Cell culture systems and methods

Abstract

This invention generally describes systems and methods for performing cell culture. Some such systems include one or more features that facilitate cell culture in a relatively advantageous manner. For example, such systems may include one or more features that facilitate cell culture in conjunction with components provided as part of or outside the system, such as incubators, imaging systems, and / or external reagent sources. As three further examples, such systems may include one or more features that facilitate use of the system in a flow-through manner, facilitate real-time sensing of one or more properties of the liquid in which cells are cultured, and / or allow the system (and / or one or more of its components) to have a relatively small volume. In some embodiments, the system has a combination of some or all of the advantages described above, which facilitates multiple experiments to evaluate the effects of different cell culture conditions on cell growth. Some of these experiments may ideally be performed without requiring substantial amounts of reagents and / or cells, and / or may ideally be performed in a single incubator. Some methods described herein may involve performing cell culture in one or more of the systems described herein.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 633536, entitled “Cell Culture Systems and Methods,” filed April 12, 2024, under 35 USC § 119(e), which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This invention generally describes systems and related methods for performing cell culture. Background Technology

[0004] In vitro cell culture can be performed using such systems. However, many of these systems exhibit one or more drawbacks, such as incompatibility with online monitoring of conditions, incompatibility with simple and / or automated reagent delivery, and / or the need to operate at undesirable high volumes.

[0005] Therefore, it would be beneficial to develop new systems and methods to address these issues. Summary of the Invention

[0006] This disclosure generally describes systems and methods. In some cases, the subjects described herein relate to interrelated products, alternative solutions to specific problems, and / or multiple uses of one or more systems and / or items.

[0007] Paragraph 1: In some embodiments, a system for performing cell culture is provided. The system includes a valve, a cell culture chamber, and a sensor. The valve is in fluid communication with a plurality of ports. The cell culture chamber includes a cell filter. At least a portion of the surface of the cell culture chamber is transparent to light of at least one wavelength. The cell culture chamber has a volume of less than or equal to 500 microliters. The system also includes a mixing chamber, a mixing element, and / or a flow path configured to facilitate mixing.

[0008] Paragraph 2: In some embodiments, a method for performing cell culture is provided. The method includes flowing liquid through a cell culture chamber and retaining cells in the cell culture chamber as the liquid flows through it. The cell culture chamber contains cells. The cell culture chamber has a volume of less than or equal to 500 microliters. The cell culture chamber is present in a system for performing cell culture. The system also includes a pump, valves, and sensors. The system further includes a mixing chamber, mixing elements, and / or flow paths configured to facilitate mixing. The valves are in fluid communication with a plurality of ports. At least a portion of the surface of the cell culture chamber is transparent to light of at least one wavelength.

[0009] Paragraph 3: In some embodiments, a system for performing cell culture includes a pump, a valve, a cell culture chamber, a sensor, an optical detector, and a breathable membrane. The valve is in fluid communication with multiple ports. The cell culture chamber includes a cell filter. The cell culture chamber has a volume of less than or equal to 500 microliters. The system also includes a mixing chamber, mixing elements, and / or flow paths configured to facilitate mixing.

[0010] Paragraph 4: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system is located on a substrate.

[0011] Paragraph 5: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the substrate is a chip.

[0012] Paragraph 6: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is a microfluidic chip.

[0013] Paragraph 7: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the surface of the chip is transparent to light of at least one wavelength.

[0014] Paragraph 8: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is transparent to light of at least one wavelength.

[0015] Paragraph 9: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is formed of a biocompatible material.

[0016] Paragraph 10: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is formed of a sterilizable material.

[0017] Paragraph 11: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is formed of plastic.

[0018] Paragraph 12: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the plastic includes polystyrene, PMMA, cyclic olefin copolymers, polycarbonate, polysulfone, polyetherimide, polyurethane, and / or 3D printing resin.

[0019] Paragraph 13: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is formed from PDMS.

[0020] Paragraph 14: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is formed of glass.

[0021] Paragraph 15: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the chip is disposable.

[0022] Paragraph 16: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes an incubator.

[0023] Paragraph 17: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is located within the incubator.

[0024] Paragraph 18: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a pump.

[0025] Paragraph 19: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the pump is a rotary membrane pump, peristaltic pump, rotary pump, piston pump, or diaphragm pump.

[0026] Paragraph 20: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the pump is a rotary membrane pump.

