Variable resonant systems and methods for cell culture

Abstract

Systems, methods, and devices described herein relate to cell culture systems configured to suspend a plurality of cells for cell culture and expansion. The cell culture systems may include a culture compartment configured to culture and expand a first plurality of cells having a first cell density to produce a second plurality of cells having a second cell density, where the second cell density is greater than the first cell density. Further, the cell culture systems comprise an acoustic actuation device includes a first element and a second element where a distance between the first element and the second element is adjustable, such that a given volume of a resonant cavity defined within the chamber by the first and second element is configured to be adjustable between a first volume and a second volume, where the first volume is different from the second volume.

Description

VARIABLE RESONANT SYSTEMS AND METHODS FOR CELL CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 549,947, filed on February 5, 2024, which is incorporated by reference in its entirety herein.BACKGROUND

[0002] Bioreactors are a promising candidate for cell culture of mammalian cells used to grow monocellular organisms such as cells, yeast, and bacteria under controlled conditions. Traditional systems for promoting proliferation of cells in a cell culture environment include monolayers of cells grown on substrates and those grown in bioreactors.

[0003] Monolayers of cells grown on substrates may face challenges associated with the high surface area and labor costs needed to grow a high number of cells. Further, monolayers of cells are traditionally grown on a substrate of either plastic or glass for monolayer cell culture which can influence cell behavior. For example, cells may interact differently with these artificial surfaces compared to the cells’ native environment. For example, mammalian cartilage cells grown on an artificial substrate may interact differently than they do in the natural extracellular matrix found in tissues, potentially impacting cell phenotype, homogeneity, genotype, and differentiation.

[0004] Traditional cell culture in bioreactors include suspending cells within cell media to promote cell expansion through proliferation. However, the mixing required to maintain cells in suspension in traditional cell culture bioreactors may damage cells or lead to changes in cell behavior. For example, some bioreactor systems generate shear stress and mechanical forces that may affect certain cell types, especially sensitive cells or those requiring a more static environment. Further, in some cases the phenotype and / or genetic drift of cells is impacted by a given cell's proximity to neighboring cells and / or its ability to communicate with other cells. Thus, cells suspended in a fluid within traditional bioreactor systems therefore may have little interaction with other neighboring cells thereby impacting cell behavior, phenotype, and genotype. Further, traditional bioreactors are limited in cell density in order to allow for sufficient exchange of nutrients.BRIEF SUMMARY

[0005] In one aspect, embodiments described herein relate to a cell culture system, including a culture compartment configured to culture and expand a first plurality of cells having a first cell density to produce a second plurality of cells having a second cell density. In some embodiments, the second cell density is greater than the first cell density. In some embodiments, the culture compartment comprises an interior chamber configured to receive the first and second plurality of cells. The cell culture system may include a flow compartment configured to provide cell culture media to the culture compartment to support the expansion of the first plurality of cells into the second plurality of cells. The cell culture system may include a membrane disposed at least partially between the culture compartment and the flow compartment. The membrane may be configured to be permeable to the cell culture media provided to the flow compartment. The cell culture system also includes an acoustic actuation device. The acoustic actuation device has a first element disposed about a distal end of the culture compartment and a second element disposed proximal and opposite to the first element. The distance between the first element and the second element is adjustable, such that a given volume of a resonant cavity defined within the chamber by the first and second element is configured to be adjustable between a first volume and a second volume. The first volume is different from the second volume. In some embodiments, the second element is disposed within the chamber. In some embodiments, the second element is configured to translate along a longitudinal axis of the chamber in a proximal or distal direction. In some embodiments, the second element is coupled to a mount that is slidable along a translation rod so as to translate the second element along the longitudinal axis. In some embodiments, the movement of the mount along the translation rod is actuated via a motor system, a hydraulic system, an electromagnetic system, or any combination thereof. In some embodiments, the cell culture system includes one or more arms extending from the mount to a piston. In some embodiments, the second element is coupled to the chamber rod at a distal end of the piston. In some embodiments, the piston is disposed through the proximal end of the culture chamber. In some embodiments, the translation of the second element within the culture chamber is automatic. In some embodiments, the cell culture system includes a distance control system comprising an actuator configured to translate the second element along the longitudinal axis. In some embodiments, the distance control system includes a processor configured to determine a target distance between the first element and the second element and an actuator configured to move the mount to position the second element to a position spaced apart from the first element by the target distance. In someembodiments, the target distance is determined based on a desired wavelength of an acoustic wave between the first and second elements. In some embodiments, the target distance correlates to a distance adapted to produce a standing wave between the first and second elements. In some embodiments, the standing wave is configured to suspend the one or more of the first plurality of cell and the second plurality of cells within the culture chamber. In some embodiments, the distance control system is in communication with the acoustic actuation device, such that the distance control system receives input relating to the wavelength of the acoustic wave emitted by the first or the second element. In some embodiments, the cell culture system further comprises one or more sensors configured to transmit signals to a processor configured to determine a wavelength of the acoustic wave, measure a distance between the first and second element, determine a voltage between first and second elements, measure an amplitude of a pressure field within the chamber, determine a capacitance between the first element and the second element, electrical potential between the first element and the second element, or any combination thereof. In some embodiments, the distance control system further comprises a processor configured to determine the target distance using one or more inputs from the one or more sensors, and an actuator configured to move the second element. In some embodiments, the target distance is determined by a processor using one or more inputs from one or more sensors. In some embodiments, the first element is an acoustic emitter, and the second element is an acoustic second element.

[0006] In another aspect, embodiments described herein relate to methods of culturing cells. The method may include disposing a first plurality of cells having a first cell density in a chamber of a culture compartment. The method may include actuating an acoustic actuation device to generate an acoustic field within the chamber. The acoustic actuation device emits an acoustic wave from an emitter to a second element, the emitter disposed at a distal end of the chamber, the second element disposed proximal and opposite to the emitter. The method may include adjusting a distance between the emitter and second element towards a target distance therebetween, so as to produce or substantially produce a standing wave within the chamber to maintain the first plurality of cells and expand the first plurality of cells to a second plurality of cells having a second cell density, wherein the second cell density is greater than the first cell density.

[0007] In another aspect, embodiments described herein relate to systems for continuously producing a standing wave. The system may include an acoustic emitter disposed at a distal end of a chamber within a housing, an acoustic second element disposed proximal and opposite to the acoustic emitter. The acoustic second element may be configured to betranslated within and along a longitudinal axis of the chamber such that a distance between the acoustic emitter and the acoustic second element is adjustable. The system may include a distance control system including a processor configured to determine a wavelength of an acoustic wave emitted by the acoustic emitter, a measured distance between the acoustic emitter and the acoustic second element, and a target distance therebetween that correlates to a standing wave. In some embodiments, the target distance is based on the wavelength of the acoustic wave. In some embodiments, the distance control system includes an actuator configured to translate the acoustic second element within the chamber to a position the target distance away from the acoustic emitter.

[0008] In some embodiments, the cell culture systems and methods provided herein are effective in cell engineering. The cell engineering may include the cell culture systems and methods noted and described herein. The cell engineering may include the cell culture systems and methods noted and described for tissue engineering.

[0009] As used herein, the terms “substantially” or "about" means within ±10% of the value it modifies. For example, "about 1" means "0.9 to 1.1", "about 2%" means" 1.8% to 2.2%", "about 2% to 3%" means" 1.8% to 3.3%", and "about 3% to about 4%" means "2.7% to 4.4%." Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about".

[0010] As used herein, the term “automatic” means the system's ability to operate without direct human intervention once it has been set up and parameters defined. For example, automatic systems are designed to perform tasks and make adjustments based on predefined parameters, feedback, or input signals, without the need for continuous manual control.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0011] FIG. 1 illustrates an example of a cell culture system, according to embodiments described herein.

[0012] FIG. 2A illustrates another example of a cell culture system, according to embodiments described herein.

[0013] FIG. 2B illustrates a cross-sectional view of the exemplary cell culture system of FIG. 2A.

[0014] FIG. 3A illustrates an isometric cross-sectional view and FIG. 3B illustrates a planar cross-sectional view of a culture compartment, according to an embodiment described herein.

[0015] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D illustrate a planar, isometric planar, isometric cross-sectional, and planar control system view respectively of a cell culture system having a compressible culture compartment according to an embodiment described herein.

[0016] FIG. 5 A illustrates an example of a second element containing air and located within a culture compartment, according to embodiments described herein.

