-based model was developed to quantify properties relating to the pathophysiology of disease (e.g., sickle cell disease (SCD), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, and anemia) under environments that mimic physiological conditions.
  • SCD sickle cell disease
  • spherocytosis spherocytosis
  • ovalocytosis ovalocytosis
  • alpha thalassemia beta thalassemia
  • delta thalassemia delta thalassemia
  • the devices provided herein may be used to mimic shear stress imposed by liquid flow, ambient gas partial pressures, gas content, osmolality, biophysical interaction between cells or between a cell and a substrate (e.g., a protein coated microchannel wall).
  • a substrate e.g., a protein coated microchannel wall.
  • This in vitro model enabled quantitative investigations of the kinetics of cell adhesion (e.g., attachment and detachment), cell morphology changes, such as cell sickling and unsickling, and cell deformability, for example of an adhered cell in response to a shear stress and/or in response to a change in a gas content.
  • the Examples provided herein relate to SCD in order to demonstrate the effectiveness of the devices, systems and methods described herein.
  • the invention is not limited to SCD.
  • Other pathologies, for example cancer, may be examined using the devices, methods and systems provided herein.
  • the Examples demonstrate that the devices, systems and methods can be used to sequentially (i) identify and classify a cell shape and/or type within a cell population, (ii) assess cell attachment, (iii) assess adherent cell deformability, and (iv) assess cell detachment with "on demand" cell microenvironment modulation. For example under conditions of normoxia and hypoxia.
  • Devices are provided herein for evaluating, characterizing, and/or assessing properties of cells, such as cell adhesion, cell deformability and/or cell shape, under controlled gas conditions.
  • devices are provided for measuring, evaluating and characterizing dynamic mechanical responses of biological cells, e.g. , red blood cells, to changes in the level of a gas (e.g., oxygen) and or in response to contacting a substrate comprising a protein (e.g., a laminin coated surface).
  • the devices are typically designed and configured to permit measurements of cell shape, adhesion, and/or deformability in a high throughput manner. For example, by measuring the shape of a cell adhered to a wall of the microfluidic device in response to changes in temperature, osmolality, gas concentration or content, and/or fluid pressure.
  • the devices typically include a structure defining a microfluidic channel through which a fluid that comprises one or more cells may pass.
  • the microfluidic channel of the devices typically include a wall, where at least a portion of the wall is coated with at least one protein. Any suitable protein may be coated onto at least a portion of the wall of the microfluidic channel. The protein used to coat at least a portion of the wall of the
  • microfluidic channel may depend on a number of factors, including but not limited to, the intended use of the device. It should be appreciated that methods for adhering (e.g., coating) a protein onto a substrate (e.g. , a wall of a microfluidic device) Any suitable protein may be used in accordance with this disclosure. In some embodiments, at least a portion of a wall of any of the microfluidic devices, provided herein, is coated with at least one protein. A wall of the microfluidic channel may be coated with any suitable protein or molecule comprising a protein. For example, the protein can be any protein that interacts (e.g., binds to) with one or more cells within the microfluidic channel of the microfluidic device.
  • the protein is a naturally-occurring protein (e.g., fibronectin or laminin).
  • the protein is a non-naturally occurring protein, for example, a variant of a naturally-occurring protein (e.g., fibronectin fused to a FITC molecule).
  • the non-naturally occurring protein comprises a naturally-occurring protein or a portion of a naturally-occurring protein with additional moieties (e.g., a protein tag, a sugar moiety, or a fluorescent molecule), which yields a protein that does not occur in nature.
  • the protein is fibronectin fused to FITC.
  • the protein is laminin fused to rhodamine.
  • ECM proteins any number of ECM proteins, or variants thereof, may be used in accordance with this disclosure.
  • at least a portion of a wall of any of the microfluidic devices, provided herein is coated with at least one ECM protein.
  • at least a portion of a wall is coated with an extracellular matrix (ECM) protein or variant thereof.
  • ECM extracellular matrix
  • extracellular matrix protein, or ECM protein refers to a protein that typically occupies an extracellular space of a tissue (e.g., in an animal).
  • ECM proteins are a collection of extracellular molecules secreted by cells that provide structural or biochemical support to the surrounding cells.
  • the ECM protein is an ECM protein from an animal, for example, an interstitial matrix protein or a basement membrane protein.
  • the ECM protein is an interstitial matrix protein.
  • interstitial matrix proteins are found in the interstitial matrix, which is present between various animal cells (e.g., the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space where they can act as a compression buffer against a stress placed on an extracellular matrix (ECM).
  • ECM protein is a basement membrane protein.
  • basement membrane proteins are proteins found in sheet-like depositions of ECM on which cell types (e.g., epithelial cells) rest.
  • at least a portion of a wall of the microfluidic device is coated with an ECM protein that comprises a carbohydrate polymer, (e.g., a glycosaminoglycan).
  • the ECM protein is a proteoglycan.
  • the ECM protein comprises a glycosaminoglycan.
  • polysaccharides that may be bound to an ECM protein include, without limitation, heparan sulfate, chondroitin sulfate and keratin sulfate.
  • the ECM protein comprises heparan sulfate.
  • Heparan sulfate is a linear polysaccharide found in all animal tissues. It typically occurs as a proteoglycan in which two or three heparan sulfate chains are attached in close proximity to cell surface or ECM proteins.
  • the ECM protein comprises chondroitin sulfate.
  • Chondroitin sulfate is a sulfated glycosaminoglycan comprised of a chain of alternating sugars (e.g. , N-acetylgalactosamine) and is typically found attached to proteins as part of a proteoglycan.
  • the ECM protein comprises keratan sulfate. Keratan sulfate typically has a variable sulfate content and does not contain uronic acid. Generally, karatan sulfates are found in the cornea, cartilage, bones, and horns of animals.
  • the ECM protein refers to a group of proteins present in an extracellular matrix as fibrous proteins, that typically provide structural support to resident cells.
  • the ECM protein is a collagen.
  • collagen is exocytosed as a precursor form (e.g., procollagen), which is cleaved by a protease , thereby allowing extracellular assembly.
  • the collagen is a fibrillar collagen.
  • the collagen may be a type I collagen, at type II collagen, a type III collagen, a type V collagen, or a type XI collagen.
  • the collagen is a facit collagen.
  • the collagen may be a type IX collagen, a type XII collagen, or a type XIV collagen.
  • the collagen is a short chain collagen.
  • the collagen may be a type VIII collagen, or a type X collagen.
  • the collagen is a basement membrane collagen.
  • the collagen may be a type IV collagen.
  • additional types of collagen may be used, for example type VI collagen, type VII collagen, and type XIII collagen, which are within the scope of this disclosure.
  • the protein is an elastin, which is a type of protein involved in modulating an elastic property of a tissue.
  • fibronectins are proteins that connect cells with collagen fibers in the ECM. Typically, fibronectins bind collagen and cell-surface integrins, which may facilitate cell movement.
  • the fibronectin comprises the amino acid sequence as set forth in (SEQ ID NO: 1).
  • the fibronectin comprises a tag.
  • the fibronectin comprises a fluorescent tag.
  • the fibronectin comprises a fluorescent tag.
  • the fibronectin comprises a FITC tag.
  • laminins are proteins found in the basal laminae of animals. Typically, laminins form networks of web-like structures that resist tensile forces in the basal lamina.
  • the laminin comprises the amino acid sequence as set forth in (SEQ ID NOs: 2-4).
  • the laminin comprises a tag.
  • the laminin comprises a fluorescent tag.
  • the laminin comprises a fluorescent tag.
  • the laminin comprises a rhodamine tag.
  • variants refers to a portion of a protein retaining at least one functional i.e. binding or interaction ability and/or therapeutic property thereof. The level or degree of which the property is retained may be reduced relative to the wild type protein but is typically the same or similar in kind. Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the protein described herein.
  • the variant proteins may comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a protein such as an ECM protein.
  • Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding a protein such as an ECM protein under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6.times. Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2. times.
  • SSC 0.1% SDS at about 50-65 degrees Celsius
  • highly stringent conditions e.g., hybridization to filter bound DNA in 6.times. sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1. times. SSC, 0.2% SDS at about 68 degrees Celsius
  • other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3).
  • polypeptide having an amino acid sequence at least, for example, 95%
  • amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a protein such as an ECM protein, can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is expressed as percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
  • cell surface proteins may be used in accordance with this disclosure.
  • at least a portion of a wall of any of the microfluidic devices, provided herein is coated with at least one cell surface protein.
  • the protein is a cell surface protein or a variant thereof.
  • a "cell surface protein” refers to a protein that is embedded in or spans a layer of a cell plasma membrane in one or more organisms. Generally, cell surface proteins are exposed to an external side of the cell membrane, for example by embedding in or spanning the plasma membrane of a cell, which separates the inside of a cell from the external environment. Any number of cell surface proteins may be used in accordance with this disclosure.
  • the protein is a cell adhesion molecule (CAM) or a variant thereof.
  • CAM cell adhesion molecule
  • the cell surface protein is a cell adhesion molecule (CAM) or variant thereof.
  • CAM cell adhesion molecule
  • the term "cell adhesion molecule” refers to a protein that is typically located on the cell surface that is involved in binding with other cells or with the extracellular matrix (ECM).
  • Cell adhesion molecules typically include four protein families, the immunoglobulin superfamily (IgSF CAMs), the integrins, the cadherins, and the selectins.
  • the protein is an IgSF, an integrin, a cadherin, or a selectin.
  • Cell adhesion molecules may be classified as calcium-independent CAMs or calcium-dependent CAMs.
  • the protein is a calcium-independent CAM.
  • the calcium- independent CAM may be an IgSF protein, or a lymphocyte homing receptor ⁇ e.g., CD34 or GLYCAM-1).
  • additional non-limiting examples of calcium-independent CAMs include N- CAM ⁇ e.g., Myelin protein zero), ICAM ⁇ e.g., ICAM1, or ICAM5), VCAM-1, PE-CAM Ll- CAM, or Nectin ⁇ e.g., PVRL1, PVRL2, or PVRL3).
  • the protein is in immunoglobulin ⁇ e.g., an antibody). Exemplary antibodies that may be used in accordance with this disclosure are described in more detail below.
  • the protein is a calcium-dependent CAM.
  • the calcium-dependent CAM may be a cadherin, a selectin, or an integrin.
  • the protein is a cadherin.
  • the cadherin is a classical cadherin, for example CDH1, CDH2, or CDH3.
  • the cadherin is a desmosomal cadherin, for example desmoglein (e.g., DSG1, DSG2, DSG3, or DSG4), or desmocollin (e.g. , DSC 1, DSC2, or DSC3).
  • the cadherin is a protocadherin, for example PCDH1, or PCDH15.
  • the cadherin may be an unconventional cadherin, for example T-cadherin, CDH4, CDH5, CDH6, CDH8, CDH11, CDH12, CDH15, CDH16, CDH17, CDH9, or CDH10.
  • T-cadherin CDH4, CDH5, CDH6, CDH8, CDH11, CDH12, CDH15, CDH16, CDH17, CDH9, or CDH10.
  • additional cadherins would be apparent to the skilled artisan and are within the scope of this disclosure.
  • the protein is a selectin.
  • Selectins are single-chain
  • selectins bind to sugar moieties and so are considered to be a type of lectin, cell adhesion proteins that bind sugar polymers.
  • the selecting is an E-selectin, an L-selectin, or a P-selectin.
  • E-selectin an E-selectin
  • L-selectin an L-selectin
  • P-selectin a P-selectin
  • the protein is an integrin.
