# US 9,752,113 B2 · Acoustic perfusion devices > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,752,113 B2 - Document ID: US9752113B2 - Status: Granted - Kind: B2 - Application number: 15/139,187 - Filed: 2016-04-26 - Published: 2016-08-18 - Granted: 2017-09-05 - Earliest claimed: 2012-03-15 - Assignees: FloDesign Sonics, Inc. - Applicants: FloDesign Sonics, Inc. - Inventors: Bart Lipkens; Erik Miller; Benjamin Ross-Johnsrud; Walter M. Presz, Jr.; Kedar Chitale; Thomas J. Kennedy, III; Lauren Winiarski - Web page: https://patentgrep.com/patent/US9752113B2 ## 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 - H10N 30/2047: {Membrane type} - 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) - H10N: ELECTRIC SOLID-STATE DEVICES NOT OTHERWISE PROVIDED FOR ## Abstract Acoustic perfusion devices for separating biological cells from other material in a fluid mixture are disclosed. The devices include an inlet port, an outlet port, and a collection port that are connected to an acoustic chamber. An ultrasonic transducer creates an acoustic standing wave in the acoustic chamber that permits a continuous flow of fluid to be recovered through the collection port while keeping the biological cells within the acoustic chamber to be returned to the bioreactor from which the fluid mixture is being drawn. ## Claims ### Claim 1 An acoustic perfusion device, comprising: an acoustic chamber; an inlet port and an inlet flow path leading from the inlet port to the acoustic chamber; an outlet port and an outlet flow path leading from the acoustic chamber to the outlet port for recirculating a fluid mixture; at least one collection port for collecting harvest fluid from the fluid mixture; and at least one ultrasonic transducer and at least one reflector opposite the at least one ultrasonic transducer, wherein the at least one ultrasonic transducer includes a piezoelectric material configured to be driven by a voltage signal to create a first multi-dimensional acoustic standing wave across a collection flow path leading from the acoustic chamber to the at least one collection port; and a secondary flow chamber configured to permit the harvest fluid to pass through a second acoustic standing wave with a frequency different from or equal to the first multi-dimensional acoustic standing wave to further clarify the harvest fluid. ### Claim 2 (depends on 1) The device of claim 1, wherein the acoustic chamber is below the at least one collection port. ### Claim 3 (depends on 1) The device of claim 1, wherein the inlet flow path is shaped to generate a tangential flow path below an acoustic field generated by the acoustic standing wave. ### Claim 4 (depends on 1) The device of claim 1, wherein a pressure rise and an acoustic radiation force on cells are generated at an upstream interface region of the acoustic standing wave to clarify fluid passing through the acoustic standing wave. ### Claim 5 (depends on 1) The device of claim 1, wherein the at least one reflector is made of a transparent material. ### Claim 6 (depends on 1) The device of claim 1, wherein the outlet port is below the inlet port. ### Claim 7 (depends on 1) The device of claim 1, having a total of two or more ultrasonic transducers located on multiple sides of the collection flow path. ### Claim 8 (depends on 7) The device of claim 7, where the at least one reflector is located between the two or more ultrasonic transducers. ### Claim 9 (depends on 7) The device of claim 7, wherein the two or more ultrasonic transducers are within the collection flow path and the at least one reflector is opposite the two or more ultrasonic transducers in a wall of the device. ### Claim 10 (depends on 1) The device of claim 1, wherein the at least one transducer is an array. ### Claim 11 (depends on 1) The device of claim 1, wherein the first multi-dimensional acoustic standing wave results in an acoustic radiation force having an axial force component and a lateral force component that are of the same order of magnitude. ### Claim 12 (depends on 1) The device of claim 1, having two or more collection ports spaced apart from each other on the top end of the device. ### Claim 13 (depends on 1) The device of claim 1, wherein both a planar acoustic standing wave and the first multi-dimensional acoustic standing wave are created across the collection flow path. ### Claim 14 (depends on 1) The device of claim 1, further comprising a recirculation flow path between the inlet port and the outlet port that does not enter the acoustic chamber, wherein the recirculation flow path is located below the acoustic chamber. ### Claim 15 (depends on 14) The device of claim 14, wherein the inlet flow path travels through a different passage