[0017] FIG. 5B illustrates an example of FIG. 5 A, with a floating second element according to embodiments described herein.

[0018] FIG. 6 illustrates an example of an acoustic actuation device, according to embodiments described herein.

[0019] FIG. 7 illustrates an example of a culture compartment having a column of cell layers, according to embodiments described herein.

[0020] FIG. 8 illustrates an exemplary depiction of relative pressure within a culture compartment including a column of cell layers disposed between a first element and a second element, according to an embodiment described herein.

[0021] FIG. 9 illustrates another example of relative pressure distribution within a culture compartment when an acoustic wave is emitted from one end and reflected from a second opposite end.DETAILED DESCRIPTION

[0022] Systems, methods, and devices described herein relate to cells which may be cultured, expanded, manipulated, and / or modified within a bioreactor system while suspended by an acoustic wave, which itself may be generated by an acoustic actuation device. Suspending cells within layers in a bioreactor may provide advantages over traditional bioreactors such as lower shear forces of mixing applied to the cells, higher cell densities due to efficient exchange of nutrients between cell layers, and / or providing an adherent cell environment within individual layers thereby permitting communication between neighboring cells.

[0023] The cell culture systems and methods provided herein are effective in cell engineering. Cell engineering may include the cell culture systems and methods noted and described herein. The cell culture systems and methods provided herein are effective in tissue engineering or using the cells cultured to create tissue replacements, and / or biological products. The cell culture systems and methods provided herein are effective in bioprocess engineering or using cells to create biological products. The cell engineeringusing the culture systems and methods described herein are effective in creating vaccines or other therapeutics, tissue repair, organ replacement, skin repair, or creating new blood vessels or other tissue systems.Cells

[0024] In some embodiments, the cells are selected from a group consisting of (human) stem cells, immune cells (such as regulatory T-cells, NK cells, Jurkat T-cells, CAR-T cells, B cells), peripheral blood mononuclear cells (PBMCs), cell lines such as HEK293, Vero, (mammalian) BHK, CHO, (insect) SF9, SF21, algae, plant cells, bacteria, yeast, and any combination thereof. In some embodiments, the cells are suspended within the system. In some embodiments, the cells are selected from the group consisting of cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells, monocytes, neutrophils, macrophages, ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, hybrids thereof, and combinations thereof. In some embodiments, the cells are attached to micro carriers.

[0025] In general, cells to be used in accordance with the present invention are any types of cells. In general, the cells should be viable when initially seeded in the culture chamber. In some embodiments, cells that can be seeded into the culture chamber in accordance with the present invention include, but are not limited to, mammalian cells (e.g., human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some embodiments, exemplary cells that may be seeded into the culture chamber include stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells. In some embodiments, exemplary cells that can be seeded into the culture chamber in accordance with the present invention include, but are not limited to, primary cells and / or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g., monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and / or hybrids thereof, may be seeded into the culture chamber in accordance with the present invention. Other types of cells that may be used herein include AAV, LV, HSV, and other types of production cells (e.g., other types for viral vector production, antibody production, etc.).

[0026] Exemplary mammalian cells that may be seeded into the culture chamber in accordance with the present invention include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Madin-Darby canine kidney (MDCK) cells, baby hamster kidney (BHK cells), NSO cells, MCF-7 cells, MDA-MB-438 cells, U87 cells, A172 cells, HL60 cells, A549 cells, SP10 cells, DOX cells, DG44 cells, HEK 293 cells, SHSY5Y, Jurkat cells, BCP-1 cells, COS cells, Vero cells, GH3 cells, 9L cells, 3T3 cells, MC3T3 cells, C3H-10T1 / 2 cells, NIH-3T3 cells, and C6 / 36 cells.

[0027] Suitable cells for use in the disclosed methods and apparatus include prokaryotic and eukaryotic cells, preferably mammalian cells. Representative cells include mammalian, preferably human, cells, embryonic or adult stem cells; pluripotent or totipotent cells obtained from placenta, cord blood, adipose tissue, neural tissue, muscle tissue, cardiac tissue, parenchymal tissues, epidermal tissue, or bone marrow; or somatic cells optionally corresponding to a tissue or organ to be treated. The cells can be derived from mesoderm, endoderm, or ectoderm. Representative cells include mesenchymal cells, especially fibroblasts, interstitial cells, endothelial cells, smooth or skeletal muscle cells, heart cells, myocytes (muscle cells), chondrocytes, adipocytes, fibromyoblasts, and ectodermal cells, including ductile and skin cells, hepatocytes, Islet cells, cells present in the intestine and other parenchymal cells, osteoblasts and other cells forming bone or cartilage, bone marrow cells and blood cells. Cells genetically engineered to avoid the need for immunosuppression in a host receiving the tissue construct may also be used.

[0028] A population of cells to be cultured can be a single type of cell, cells from a single type of tissue (for example a population of cardiac cells), or a combination of different types of cells (for example endothelial cells in combination with cardiac myocytes). The cells can be autologous, heterologous, allogenic, xenogeneic, or a combination thereof.

[0029] Suitable cells also include recombinant cells that express one or more heterologous nucleic acids. The heterologous nucleic acid can express a protein involved in wound healing or tissue regeneration, for example an extracellular matrix protein, matrix metalloprotease or GAG synthase, or a fusion protein thereof. Alternatively, the cell can be genetically engineered to secrete paracrine factors including, but not limited to growth factors such as fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial cell growth factor, and insulin-like growth factor, bone morphogenic proteins and combinations thereof. The cells can also be engineered to express inhibitors of complement or inflammation, such as CD46 or CD59. Methods and protocols for producing recombinant cells are known in the art.

[0030] In some embodiments, cells are obtained by biopsy and dissociated using standard techniques, such as digestion with a collagenase, trypsin or other protease solution. Cells can be easily obtained through a biopsy anywhere in the body, for example, skeletal muscle biopsies can be obtained easily from the arm, forearm, or lower extremities, smooth muscle can be obtained from the area adjacent the subcutaneous tissue throughout the body, and bone marrow can be obtained from the iliac crest. The biopsy can be readily obtained with the use of a biopsy needle, a rapid action needle which makes the procedure extremely simple and almost painless. Cells may also be procured from, for example, cardiac tissue, blood vessels, blood, such as umbilical cord blood or adult blood, valves and discarded tissues, such as placenta and tissue obtained during orthopaedic, reconstructive, aesthetic or cosmetic surgical procedures.

[0031] For example, the dermal layer of a skin biopsy can be digested with collagenase or other proteases. After the digestion of the dermal fragments, mesenchymal cells can be harvested following centrifugation and expanded in cell culture media. Alternatively, dermal fibroblasts or adventitial fibroblasts may be used. Fibroblasts are easily available, and they are the primary collagen secreting cells in connective tissues. Dermal fibroblasts are typically harvested from normal adult skin specimens removed during reductive breast surgery, or from neonatal foreskin.

[0032] The frequencies and amplitudes of an emitted acoustic wave and corresponding reflected wave, which may result in a constructed standing wave, may impact the interlayer pressure exerted on each layer of cell, the number of layers within a bioreactor, and the movement of a column of cell layers for nutrient and waste exchange.Frequency, Wavelength, and Trapping Sites

[0033] In some embodiments, the acoustic wave in the culture compartment is controlled by altering the distance between an acoustic emitter emitting an acoustic wave and an acoustic reflector reflecting the acoustic wave to form a standing wave, wherein the acoustic emitter and the acoustic reflector are part of the acoustic actuation device. The cells in a culture compartment of a bioreactor (as described herein), not being bound to a mechanical point, may be manipulated in the volume of the culture compartment by adjusting the phase and / or frequency of the acoustic wave generated by the acoustic actuation device.

[0034] The ability to control both wavelength and pressure of the generated acoustic wave is important in being able to control the acoustic force (correlated to acoustic pressure and acoustic wavelength) exerted on cells within a culture compartment, which helpscontrol the manner in which the cells are suspended. Furthermore, control of wavelength and pressure and thus ability to control the acoustic force, exerted on cells within a culture compartment, may aid in controlling the manner in which cells grow, proliferate, produce biomolecules, be manipulated, modified, and / or engineered.