  • Integrins are transmembrane receptors that typically bridge cell-cell and cell-extracellular matrix (ECM) interactions.
  • ECM cell-extracellular matrix
  • the integrin is an ⁇ , ⁇ 2 ⁇ 1, ⁇ 4 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ ⁇ , ⁇ 2, ⁇ 2, ⁇ 3 ⁇ 4 ⁇ 3, ⁇ 3, ⁇ 5, ⁇ , or a ⁇ 6 ⁇ 4 integrin.
  • Additional examples of integrins include, without limitation LFA- 1 (e.g., CDl la and CD18), Integrin alphaXbeta2 (e.g., CD 11c and CD 18) Macrophage- 1 antigen (e.g.
  • VLA-4 e.g., CD49d and CD29
  • Glycoprotein Ilb/IIIa e.g., ITGA2B and ITGB3
  • the protein is a receptor tyrosine kinase, or a G-protein coupled receptor.
  • the receptor tyrosine kinase is a an ErbB family receptor (e.g., EGF receptor), insulin receptor, PDGF receptor, FGF receptor, VEGF receptor, HGF receptor, Trk receptor, Eph receptor, AXL receptor, LTK receptor, TIE receptor, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, or MuSK receptor.
  • the G-protein coupled receptor is a rhodopsin-like receptor, the secretin receptor, metabotropic glutamate/pheromone receptor, cyclic AMP receptor, frizzled/smoothened receptor, CXCR4, CCR5, or beta-adrenergic receptor. It should be appreciated that additional tyrosine kinase receptors and G-protein coupled receptors would be apparent to the skilled artisan and are within the scope of this disclosure.
  • the protein is a protein of a vessel wall (e.g., a blood vessel wall).
  • the protein is selectin, vascular cell adhesion molecule- 1, alpha -V Beta-3 integrin, CD36, fibronectin, thrombospondin, Von Willebrand Factor, or laminin.
  • selectin vascular cell adhesion molecule- 1, alpha -V Beta-3 integrin, CD36, fibronectin, thrombospondin, Von Willebrand Factor, or laminin.
  • additional proteins found on the walls of blood vessels are known and would be apparent to the skilled artisan.
  • immunoglobulin proteins e.g. antibodies
  • at least a portion of a wall of any of the microfluidic devices, provided herein is coated with at least one antibody.
  • an "antibody” encompasses not only intact (e.g. , full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g.
  • bispecific antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g.
  • immunoglobulins The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • any of the antibodies, provided herein may be designed to bind to any antigen of interest, for example, and antigen on the surface of a cell.
  • the antibody binds to an antigen on the surface of a cell.
  • the antibody binds to an antigen on the surface of a specific cell type.
  • the antibody may be designed to bind an erythrocyte (i.e. , a red blood cell).
  • the antibody binds to an antigen on a differentiated cell.
  • the antibody binds to an antigen on a blood or immune cell.
  • the antibody binds to an antigen on an erythrocyte (e.g., a red blood cell), a megakaryocyte (e.g. , a platelet precursor), a monocyte (e.g. , a white blood cell), a connective tissue macrophage, an epidermal
  • an erythrocyte e.g., a red blood cell
  • a megakaryocyte e.g. , a platelet precursor
  • monocyte e.g. , a white blood cell
  • connective tissue macrophage e.g., an epidermal
  • an osteoclast e.g., in bone
  • a dendritic cell e.g., in lymphoid tissues
  • a microglial cell e.g., in central nervous system
  • a neutrophil granulocyte an eosinophil granulocyte, a basophil granulocyte, a hybridoma cell, a mast cell, a helper T cell, a suppressor T cell, a cytotoxic T cell, a natural killer T cell, a B cell, or a reticulocyte.
  • any of the antibodies provided herein may bind to an antigen on a cultured cell or a cell from a subject (e.g. , a human subject).
  • the antibody binds to an antigen on a mammalian cell, examples of mammalian cells include but are not limited to, a cell from a rodent, a mouse, a rat, a hamster, or a non-human primate.
  • the target cell may also be from a human.
  • the cell may be also from an established cell line (e.g., a 293T cell), or a primary cell cultured ex vivo (e.g., cells obtained from a subject and grown in culture).
  • the antibody may bind to an antigen on a hematologic cell (e.g. , a hematopoietic stem cell, a leukocyte, a thrombocyte, or erythrocyte), or on a cell from a solid tissue, such as a liver cell, a kidney cell, a lung cell, a heart cell, a bone cell, a skin cell, a brain cell, or any other cell found in a subject.
  • a hematologic cell e.g. , a hematopoietic stem cell, a leukocyte, a thrombocyte, or erythrocyte
  • a cell from a solid tissue such as a liver cell, a kidney cell, a lung cell, a heart cell, a bone cell, a skin cell, a brain cell, or any other cell found in a subject.
  • a hematologic cell e.g. , a hematopoietic stem cell, a leukocyte,
  • any of the antibodies provided herein may bind to an antigen on a stem cell, such as a pluripotent stem cell or a totipotent stem cell.
  • a stem cell such as a pluripotent stem cell or a totipotent stem cell.
  • antigens specific to pluripotent stem cells include Oct4 and Nanog, which were the first proteins identified as essential for both early embryo development and pluripotency maintenance in embryonic stem cells.
  • Stem cell antigens are known in the art and have been described previously, for example in Nichols J, et al. "Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.", Cell. 95:379-91, 1998; the contents of which are hereby incorporated by reference).
  • any of the antibodies provided herein may bind to an antigen on a tumor or cancer cell. In some embodiments, any of the antibodies provided herein may bind to an antigen that is unique to a tumor or cancer cell, for example, as compared to a non- tumor or non-cancer (e.g., normal) cell.
  • the protein is a protein that binds a tumor associated or tumor specific antigen. In some embodiments, the protein is an antibody that binds a tumor associated or tumor specific antigen.
  • tumor antigens include, CA19-9, c-met, PD-1, CTLA-4, ALK, AFP, EGFR,
  • immunoglobulins that bind other tumor antigens would be apparent to the skilled artisan and are within the scope of this disclosure.
  • tumor specific antigens have been described in Bigbee W., et al. "Tumor markers and immunodiagnosis.”, Cancer Medicine . 6th ed. Hamilton, Ontario, Canada: BC Decker Inc., 2003.; Andriole G, et al.
  • any of the antibodies used in accordance with this disclosure may be produced by any suitable method. Numerous methods may be used for obtaining antibodies, or antigen binding fragments thereof, of the disclosure. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g.
  • ELISA enzyme-linked immunosorbent assay
  • surface plasmon resonance e.g.
  • any form of the specified antigen may be used as the immunogen, e.g. , recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof (e.g. , any of the epitopes described herein as a linear epitope or within a scaffold as a
  • One exemplary method of making antibodies includes screening protein expression libraries that express antibodies or fragments thereof (e.g. , scFv), e.g. , phage or ribosome display libraries.
  • Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol.
  • the specified antigen e.g. , a blood cell antigen
  • a non-human animal e.g. , a rodent, e.g. , a mouse, hamster, or rat.
  • the non-human animal is a mouse.
  • a monoclonal antibody is obtained from the non-human animal, and then modified, e.g. , chimeric, using suitable recombinant DNA techniques.
  • modified e.g. , chimeric
  • suitable recombinant DNA techniques e.g., DNA sequences, DNA sequences, and fragments thereof.
  • the proteins provided herein may be from any organism.
  • the protein may be a naturally-occurring protein from an animal.
  • the protein is a naturally-occurring protein from a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, a pig, or a fish.
  • proteins from other organisms would be apparent to the skilled artisan and are within the scope of this disclosure.
  • the disclosure provides microfluidic devices that are coated with cells.
  • at least a portion of a wall of any of the microfluidic devices, provided herein is coated with at least one cell.
  • the cell may be attached to a wall of the device covalently or non-covalently.
  • any of the proteins provided herein are comprised in a cell.
  • the cell is a cultured cell (e.g., from a cell line).
  • the cell is a primary cell, from a subject, which may, or may not be taken directly from a subject, or may be cultured after being obtained from the subject.
  • the one or more walls of any of the microfluidic devices provided herein may be coated with one or more proteins, which may be naturally-occurring (e.g. , naturally produced by a cell), or non-naturally occurring, meaning that the protein does not occur in nature.
  • a non-naturally occurring protein may comprise a naturally-occurring protein.
  • a naturally occurring human fibronectin protein fused to a protein tag e.g. a FLAG tag
  • the disclosure provides proteins that are variants of or homologous to any naturally-occurring protein.
  • the protein is homologous to any of the proteins provided herein.
  • proteins are considered to be “homologous” to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. In some embodiments, proteins are considered to be “homologous” to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (e.g., two amino acid sequences).
  • Devices containing a microfluidic channel further contain a first wall adjacent to the microfluidic channel, where at least a portion of the first wall is coated with at least one protein and a second wall adjacent to the microfluidic channel, where at least a portion of the second wall comprises a gas permeable membrane or film.
  • adjacent to refers to a physical proximity to the channel such that at least a portion of a wall (e.g., a first wall or a second wall) and at least a portion of the channel are in physical contact or are separated by a space that contains the gas. "Adjacent to” could mean that the wall defines a surface of the channel.
  • Adjacent to could also mean that the wall defines an inner surface and/or outer surface of the microfluidic device.
  • the microfluidic channel may have a top surface, bottom surface, side surface or end surface that contacts and/or contains a fluid that is flowed through one or more of the microfluidic channels.
  • the gas permeable portion of a wall (e.g., a second wall), which can be, for example a gas permeable membrane or film e.g., polydimethylsiloxane (PDMS), permits the control of the level of a gas in the microfluidic device.
  • a wall e.g., a second wall
  • PDMS polydimethylsiloxane
  • the gas permeable film has a thickness ranging from 5 ⁇ to 500 ⁇ .
  • the gas permeable film has a thickness ranging from 5 ⁇ to 20 ⁇ , from 5 ⁇ to 50 ⁇ , from 5 ⁇ to 100 ⁇ , from 5 ⁇ to 150 ⁇ , from 5 ⁇ to 200 ⁇ , from 5 ⁇ to 250 ⁇ , from 5 ⁇ to 300 ⁇ , from 5 ⁇ to 400 ⁇ , from 5 ⁇ to 500 ⁇ , from 50 ⁇ to 100 ⁇ , from 50 ⁇ to 150 ⁇ , from 50 ⁇ to 200 ⁇ , from 50 ⁇ to 300 ⁇ , from 50 ⁇ to 400 ⁇ , from 50 ⁇ to 500 ⁇ , from 100 ⁇ to 200 ⁇ , from 100 ⁇ to 300 ⁇ , from 100 ⁇ to 400 ⁇ , from 100 ⁇ to 500 ⁇ , from 200 ⁇ to 300 ⁇ , from 200 ⁇ to 400 ⁇ , from 200 ⁇ to 500 ⁇ , from 300 ⁇ to 400 ⁇ , from 300 ⁇ to 500 ⁇ or from 400 ⁇ to 500 ⁇ .
  • the gas permeable film has a thickness of aboutl50 ⁇ . It should be appreciated that the gas permeable membrane or film may make up an entire wall (e.g., a second wall) or a portion of a wall (e.g., a second wall) of the microfluidic channel.
  • the gas permeable membrane makes up from 1% to 100% of the surface area of a wall (e.g., the second wall) of the device. In some embodiments, the gas permeable membrane makes up from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 30%, from 1% to 50%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 30%, from 5% to 50%, from 5% to 60%, from 5% to 5% to
  • a wall e.g., a second wall
  • one or more walls of the microfluidic device may have at least a portion of a wall (e.g., a second wall) that is made of a gas permeable membrane or film.
