# US 8,771,933 B2 · Continuous-flow deformability-based cell separation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,933 B2 - Document ID: US8771933B2 - Status: Granted - Kind: B2 - Application number: 12/899,197 - Filed: 2010-10-06 - Published: 2011-04-07 - Granted: 2014-07-08 - Earliest claimed: 2009-10-06 - Assignees: Massachusetts Institute of Technology - Applicants: Jongyoon Han; Hansen Bow; Patrick Abgrall - Inventors: Jongyoon Han; Hansen Bow; Patrick Abgrall - Web page: https://patentgrep.com/patent/US8771933B2 ## CPC - C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves - C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)} - C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)} - G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics} - G01N 33/5091: {for testing the pathological state of an organism} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract This invention provides methods utilizing a microfluidic device that can quickly and accurately discern differences in deformability between individual cells and sets of cells and continuously fractionate populations of cells based on their deformability. This information may be important in disease diagnosis and treatment efficacy monitoring. For example such a device may be able to determine the stage of malarial infection by using red blood cell deformability. Additionally, methods of the invention may be used as a tool to screen drugs that can make cells more flexible in diseases such as sickle cell anemia that causes sickle cell crises. The relatively low manufacturing and operation costs of methods of the invention enable this device to be used in resource-limited settings to diagnose and monitor disease. ## Claims ### Claim 1 A method of sorting a fluid mixture comprising a plurality of cells, said method comprising the steps of: a) loading the fluid mixture comprising a plurality of deformable and non-deformable cells in a microfluidic cell sorter comprising: i. a first substrate comprising plurality of parallel trenches, arranged at intervals, the trenches each have a contour of a pair of walls and a bottom; ii. a second substrate positioned parallel to said first substrate, such that a spacing is formed in the form of a plurality of shallow regions or slits between said first and said second substrates to allow deformable cells to pass through one trench to another, and wherein a supporting structure holds said second substrate suspended over said first substrate; iii. a sample inlet to said sorter; iv. a sample outlet from said sorter; b) applying a force field diagonally with respect to the length of said trenches, whereby applying said force field allows diagonal fluid flow with respect to the shallow regions or slits for separation of said plurality of cells, wherein said deformable cells deform while crossing from one trench to another through said shallow regions or slits, and said non-deformable cells slide along said shallow regions or slits in a direction parallel to said trenches resulting in a continuous sorting of said deformable and non-deformable cells, and ensuring that said spacing is smaller than the smallest dimension of the cells; and c) collecting separated cells obtained in (b) from said sample outlet. ### Claim 2 (depends on 1) The method of claim 1, wherein said separation is a result of the difference in cell deformability of different cells in said plurality of cells. ### Claim 3 (depends on 1) The method of claim 1, wherein said separation is a result of the difference in cell stiffness of different cells in said plurality of cells. ### Claim 4 (depends on 1) The method of claim 1, wherein a sub-population of cells is isolated. ### Claim 5 (depends on 4) The method of claim 4, wherein said sub-population of cells are cancer cells. ### Claim 6 (depends on 1) The method of claim 1, wherein cell separation rate is less than 0.1 seconds per cell. ### Claim 7 (depends on 1) The method of claim 1, wherein cell separation rate ranges between 0.1-1 seconds per cell. ### Claim 8 (depends on 1) The method of claim 1, wherein said method reduces or eliminates clogging of said sorter. ### Claim 9 (depends on 1) The method of claim 1, wherein said diagonal force field applied with respect to the length of said trenches is an electrostatic force field. ### Claim 10 (depends on 9) The method of claim 9, wherein said electrostatic force field provides an electroosmotic driving force for said fluid. ### Claim 11 (depends on 1) The method of claim 1, wherein said fluid has an ionic strength of about 1-1000 mM. ### Claim 12 (depends on 1) The method of claim 1, wherein said sorting is deformability-based. ### Claim 13 (depends on 1) The method of claim 1, wherein said sorting is size-based. ### Claim 14 (depends on 1) The method of claim 1, wherein said sorting is charge-based. ### Claim 15 (depends on 1) The method of claim 1, wherein the depth of said trenches ranges between 10-10,000 nm. ### Claim 16 (depends on 1) The method of claim 1, wherein the spacing between said second substrate and said first substrate ranges between 10-5000 nm. ### Claim 17 (depends on 1) The method of claim 1, wherein the width of said trenches ranges between 10-100,000 nm. ### Claim 18 (depends on 1) The method of claim 1, wherein the length of said trenches ranges between 10 nm and 1000 μm. ### Claim 19 (depends on 1) The method of claim 1, wherein said sample inlet, said sample outlet or a combination thereof are in fluid communication with a reservoir. ### Claim 20 (depends on 19) The method of claim 19, wherein voltage is applied to said reservoir. ### Claim 21 (depends on 20) The method of claim 20, wherein said applied voltage is less than 1000 V. ### Claim 22 (depends on 20) The method of claim 20, wherein pressure is applied to said reservoir. ### Claim 23 (depends on 1) The method of claim 1, wherein said fluid mixture comprises a cell mixture having more than one type of cells. ### Claim 24 (depends on 1) The method of claim 1, wherein said fluid mixture comprises a buffered solution. ### Claim 25 (depends on 1) The method of claim 1, further comprising the step of sorting a sample of said fluid mixture two or more times, wherein the pH or ionic strength of said buffered solution is varied at the time of said sorting. ### Claim 26 (depends on 1) The method of claim 1, wherein said trenches comprise a material having a Young's Modulus of at least 500 kPa. ### Claim 27 (depends on 1) The method of claim 1, wherein the ratio between length of said trenches and the spacing between the first and second substrates is at least 3:1. ### Claim 28 (depends on 1) The method of claim 1, wherein the ratio between length of said trenches and the spacing between the first and second substrates ranges between 10:1 and 100:1. ### Claim 29 (depends on 1) The method of claim 1, wherein the spacing between said second substrate and said first substrate is less than 50% of the spacing between the second substrate and the bottom of said trenches of said first substrate. ### Claim 30 (depends on 1) The method of claim 1, wherein the spacing between said second substrate and said first substrate ranges between 10% and 70% of the spacing between the second substrate and the bottom of said trenches of said first substrate. ### Claim 31 (depends on 1) The method of claim 1, wherein said spacing between said first and said second substrate ranges between 0.1-10 μm. ### Claim 32 (depends on 1) The method of claim 1, wherein said force field is an electric field. ### Claim 33 (depends on 1) The method of claim 1, wherein said sample inlet comprises sample loading ports. ### Claim 34 (depends on 1) The method of claim 1, wherein said sample outlet comprises sample collection ports. ### Claim 35 (depends on 1) The method of claim 1, wherein said first substrate, said second substrate, portions thereof or a combination thereof comprises polydimethylsiloxane, polyurethane, glass, silicon, SiO2 or a combination thereof. ### Claim 36 (depends on 1) The method of claim 1, wherein said first substrate, said second substrate, portions thereof or a combination thereof comprises a transparent material. ### Claim 37 (depends on 1) The method of claim 1, wherein the surface of said first substrate, said second substrate, portions thereof or a combination thereof are coated to reduce cell adhesion. ### Claim 38 (depends on 1) The method of claim 1, wherein said sorter is part of a microchip. ### Claim 39 (depends on 38) The method of claim 38, wherein said microchip is disposable.