than the outlet flow path. ### Claim 16 (depends on 14) The device of claim 14, wherein the inlet flow path and the outlet flow path travel through a common passage. ### Claim 17 (depends on 1) The device of claim 1, wherein the fluid mixture is made up of a primary fluid and particles. ### Claim 18 (depends on 17) The device of claim 17, where the particles comprise mammalian cells, bacteria, cell debris, fines, proteins, exosomes, vesicles, viruses, or insect cells. ### Claim 19 A method for separating biological cells from a fluid mixture, comprising: flowing the fluid mixture containing the biological cells through an acoustic perfusion device, the device comprising: an acoustic chamber; an inlet port, an inlet flow path leading from the inlet port to the acoustic chamber; an outlet port for recirculating the fluid mixture and the biological cells; at least one collection port for collecting harvest fluid; and at least one ultrasonic transducer below the at least one collection port and at least one reflector opposite the at least one ultrasonic transducer, wherein the at least one ultrasonic transducer includes a piezoelectric material driven by a voltage signal to create a first multi-dimensional acoustic standing wave across a collection flow path leading from the acoustic chamber to the at least one collection port; and a secondary flow chamber in which the harvest fluid passes through a second acoustic standing wave with a frequency different from or equal to the first multi-dimensional acoustic standing wave to further clarify the harvest fluid; driving the at least one ultrasonic transducer to create the acoustic standing wave; and collecting a fluid mixture enriched in cells from the outlet port and collecting a harvest fluid mixture depleted in cells from the at least one collection port. ### Claim 20 (depends on 19) The method of claim 19, wherein the outlet port is below the inlet port. ### Claim 21 (depends on 19) The method of claim 19, wherein a flow rate through the collection flow path is at least one order of magnitude smaller than a flow rate through the inlet flow path. ### Claim 22 (depends on 19) The method of claim 19, wherein the inlet flow path leads from the inlet port downwards towards the outlet port and then upwards to the acoustic chamber, creating a recirculating fluid stream that is locally substantially tangential to the at least one acoustic standing wave. ### Claim 23 (depends on 22) The method of claim 22, wherein the recirculating fluid stream transports away cells that are constantly held back at an interface region of the acoustic standing wave. ### Claim 24 (depends on 19) The method of claim 19, wherein a pressure rise and an acoustic radiation force on cells are generated at an upstream interface region of the acoustic standing wave to clarify fluid passing through the acoustic standing wave. ### Claim 25 (depends on 19) The method of claim 19, wherein a flow rate of the fluid mixture entering the device through the inlet port is about 1 liter per minute and a flow rate of the harvest fluid depleted in cells exiting the device through the at least one collection port is about 10 milliliters per minute. ### Claim 26 (depends on 19) The method of claim 19, further comprising passing the harvest fluid through the secondary flow chamber to further clarify the harvest fluid. ### Claim 27 A flow device, comprising: at least one inlet for receiving a flowing mixture of a primary fluid and cells, a first ultrasonic transducer configured to produce a first multi-dimensional acoustic standing wave and a pressure rise and an acoustic radiation force on cells, generated at an upstream interface region of the first multi-dimensional acoustic standing wave, to separate the flowing mixture into a primary high cell concentration fluid stream and a secondary harvest fluid stream; an outlet port for the primary high cell concentration fluid stream; a bleed port for extracting a concentrated fluid/cell mixture; and at least one collection port for the secondary harvest fluid stream; and a secondary flow chamber containing a second ultrasonic transducer that produces a second acoustic standing wave with a frequency different from or equal to the first multi-dimensional acoustic standing wave to further clarify the secondary harvest fluid stream. ### Claim 28 (depends on 27) The device of claim 27, where the secondary flow chamber is configured to further clarify the secondary harvest fluid stream by trapping and subsequently clumping, clustering, or agglomerating materials with a size of about 10 microns or less, such that the clumped, clustered, or agglomerated material separates from the secondary harvest fluid stream due to gravity.