  • the gas permeable membrane or film may be permeable to any number of gases that are supplied to the gas permeable membrane or film.
  • the membrane or film may be permeable to gasses including but not limited to oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide and/or methane.
  • the membrane or film is permeable to oxygen.
  • the gas permeable membrane or film may be constructed of any suitable material that is permeable to any of the gases, described herein.
  • the gas permeable membrane or film may be made of a material including but not limited to polydimethylsiloxane (PDMS), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose triacetate (CTA), or poly(methyl methacrylate) (PMMA).
  • PDMS polydimethylsiloxane
  • HPMC hydroxypropyl cellulose
  • HPMC hydroxypropyl methylcellulose
  • CTA cellulose triacetate
  • PMMA poly(methyl methacrylate)
  • Other gas permeable membranes or films are known in the art, such as those disclosed in Budd et al. (Peter M. Budd and Neil B. McKeown, Highly permeable polymers for gas separation membranes, Polym. Chem., 2010, 1, 63-68; the entire contents of which are hereby incorporated by reference).
  • the gas permeable film is made of PDMS.
  • the gas permeable film or membrane comprises PDMS (Dow Corning Sylgard elastomer 184).
  • the PDMS membrane or film can be produced by mixing different ratios of the pre-polymer base with the crosslinking curing agent.
  • the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) from 1 : 1 to 20: 1.
  • the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) from 1 : 1 to 2: 1, from 1 : 1 to 4: 1, from 1 : 1 to 6: 1, from 1 : 1 to 8: 1, from 1 : 1 to 10: 1, from 1 : 1 to 12: 1, from 1 : 1 to 14: 1, from 1 : 1 to 18: 1, or from 1 : 1 to 20: 1.
  • the PDMS pre-polymer base is mixed with the crosslinking curing agent at a ratio (PDMS pre-polymer : crosslinking curing agent) of 5: 1, 10: 1, 15: 1 or 20: 1. It should be appreciated that the ratio may be modified to control one or more properties of the PDMS membrane or film produced.
  • a portion of a wall e.g. , a first wall
  • an entire wall e.g. , a first wall
  • at least one protein e.g., fibronectin
  • at least a portion of a wall may be coated with at least one protein, for example, from 1% to 100% of the surface area of a wall (e.g. , a first wall) may be coated with at least one protein.
  • the walls may be coated with any suitable amount of protein, for example, an amount sufficient to permit or facilitate binding of a cell to a wall of the device.
  • a wall of the device may be coated with a given amount of protein in a given surface area.
  • a protein may be densely coated or thinly coated onto a wall of the device.
  • a wall or a portion of a wall is coated with from 0.0 ⁇ g/in to 50 ⁇ g/in of protein.
  • a wall or a portion of a wall is coated with
  • the proteins provided herein may be coated onto a wall of the device using any suitable method, for example a method that would be apparent to the skilled artisan.
  • the protein is bound to the surface of a wall.
  • the protein is non-covalently bound to the surface of a wall.
  • the protein is covalently bound to the surface of a wall.
  • the protein may be bound directly to the wall, or indirectly to the wall, e.g., via a linker.
  • linker refers to a chemical group or a molecule linking two molecules or moieties, e.g., a wall (e.g., a coated wall) to a protein, such as laminin.
  • the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two.
  • the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein).
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker is 5- 100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60- 70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
  • any of the devices, described herein, may also contain a gas channel.
  • This gas channel may be used to supply a gas to the gas permeable membrane or film of the device in order to regulate the gas content of the fluid in the device.
  • the gas channel may encase the gas permeable membrane or film on a wall of the microfluidic channel such that gas exchange can occur between the gas in the gas channel and the fluid in the microfluidic channel through the gas permeable membrane or film.
  • An exemplary microfluidic device with a gas channel encasing a gas permeable layer is shown in Figs. 1 and 1 1 - 16.
  • the gas channel is separated from the microfluidic channel by a gas permeable membrane to allow gas exchange between the gas channel and a fluid in the microfluidic device.
  • the gas channel may be any size or shape suitable for supplying a gas to the gas permeable membrane or film of the microfluidic device.
  • the gas channel is between 10 ⁇ and 10mm in height. In one specific embodiment, the gas channel is 100 ⁇ in height.
  • any of the microfluidic devices may comprise one or more gas channels to deliver one or more gasses to any portion of the microfluidic device with a gas permeable membrane or film.
  • the gas channel comprises at least one inlet (e.g. for gas to enter the gas channel) and/or at least one outlet (e.g., for gas to exit or flow out of).
  • a gas or gas mixture may be supplied to the inlet of the gas channel from one or more tanks containing the gas or gas mixture.
  • the gas supplied to the gas channel is oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide, methane, or any combination thereof.
  • the gas supplied to the gas channel contains oxygen.
  • the gas supplied contains between 1% and 100 % oxygen.
  • the gas supplied contains from 1% to 2%, from 1% to 3%, from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 40%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 40%, from 5% to 60%, from 5% to 80%, from 5% to 100%, from 20% to 40%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 40% to 60%, from 40% to 80%, from 40% to 100%, from 60% to 80%, from 60% to 100% or from 80% to 100% oxygen.
  • the gas supplied to the gas channel contains about 2%, about 5%, or about 20% oxygen.
  • the term “about,” or “approximately” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value).
  • the gas supplied to the gas channel contains about 20% oxygen, 5% carbon dioxide and about 75% nitrogen.
  • the gas supplied to the gas channel contains about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet other embodiments the gas supplied to the gas channel contains about 2% oxygen, 5% carbon dioxide and about 93% nitrogen.
  • the gas or gas mixture may be supplied to one or more inlets of one or more gas channels using any suitable means, such as tubing or hoses.
  • the gas or gas mixture may be delivered to the gas channel continuously such that the gas enters the inlet of the gas channel and exits from the outlet of the gas channel. This ensures that the gas or gas mixture in the channel remains consistent as gas exchanges across the gas permeable membrane.
  • consistent means that the level of, or % composition of a gas in a given space (e.g., a channel) does not vary by a large amount. In some embodiments consistent means that the level of, or % composition of a gas entering the gas channel does not vary by more than 1%, 2%, 3%, 4%, 5%, 8% or 10% before exiting the gas channel.
  • the gas or gas mixture may be flowed through the gas channel at any suitable rate.
  • the gas in the gas channel may regulated at a specific pressure.
  • the pressure of the gas in the gas channel is from lpsi to lOpsi. In a specific embodiment, the pressure of the gas in the gas channel is regulated to be about 5psi.
  • the device may be configured such that the gas or gas mixture, supplied to one or more gas inlets of the device, can be switched to a different gas or gas mixture.
  • This enables the device to dynamically control the gas content of a fluid in the microfluidic channel.
  • a fluid containing cells flowing through one or more microfluidic channels of the device can be exposed to a gas with high oxygen content (e.g., 20% oxygen) for a given time through the gas channel.
  • a different gas may be supplied to the same gas channel or a different gas channel.
  • the gas delivered to the gas channel can be switched to a gas with low oxygen content (e.g., 2% oxygen). This allows for the dynamic
  • a fluid containing red blood cells is flowed through the microfluidic device where the gas delivered to the gas channel contains about 20% oxygen, about 5% carbon dioxide and about 75% nitrogen.
  • One or more measurements for example a morphological measurement (e.g., cell sickling) or a mechanical measurement (e.g., cell stretching) can be made as the cells flow through the microfluidic device under high oxygen content.
  • the gas delivered to the gas channel can then be switched to a gas having a low oxygen content (e.g., about 2% oxygen, about5% carbon dioxide and about 75% nitrogen) to regulate the oxygen content of the fluid containing red blood cells.
  • One or more additional measurements may be taken over time to dynamically observe/measure one or more cell parameters in response to low oxygen conditions. For example, cell sickling time, or adhesiveness may be determined for a given cell sample when oxygen levels decrease. It should be appreciated that the device may be used to measure a cell-scale parameter in response to any gas or gas mixture and is not limited to the examples provided herein.
  • Devices containing a microfluidic channel can further contain a substantially planar transparent wall that defines a wall of a microfluidic channel.
  • This substantially planar transparent wall which can be, for example, glass or plastic, permits observation into the microfluidic channel by microscopy so that at least one measurement of each cell that passes through one of the microfluidic channels can be obtained.
  • the transparent wall has a thickness of 0.05 mm to 2 mm.
  • the transparent wall may be a microscope cover slip, or similar component.
  • Microscope coverslips are widely available in several standard thicknesses that are identified by numbers, as follows: No. 0 - 0.085 to 0.13 mm thick, No. 1 - 0.13 to 0.16 mm thick, No.
  • any of the microfluidic channels of the present disclosure may have a height, for example from a top wall to a bottom wall, ranging from 0.5 ⁇ to 100 ⁇ .
  • the microfluidic channel of any of the devices provided herein may have a height in a range of 0.5 ⁇ to 100 ⁇ , 0.1 ⁇ to 100 ⁇ , 1 ⁇ to 50 ⁇ , 1 ⁇ to 50 ⁇ , 10 ⁇ to 40 ⁇ , 5 ⁇ to 15 ⁇ , 0.1 ⁇ to 5 ⁇ , or 2 ⁇ to 5 ⁇ .
  • the microfluidic channel may have a height of up to 0.5 ⁇ , 1 ⁇ , 1.5 ⁇ , 2.0 ⁇ , 2.5 ⁇ , 3.0 ⁇ , 3.5 ⁇ , 4.0 ⁇ , 4.5 ⁇ , 5.0 ⁇ , 5.5 ⁇ , 6.0 ⁇ , 6.5 ⁇ , 7.0 ⁇ , 7.5 ⁇ , 8.0 ⁇ , 8.5 ⁇ , 9.0 ⁇ , 9.5 ⁇ , 10 ⁇ , 20 ⁇ , 30 ⁇ , 40 ⁇ , 50 ⁇ , 75 ⁇ , 100 ⁇ , or more.
  • the microfluidic channel has a height of 15 ⁇ , or about 15 ⁇ .
  • the height of the microfluidic channel can be any suitable height, which may be based on the intended use of the microfluidic device.
  • the height of the device may be larger than a cell to allow the cell to flow through the device.
  • the height of the device may be slightly larger than a cell that is introduced into the device so that it can be squeezed between the top wall and the bottom wall, where one of the walls is a membrane or film.
  • a force e.g., pressure
  • the height of the microfluidic channel is from ⁇ . ⁇ to 100 ⁇ larger than a dimension (e.g.
  • the microfluidic channel is from ⁇ . ⁇ to 0.1 ⁇ , from ⁇ . ⁇ to 1 ⁇ , from ⁇ . ⁇ to 10 ⁇ , from ⁇ . ⁇ to 20 ⁇ , from ⁇ . ⁇ to 50 ⁇ , from ⁇ . ⁇ to 80 ⁇ , from ⁇ . ⁇ to 1 ⁇ , from ⁇ . ⁇ to 10 ⁇ , from ⁇ . ⁇ to 20 ⁇ , from ⁇ . ⁇ to 50 ⁇ , from ⁇ .
  • ⁇ to 80 ⁇ from ⁇ to 10 ⁇ , from ⁇ to 20 ⁇ , from ⁇ to 50 ⁇ , from ⁇ to 80 ⁇ , from 10 ⁇ to 20 ⁇ , from 10 ⁇ to 50 ⁇ , from 10 ⁇ to 80 ⁇ , from 20 ⁇ to 50 ⁇ , from 20 ⁇ to 80 ⁇ , or from 20 ⁇ to 80 ⁇ larger than a dimension (e.g. , diameter) of a cell in the device.