[0035] Wavelength and frequency are linked by the following equation:

[0036] where f is frequency, c is the speed of sound in the respective medium, and X is wavelength. In order to be resonant (i.e. , to build a standing wave), the distance (e.g., h, as described in the following equation) between the emitter and the reflector needs to satisfy in one dimension h=n x ( / 2), wherein n is an integer. Traditionally, frequency may be easier to control than the distance between the emitter and reflector (e.g., distance may be limited due to fixed channel geometries within a bioreactor, hard to control manufacturing tolerances vs. frequency generated through electronics), so historically the frequency may have been tuned to match with the dimension of the cavity (e.g., distance between the emitter and reflector) in other acoustophoretic applications. Changes to frequency can impact the acoustic force, the number of trapping sites (as described herein), oxygenation, nutriment replenishment, and bubble cavitation. For example, increasing the acoustic force while holding all other parameter constants may correspond to conveying more trapping force at the same acoustic pressure. In some cases, changing the frequency may increase or decrease a number of trapping sites of the cells (two sites per wavelength), thereby enabling real time control of the effective working cell density (i.e., the cell density in the culture component at a given time) and the cell density within each layer of cells (i.e., the cell density in an individual cell layer at a given time).

[0037] In the context of sound resonance or any type of wave resonance, extrema typically refer to the locations or points where the amplitude of the emitted wave and the reflected wave are at their relative extreme values along the longitudinal axis of the culture compartment. For example, where both waves are at their longitudinal minimal point they form trapping sites for cells (e.g., trapping site 602 on FIG. 6) and vary to their longitudinal maximal point (e.g., see maximum amplitude 604 on FIG. 6). Typically, these are positions where the wave exhibits constructive or destructive interference, resulting in maximum or minimum of the amplitude of the wave.

[0038] Furthermore, minima and maxima pressures exist in a latitudinal direction along the plane of the trapping sites. Depending on their relative properties to the suspending medium they are in, cells in suspension will be trapped in maxima or minima of pressure, for instance. However, there are also locations within these extrema that cells are morelikely to be located within this latitudinal plane. For example, the acoustic wave can be shaped such as the center of it is at a higher pressure than its exterior, or lateral reflection caused by the finite nature of the culture chamber building up more pressure in precise locations within it. This will cause the trapped cells, in addition to being located within extrema of the main direction of the wave, to laterally move to local extrema, referred to as the orthogonal extrema. In some embodiments, cells are trapped in either minima or maxima pressure, depending on the property differences between the cells and the medium in which the cells are suspended.

[0039] Because of orthogonal extrema, lateral displacement of the trapping sites is observed through gentle acoustic sweeping, allowing for oxygenation, nutriment replenishment, etc. In other words, the orthogonal extrema allow for a column of cell layers to shift the location of a longitudinal axis of the column of cells within a culture compartment or undergo an acoustic sweep to permit new cell media to interface with cell layers providing oxygenation and nutriment replenishment.

[0040] Bubble cavitation is the formation or the collapse of gas of vapor-filled bubbles suspended within a liquid. In some embodiments, bubble cavitation can be controlled through changes in the acoustic wave frequency, changes in the acoustic wave amplitude, and / or through the injection of preconstructed bubbles. For example, the relationship between bubble cavitation and frequency is primarily associated with the frequency of sound waves or pressure fluctuations within a fluid. Sound waves with specific frequencies can induce and influence bubble cavitation. Bubble cavitation may occur when a low frequency is generated throughout the liquid. Without being bounded by theory, low frequency pressure fluctuations may cause the pressure in the fluid to oscillate. When the pressure decreases during the troughs of the pressure wave, it can create conditions where the pressure drops below the vapor pressure of the liquid. When this occurs, small gas or vapor nuclei in the liquid can expand and form cavitation bubbles. In some embodiments, controlling bubble cavitation can be used to provide and facilitate gas and other nutrient exchange for cells within a culture compartment. For example, in some embodiments, controlling bubble cavitation can be used to facilitate permeation of the cell membranes, improve cargo transport across said membrane, and / or help rupture cells for the harvest of their production.

[0041] FIG. 1 illustrates an example of a cell culture system 102 according to embodiments described herein. In some embodiments, the cell culture system 102 comprises a cell culture apparatus 104 and a distance control system 106.

[0042] In some embodiments, the cell culture apparatus 104 comprises a piston 108 and a culture compartment 110. In some embodiments, the culture compartment 110 extends from a proximal end of the culture compartment to a distal end of the culture compartment 110. In some embodiments, the piston 108 extends at least partially through the proximal end of the culture compartment 112r of the culture compartment 110. The piston 108 may extend from a first end of the piston 114 to a second end of the piston 116. In some embodiments, the culture compartment 110 is substantially cylindrical in shape. In some embodiments, the culture compartment 110 is upright with a lateral wall 118 encircling the interior chamber 120 from the proximal end of the culture compartment 112 to the distal end of the culture compartment 122.

[0043] In some embodiments, the culture compartment 110 is configured to culture and expand a first plurality of cells having a first cell density to produce a second plurality of cells having a second cell density. In some embodiments, the second cell density is greater than the first cell density. In some embodiments, the culture compartment 110 comprises an interior chamber 120 configured to receive the first plurality of cells and / or the second plurality of cells. In some embodiments, the culture compartment 110 is at least partially filled with cell media configured to suspend the first plurality of cells and / or second plurality of cells within the interior chamber 120.

[0044] In some embodiments, the cell culture system 102 includes an acoustic actuation device, having a first element 124 and a second element 126. In some embodiments, the first element 124 is an acoustic emitter and the second element 126 is an acoustic reflector. In some embodiments, the second end of the piston 116 is coupled to a second element 126. In some embodiments, the second element 126 is a reflector and extends from a first end of the reflector 128 thereof to a second end of the reflector 130. In some embodiments, the second element 126 is substantially cylindrical in shape. In some embodiments, the second element 126 is substantially square in shape. In some embodiments, the second element 126 is substantially rectangular in shape. In some embodiments, the second element 126 is disposed within the interior chamber 120 of the culture compartment 110. In some embodiments, the second element 126 has an outer diameter smaller than the inner diameter of the interior chamber 120 of the culture compartment 110. In some embodiments, the cell culture system 102 is configured to form a single enclosed device.

[0045] In some embodiments, the culture compartment 110 comprises a flow compartment (not shown) configured to provide cell culture media to the interior chamber 120 of the culture compartment 110. As used herein, the term “cell culture media” and “cell media” may be used interchangeably. In some embodiments, the cell media is adapted to supportthe culture of cells and expansion of the first plurality of cells into the second plurality of cells. Any cell culture media components known to those skilled in the art may be used to culture and expand the cell density within the interior chamber 120 and flow through the flow compartment. In some embodiments, the culture compartment comprises one or more openings configured to receive and / or dispense the cell culture media. For example (and as described herein in other embodiments), in some cases, the culture compartment comprises an opening disposed about a proximal portion of the culture compartment, which is configured to receive cell media. In some embodiments, the culture compartment comprises an opening disposed about a

[0046] In some embodiments, the first cell density of cells seeded in the culture compartment 110 is between 50 k / mL and 500 M / mL. In some embodiments, the first cell density of cells seeded in the culture compartment 110 is between 400 k / mL and 1 M / mL In some cases, the concentration of cells seeded into the culture compartment 110 is important, because at too low a cell density, the acoustic force may be unable to prevent microflow from disrupting layers of cells. Further, at too high of a cell density, the cells may impact the interarticular effects of cell agglomeration causing interactions between layers of cells.

[0047] In some embodiments, the cells are screened for viability prior to being seeded into the culture compartment 110. In some embodiments, cells seeded into the culture compartment 110 have 95% or greater viability. In some embodiments, cells seeded into the culture compartment 110 have greater than 70%, 75%, 80%, 85%, 90%, or 95% viability. In some embodiments, the cell culture system 102 is configured to be cell agnostic such that any cell may be added and cultured within the system. In some embodiments, the cell culture system 102 has a design or specific parameters designed to promote the proliferation and / or differentiation of a specific type of cell.

[0048] In some embodiments, a membrane (not shown) is disposed around the piston 108 at a location between the first end of the piston 114 and the second end of the piston 116, and within the culture compartment 110. In some embodiments, the membrane is disposed at least partially between the interior chamber 120 and the flow compartment. In some embodiments, the membrane is configured to be permeable to cell culture media components provided to the interior chamber 120 by the flow compartment. For example, the membrane may be permeable to nutrients and proteins within the cell media while remaining impermeable to cells. In some embodiments, the membrane comprises a hermetic seal adapted to separate the interior chamber 120 from the flow compartment.

