# US 10,370,635 B2 · Acoustic separation of T cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,635 B2 - Document ID: US10370635B2 - Status: Granted - Kind: B2 - Application number: 15/417,172 - Filed: 2017-01-26 - Published: 2017-08-03 - Granted: 2019-08-06 - Earliest claimed: 2012-03-15 - Assignees: FloDesign Sonics, Inc. - Applicants: FloDesign Sonics, Inc. - Inventors: Bart Lipkens; Rudolf Gilmanshin; Brian Dutra; Benjamin Ross-Johnsrud - Web page: https://patentgrep.com/patent/US10370635B2 ## CPC - C12M 47/02: {Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02)} - B01D 17/04: Breaking emulsions - B01D 17/06: Separation of liquids from each other by electricity - B01D 21/283: {Settling tanks provided with vibrators} - B06B 1/0644: {using a single piezoelectric element (B06B1/0688 takes precedence)} - C12M 29/10: {Perfusion} - C12M 29/18: {External loop; Means for reintroduction of fermented biomass or liquid percolate (loop type reactors for chemical or physical processes B01J19/2435)} - C12M 33/08: {by vibration} - C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli} - C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves - G10K 11/28: using reflection, e.g. parabolic reflectors - 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} - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - B06B: METHODS OR APPARATUS FOR GENERATING OR TRANSMITTING MECHANICAL VIBRATIONS OF INFRASONIC, SONIC, OR ULTRASONIC FREQUENCY, {e.g.} FOR PERFORMING MECHANICAL WORK IN GENERAL (for particular applications, see the relevant subclasses, e.g. B07B1/40, B23Q17/12, B24B31/06; measurement of mechanical vibrations G01H; in direction finding, locating, distance or velocity measuring G01S; {generating seismic energy G01V1/02}; control of mechanical vibrations in general G05D; sound-producing devices, e.g. bells, sirens, whistles G10K, {e.g. methods or devices for transmitting, conducting, or directing sound in general G10K11/00}; generation of electrical oscillations H03B; electromechanical resonators in general H03H; electromechanical transducers {for communication techniques, e.g. microphones, speakers} H04R) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G10K: SOUND-PRODUCING DEVICES; METHODS OR DEVICES FOR PROTECTING AGAINST, OR FOR DAMPING, NOISE OR OTHER ACOUSTIC WAVES IN GENERAL; ACOUSTICS NOT OTHERWISE PROVIDED FOR ## Abstract Acoustophoretic devices and methods for separating biological cells (particularly T-cells) from other fluids/materials using multi-dimensional acoustic standing waves are disclosed. The devices include an inlet, at least two outlets, and a flow chamber having an ultrasonic transducer-reflector pair. Specifically, T cells, B cells, or NK cells can be separated from other blood components. A dual-pass acoustophoretic system including two acoustophoretic devices arranged in series and fluidly connected to one another is also illustrated. Means for pre-chilling the mixture prior to separation in the devices or system can be used to improve retention, concentration, and clarification and to prevent outgassing. ## Claims ### Claim 1 A method for separating biological cells from a host fluid, comprising: chilling a mixture of the host fluid and the biological cells; flowing the cooled mixture of the host fluid and the biological cells through a first acoustophoretic device, the first acoustophoretic device comprising: a flow chamber including at least one inlet and at least one outlet; at least one ultrasonic transducer coupled to the flow chamber, the at least one ultrasonic transducer including a piezoelectric material configured to be driven to create a multi-dimensional acoustic standing wave in the flow chamber; and a reflector opposite to the at least one ultrasonic transducer; and driving the at least one ultrasonic transducer to create the multi-dimensional standing wave in the flow chamber, such that at least some of the biological cells are trapped in the standing wave, and agglomerate, aggregate, clump, or coalesce together, and settle out of the host fluid due to enhanced gravitational settling forces. ### Claim 2 (depends on 1) The method of claim 1, wherein the biological cells are Jurkat T cells, B cells, or NK cells. ### Claim 3 (depends on 1) The method of claim 1, wherein the biological cells are T cells having a low ribosomal content of less than 30 wt %. ### Claim 4 (depends on 1) The method of claim 1, wherein the mixture is chilled to a temperature of about 20° C. to about 25° C. ### Claim 5 (depends on 1) The method of claim 1, wherein the chilling lowers the temperature of the mixture by from about 10° C. to about 20° C. ### Claim 6 (depends on 1) The method of claim 1, wherein the first acoustophoretic device further comprises at least one concentrate outlet located at a bottom end of the flow chamber for recovering the biological cells; and the first acoustophoretic device also further comprises a permeate outlet located at a top end of the flow chamber. ### Claim 7 (depends on 6) The method of claim 6, further comprising sending the host fluid and biological cells exiting the permeate outlet of the first acoustophoretic device to an inlet of a second acoustophoretic device. ### Claim 8 (depends on 6) The method of claim 6, wherein fluid recovered from the at least one concentrate outlet (i) has a cell concentration of at least two times an original cell concentration of the mixture of the host fluid and the biological cells; and (ii) has a volume of at least one half an original feed volume of the mixture of the host fluid and the biological cells. ### Claim 9 (depends on 1) The method of claim 1, wherein the mixture is flowed into the first acoustophoretic device at a flow rate such that the mixture has a residence time of at least 5 minutes. ### Claim 10 An acoustophoretic system for separating biological cells from a mixture of a host fluid and the biological cells, comprising: a chiller for chilling the mixture; and a first acoustophoretic device comprising: a flow chamber including at least one inlet and at least one outlet, the at least one inlet being fluidly connected to the chiller; and at least one ultrasonic transducer coupled to the flow chamber, the at least one ultrasonic transducer including a piezoelectric material configured to be driven to create a multi-dimensional acoustic standing wave in the flow chamber. ### Claim 11 (depends on 10) The system of claim 10, further comprising a reflector opposite to the at least one ultrasonic transducer. ### Claim 12 (depends on 10) The system of claim 10, wherein the chiller for chilling the mixture is a pre-chiller or a fan. ### Claim 13 (depends on 10) The system of claim 10, wherein the at least one inlet of the first acoustophoretic device is a dump diffuser. ### Claim 14 (depends on 10) The system of claim 10, wherein the first acoustophoretic device further comprises at least one concentrate outlet located at a first end of the flow chamber, and wherein the first acoustophoretic device also further comprises a permeate outlet located at a second end of the flow chamber opposite the first end. ### Claim 15 (depends on 14) The system of claim 14, further comprising a second acoustophoretic device comprising: a flow chamber including at least one inlet and at least one outlet, the at least one inlet being fluidly connected to the permeate outlet of the first acoustophoretic device; at least one ultrasonic transducer coupled to the flow chamber, the at least one ultrasonic transducer including a piezoelectric material configured to be driven to create a multi-dimensional acoustic standing wave in the flow chamber; and a reflector opposite to the at least one ultrasonic transducer. ### Claim 16 A device for separating biological cells from a host fluid, comprising: means for chilling a mixture of the host fluid and the biological cells; means for flowing the cooled mixture of the host fluid and the biological cells through a first acoustophoretic device; means for separating the biological cells from the host fluid using a multi-dimensional acoustic standing wave. ### Claim 17 (depends on 16) The device according to claim 16, further comprising means for collecting the separated biological cells. ### Claim 18 (depends on 16) The device according to claim 16, further comprising means for collecting a permeate.