  • a dimension e.g. , diameter
  • any of the microfluidic channels of the present disclosure may have a width, for example from a first side wall to a second side wall, ranging from 0.01 mm to 5 mm.
  • the microfluidic channel of any of the devices provided herein may have a width in a range of 0.01 mm to 4 mm, 0.1 mm to 3 mm, 0.1 mm to 2 mm, 0.2 mm to 2 mm, 0.5 mm to 2 mm, 0.5 mm to 1.5 mm, 0.8 mm to 1.5 mm, or 1 mm to 1.4 mm.
  • the microfluidic channel may have a width of up to 0.01 mm, 0.05 mm, 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.8 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 6 mm, 6.5 mm, 7 mm, or more.
  • the microfluidic channel has a width of 1.3 mm, or about 1.3 mm.
  • any of the microfluidic channels of the present disclosure may have a length, for example from a first end wall to a second end wall, or from a first inlet to a second inlet, ranging from 0.1 mm to 20 mm.
  • the microfluidic channel of any of the devices provided herein may have a length in a range of 0.1 mm to 19 mm, 0.1 mm to 18 mm, 0.1 mm to 15 mm, 0.2 mm to 14 mm, 0.2 mm to 12 mm, 0.5 mm to 10 mm, 0.5 mm to 5 mm, or 1 mm to 5 mm.
  • the microfluidic channel may have a length of up to 0.01 mm, 0.05 mm, 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.8 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 6 mm, 6.5 mm, 7 mm, or more.
  • the microfluidic channel has a length of 3 mm, or about 3 mm.
  • the device described above can further contain a reservoir fluidically connected with the one or more microfluidic channels, and a pump that perfuses fluid from the reservoir through the one or more microfluidic channels, and optionally, a microscope arranged to permit observation within the one or more microfluidic channels.
  • the reservoir may contain cells suspended in a fluid.
  • the fluidics connecting the reservoir to the microfluidic channel may include one or more filters to prevent the passage of unwanted or undesirable components into the microfluidic channels.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of 0.05 Pa/ ⁇ , 0.1 Pa/ ⁇ , 0.15 Pa/ ⁇ , 0.2 Pa/ ⁇ , 0.25 Pa/ ⁇ , 0.3 Pa/ ⁇ , 0.35 Pa/ ⁇ , 0.4 Pa/ ⁇ , 0.45 Pa/ ⁇ , 0.5 Pa/ ⁇ , 0.55 Pa/ ⁇ , 0.6 Pa/ ⁇ , 0.65 Pa/ ⁇ , 0.7 Pa/ ⁇ , 0.75 Pa/ ⁇ , 0.8 Pa/ ⁇ , 0.85 Pa/ ⁇ , 0.9 Pa/ ⁇ , 0.95 Pa/ ⁇ , 1 Pa/ ⁇ , 2 Pa/ ⁇ , 3 Pa/ ⁇ , 4 Pa/ ⁇ , 5 Pa/ ⁇ , 10 Pa/ ⁇ , or more.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of about 0.05 Pa/ ⁇ , 0.1 Pa/ ⁇ , 0.15 Pa/ ⁇ , 0.2 Pa/ ⁇ , 0.25 Pa/ ⁇ , 0.3 Pa/ ⁇ , 0.35 Pa/ ⁇ , 0.4 Pa/ ⁇ , 0.45 Pa/ ⁇ , 0.5 Pa/ ⁇ , 0.55 Pa/ ⁇ , 0.6 Pa/ ⁇ , 0.65 Pa/ ⁇ , 0.7 Pa/ ⁇ , 0.75 Pa/ ⁇ , 0.8 Pa/ ⁇ , 0.85 Pa/ ⁇ , 0.9 Pa/ ⁇ , 0.95 Pa/ ⁇ , 1 Pa/ ⁇ , 2 Pa/ ⁇ , 3 Pa/ ⁇ m, 4 Pa/ ⁇ m, 5 Pa/ ⁇ , 10 Pa/ ⁇ m, or more.
  • the pressure gradient may be expressed in other units, for example in mbar.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet from 50 mbar to 500 mbar, for example 60 mbar, 80 mbar, 100 mbar, 150 mbar, 200 mbar, 250 mbar, 300 mbar, 350 mbar, 400 mbar, 450 mbar, or 500 mbar.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of about 60 mbar, 80 mbar, 100 mbar, 150 mbar, 200 mbar, 250 mbar, 300 mbar, 350 mbar, 400 mbar, 450 mbar, or 500 mbar.
  • the device may be designed and configured to create a pressure gradient from a channel inlet to a channel outlet in a range of 0.05 Pa/ ⁇ to 0.1 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.3 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.5 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.8 Pa/ ⁇ , 0.5 Pa/ ⁇ to 1 Pa/ ⁇ , 1 Pa/ ⁇ to 10 Pa/ ⁇ , for example.
  • the pressure gradient may be linear or non-linear.
  • the device may be designed and configured to create a pressure (gauge pressure) in the channel of up to 5 Pa, 10 Pa, 20 Pa, 30 Pa, 40 Pa, 50 Pa, 100 Pa, 200 Pa, 300 Pa, 400 Pa, 500 Pa, 600 Pa, 700 Pa, 800 Pa, 900 Pa, 1 kPa, 2 kPa, 5 kPa, 10 kPa or more.
  • the device may be designed and configured to create a pressure (gauge pressure) in the channel in a range of 50 Pa to 200 Pa, 100 Pa to 500 Pa, 100 Pa to 800 Pa, 100 Pa to 1 kPa, 500 Pa to 5 kPa, or 500 Pa to 10 kPa.
  • the device may be designed and configured to create an average fluid velocity within the channel of up to 1 ⁇ /s, 2 ⁇ /s, 5 ⁇ /s, 10 ⁇ /s, 20 ⁇ /s, 50 ⁇ /s, 100 ⁇ /s, or more.
  • the device may be designed and configured to create an average fluid velocity within the channel in a range of 1 ⁇ /8 to 5 ⁇ /s, 1 ⁇ /s to 10 ⁇ /s, 1 ⁇ /s to 20 ⁇ /s, 1 ⁇ /s to 50 ⁇ /s, 10 ⁇ /s to 100 ⁇ /s, or 10 ⁇ /s to 200 ⁇ /s, for example.
  • the device may be configured to pulse a fluid through the channel of any of the microfluidic devices.
  • pulsing the fluid allows the gas content of the fluid within the chamber (e.g., under conditions of hypoxia) to be maintained under high pressure or flow rates.
  • the gas content of the fluid within the chamber e.g., under conditions of hypoxia
  • the device is configured so that the pulsing may be controlled.
  • the amplitude, duty cycle, and period, duration of the pulse may be controlled.
  • the device may be designed and configured to have a channel cross-sectional area, perpendicular to the flow direction, of 0.1 mm 2 , 0.5 mm 2 , 10 mm 2 , 20 mm 2 , 30 mm 2 , 40 mm 2 , 50 mm 2 , 60 mm 2 , 70 mm 2 , 80 mm 2 , 90 mm 2 , 100 mm 2 , 150 mm 2 , 200 mm 2 , 300 mm 2 , 400 mm 2 , 500 mm 2 , 600 mm 2 , 700 mm 2 , 800 mm 2 , 900 mm 2 , or 1000 mm 2 .
  • the device may be designed and configured to produce any of a variety of different shear rates (e.g., up to 1000 s "1 ).
  • the device may be designed and configured to produce a shear rate in a range of 10 s "1 to 50 s “1 , 10 s “1 to 100 s “1 , 50 s “1 to 200 s “1 , 100 s “1 to 200 s “1 , 100 s “1 to 500 s “1 , 50 s “1 to 500 s “1 , or 50 s "1 or 1000 s "1 .
  • the device described herein further contains a heat transfer element, which can maintain the fluid at a predetermined temperature (e.g. , a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C).
  • a predetermined temperature e.g. , a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C).
  • non-microfluidic devices are provided.
  • the non-microfluidic device is AFM, optical tweezers, micropipette, magnetic twisting cytometer, cytoindenter, microindenter, nanoindenter, microplate stretcher, microfabricated post array detector, micropipette aspirator, substrate stretcher, shear flow detector, diffraction phase microscope, or tomographic phase microscope.
  • the microfluidic device 1 includes a microfluidic channel 10 comprising at least one inlet 5 and one outlet 15, each connected to a reservoir (inlet reservoir 20, outlet reservoir 140).
  • a microfluidic device is shown in Fig. 1.
  • Other exemplary schematic representations of the device design can be found in FIGs. 11-16 and 20.
  • the microfluidic channel comprises a first wall 110 wherein at least a portion of this wall is coated with at least one protein and a second wall 120 adjacent to the microfluidic channel, wherein at least a portion of the second wall 120 comprises a gas permeable membrane or film 130.
  • the device may include a pump 25 that perfuses fluid from a reservoir 20.
  • the reservoir 20 further comprises a filter.
  • a single syringe 150 is used as a source of back pressure (FIG. 15).
  • a flow sensor 80 and/or pressure regulator 90 may connect the reservoir 20 to the microfluidic channel 10.
  • the device can accommodate a microscope 30 arranged to permit observation within the microfluidic channel 10.
  • the device may further comprise a heat transfer element 40 to regulate temperature within the microfluidic channel.
  • the device may comprise a gas channel 50, which contacts at least a portion of the gas permeable membrane or film 130.
  • a gas flow sensor or regulator 70 may connect the gas channel 50 to the gas source 60.
  • the device system may also comprise a computer 100 in electronic communication with the microscope 30, heat transfer element 40, gas flow sensor or regulator 70, the pressure regulator 90, the fluid flow sensor 80, and/or the pump 25.
  • Described herein are devices and methods for assessing cell properties under controlled gas environments. Accordingly, a microfluidics-based model was developed to quantify cell-level processes modulating the pathophysiology of disease ⁇ e.g., sickle cell disease (SCD), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria and anemia).
  • SCD sickle cell disease
  • spherocytosis ovalocytosis
  • alpha thalassemia beta thalassemia
  • delta thalassemia malaria and anemia
  • This in vitro model enabled quantitative investigations of the kinetics of cell processes and transformations such as cell sickling, unsickling and cell rheology. Examples of the use of the devices of the invention are included in the Examples below.
  • the methods may be carried out in a high throughput manner.
  • methods are provided that are useful for diagnosing, assessing, characterizing, evaluating, and/or predicting disease based on transit characteristics of cells, e.g. , red blood cells, platelets, cancer cells, and tissues, e.g. , blood in microfluidic devices.
  • the present disclosure includes a high throughput method of measuring a morphological and/or mechanical property of an individual cell under controlled gas conditions including: flowing a fluid comprising a plurality of cells through a channel comprising a wall, wherein at least a portion of the wall is coated with at least one protein, obtaining at least one measurement of a cell in the fluid; and regulating a level of gas in the fluid.
  • the protein may be any protein provided herein.
  • the method measures morphological properties, including cell shape, such as round, disk shaped, biconcave, oblong, or sickle shaped.
  • the method may be used to assess normal or abnormal cell textures, including smooth, coarse, or spiky textures.
  • the method may measure a fraction of cells with an abnormal shape or texture by examining the delay time of recovery from an abnormal shape change.
  • the cell shape change is between sickling and unsickling.
  • the cells are bound to the portion of the wall coated with at least one protein, and in other embodiments, the cells are not bound to the portion of the wall coated with at least one protein.