[0049] In some embodiments, the flow compartment and the interior chamber 120 each individually are defined by interior surfaces or walls. In some embodiments, at least a portion of the walls defining the flow compartment and / or the interior chamber 120 comprise the membrane, which physically separates the flow compartment from the interior chamber 120. In some embodiments, the flow compartment and the interior chamber 120 are separated by the membrane, wherein the membrane at least partially comprises walls separating the flow compartment and the interior chamber 120. For example, the walls separating the flow compartment and the interior chamber 120 may be biocompatible, configured to retain particles suspended in the liquid with which they are in contact, are acoustically transparent, and / or are manufacturable by injection or molding. For instance, the walls may comprise polypropylene, polystyrene, and / or be obtained from polymer resin adapted for stereolithography. In some embodiments, the membrane is also acoustically transparent.

[0050] In some embodiments, as described herein, the cell culture apparatus 104 comprises a first element 124 and the second element 126. In some embodiments, the first element 124 and / or the second element 126 is disposed within or adjacent to the culture compartment 110. As described herein, in some embodiments, the first element 124 is an acoustic emitter. In some embodiments, the first element 124 is disposed about or adjacent to a distal end of the culture compartment 122. As described herein, in some embodiments, the second element 126 is a reflector. In some embodiments, the second element 126 is disposed proximally and spaced apart from the first element 124. For example, the second element may be located within the interior chamber 120 at a location opposite to the first element 124. In some embodiments, the second element 126 is an acoustic reflector. In some embodiments, the second element 122 is another acoustic emitter. In some embodiments, the second element 122 is configured to translate along a longitudinal axis of the interior chamber 118 in a proximal or distal direction.

[0051] In some embodiments, the acoustic actuation device comprises a plurality of first elements, and a plurality of corresponding second elements. For example, in some embodiments, the acoustic actuation device comprises a plurality of emitters and a plurality of corresponding reflectors or additional emitters. In some embodiments, each second element of the plurality of second elements is configured to be translated along a longitudinal axis of the interior chamber 118 in a proximal or distal direction. In some embodiments, the plurality of second elements are configured to be translated together. For example, in some embodiments, the plurality of second elements may be coupled to a similar piston or moved via a similar actuator (as described herein). In some embodiments,each of the plurality of second elements are configured to be individually translated along the longitudinal axis of the interior chamber 118, such that in some cases, one or more second elements may be at a different longitudinal position within the interior chamber as with another second element. In some embodiments, each second element of the plurality of elements is configured to be translated using a similar or the same height control system as described herein for FIG. 1. In some embodiments, each of the plurality of first elements are spaced apart from each other. In some embodiments, each of the plurality of second elements are spaced apart from each other.

[0052] In some embodiments, any given first element and / or second element described herein comprises an array of emitters. In some embodiments, each emitter in the array of emitters is configured to be individually controlled.

[0053] In some embodiments, any cell culture system described herein comprises an actuation device configured to emit an acoustic wave in a direction that may be perpendicular to a longitudinal axis of a culture compartment (as described herein), and / or in a direction that may be oblique to a longitudinal axis of a culture compartment (as described herein). For example, in some embodiments, an emitter of an actuation device is disposed on lateral wall (e.g., ref. char. 118 in FIG. 1) of the culture compartment, while an opposing emitter or reflector is either located about an opposite lateral wall or within the interior chamber but opposite to the emitter of the first element. Accordingly, the lateral wall actuation device will generate an acoustic wave that travels perpendicular to the longitudinal axis of the culture compartment. In some embodiments, any number of actuation devices can be included with the cell culture system to provide acoustic waves in multiple dimensions. For example, in some embodiments, a cell culture system described herein includes a first acoustic actuation device (e.g., first element 124 in FIG. 1), and a second actuation device (e.g., second element 126 in FIG. 1) disposed on a lateral wall that generates an acoustic wave in a direction perpendicular to the direction of the acoustic wave generated by the first acoustic actuation device. In some embodiments, a second element of each acoustic actuation device is configured to have its position individually adjusted, as described herein.

[0054] In some embodiments, a distance between the first element 124 and the second element 126 is adjustable, such that a measured volume of the interior chamber 120 is defined within the chamber. In some embodiments, the measured distance between the first element 124 and the second element 126 is adjustable between a first distance and a second distance. In some embodiments, the distance between the first element 124 and the second element 126 is correlated with a measured volume of the interior chamber 120. In someembodiments, the measured volume of the interior chamber 120 is configured to be adjusted from a first volume to a second volume. For example, if the membrane within the interior chamber 120 moved towards the distal end of the culture compartment 122 the volume within the interior chamber 120 would decrease. In some embodiments, the culture compartment 110 includes at least one port 134 configured to transport fluid into the interior chamber 120 and / or permit fluid to leave the interior chamber 120.

[0055] In some embodiments, the membrane is configured to remain fixed at a position separating at least a portion of the interior chamber 120 from the flow compartment. For example, if the second element 122 within the interior chamber 120 moved (e.g., via the piston 116) towards the distal end of the culture compartment 136, the volume within the interior chamber 120 would decrease. In some embodiments, the culture compartment comprises flexible lateral walls, such that as the position of the second element is modulated, the lateral walls correspondingly expand outward or compress inwards, such that the measured volume of the interior chamber 120 remains the same or substantially the same. For example, the cell culture system 402 depicted in FIGS. 4A-4B includes a culture compartment 404 having flexible lateral walls (e.g., flexible outer wall 406). In some embodiments, for a cell culture system having flexible lateral walls, and wherein the second element of an actuation device is located therein, as the second element moves distally towards the first element, the flexible lateral walls will expand accordingly to keep the measured volume the same or substantially the same. The opposite effect will occur if the second element moves proximally away from the first element (the flexible lateral walls will compress inwards).

[0056] In some embodiments, the membrane is configured to remain fixed at a position separating at least a portion of the interior chamber 120 from the flow compartments 154. For example, the membrane may have a center passageway configured to permit the piston 108 to slidably pass therethrough. In some embodiments, the membrane comprises a first sealing member configured to form a seal between an outer circumference of the membrane with the inner surface of the lateral wall 118. In some embodiments, the membrane comprises a second sealing member (not shown) configured to form a seal between the inner surface of the center passageway of the membrane and the outer surface of the piston 108.

[0057] In some embodiments, the second element 126 is configured to translate along a longitudinal axis of the culture compartment 110 in a proximal or distal direction. For example, the second element 126 shown in FIG. 1 is a reflector and may be coupled with a piston 108. The second element 126 may be coupled to the second end of the piston 116 ofthe piston 108. The piston 108 may be fixedly attached, or removably coupled to the second element 126. As described herein, the piston 108 may be disposed through a proximal end of the culture compartment 112.

[0058] In some embodiments, the distance control system 106 comprises a platform 138, a translation rod 136, and a mount 140. In some embodiments, the translation rod 136 is coupled to the platform 138. For example, the translation rod 136 may attach to a translation surface 142 and extend along a longitudinal axis parallel to that of the platform 138. In some embodiments, the translation rod 136 is substantially cylindrical shaped. In some embodiments, the translation rod 136 comprises a track system. In some embodiments, the mount 140 is coupled with the translation rod 136 such that it is configured to move about the longitudinal axis of the translation rod 136. In some embodiments, the second element 126 is coupled to the mount 140 and is configured to slide along the translation rod 136, thereby enabling the second element 126 to translate proximally and distally with respect to the culture compartment 110. In some embodiments, the movement of the mount 140 along the translation rod 136 is actuated by an actuator (not shown). For example, the actuator may be a motor system, a hydraulic system, an electromagnetic system, or any combination thereof. In some embodiments, the translation of the second element 126 within the interior chamber 120 is automatic.

[0059] In some embodiments, the distance control system 106 comprises at least one actuator configured to translate the mount 140 about the longitudinal axis of the translation rod 136. In some embodiments, the actuator is a linear actuator. In some embodiments, a stepper is configured to rotate an endless screw (not shown) that moves the second element 126 holder to translate the second element 126 in a direction proximal or distal along the culture compartment 110 longitudinal axis.

[0060] In some embodiments, the second element 126 is configured to be angled relative to the first element 124. For example, the cell culture system may include a second actuator to angle the second element 126, the piston 108, or both, relative to the first element 124. In some embodiments, a processor is configured to transmit one or more signals to the second actuator to direct the actuator to tilt the second element 126, piston 108, or both, into a desired angle between the first and second elements. In some embodiments, the angle of tilt correlates with generating a standing wave between the first and second elements.