  • the measurement may be used to determine a proportion of cells having an abnormal shape and/or texture at a certain temperature, flow rate and/or gas concentration.
  • the property may be a mechanical property, such as adhesiveness.
  • the method includes measuring the adhesiveness of the sample to the portion of the wall coated with at least one protein. Cells may slide, roll, or tumble along the portion of the wall that is coated with at least on protein, and the device may measure the number and/or fraction of cells that bind to the portion of the wall coated with at least one protein.
  • measurements contemplated include: the rate at which the cells bind to the portion of the wall coated with at least one protein, the speed that the cells move along the portion of the wall coated with at least one protein, and the number and/or fraction of cells that detach from a portion of the wall that is coated with at least one protein.
  • Each measurement can be conducted at a certain temperature, flow rate, and/or gas concentration and average rates, speeds, and distances can be determined from the measurements.
  • the measurement may be used to determine the number of and/or fraction of cells that detach from the wall that is coated with at least one protein at a certain temperature, flow rate, and/or gas concentration.
  • the method also may include contacting the cell with the wall that is coated with at least one protein while fluid flows through the channel.
  • the flow of the fluid through the channel may be stopped.
  • the cell may contact the wall that is coated with at least one protein when the fluid is stopped, and the fluid may pass through the channel after it had been stopped.
  • the cell may be bound to a fixed position on the portion of the wall coated with at least one protein, where its deformability may be measured.
  • Deformability can be measured as the distance that a cell stretches or the ratio of the length versus width of a cell, The
  • a cell deform measurement can be used to determine the amount, or average amount, a cell deforms under certain temperatures, flow rates, and/or gas concentrations.
  • additional indices may be used in the assessment of cell deformability. For example, cell location, cell adhesion sites, cell area (projected), and cell shape parameters, such as elongation ratio, maximum Feret diameter ratio, and Convex Hull etc.
  • additional indices for assessing cell deformability would be apparent to the skilled artisan and are within the scope of this disclosure.
  • Some aspects of the disclosure provide devices and methods for measuring the fragility of one or more objects (e.g., cells).
  • the fragility of cells e.g., red blood cells
  • the mechanical fragility of one or more cells is determined.
  • the mechanical fragility index of one or more cells is determined.
  • the mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion.
  • compressive stress force/unit area
  • when the cells are compressed with the membrane their surface area increases (2) applied load vs time curve; (3) applied compressive stress vs time curve; and (4) applied compressive force vs area increase.
  • the cells are from a subject.
  • Such cells can be obtained from a blood sample, and may comprise red blood cells, white blood cells, stem cells or epithelial cells.
  • the cells comprise one or more tumor cells.
  • the gas channel comprises at least one inlet and/or at least one outlet.
  • a gas or gas mixture may be supplied to the inlet of the gas channel from one or more tanks containing the gas or gas mixture.
  • the gas supplied to the gas channel is oxygen, nitrogen, carbon dioxide, nitric oxide, carbon monoxide, nitrous oxide, nitrogen dioxide, methane, or any combination thereof.
  • the gas supplied to the gas channel contains oxygen. In some embodiments the gas supplied contains between 1% and 100 % oxygen.
  • the gas supplied contains from 1% to 2%, from 1% to 3%, from 1% to 5%, from 1% to 10%, from 1% to 20%, from 1% to 40%, from 1% to 60%, from 1% to 80%, from 1% to 100%, from 5% to 10%, from 5% to 20%, from 5% to 40%, from 5% to 60%, from 5% to 80%, from 5% to 100%, from 20% to 40%, from 20% to 60%, from 20% to 80%, from 20% to 100%, from 40% to 60%, from 40% to 80%, from 40% to 100%, from 60% to 80%, from 60% to 100% or from 80% to 100% oxygen.
  • the gas supplied to the gas channel contains about 2%, about 5%, or about 20% oxygen.
  • the term “about,” or “approximately” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value).
  • the gas supplied to the gas channel contains about 20% oxygen, 5% carbon dioxide and about 75% nitrogen.
  • the gas supplied to the gas channel contains about 5% oxygen, 5% carbon dioxide and about 90% nitrogen. In yet other embodiments the gas supplied to the gas channel contains about 2% oxygen, 5% carbon dioxide and about 93% nitrogen.
  • the gas or gas mixture may be supplied to one or more inlets of one or more gas channels using any suitable means, such as tubing or hoses.
  • the level of gas in the fluid is regulated to be at a concentration of less than 5%. In other embodiments, the level of the gas in the fluid is regulated to be at a concentration from 5% to 10%, 5% to 15%, 5% to 20%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%, 40% to 45%, 40% to 50%, 40% to 55%, 40% to 60%, or to a level greater than 60%. In some embodiments, the level of gas in the fluid is regulated to be at a concentration of about 5%, about 4%, about 3%, or about 2%.
  • the property is measured at two or more different gas concentrations.
  • the gas concentration may be increased and/or decreased, and the property may be measured as a function of time and as a function of gas concentration.
  • test agent may be obtained from the subject that has a material property (e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.) that is indicative of the condition or disease.
  • material property e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.
  • the condition or disease to be detected may be, for example, a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious mononucleosis, HIV, malaria, leishmaniasis, babesiosis, monoclonal gammopathy of undetermined significance or multiple myeloma.
  • a fetal cell condition such as a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious
  • hematological cancer examples include, but are not limited to, Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AILT), multiple myeloma, Waldenstrom macroglobulinemia, plasmacytoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B cell CLL, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL) and
  • the fluid comprising the cells is flowed at a predetermined flow rate.
  • the device may be designed and configured to create a flow rate in a range of about 0.01 ⁇ / ⁇ to 0.1 ⁇ / ⁇ , 0.1 ⁇ / ⁇ to 1 ⁇ / ⁇ , 0.1 ⁇ 7 ⁇ to 10 ⁇ / ⁇ , 0.1 ⁇ 7 ⁇ to 20 ⁇ / ⁇ , 0.1 ⁇ to 30 ⁇ 7 ⁇ , 0.1 to 40 ⁇ 7 ⁇ , 0.1 ⁇ 7 ⁇ to 50 ⁇ , 0.1 ⁇ 7 ⁇ to 60 ⁇ , 0.1 ⁇ to 70 ⁇ 7 ⁇ , 0.1 ⁇ 7 ⁇ to 80 ⁇ , 0.1 ⁇ 7 ⁇ to 90 ⁇ , or 0.1 ⁇ to 100 ⁇ 7 ⁇ .
  • the fluid comprising the cells is flowed at a predetermined pressure gradient.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet of 0.01 Pa/ ⁇ , 0.05 Pa/ ⁇ , 0.1 Pa/ ⁇ , 0.15 Pa/ ⁇ , 0.2 Pa/ ⁇ , 0.25 Pa/ ⁇ , 0.3 Pa/ ⁇ , 0.35 Pa/ ⁇ , 0.4 Pa/ ⁇ , 0.45 Pa/ ⁇ , 0.5 Pa/ ⁇ , 0.55 Pa/ ⁇ , 0.6 Pa/ ⁇ , 0.65 Pa/ ⁇ , 0.7 Pa/ ⁇ , 0.75 Pa/ ⁇ , 0.8 Pa/ ⁇ , 0.85 Pa/ ⁇ , 0.9 Pa/ ⁇ , 0.95 Pa/ ⁇ , 1 Pa/ ⁇ , 2 Pa/ ⁇ , 3 Pa/ ⁇ , 4 Pa/ ⁇ , 5 Pa/ ⁇ , 10 Pa/ ⁇ , or more.
  • the device may be designed and configured to create a pressure gradient from the channel inlet to the channel outlet in a range of 0.01 Pa/ ⁇ to 0.05 Pa/ ⁇ , 0.05 Pa/ ⁇ to 0.1 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.3 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.5 Pa/ ⁇ , 0.1 Pa/ ⁇ to 0.8 Pa/ ⁇ , 0.1 Pa/ ⁇ to 2 Pa/ ⁇ , 0.1 Pa/ ⁇ to 5 Pa/ ⁇ , 0.5 Pa/ ⁇ to 1 Pa/ ⁇ , 1 Pa/ ⁇ to 10 Pa/ ⁇ , for example.
  • the pressure gradient may be ceased, continuous, or not continuous.
  • the methods provide regulating the pressure of a gas or fluid in the gas channel of any of the devices provided herein. It should be appreciated that, as one example, pressurizing the gas channel may exert a force on the membrane of any of the devices provided herein to narrow the microfluidic channel, thereby squeezing one or more objects (e.g., cells) in the microfluidic channel.
  • the gas or fluid is supplied to the gas channel via one or more inlets or outlets of the gas channel.
  • the gas or fluid is at a pressure from 0.1 psi to 100 psi in the gas channel.
  • the gas or fluid is at a pressure from 0.1 psi to 1 psi, from 0.1 psi to 2 psi, from 0.1 psi to 5 psi, from 0.1 psi to 10 psi, from 0.1 psi to 20 psi, from 0.1 psi to 40 psi, from 0.1 psi to 60 psi, from 0.1 psi to 80 psi, from 1 psi to 2 psi, from 1 psi to 5 psi, from 1 psi to 10 psi, from 1 psi to 20 psi, from 1 psi to 40 psi, from 1 psi to 60 psi, from 1 psi to 80 psi, from 2 psi to 5 psi, from 2 psi to 10 psi, from 2 psi to 20 psi, from 1
  • the methods provide regulating the pressure of a gas or fluid in the gas channel of any of the devices provided herein.
  • the pressure of the gas or fluid is increased.
  • the pressure of the gas or fluid is decreased.
  • the pressure of the gas or fluid is increased at a rate from 1 psi/min to 100 psi/min.
  • the pressure of the gas or fluid is increased at a rate from 1 psi/min to 5 psi/min, from 1 psi/min to 10 psi/min, from 1 psi/min to 15 psi/min, from 1 psi/min to 20 psi/min, from 1 psi/min to 40 psi/min, from 1 psi/min to 60 psi/min, from 1 psi/min to 80 psi/min, from 5 psi/min to 10 psi/min, from 5 psi/min to 15 psi/min, from 5 psi/min to 20 psi/min, from 5 psi/min to 40 psi/min, from 5 psi/min to 60 psi/min, from 5 psi/min to 80 psi/min, from 10 psi/min to 15 psi/min,
  • the pressure of the gas or fluid is decreased at a rate from 1 psi/min to 100 psi/min. In some embodiments, the pressure of the gas or fluid is decreased at a rate from 1 psi/min to 5 psi/min, from 1 psi/min to 10 psi/min, from 1 psi/min to 15 psi/min, from 1 psi/min to 20 psi/min, from 1 psi/min to 40 psi/min, from 1 psi/min to 60 psi/min, from 1 psi/min to 80 psi/min, from 5 psi/min to 10 psi/min, from 5 psi/min to 15 psi/min, from 5 psi/min to 20 psi/min, from 5 psi/min to 40 psi/min, from 5 psi/min to 60 psi/min.
  • the device may be designed and configured to measure the designated property after one or more reoxygenation (ReOxy) and/or deoxygenation (DeOxy) cycles.
  • ReOxy reoxygenation
  • DeOxy deoxygenation
  • Methods are provided herein for evaluating, characterizing, and/or assessing mechanical, morphological, kinetic, rheological or hematological properties of cells under controlled gas conditions.
  • methods are provided for measuring, evaluating and/or characterizing dynamic mechanical responses of biological cells, e.g. , red blood cells, white blood cells,
  • the methods typically involve obtaining measurements of cell deformability, cell velocity and cell shape. Measurements of cell deformability often involve an assessment of the transit time of one or more cells through one or more constrictions within a fluid channel of a microfluidic device, or an assessment of another parameter indicative of a resistance to deformation. In further aspects, methods are provided that are useful for measuring changes in cell properties or characteristics in response to changes in the concentration of one or more gasses. As one example, the transit characteristics of a red blood cell through one or more constrictions of a microfluidic device are measured at high oxygen content (e.g., 20% oxygen) and low oxygen content (e.g., 2% oxygen).
  • high oxygen content e.g. 20% oxygen
  • low oxygen content e.g., 2% oxygen
  • Some aspects of the disclosure relate to determining cell properties in response to repetitive or cyclical changes in the concentration of one or more gases (e.g., alternating between relatively high and low concentrations of a gas in a fluid).
  • methods are provided that are useful for measuring changes in cell properties or characteristics in response to one or more cycles of a gas concentration.
  • one or more changes in cell properties or characteristics are measured in response to one or more cycles of an oxygen, a nitrogen, a carbon dioxide, a carbon monoxide, a nitric oxide, a nitrous oxide, a nitrogen dioxide, or a methane gas concentration.
  • cell properties or characteristics may be determined in response to one or more cycles of any suitable gas concentration.
  • one or more changes in cell properties or characteristics are measured in response to one or more changes in oxygen concentration.
  • a cycle of a gas concentration refers to a change from a relatively high gas concentration (e.g. , 20% oxygen) to a relatively low gas concentration (e.g. , 2% oxygen) and back to a relatively high gas concentration.
  • a cycle of a gas concentration also refers to a change from a relatively low gas concentration (e.g. , 2% oxygen) to a relatively high gas concentration (e.g. , 20% oxygen) and back to a relatively low gas concentration.
  • a cycle of a gas concentration refers to a change from a relatively high oxygen concentration to a relatively low oxygen concentration and back to a relatively high oxygen concentration, referred to herein as a deoxygenation (DeOxy) cycle.
  • DeOxy deoxygenation
  • a cycle of a gas concentration refers to a change from a relatively low oxygen concentration to a relatively high oxygen concentration and back to a relatively low oxygen concentration, referred to herein as a reoxygenation (ReOxy) cycle.
  • a change from a relatively high gas concentration e.g.
  • a change from a relatively low gas concentration (e.g. , of oxygen) to a relatively low gas concentration refers to a decrease in gas concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or at least 100% in a gas or fluid.
  • concentration refers to an increase in gas concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% in a gas or fluid.
  • Some aspects of the disclosure relate to determining cell properties in response to one or more cycles of a gas concentration.
  • one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000 cycles of a gas concentration.
  • one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000
  • deoxygenation (DeOxy) cycles In some embodiments, one or more cell properties are determined after being exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or at least 1000 reoxygenation (ReOxy) cycles.
  • the cycles of a gas concentration provided herein may be performed for any suitable duration of time, which may depend on, among other factors, the intended purpose or the nature of the cells (e.g., healthy or diseased cells). In some embodiments, the duration of two or more consecutive cycles are the same.
  • two or more consecutive cycles may be 360 seconds long.
  • the length of two or more consecutive cycles are different.
  • a first cycle may be 360 seconds long and a second cycle may be 400 seconds long.
  • the length of two or more consecutive cycles may be increased.
  • the length of two or more consecutive cycles may be decreased.
  • a cycle is from 5seconds (5s) to 1 hour (lh) long.
  • a cycle may be any suitable duration and any exemplary cycle durations provided herein are not intended to be limiting.
  • a cycle is from 5s to 20s, from 5s to 100s, from 5s to 200s, from 5s to 400s, from 5s to 600s, from 5s to 1000s, from 5s to 20min, from 5s to 30min, from 5s to 40min, from 5s to 50min, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 400s to 1000s, from 400s to 20min, from 400s to 30min, from 400s to 40min, from 400s to 50min from 200s to lh, from 400s to 600s, from 400s to 1000
  • the concentration, within a cycle may vary.
  • the duration of time that a gas is at a relatively low concentration ,within a cycle may vary.
  • the duration of time at which a gas is at a relatively high concentration and the duration of time at which a gas is at a relatively low concentration, within a cycle is the same.
  • the duration of time at which a gas is at a relatively high concentration and the duration of time at which a gas is at a relatively low concentration, within a cycle is different.
  • the duration of time at which a gas is at a relatively high concentration is greater than the duration of time at which a gas is at a relatively low concentration, within a cycle.
  • the duration of time at which a gas is at a relatively high concentration is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, or at least 5000% greater than the duration of time at which a gas is at a relatively low concentration, within a cycle.
  • the duration of time at which a gas is at a relatively high concentration, within a cycle is from Is to 20s, from Is to 100s, from Is to 200s, from Is to 400s, from Is to 600s, from Is to 1000s, from Is to 20min, from Is to 30min, from Is to 40min, from Is to 50min, from Is to lh, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min,
  • the duration of time at which a gas is at a relatively high concentration is less than the duration of time at which a gas is at a relatively low concentration, within a cycle.
  • the duration of time at which a gas is at a relatively high concentration is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, or at least 5000% less than the duration of time at which a gas is at a relatively low concentration, within a cycle.
  • the duration of time at which a gas is at a relatively low concentration, within a cycle is from Is to 20s, from Is to 100s, from Is to 200s, from Is to 400s, from Is to 600s, from Is to 1000s, from Is to 20min, from Is to 30min, from Is to 40min, from Is to 50min, from Is to lh, from 100s to 200s, from 100s to 400s, from 100s to 600s, from 100s to 1000s, from 100s to 20min, from 100s to 30min, from 100s to 40min, from 100s to 50min from 100s to lh, from 200s to 400s, from 200s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min, from 200s to 50min from 200s to lh, from 400s to 600s, from 2s to 1000s, from 200s to 20min, from 200s to 30min, from 200s to 40min,
  • the device described herein further contains a heat transfer element, which can maintain the fluid at a predetermined temperature (e.g., a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C).
  • a predetermined temperature e.g., a physiologically relevant temperature (e.g., a temperature that would be found in vivo in a healthy or diseased subject or one with a particular condition as provided herein), such as 30 °C to 45 °C, preferably 37 °C, 40 °C or 41 °C).
  • the fluid comprising the cells is flowed at a predetermined temperature, preferably a physiological temperature.
  • the method includes a protein, such as a cell surface protein or extracellular matrix (ECM) protein.
  • the cell surface protein may be a cell adhesion molecule, such as an integrin, a cadherin, a selectin, a receptor tyrosine kinase, or a G-protein coupled receptor.
  • the ECM protein in some embodiments, may be collagen, elastin, laminin, or fibronectin.
  • the protein may comprise an antibody.
  • the fluid comprising a plurality of cells is flowed through the device of the present disclosure.
  • Methods are also provided for testing candidate therapeutic agents for treating a condition or disease in a subject.
  • the methods typically involve: (a) perfusing a fluid comprising one or more cells from the subject through the any of the microfluidic devices, described herein, where the level of one or more gases is regulated, (b) administering one or more compounds to the fluid of (a), or wherein the fluid comprises the one or more compounds; (c) determining a property of one or more of the cells; and (d) comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject.
  • the two or more compounds may be administered to the fluid sequentially or simultaneously.
  • An effective therapeutic agent may be identified based on the comparison in (d).
  • the cells may be from a subject, and the effective therapeutic agent may be administered to the subject.
  • the compounds may be from a library of compounds, and in some
  • embodiments are candidate therapeutic agents.
  • this method may be used to identify candidate therapeutic agents that improve blood flow in subjects with circulation problems such as sickle cell disease, leg ulcers, pain from diabetic neuropathy, eye and ear disorders, and altitude sickness. Similarly for subjects with aggregation or clotting disorders of cells or insufficient delivery of essential chemicals such as oxygen to the brain in subjects with strokes from blood clots.
  • a method for analyzing, diagnosing, detecting, or determining the severity of a condition or disease in a subject includes (a) perfusing a fluid comprising one or more cells from the subject through the any of the microfluidic devices, described herein, where the level of one or more gases is regulated, (b) determining a property of one or more of the cells; and (c) comparing the property to an appropriate standard, wherein the results of the comparison are indicative of the status of the condition or disease in the subject.
  • the results of the comparison are typically indicative of the status of the condition or disease in the subject.
  • the device is a microfluidic channel with a gas permeable membrane or film.
  • the device is a microfluidic channel with a gas permeable membrane or film and a gas channel.
  • the deformable object in this example, typically has a mechanical property, the value of which is indicative of the presence of sickle cell disease.
  • the method is used to determine the severity of the disease based on differences in mechanical properties.
  • the method is used to predict the likelihood that a subject will undergo vaso-occlusion crisis based on differences in mechanical properties. In such methods, the methods may be performed under different regulated gas conditions.
  • the property may be adhesiveness and/or detachment.
  • certain methods of the invention provide for measurement of cytoadhesive properties of a cell population, in combination with or separate from measurement of the deformability of the cell population. The combination of determining cytoadhesive properties and the
  • deformative properties of a cell population may be used to generate a "Health Signature" that comprises an array of properties that can be tracked in a subject over a period of time.
  • a Health Signature may facilitate effective monitoring of a subject's health over time. Such monitoring may lead to an early detection of potential acute or chronic infection, or other disease, disorder, fitness, or condition.
  • knowledge of the overall rheology of a material, along with either the deformative or cytoadhesive property of a cell allows the determination of the other property.
  • An "appropriate standard” is a parameter, value or level indicative of a known outcome, status or result (e.g. , a known disease or condition status).
  • An appropriate standard can be determined (e.g. , determined in parallel with a test measurement) or can be preexisting (e.g. , a historical value, etc.).
  • the parameter, value or level may be, for example, a transit characteristic (e.g. , transit time), a value representative of a mechanical property, a value representative of a rheological property, etc.
  • an appropriate standard may be the transit characteristic of a test agent obtained from a subject known to have a disease, or a subject identified as being disease-free.
  • a lack of a difference between the transit characteristic and the appropriate standard may be indicative of a subject having a disease or condition.
  • the presence of a difference between the transit characteristic and the appropriate standard may be indicative of a subject having a disease or condition.
  • the appropriate standard can be a mechanical property or rheological property of a cell obtained from a subject who is identified as not having the condition or disease or can be a mechanical property or rheological property of a cell obtained from a subject who is identified as having the condition or disease.
  • the magnitude of a difference between a parameter, level or value and an appropriate standard that is indicative of known outcome, status or result may vary. For example, a significant difference that indicates a known outcome, status or result may be detected when the level of a parameter, level or value is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000% higher, or lower, than the appropriate standard.
  • a significant difference may be detected when a parameter, level or value is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or more higher, or lower, than the level of the appropriate standard.
  • Significant differences may be identified by using an appropriate statistical test. Tests for statistical significance are well known in the art and are exemplified in Applied Statistics for Engineers and Principles by Petruccelli, Chen and Nandram Reprint Ed. Prentice Hall (1999).
  • test agent may be obtained from the subject that has a material property (e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.) that is indicative of the condition or disease.
  • material property e.g. , deformability, shear modulus, viscosity, Young's modulus, etc.
  • the condition or disease to be detected may be, for example, a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious mononucleosis, HIV, malaria, leishmaniasis, babesiosis, monoclonal gammopathy of undetermined significance or multiple myeloma.