[0061] In some embodiments, the second element 126 is a reflector which has a concave or a convex curvature configured to reflect acoustic caves in a more concentrated form relative to a flat reflector. In some embodiments, the second element 126 is configured to atleast partially transition from a concave curvature to a flat or convex curvature. In some embodiments, the second element 126 is configured to transition from a convex curvature to a flat or concave curvature. In some embodiments, the second element 126 is configured to transition from a flat curvature to an at least partially concave or convex curvature. In some embodiments, the surface of the second element 126 is textured. For example, the texture of the second element 126 may be a pattern of grooves and domes configured to maximize the frequency sweeping of cells. In some embodiments, the frequency of the sweeping of cells is configured to be between 2-4 mHz.

[0062] In some embodiments, the distance control system 106 comprises at least one processor (not shown) configured to transmit signals to direct the actuator to move the mount 140. In some embodiments, the processor is external from the distance control system 106. In some embodiments, the processor receives signals from the distance control system 106 wirelessly and / or manually coupled thereto. In some embodiments, the processor is located within the mount 140.

[0063] In some embodiments, the mount 140 extends from a first end of the mount 144 to a second end of the mount 146. In some embodiments, a plate 148 is coupled to the mount 140. In some embodiments, the plate 148 is oriented perpendicular to and extends outwardly from the first end of the mount 144 and the second end of the mount 146. In some embodiments, a first arm 150 extends from the plate 148 or the mount 140. In some embodiments, a second arm 152 extends from the plate 148 or the mount 140. In some embodiments, at least one of the first arm 150 and the second arm 152 are configured to couple with the piston 108 such that as the mount 140 moves in a proximal or distal direction about the longitudinal axis of the translation rod 136, the piston 108 also moves in a corresponding proximal or distal direction.

[0064] Accordingly, in some embodiments, the distance control system 106, for example using one or more of the sensors described herein, is configured to determine a target distance (as described herein) so as to generate a standing wave between the first element 124 and the second element 126. As described herein, generating a standing wave allows the first and / or second plurality of cells to be suspended along the standing wave, thereby helping the cells exchange nutrients and other material via the cell culture media (e.g., as provided by the flow compartment). In some embodiments, the distance control system is further configured to move the mount 140 (e.g., via an actuator as described herein), so as to position the second element within the interior chamber at a location that is spaced apart from the first element by the target distance. In some embodiments, the distance control system 106 comprises at least one processor (not shown) configured to determine a targetdistance between the first element 124 and the second element 126. In some embodiments, the distance control system 106 comprises an actuator (not shown) configured to move the mount 140 to move the second element 126 to a position spaced apart from the first element 124 by the target distance. In some embodiments, the target distance is determined based on a desired wavelength of an acoustic wave between the first element 124 and the second element 126. In some embodiments, the target distance correlates to a distance adapted to produce a standing wave between the first and second elements. In some embodiments, the standing wave is configured to suspend the one or more of the first plurality of cell and the second plurality of cells within the culture chamber. In some embodiments, the distance control system 106 is in communication with the first element 124 (i.e. an acoustic actuation device), such that a processor of the distance control system 106 receives inputs relating to the wavelength of the acoustic wave emitted by the first or the second element. In some embodiments, the processor is located within or adjacent to the mount 140. In some embodiments, the processor is external to the distance control system 106. In some embodiments, the processor is configured to wirelessly receive signals from one or more sensors of the distance control system 106.

[0065] In some embodiments, the distance control system 106, through, for example, one or more of the sensors described herein, is configured to adjust the position of the second element 126 (e.g., within the interior chamber 120) in real-time so as to maintain the standing wave between the first element 124 and the second element 126. In some embodiments, the cell culture system 102 further configured to transmit signals (e.g., via one or more sensors, as described herein) to a processor. In some embodiments, the processor is configured to determine at least one of a wavelength of the acoustic wave, a distance between the first element 120 and the second element 122, a voltage between the first element 120 and the second element 122, an amplitude of a pressure field within the interior chamber 118, a capacitance between the first element 120 and the second element 122, an electrical potential between the first element 120 and the second element 122, an acoustic pressure within the interior chamber 118, or any combination thereof. In some embodiments, the distance control systems 106 comprises one or more processors (e.g., as described herein) configured to determine the target distance using one or more inputs from the one or more sensors and / or an actuator configured to move the second element 122. In some embodiments, the target distance is determined by a processor using one or more inputs from one or more sensors. In some embodiments, the first element 120 is an acoustic emitter, and the second element 122 is an acoustic reflector. As described herein, in some embodiments, the first element 120 and the second element 122 are both acoustic emitters.In some embodiments, the acoustic waves generated by both acoustic emitters may be combined to form a constructive standing wave as described herein.

[0066] In some embodiments, the one or more sensors are a simple switch configured to trigger movement of the mount 140 when one or more threshold levels have been exceeded. For example, the one or more sensors may be a capacitance sensing switch. In this example, when the capacitance between the first element 124 and the second element 126 is below the threshold value, the switch allows electric current to flow through the circuit. When the capacitance is interrupted, such as through an increased cell density within the interior chamber 120 decreasing capacitance beyond a threshold value, the electrical current would be interrupted. Accordingly, in some embodiments, the cell culture system 102, for example through the distance control system 106, may be configured to translate the second element 126 in response to a sensor detecting an input above or below a threshold value.

[0067] In another aspect, embodiments described herein relate to methods of culturing cells. The method may include disposing a first plurality of cells having a first cell density in a chamber of a culture compartment 110. In some embodiments, the method includes actuating an acoustic actuation device 138 to generate an acoustic field within the interior chamber 118. In some embodiments, the acoustic actuation device 138 emits an acoustic wave 602 (see e.g., FIG. 6A) from a first element 120 such as an emitter to a second element 122 such as a reflector. In some embodiments, the first element 120 (e.g., an emitter) may be disposed at or about a distal end of the culture compartment 136, while the second element 122 (e.g., a reflector) may be disposed proximal and opposite to the emitter. In some embodiments, the method comprises adjusting a distance between the emitter and second element 122 towards a target distance therebetween, so as to produce or substantially produce a standing wave within the interior chamber to help maintain the first plurality of cells in a suspension, and expand the first plurality of cells to a second plurality of cells having a second cell density, wherein the second cell density is greater than the first cell density. In some embodiments, the method includes pausing or terminating the transmission of the acoustic wave thereby causing the cells (first and / or second plurality of cells) to settle at the bottom of the culture compartment 110. In some embodiments, the method includes determining the second density of cells within the culture compartment 110. In some embodiments, the method includes harvesting cells when the second cell density has exceeded a threshold value between 1-10 M / mL. In some embodiments, the method includes harvesting cells when the second cell density has exceeded a threshold value between 1-100 M / mL. In some embodiments, the method includes harvesting cells when the second cell density has exceeded a threshold value of 50 M / mL.

[0068] FIG. 2A illustrates an example of another cell culture system 202 according to embodiments described herein. In some embodiments, the cell culture system 202 includes similar features to cell culture system 102, aside from the features described herein. In some embodiments, a distance control system 204 comprises a height control system 206 comprising a mount 208 coupled to an arm 210 connecting the mount 208 to a hand 212. In some embodiments, the hand 212 has a cylindrical body with an open lumen extending at least partially therethrough. In some embodiments, the mount 208 extends from a first end of the mount 214 to a second end of the mount 216 along a longitudinal axis of the mount 208 and the arm 210 extends outwardly from and normal to the longitudinal axis of the mount 208. In some embodiments, the hand 212 is configured to have an inner diameter (not shown) of the lumen greater than the piston 218 thereby permitting the piston 218 to pass through the center lumen of the hand 212. In some embodiments, the mount 208 is configured to translate the hand 212 about a longitudinal axis of the piston 218.

[0069] In some embodiments, any cell culture system described herein comprises an actuation device configured to emit an acoustic wave in a direction that may be perpendicular to a longitudinal axis of a culture compartment (as described herein), and / or in a direction that may be transverse to a longitudinal axis of a culture compartment (as described herein). For example, in some embodiments, an emitter of an actuation device is disposed on a lateral wall (e.g., lateral wall 118 on FIG. 1) of the culture compartment, while an opposing emitter or reflector is either located about an opposite lateral wall or within the interior chamber but opposite to the emitter of the first element. Accordingly, the lateral wall actuation device will generate an acoustic wave that travels perpendicular to the longitudinal axis of the culture compartment. In some embodiments, any number of actuation devices can be included with the cell culture system to provide acoustic waves in multiple dimensions. For example, in some embodiments, a cell culture system described herein includes a first acoustic actuation device (e.g., first element 124 in FIG. 1), and a second actuation device disposed on a lateral wall that generates an acoustic wave in a direction perpendicular to the direction of the acoustic wave generated by the first acoustic actuation device. In some embodiments, a second element of each acoustic actuation device is configured to have its position individually adjusted, as described herein. FIG. 2A illustrates an example of a cell culture system 202 according to embodiments described herein. In some embodiments, a distance control system 204 comprises a height control system 206 comprising a mount 208 coupled to an arm 210 connecting the mount 208 to a hand 212. In some embodiments, the hand 212 has a cylindrical body with an open lumen extending at least partially therethrough. In some embodiments, the mount 208 extendsfrom a first end of the mount 214 to a second end of the mount 216 along a longitudinal axis of the mount 208 and the arm 210 extends outwardly from and normal to the longitudinal axis of the mount 208. In some embodiments, the hand 212 is configured to have an inner diameter (not shown) of the lumen greater than the piston 218 thereby permitting the piston 218 to pass through the center lumen of the hand 212. In some embodiments, the mount 208 is configured to translate the hand 212 about a longitudinal axis of the piston 218.

[0070] In some embodiments, the hand 212 is coupled with the piston 218 so as to translate the piston and a second element 220 (that may be coupled with the piston, as described herein) within a culture compartment 222 to be a desired distance from the first element 224. In some embodiments, a bearing 226 extends proximally from the proximal end of the culture compartment 228 towards the hand 212. In some embodiments, the bearing 226 comprises a linear ball bearing.

[0071] In some embodiments, the cell culture apparatus 230 and the distance control system 204 are mounted on a base 232. In some embodiments, the first element 224 is at least partially disposed in the surface of the base 232. In some embodiments, the cell culture apparatus 230 and the distance control system 204 are each loaded on respective bases 232 separated by a distance.

[0072] In some embodiments, the cell culture system 202 comprises a heating j acket (not shown) disposed at least partially around the culture compartment 222 and configured to maintain the temperature within the culture compartment 222 at a desired temperature for cell growth and division. For example, the heating j acket may be configured to maintain the interior of the culture compartment 222 between 34-80 °C. In some embodiments, the heating jacket may be configured to maintain the interior of the culture compartment 222 between 25-80 °C.

[0073] FIG. 2B illustrates a cross sectional view of the exemplary cell culture system 202 of FIG. 2A, according to an embodiment described herein. The second element 220 is disposed within the interior chamber 234 of the culture compartment 222 and configured to be moved to a desirable distance from the first element 224.

[0074] In some embodiments, the bearing 226 incudes a bearing groove 236 configured to at least partially receive a first sealing member (not shown). For example, the first sealing member may be an O-ring. In some embodiments, the bearing groove 236 is configured to receive the first sealing member so as to form a fluid-tight seal between a first portion ofthe bearing 226 disposed at least partially within the interior chamber 234 and a portion of the walls defining the interior chamber 234.

[0075] In some embodiments, the bearing 226 is configured to receive a second sealing member 238 at least partially within a bearing lumen 240. In some embodiments, the second sealing member 238 is configured to form a fluid tight seal between at least a portion of the walls defining the bearing lumen 240 and the piston 218. In some embodiments, the second sealing member 238 is configured to allow the piston 218 to slide in a proximal and / or distal direction thereof.

[0076] In some embodiments, a heat transfer element 242 is disposed at a location beneath the first element 224. In some embodiments, the heat transfer element 242 is configured to provide heating and / or cooling to the culture compartment 222. In some embodiments, the base support 244 is disposed at a location beneath the heat transfer element 242 and configured to provide support to the culture compartment 222.

[0077] FIG. 3A illustrates an isometric cross-sectional view and FIG. 3B illustrates a planar cross-sectional view of a culture compartment 222, according to an embodiment described herein. In some embodiments, the culture compartment 222 has a first port 302 and a second port 304. In some embodiments, at least one of the first port 302 and the second port 304 form an opening through the walls defining the interior chamber 234.

[0078] In some embodiments, the culture compartment 222 comprises a dividing wall 306 at least partially isolating a section of the interior chamber 234. In some embodiments, the dividing wall 306 separates a first portion of the interior chamber 234 having a second element 220 from a second portion of the interior chamber 234 having the first port 302 and the second port 304.

[0079] In some embodiments, the dividing wall 306 at least partially comprises a membrane. In some embodiments, the membrane of the dividing wall 306 is configured to be permeable to cell culture media components provided to the interior chamber 234 by the flow compartment 308. For example, the membrane of the dividing wall 306 may be permeable to nutrients and proteins within the cell media while remaining impermeable to cells. In some embodiments, the membrane comprises a hermetic seal adapted to separate the interior chamber 234 from the flow compartment 308.

[0080] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D illustrate a planar, isometric planar, isometric cross-sectional, and planar control system view, respectively, of a cell culture system 402 having a compressible culture compartment 404 according to an embodiment described herein. As shown in FIG. 4A, for example, the culture compartment 404 maycomprise a flexible outer wall 406 configured to be compressed when pressure from a first element 420 coupled to a distal end of a piston 410 is applied. In some embodiments, the volume within the culture compartment 404 may be controlled by compressing the culture compartment 404 between the first element 420 and second element 408. In some embodiments, the culture compartment 404 is configured to transition between a compressed configuration having a first volume and an extended configuration have a second volume greater than the first volume.

[0081] In some embodiments, the culture compartment 404 is located in contact with a pedestal 412. In some embodiments, the pedestal 412 is a heat transfer element. For example, the pedestal 412 may have a first port 414 configured to permit fluid of a desired temperature to enter the pedestal 412 and a second port 416 configured to permit the fluid entering the first port 414 to exit the pedestal 412. In some embodiments, fluid injected into the pedestal 412 is configured to heat and / or cool the culture compartment 404 to a desired temperature for cell growth.

[0082] As shown in FIG. 4C, for example, the culture compartment 404 may comprise an interior chamber 418 surrounded by the flexible outer wall 406 that is configured to be deformed to decrease the height and / or volume of the culture compartment 404. As shown in FIG. 4D, in some embodiments, the cell culture system 402 is located with the distance control system 204.

[0083] FIG. 5 A illustrates an example of a culture compartment 502 according to an embodiment described herein. In some embodiments, the culture compartment 502 includes a height control system 504, a first element 506, and a second element 508. In some embodiments, the first element 506 is an acoustic transducer. In some embodiments, the first element 506 is the first element 124 of FIG. 1 and / or a first element 606 of FIG. 6. In some embodiments, the second element 508 is the second element 126 or FIG. 1 and / or a second element 608 of FIG. 6. In some embodiments, the second element 508 is a reflector.

[0084] In some embodiments, the second element 508 comprises one or more acoustic compartments 510. In some embodiments, the second element 508 comprises a plurality of acoustic compartments 510 separated by acoustic walls. In some embodiments, one or more acoustic compartments 510 acts as a reflector for acoustic waves transmitted by the first element 506. For example, the second element 508 shown in FIG. 5A has a first acoustic wall 510, a second acoustic wall 512, a third acoustic wall 514, a fourth acoustic wall 516, and a fifth acoustic wall 518. In some embodiments, each acoustic compartment 520 is configured to be liquid tight.

[0085] In some embodiments, the second element 508 is made of a material configured to provide sufficient rigidity to withstand an acoustic field. In some embodiments, the second element 508 comprises a poly-methyl-methacrylate (PMMA) or a polylactic acid (PLA) material. In some embodiments, the acoustic compartment(s) are configured to hold air, or other types of fluids known to those skilled in the art. In some embodiments, the material used to form the second element 508 provides substantially no reflection compared to the reflection by the medium within the acoustic compartment(s) (e.g., air) of the second element 508. In some embodiments, the second element 508 is about 2 mm thick.

[0086] In some embodiments, as described herein, the acoustic compartment 520 is filled with air. In some embodiments, the second element 508 is an air reflector configured to modify, by reflection, the spatial distribution of the flow of acoustic radiation. Different materials have different acoustic reflection coefficients. In particular, using a reflector filled with air allows for controlled deformability and tunable shapes of the reflector, thereby providing further control over the formed acoustic field. In some embodiments, the second element 508 being an air reflector may be configured to modify, by reflection, the spatial distribution of the flow of acoustic radiation.