  • a fetal cell condition such as a fetal cell condition, HPV infection, or a hematological disorder, such as sickle cell disease, sickle cell trait (SCT), spherocytosis, ovalocytosis, alpha thalassemia, beta thalassemia, delta thalassemia, malaria, anemia, diabetes, leukemia, hematological cancer, infectious
  • hematological cancer examples include, but are not limited to, Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, anaplastic large cell lymphoma, splenic marginal zone lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AILT), multiple myeloma, Waldenstrom macroglobulinemia, plasmacytoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B cell CLL, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL) and
  • the appropriate standard is the value of a transit characteristic for a test agent at a regulated gas concentration that has been contacted with a control therapeutic agent (e.g., hydroxyurea or 5 -hydroxy methy If urfural).
  • a control therapeutic agent e.g., hydroxyurea or 5 -hydroxy methy If urfural.
  • a control therapeutic agent is a molecule that has a known effect on deformability of a test agent and that is effective for treating the condition or disease.
  • a characteristic of a candidate therapeutic agent with that of a control therapeutic agent provides a basis for identifying candidate therapeutic agents that are likely to be useful for treating the disease or condition. For example, a candidate therapeutic agent that results in the same or a similar value for a particular transit characteristic as that of a control therapeutic agent that is known to be effective for treating the disease or condition is likely to be an agent that is also effective for treating the disease or condition.
  • the therapeutic agent or candidate therapeutic agent is a small molecule or pharmaceutical agent.
  • Small molecule refers to organic compounds, whether naturally- occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Small molecules are typically not polymers with repeating units. In certain embodiments, a small molecule has a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the polymer is less than about 1000 g/mol. Also, small molecules typically have multiple carbon-carbon bonds and may have multiple stereocenters and functional groups.
  • “Pharmaceutical agent,” also referred to as a “drug,” is used herein to refer to an agent that is administered to a subject to treat a disease, disorder, or other clinically recognized condition that is harmful to the subject, or for prophylactic purposes, and has a clinically significant effect on the body to treat or prevent the disease, disorder, or condition.
  • Therapeutic agents include, without limitation, agents listed in the United States
  • the methods herein also provide for monitoring and/or determining the effectiveness of a therapeutic agent.
  • One method for monitoring the effectiveness of a therapeutic agent for treating a disease or condition in a subject includes (a) perfusing a fluid comprising one or more cells from the subject through the microfluidic device described above; (b) determining a property of one or more of the cells; (c) treating the subject with the therapeutic agent; and (d) repeating steps (a) and (b) at least once wherein a difference in the property of one or more cells is indicative of the effectiveness of the therapeutic agent.
  • Determining the effectiveness of a therapeutic includes (a) obtaining a biological sample from a subject comprising a cell; (b) perfusing a fluid comprising one or more cells from the subject through the microfluidic device described above; (c) determining a property of one or more of the cells; (d) contacting the biological sample comprising a cell with the therapeutic; (e) perfusing a fluid comprising the product of (d) through the microfluidic device; (f) determining a property of one or more of the cells from (e); and (g) comparing the property of one or more cells from (c) with the property of one or more cells from (f), wherein the results of the comparison are indicative of the effectiveness of the therapeutic.
  • Such methods may be used for a therapeutic to treat sickle cell disease, for example.
  • the therapeutic is hydroxyurea (HU) or 5-hydroxymethylfurfural (Aes-103).
  • a method includes (a) perfusing a fluid comprising one or more blood cells through a microfluidic described above, wherein the fluid has a first level of gas; (b) determining a property of one or more of the cells from (a); (c) perfusing a fluid comprising one or more cells through the microfluidic device described above; wherein the fluid has a second level of gas that is different from the first level; (d) determining a property of one or more of the cells from (c); and (e) quantifying the cell morphological kinetics of the cells from (b) and (d).
  • the method may be used to examine cell sickling and/or unsickling kinetics, such as cell sickling in response to low oxygen concentrations.
  • the methods provided also allow for the real-time observation of reoxygenation induced cell shape recovery. For example the transition of sickled cells into normal disk shaped red blood cells.
  • a morphological characteristic such as a cell shape change (e.g., sickling, a sphericity change, and aspect ratio change or a texture change), of one or more cells is measured at one or more gas concentrations.
  • a morphological characteristic of one or more red blood cells from a subject is measured at a low oxygen concentration (e.g., 2% oxygen).
  • Another transient characteristic of one or more cells can be measured.
  • the measurements of cell morphology of the cells may be used to determine the fraction of abnormally shaped (e.g., sickled) cells.
  • methods provided herein allow for the real-time observation of hypoxia- induced changes in cell morphology. For example, cell sickling in response to low oxygen concentrations.
  • the methods provided also allow for the real-time observation of reoxygenation induced cell shape recovery. For example the transition of sickled cells, cells with a rough texture, or spiky cells into normal disk shaped red blood cells.
  • the methods, described herein allow for the simultaneous measurement of cell shape changes over time and cell transit characteristics in response to changes in gas concentration, for example, cell sickling delay time and sickled fraction can be simultaneously measured in real-time in response to decreased oxygen concentration.
  • the methods, described herein, may be used to determine the fraction of obstructed cells, the fraction of cells with an abnormal shape and/or texture, the capillary obstruction ratio, the delay time of an abnormal cell shape change, and/or the delay time of recovering from an abnormal cell shape change.
  • Example 1 Microfluidic-based multiplex Cell Assay for Drug Compound Testing
  • the optically transparent microfluidic-based polymeric platform utilizes a combination of geometric, physical, chemical and biological means to quantify cell biophysical properties in vitro to mimic the in vivo microenvironment.
  • Effective integration of the pathological processes within a single system offers a) the quantitative investigation of microvascular occlusion, thrombosis and other hematologic diseases and b) In silico cell modeling development.
  • the platform may be used as a vehicle for both drug testing experimentation and for the manifestation of underlying microvascular-related mechanisms.
  • microenvironment of the cells is mimicked through precise shear stress modulation imposed by liquid flow profile, ambient gas partial pressure modulation
  • osmolality control constant or modulated osmolality
  • the following assays can be performed sequentially with "on-demand" cell microenvironment modulation: a) identification and classification of the cell shape/type population, b) cell (specific binding) attachment assay; and/or actively control the rectangular microchannel hydraulic diameter geometry by manipulation of free standing polymeric membranes (% degree of constriction), c) adherent cell deformability cytometer d) cell detachment.
  • the assays may all be performed under hypoxia and normoxia conditions:
  • A) Identification and classification of the cell shape/type population Image processing.
  • cytometry Application and release of a shear stress range to effectively measure the cell shear modulus and viscosity via a dynamic shape recovery process; under hypoxia and normoxia conditions. (Figs. 8A-8B, Fig. 9, Figs. 10A-10B, and Table 1) Tethering formation and breakup as a function of shear rate and hypoxia (Fig. 5).
  • Cell detachment assay Suitable laminar flow profile (pulsatile, continuous, sinus etc.) to modulate the shear stress on the adherent cells and quantify detachment over shear rate range, protein type; under hypoxia and normoxia conditions.
  • Figs. 4A-4B, Figs. 6A-6B Cell (specific binding ) attachment assay
  • Oxygenated and deoxygenated conditions were controlled using gas mixtures as described above prior to initiation of attachment assay. Attachment was quantified as percentage of cells remaining attached after low flow washing per mm . Multiple fields of view were observed.
  • Static and dynamic attachment was studied using functionalized microfluidic devices.
  • static attachment assays cell suspensions were loaded into the fluid channel of BSA passivated protein functionalized devices. After loading, cells were incubated at 37°C without flow for 10-15 min. Following incubation, very low flow (0.1-1 ⁇ /min) was introduced for 15 s to quantify how many cells had indeed attached.
  • dynamic attachment assays a similar protocol was followed. However, instead of static incubation for 10-15 min at 37°C, cells were perfused continuously at very low flow rates (0.1-1 ⁇ /min), to simulate the vasoocclusion microenvironment.
  • a detachment assay was conducted following the static or dynamic adhesion incubation period described above. Using the Elveflow flow sensor to provide real-time information on flow rates, the flow rate was increased for specified durations until all cells had detached. The flow rates at which cells detached indicated the strength of the adhesive bond between the cell and the functionalized protein. Detachment was quantified as percentage of cells detached after specified flow rate per mm . Multiple fields of view were observed.
  • a microfluidic device was designed with comparable geometry to post-capillary venules, the site often associated with vasoocclusions in the sickle cell patients.
  • Devices included cell channels with 15 ⁇ height, 1.3 mm width and 3 mm length dimensions. This width and length were selected to enable imaging of higher number of cells and prevent entrance effects, respectively. The channels were casted in the silicone elastomer
  • PDMS polydimethylsiloxane
  • the cell channel was functionalized (e.g., coated ) with proteins known to be relevant for adherent sickle RBCs.
  • the extra-cellular matrix proteins fibronectin and laminin were chosen as both have been well characterized in promoting sickle RBC adhesion to the endothelium.
  • Fibronectin binds to sickle RBCs through ⁇ 4 ⁇ 1 integrins while laminin interacts with the basal cell adhesion molecule/Lutheran protein (B-CAM/LU) receptor 5 ' 6 .
  • fluorescently tagged versions of these proteins were selected to aid in imaging and measurement of surface concentration prior to and during the course of the studies.
  • APTMS gas vapor deposition was used followed by protein
  • the heterogeneity of the sickle RBC population in terms of morphology and density add a level of complexity to the events leading up to vaso-occlusion. Indeed, several studies have demonstrated higher levels of adhesion in lighter density and irregularly shaped discoid cells and lower adhesion for high density irreversibly sickled cells to endothelial cells 7—10.
  • a low flow adhesion scheme under oxygenated conditions was utilized in the fibronectin (50 ⁇ g/ml) functionalized microfluidic devices to study the role density played in adhesion. Shear rate may be calculated and related to a physiological flow rate.
  • a density gradient fractionation technique was used, which has been previously published.
  • cells in fractions exhibited various behaviors prior to attachment.
  • interleukin-6 has been shown to induce greater amounts of fibronectin synthesis by monocytes and hepatocytes 5 .
  • Increased laminin exposure has also been shown under high concentrations of interleukin- 1 and tumor necrosis factor due to endothelial cell retraction 6 .
  • inflammation is not uncommon in sickle cell disease patients.
  • delineating the role each protein plays in adhesion can help unravel the more complicated vasocclusive cascade.
  • a series of static adhesion studies under oxygenated conditions were performed.
  • the platform can characterize both the adhesive and mechanical properties of sickle RBCs, offering an added benefit.
  • Future elongation ratio and relaxation time analyses of larger number of adherent cells is necessary to understand deformability properties of cell types.
  • Deoxygenation conditions were pursued with the closed loop gravitational potential driven setup but were unable to maintain the deoxygenated microenvironment after ⁇ 9ul/min flow rate. For this reason, an alternate system design was pursued.
  • One way to address this issue is to pulse the fluid through the device, which allows the fluid within the device to maintain a deoxygenated microenvironment under high pressures or flow rates.
  • Characterizing the role adhesion plays in a deoxygenated microenvironment may help in understanding the vasoocclusive cascade of events and possibly aid in predicting when these painful crises occur.
  • the ability to precisely tune the microenvironment with respect to geometry, oxygen content, proteins present and fluid flow in an in vitro device offers tremendous potential for patient-specific therapeutic applications.
  • Examples of the types of cell specific adhesion dysfunction that may be detected by the methods, devices, and systems provided herein are numerous.