[0087] The acoustic reflection coefficient stems from the difference in mechanical properties between the liquid, such as the cell culture media, and the reflector. Using different compositions for the cell culture media may affect the reflection coefficient. Different compositions of cell media may have a substantially smaller effect on the reflection coefficient compared to the material used for the reflector itself (e.g., a metal reflector or an air reflector versus cell culture media suspending the cells).

[0088] For example, the distance between the first element 506 and the second element 508 may be adjusted to a desired distance by moving the second element 508 in a distal or proximal direction using the height control system 504. In some embodiments, a circuit 522 comprising a sensor and a processor is configured to detect the distance between the first element 506 and second element 508 and / or transmit a signal to the height control system 504 to direct the height control system 504 to move in a proximal or distal direction to achieve a desired distance between the first element 506 and the second element 508. In some embodiments, the circuit 522 includes a sensor configured to detect resonant distance, which may correspond to a target distance as described herein that corresponds to forming a standing wave. In some embodiments, the processor determines when the sensor has detected a resonant distance and / or an optimal distance by using a lookup table.

[0089] FIG. 5B illustrates an example of FIG. 5 A with a floating second element 508 according to embodiments described herein. In some embodiments, the culture compartment 502 comprises a height control system 504 that modulates the level of fluid within the culture compartment to adjust the position of the second element 508 (since it is configured to float). For example, the second element 508 may be configured to float at least partially on top of the liquid within the interior chamber 524 of the culture compartment 502. In some embodiments, the culture compartment 502 comprises one or more openings thereon (e.g., 526, 528 ) configured to receive and / or dispense liquid from therein. In some embodiments, the first port 526 may be used to provide fresh cell culture media to the interior chamber 524 and / or to remove waste components from the interior chamber 524 of the culture compartment 502. In some embodiments, the culture compartment 502 comprises a first port 526 and a second port 528. In some embodiments, one of the first port 526 and the second port 528 is configured to transport fluid such as fresh cell media to the culture compartment 502 and the other port is configured to transport fluid such as waste or cells out of the culture compartment 502.

[0090] In some embodiments, the culture compartment 502 comprises a first opening (e.g., 526) configured to receive fluid (e.g., cell culture media as described herein). In some embodiments, the culture compartment comprises a second opening (e.g., 528) configured to dispense the fluid therein (e.g., cell culture media). In some embodiments, a supply pump is used to supply the fluid to the culture compartment. In some embodiments, a withdrawal pump is used to draw fluid out from the culture compartment. In some embodiments, no pump is used to draw fluid out from the culture compartment, but instead the fluid flows out due to pressure from the static head of the fluid.

[0091] In some embodiments, the height control system for the culture compartment 502 uses similar processor(s) and sensors in determining a target distance between the first and second element, and for determining a current distance between the first and second elements. In some embodiments, the height control system is configured to control a pump flow rate for a supply pump and / or a withdrawal pump (as described herein) so as to modulate a height of the fluid within the culture chamber, and thereby modulate a distance between the first and second elements. Accordingly, if the height control system for culture compartment 502 detects a distance between the first and second elements deviating from and / or not being at a distance that corresponds to a standing wave distance, a controller (e.g., a valve, or other flow restriction device) on a channel for the fluid in fluid communication with one or both of a supply pump and / or a withdrawal pump is modulated so as to allow either more fluid to be entered in the culture compartment to raise the fluidlevel (thereby increasing the distance between the first and second elements), or more fluid to be withdrawn (e.g., dispensed) from the culture compartment so as to lower the fluid level (thereby decreasing the distance between the first and second elements). Once the target distance between the first and second elements is achieved, the height control system is configured to adjust the flow rates of the fluid entering and / or exiting the culture compartment 502 to be at or substantially at steady-state.

[0092] In some embodiments, the second port 528 is configured to transport cells out of the second port 528 such as to be harvested. In some embodiments, the cells are harvested from the second port 528 using a syringe or sampling pouch using a pump. In some embodiments, the pump is a peristaltic pump.

[0093] FIG. 6 illustrates an example of an acoustic actuation device 610 according to embodiments described herein. In some embodiments, the acoustic actuation device 610 includes an interior chamber 612, a first element 606, a second element 608, and a signal generator and sensor 614. In some embodiments, the second element 608 is a reflector. In some embodiments, the second element 608 is the second element 126 of FIG. 1. In some embodiments, the first element 606 is the first element 124 of FIG. 1. In some embodiments, the first element 606 comprises a piezoelectric ceramic.

[0094] In some embodiments, an electrical wave is generated by a signal generator and sensor 614 and converted by a first element 606 into an emitted acoustic wave 616. In some embodiments, the acoustic wave 616 propagates through the liquid medium, reflects on the second element 608 acting as a reflector to form a reflected wave 618 that pushes back against the first element 606. In some embodiments, the quality and / or strength of the reflected wave 618 is monitored by measuring the first element 606 voltage. In some embodiments, one or more hydrophones (not shown) are used to measure the pressure of the reflected wave 618.

[0095] In some embodiments, a baseline is determined prior to adding a plurality of cells to the resonance cavity of the acoustic actuation device 610 to determine zero distance between the first element 606 and the second element 608. For example, the second element 608 may be lowered to abut the first element 606 such that the distance between the second element 608 and the first element 606 is zero. A processor (as described herein) may determine the voltage of the first element 606 or signals measured by one or more sensors at this distance and set the measured values to correlate to a baseline zero distance.

[0096] In some embodiments, the potential wavelength of an emitted acoustic wave is continuously and / or automatically computed by a processor of a control system (e.g.,distance control system as described herein) configured to receive inputs from the signal generator and sensor 614. For example, the equation (X=c / f) may be used to determine a wavelength of the acoustic wave by inputting the frequency generated and measuring or inferring the interior chamber 612 temperature. The processor (e.g., as described herein with the distance control system) may transmit one or more signals to a controller to adjust the distance between the first element 606 and the second element 608 to optimize the amplitude of the reflected wave 618. In some embodiments, the system may be configured to continuously and / or automatically adjust the distance between the first element 606 and the second element 608 for producing a reflected wave 618. In some embodiments, the system may be configured to produce a reflected wave 618 of a predetermined amplitude or frequency by adjusting the distance between the first element 606 and the second element 608.

[0097] In some embodiments, the amplitude of the emitted acoustic wave 616 is configured to maintain cells within one or more layers. In some embodiments, the system is configured to mismatch the amplitude of the emitted acoustic wave 616 thereby causing a column of cell layers to shift slightly. For example, the shifting column of cells may enhance nutrient exchange for cells.

[0098] In some embodiments, a plurality of cell layers are stacked along the reflected wave 618 and the acoustic wave 616 such that each layer of cells is located at the intersection of the acoustic wave 616 and the reflected wave 618.

[0099] FIG. 7 illustrates an example of a culture compartment 702 having a column of cell layers 704 according to an embodiment described herein. In some embodiments, cells within the culture compartment 702 form one or more cell layers 706 in response to acoustic waves. In some embodiments, a plurality of cell layers 706 are substantially stacked on top of one another about a longitudinal axis of the culture compartment 702 to form a column of cell layers 704. In some embodiments, each cell layer 706 in the column of cell layers 704 is separated by a gap 708. In some embodiments, the culture compartment 702 is configured to form one or more cell layers 706 where the incident wave (e.g., emitted acoustic wave) and reflected wave intersect to form the standing wave. In some embodiments, the gap 708 is between 0.2-0.4 mm. In some embodiments, the gap 708 may be configured to be between 50 pm -2 mm. In some embodiments, the cell layer 706 is between about 1-5 cells thick. In some embodiments, the cell layer 706 is between about 1- 20 cells thick. In some embodiments, the cell layer 706 is three cells thick. In some embodiments, the cell density within a given cell layer 706 is greater 500 M cells / mL. Insome embodiments, a given cell layer 706 has between 0.1-1.0 million cells. In some embodiments, a given cell layer 706 has between 10 thousand to 10 million cells.

[0100] In some embodiments, the culture compartment 702 is configured to form one or more cell layers 706 at a trapping site 710 where cells are concentrated. In some embodiments, as the frequency of the acoustic wavelength within the culture compartment 702 is increased, the number of cell layers 706 increases and the concentration of cells in each layer decreases.