  • (C) deoxygenated sickle cells exhibit increased adhesion as compared to oxygenated sickle cells in both continuous flow and push/pull systems
  • (D) fibronectin exhibits stronger adhesive bonding to sickle cells than laminin at identical concentrations, evident through increased shear rate necessary for detachment.
  • EDTA ethylenediaminetetraacetic acid
  • pRBCs Packed red blood cells
  • D-PBS D-PBS
  • BSA bovine serum albumin
  • Sickle red blood cells were fractionated into four fractions of increasing density using methods described (Du PNAS 2015).
  • solutions of increasing density 1.081, 1.091, 1.100 and 1.111 g/ml
  • Optiprep density gradient medium Sigma Aldrich, St. Louis, MO
  • D-PBS D-PBS
  • 2.5 ml volumes of solutions were then layered such that the densest fraction (1.111 g/ml) was on the bottom of the gradient.
  • Washed pRBCs were resuspended in D-PBS to reach a 70-80% hematocrit and layered on top of the gradient. Cells were then centrifuged for 30 minutes at 821 g and 21°C. Cells between density fractions were removed carefully and washed twice for 5 minutes at 821 g and 21°C. Fractions were then resuspended in the RPMI solution described above.
  • Devices for adhesion study were fabricated using standard photolithography and polydimethylsiloxane (PDMS) methods.
  • the microfluidic platform consisted of a two layer device with a fluidic channel for cells and a gas channel to allow control of the amount of oxygen within the microenvironment.
  • Device design was similar to the cell sickling device as it has been described in Du et al. (Du PNAS 2015). Channels were separated by a 150 ⁇ thick PDMS gas permeable membrane. The amount of oxygen and its diffusion rate into the fluidic channel was regulated by the gas and gas pressure in the gas channel and thickness of the PDMS membrane.
  • hypoxic conditions were created by using a 2% 0 2 , 5% C0 2 , 93% N 2 gas mixture while normoxic conditions were mimicked using a 20% 0 2 , 5% C0 2 , 75% N 2 gas mixture.
  • Both fluidic and gas channels were 3 mm in length and 1.326 mm in width. The height of the fluidic and gas channel were 15 ⁇ and 100 ⁇ , respectively.
  • Standard photolithography techniques were used to create SU-8 masters on 6 in silicon wafers. To aid in removal of PDMS from masters, wafers were passivated using tridecafluoro-1,1,2,2- tetrahydrooctyl)-l-trichlorosilane for 2 h under vacuum.
  • a polymer to curing agent ratio of 10 to 1 (w/w) of Sylgard® 184 silicone elastomer (Dow Corning, Auburn, MI) was mixed, and poured onto wafers after all bubbles from mixture had been removed.
  • PDMS was cured for at least 2 h at 80°C before removal.
  • mixed PDMS was spincoated onto the wafer to attain a thickness of 150 ⁇ and then cured as described.
  • An inlet and outlet were created in both gas and cell channels using a 1.5 mm diameter biopsy punch, 1.2 mm away from the middle. Both channels were bonded to cleaned cover glasses using plasma treatment.
  • ATMS (3-Aminopropyl)- trimethoxysilane
  • Fibronectin was selected for adhesion studies as its presence on the vessel lining and promotion of sickle RBC binding to the endothelium via a4bl integrins have been well-documented (Kasschau et al Blood 1996).
  • Laminin is another ECM protein, known to bind to the basal cell adhesion molecule/Lutheran protein (B-CAM/LU) receptor which is overexpressed on sickle RBCs (Zen et al AJH 2004).
  • Gently washed devices were passivated with a 3% (w/v) BSA (EMD Millipore, Billerica, MA) in PBS solution over night at 4°C and used within 24-48 h. Washing and blocking of devices were performed with a low flow rate using a syringe pump to minimize disturbance to functionalized protein.
  • Microfluidic devices were imaged using Zeiss Axiovert 200 and Olympus 1X71 inverted microscopes.
  • a 414/446-nm band-bass filter (Semrock, Rochester, NY) was used.
  • a Hitachi HAL100 camera (Tarrytown, NY) and— were used for image acquisition for the Zeiss and Olympus microscopes, respectively. All testing was performed at 37°C using a heating incubator (ibidi heating system, ibidi USA, Madison, WI).
  • the height of liquid in columns connected to the device reservoirs via Tygon tubing (0.02 in inner diameter x 0.06 in outer diameter) was used to regulate the flow.
  • a system using pressurized air to control fluid flow was also used in specified experiments.
  • Hypoxic and normoxic conditions were mimicked using gas mixtures of 2% oxygen, 5% carbon dioxide, 93% nitrogen and 20% oxygen, 5% carbon dioxide, 75% nitrogen, respectively.
  • Gas mixtures were connected to the gas channel in microfluidic devices using Tygon tubing (0.02 in inner diameter x 0.06 in outer diameter). Gas mixtures then diffused through the PDMS permeable membrane to provide the desired environment to cell channel.
  • An Elveflow flow sensor (Paris, France) was connected in-line with the tubing controlling fluid flow and gas flow to provide a real-time measurement of flow rate and gas pressure and ensure consistency. Flow rates of 0.1-1 ⁇ /min were used for dynamic infusion studies and detachment rates up to 80 ⁇ /min were used for detachment studies.
  • Example 2 Microfluidic-based assay for testing mechanical fragility.
  • MF microfluidic mechanical fragility
  • MF mechanical fragility
  • MFI mechanical fragility index
  • Methods provided herein utilize a poly(dimethylsiloxane) (PDMS) thin elastomeric membrane in direct contact with the RBCs surface via compressive loading up to the point of cell lysing. This process is monitored and recorded (compression versus cell response) hence compressive loading-unloading curves can also reflect the sub-hemolytic RBC damage.
  • the assay can be designed to handle 20- 100 cells per test; however due to inherent advantages of microfluidic processability utilizing automated RBC positioning and fluidic multiplexing, it can be extended with further engineering development for hemolytic screening of 2,000- 10,000 cells in less than an hour.
  • devices described herein involve mass production- amenable technology to fabricate optically transparent Lab-on-a-Chip polymeric microfluidic devices that feature a flexible poly(dimethylsiloxane) (PDMS) permeable membrane; acting as a mechanical actuator for compressive loading-unloading.
  • PDMS poly(dimethylsiloxane)
  • This platform is advantageous due to, at least in part: ease of process ability (multilayer soft lithography), gas permeability, biocompatibility, optical transparency and low-cost processing for translational device fabrication. Consequently, the proposed platform uniquely utilizes a combination of geometric, physical, chemical and biological means to quantify ex vivo red blood cell fragility ⁇ i.e., susceptibility to hemolysis) at single-cell level via application of compressive load.
  • micro-hemolytic device is based on the deflection of a PDMS membrane that imposes compressive load in direct-contact with the RBCs.
  • the membrane is free-standing within a dual-layer microchannel construction.
  • a schematic representation of an exemplary microfluidic ⁇ e.g., micro-hemolytic) device is shown in
  • the dual-layer micro-hemolytic device comprises a "flow channel” in which there is flow of a cell ⁇ e.g., red blood cell (RBC)) suspension; and a "control microchannel,” which may also be referred to as a "gas channel”, for pressure regulation.
  • a cell e.g., red blood cell (RBC)
  • a control microchannel which may also be referred to as a "gas channel”
  • Both channels may be connected with in-line pressure and flow sensors (with feedback loop) for precise PDMS membrane deflection position under compressive loading.
  • pressurized gas or liquid can be delivered via a high precision pneumatic pump.
  • both pressure and vacuum control may be regulated in the control channel for precise membrane placement and initialization prior to each MF test; to minimize fabrication device dependent discrepancies.
  • Membrane deflection can be directly observed via the change in channel height filled with fluorescein solution. Fluorescence intensity change can be measured and correlated with the applied pressure under compressive loading/unloading (while
  • the RBC microenvironment and positioning within the micro-hemolytic device region of interest can be tuned independently by: precise RBC x-positioning imposed by the liquid flow profile, ambient gas partial pressure modulation (diffusion rate control), gas mixture, temperature and PDMS membrane geometry and elasticity.
  • the pneumatic pumps that control both the control and the flow channels are fully programmable and can be integrated with the microscope's motorized stage and camera for fully automatic operation, improving assay reproducibility and robustness.
  • the assay can be carried out sequentially as follows:
  • Cells may be lysed under varying gas conditions.
  • Figure 21 shows a RBC under the same experimental conditions as described for Figs. 18 and 19, but under hypoxic (DeOxy) conditions(gas mixture of 2% 0 2 , 5% C0 2 , 93% N 2 ).
  • Panels a-j show high speed imaging snapshots of RBC lysis over time (Fig. 21). Abrupt sickled RBC membrane rupture and consequent release of visible clusters of polymerized HbS and hemolysis products under steady state deoxygenation are shown.
  • Borovetz HS In vitro evaluation of hemolysis and sublethal blood trauma in a novel
  • the methods and devices provided herein provide the opportunity to study for the first time, simultaneous adhesion and sickle cell polymerization under hypoxia in cells, for
  • HbS polymer fiber growth outwards of the cell In very young cells (reticulocytes) there is apparent reorganization of the polymer content that grows even outwards of the cell and on the fibronectin (FN)-coated surface (reticulocyte in Fig. 25); polymer HbS polymer fibers growth can be monitored as well as apparent adherent contact area change (up to 15%) (Fig. 25)
  • reticulocytes In very young cells (reticulocytes) there is apparent reorganization of the polymer content that grows even outwards of the cell and on the fibronectin (FN)-coated surface (reticulocyte in Fig. 25); polymer HbS polymer fibers growth can be monitored as well as apparent adherent contact area change (up to 15%) (Fig. 25)
  • Cell membrane change at the contact line In light-density, highly-deformable cells, where cell membrane is evidently separated from the bulk of the intracellular polymerized content when cell membrane attaches to the surface cell contact line appears spiky (

Abstract

Selon certains aspects, l'invention se rapporte à des procédés et à des dispositifs à rendement élevé pour évaluer des propriétés mécaniques, morphologiques, cinétiques, rhéologiques ou hématologiques de cellules, telles que des cellules sanguines dans des conditions de fixation de cellules et des conditions gazeuses régulées. Dans certains aspects, l'invention concerne des procédés d'évaluation de l'adhésion cellulaire dans des conditions régulées, par exemple la concentration de gaz, la température, la force de cisaillement et la fixation aux protéines. Dans certains aspects, l'invention concerne des procédés et des dispositifs d'identification d'agents thérapeutiques. Selon certains aspects, l'invention se rapporte à des procédés et à des dispositifs pour diagnostiquer et/ou caractériser un état ou une maladie chez un sujet, par exemple, par mesure d'une propriété d'une cellule provenant du sujet dans des conditions contrôlées.
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WO2021247868A1 (fr) * 2020-06-03 2021-12-09 Case Western Reserve University Classification de cellules sanguines
WO2021249543A1 (fr) * 2020-06-12 2021-12-16 杭州准芯生物技术有限公司 Cuve de détection de liquide et système d'analyse de liquide

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US20020166585A1 (en) * 2000-11-06 2002-11-14 Nanostream, Inc. Microfluidic regulating device
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US20040072278A1 (en) * 2002-04-01 2004-04-15 Fluidigm Corporation Microfluidic particle-analysis systems
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Publication number Priority date Publication date Assignee Title
WO2021247868A1 (fr) * 2020-06-03 2021-12-09 Case Western Reserve University Classification de cellules sanguines
WO2021249543A1 (fr) * 2020-06-12 2021-12-16 杭州准芯生物技术有限公司 Cuve de détection de liquide et système d'analyse de liquide

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