[0101] In some embodiments, the culture compartment 702 comprises a plurality of column of cell layers 704 comprising a plurality of cell layers 706.

[0102] FIG. 8 illustrates an exemplary depiction of relative pressure within a culture compartment 802 including a column of cell layers 804 disposed between a first element 806 and a second element 808, according to an embodiment described herein. In some embodiments, the first element 806 is an acoustic emitter or an acoustic reflector. In some embodiments, the second element 808 is an acoustic emitter or an acoustic reflector. The layers of minimum pressure 810 and maximum pressure 812 may each be separated by a gap 814. In some embodiments, the layers of the maximum pressure 812 and the minimum pressure 810 alternate through a longitudinal axis of the column of cell layers 804. In some embodiments, cells relocate to minimum pressure 810 layers (i.e., trapping sites) when a standing wave is formed within the culture compartment 802.

[0103] Furthermore, within each layer of minimum pressure 810, reflection of the acoustic wave in a direction perpendicular to the longitudinal axis of the column of cell layers 804 (i.e., latitudinal direction) create latitudinal minimum pressure 816 depicted with thicker lines and latitudinal maximum pressure 818 depicted with thinner lines. In some embodiments, a first concentration of cells located in the latitudinal minimum pressure 816 is greater than a second concentration of cell located in the latitudinal maximum pressure 818. In some embodiments, a first cell density located in the latitudinal minimum pressure 816 is greater than a second cell density located in the latitudinal maximum pressure 818.

[0104] FIG. 9 illustrates another example of relative trapping force distribution within a culture compartment 802 when an acoustic wave is emitted from one end and reflected from a second opposite end. The trapping force as described herein may help to maintain cells at or about a particular position. FIG. 9 includes a scale 902 illustrating increasing trapping force corresponding with color. In this example, the lower the number represents a lower trapping force and a higher number on the scale 902 represents a higher trapping force. For example, within a given cell layer a latitudinal maximum trapping force 904 and alatitudinal minimum trapping force 906 may be formed. In some embodiments, a first concentration of cells located in the latitudinal minimum trapping force 906 is greater than a second concentration of cell located in the latitudinal maximum trapping force 904. In some embodiments, a first cell density located in the latitudinal minimum trapping force 906 is greater than a second cell density located in the latitudinal maximum trapping force 904. In some embodiments, cells and / or cell density accumulates within a cell layers first and then laterally within trapping sites.

[0105] Certain examples of the present disclosure were described above. It is, however, expressly noted that the present disclosure is not limited to those examples, but rather the intention is that additions and modifications to what was expressly described herein are also included within the scope of the disclosed examples. Moreover, it is to be understood that the features of the various examples described herein were not mutually exclusive and may exist in various combinations and permutations, even if such combinations or permutations were not made express herein, without departing from the spirit and scope of the disclosed examples. In fact, variations, modifications, and other implementations of what was described herein will occur to those of ordinary skill in the art without departing from the spirit and the scope of the disclosed examples. As such, the disclosed examples are not to be defined only by the preceding illustrative description.

[0106] In the appended claims, the terms "including" and "in which" are used as the plain- English equivalents of the respective terms "comprising" and "wherein," respectively. Moreover, the terms "first," "second," "third," and so forth, are used merely as labels and are not intended to impose numerical requirements on their objects.

[0107] The foregoing description of examples has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the present disclosure be limited not by this detailed description, but rather by the claims appended hereto. Future filed applications claiming priority to this application may claim the disclosed subject matter in a different manner and may generally include any set of one or more limitations as variously disclosed or otherwise demonstrated herein.

Claims

CLAIMSWhat is claimed is:

1. A cell culture system, comprising: a culture compartment configured to culture and expand a first plurality of cells having a first cell density to produce a second plurality of cells having a second cell density, wherein the second cell density is greater than the first cell density, and wherein the culture compartment comprises an interior chamber configured to receive the first and second plurality of cells: a flow compartment configured to provide cell culture media to the culture compartment to support the expansion of the first plurality of cells into the second plurality of cells; a membrane disposed at least partially between the culture compartment and the flow compartment, the membrane configured to be permeable to the cell culture media; and an acoustic actuation device comprising: a first element disposed about a distal end of the culture compartment; and a second element disposed proximal and opposite to the first element, wherein a distance between the first element and the second element is adjustable, such that a given volume of a resonant cavity defined within the interior chamber between the first and second element is configured to be adjustable between a first volume and a second volume, wherein the first volume is different from the second volume.

2. The cell culture system of claim 1, wherein the second element is disposed within the interior chamber.

3. The cell culture system of claim 1 or claim 2, wherein the second element is configured to translate along a longitudinal axis of the interior chamber in a proximal or distal direction.

4. The cell culture system of any previous claim, wherein the second element is coupled to a mount that is slidable along a translation rod so as to translate the second element along the longitudinal axis.

5. The cell culture system of any previous claim, wherein movement of the mount along the translation rod is actuated via a motor system, a hydraulic system, an electromagnetic system, or any combination thereof.

6. The cell culture system of any previous claim, wherein the cell culture system further comprises one or more arms extending from the mount to a piston, wherein the second element is coupled to a distal end of the piston, and wherein the piston is disposed through a proximal end of the culture chamber.

7. The cell culture system of any previous claim, wherein translation of the second element within the culture chamber is automatic.

8. The cell culture system of any previous claim, wherein the cell culture system further comprises a distance control system comprising an actuator configured to translate the second element along the longitudinal axis.

9. The cell culture system of any previous claim, wherein the distance control system comprises a processor configured to determine a target distance between the first element and the second element, and an actuator configured to move the mount to position the second element to a position spaced apart from the first element by the target distance.

10. The cell culture system of any previous claim, wherein the target distance is determined based on a desired wavelength of an acoustic wave between the first and second elements.

11. The cell culture system of any previous claim, wherein the target distance correlates to a distance adapted to produce a standing wave between the first and second elements.

12. The cell culture system of any previous claim, wherein the standing wave is configured to suspend the one or more of the first plurality of cell and the second plurality of cells within the interior chamber.

13. The cell culture system of any previous claim, wherein the distance control system is in communication with the acoustic actuation device, such that the distance control system receives input relating to the wavelength of the acoustic wave emitted by the first or second element.

14. The cell culture system of any previous claim, wherein the cell culture system further comprises one or more sensors configured to transmit signals for i) determining a wavelength of the acoustic wave, ii) measuring a distance between the first and second element, ii) determining a voltage between the first and second elements, iv) measuring an amplitude of a pressure field within the chamber, v) determining a capacitance between thefirst element and the second element , vi) determining an electrical potential between the first element and the second element, or any combination thereof.

15. The cell culture system of any previous claim, wherein the distance control system further comprising i) a processor configured to determine the target distance using the one or more sensors, and ii) the actuator configured to move the second element.

16. The cell culture system of any previous claim, wherein the target distance is determined by a processor using one or more inputs from one or more sensors.

17. The cell culture system of any previous claim, wherein the first element is an acoustic emitter, and the second element is an acoustic reflector.

18. A method of culturing cells, comprising: disposing a first plurality of cells having a first cell density in a chamber of a culture compartment; actuating an acoustic actuation device to generate an acoustic field within the chamber, wherein the acoustic actuation device emits an acoustic wave from an emitter to a reflector, the emitter disposed at a distal end of the chamber, the reflector disposed proximal and opposite to the emitter; and adjusting a distance between the emitter and reflector towards a target distance therebetween, so as to produce or substantially produce a standing wave within the chamber to maintain the first plurality of cells and expand the first plurality of cells to a second plurality of cells having a second cell density, wherein the second cell density is greater than the first cell density.

19. A system for continuously producing a standing wave, the system comprising: an acoustic emitter disposed at a distal end of a chamber within a housing; an acoustic second element disposed proximal and opposite to the acoustic emitter, the acoustic second element configured to be translated within and along a longitudinal axis of the chamber such that a distance between the acoustic emitter and the acoustic second element is adjustable; a distance control system comprising a processor configured to determine i) a wavelength of an acoustic wave emitted by the acoustic emitter, ii) a measured distance between the acoustic emitter and the acoustic second element, and iii) a target distance therebetween that correlates to a standing wave; wherein, the target distance is based on the wavelength of the acoustic wave; andwherein, the distance control system comprises an actuator configured to translate the acoustic second element within the chamber to a position spaced apart from the acoustic emitter by the target distance.

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