[0027] Paragraph 21: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the pump is a roller pump.

[0028] Paragraph 22: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes using the pump to flow the liquid through the cell culture chamber.

[0029] Paragraph 23: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the valve is a pneumatic valve, a rotary valve, a rotary diaphragm valve, a shape memory alloy valve, and / or a solenoid valve.

[0030] Paragraph 24: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the valve is a rotary diaphragm valve.

[0031] Paragraph 25: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein at least one port is in fluid communication with a reagent source.

[0032] Paragraph 26: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein at least one port is in fluid communication with a waste chamber.

[0033] Paragraph 27: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein flowing the liquid through the cell culture chamber includes flowing the liquid from the reagent source through a port and then into the cell culture chamber.

[0034] Paragraph 28: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein flowing the liquid through the cell culture chamber includes flowing the liquid out of the cell culture chamber, through a port, and then into the waste chamber.

[0035] Paragraph 29: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes allowing fluid to flow through a port, through the valve, and into the cell culture chamber.

[0036] Paragraph 30: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes allowing fluid to flow out of the cell culture chamber, through the valve, and through a port.

[0037] Paragraph 31: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the fluid comprises a cell culture medium.

[0038] Paragraph 32: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the valve enables two or more ports to be in fluid communication with the first mixing chamber simultaneously.

[0039] Paragraph 33: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the valve enables two or more ports to be in simultaneous fluid communication with the second mixing chamber.

[0040] Paragraph 34: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes pumping fluid from a port through the valve and into the first mixing chamber.

[0041] Paragraph 35: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, further comprising simultaneously pumping a first fluid from a first port through the valve and into the first mixing chamber, and pumping a second fluid from a second port through the valve and into the first mixing chamber.

[0042] Paragraph 36: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes pumping fluid from a port through the valve and into the second mixing chamber.

[0043] Paragraph 37: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber contains cells.

[0044] Paragraph 38: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cells include adherent cells.

[0045] Paragraph 39: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the adherent cells include HeLa cells, HEK293 cells, iPSC cells, and / or hMSC cells.

[0046] Paragraph 40: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cells include suspended cells.

[0047] Paragraph 41: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the suspended cells include CHO cells, iPSC aggregates, T cells, HEK 293S cells, and / or Jurkat cells.

[0048] Paragraph 42: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is substantially composed of channels.

[0049] Paragraph 43: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the channel is a microfluidic channel.

[0050] Paragraph 44: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber has a volume greater than or equal to 30 microliters and less than or equal to 500 microliters.

[0051] Paragraph 45: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the surface of the cell culture chamber is transparent to light of at least one wavelength.

[0052] Paragraph 46: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein at least a portion of the cell culture chamber is transparent to light of at least one wavelength.

[0053] Paragraph 47: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is transparent to light of at least one wavelength.

[0054] Paragraph 48: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the wavelength of the light is greater than or equal to 400 nm and less than or equal to 700 nm.

[0055] Paragraph 49: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the transparency is greater than or equal to 70%.

[0056] Paragraph 50: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is formed of a biocompatible material.

[0057] Paragraph 51: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the cell culture chamber is formed of a sterilizable material.

[0058] Paragraph 52: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is formed of plastic.

[0059] Paragraph 53: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the plastic comprises polystyrene and / or PMMA.

[0060] Paragraph 54: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the fluid dead zone occupies less than or equal to 20% of the volume of the cell culture chamber.

[0061] Paragraph 55: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the fluid dead zone occupies less than or equal to 5% of the volume of the cell culture chamber.

[0062] Paragraph 56: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the system includes a cell filter, which is a filter, a helical inertial separator, a hollow fiber membrane, and retains cells by sedimentation, by centrifugal force, by dielectric electrophoresis, by acoustic separation, and / or by deterministic lateral displacement.

[0063] Paragraph 57: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a cell filter as a filter.

[0064] Paragraph 58: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the filter is a microfiltration membrane or a hollow fiber membrane.

[0065] Paragraph 59: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the filter is a microfiltration membrane.

[0066] Paragraph 60: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the filter is flat.

[0067] Paragraph 61: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the filter is located on the valve.

[0068] Paragraph 62: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cells are retained with an efficiency of 70% or greater.

[0069] Paragraph 63: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes the mixing chamber.

[0070] Paragraph 64: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing chamber is in fluid communication with the cell culture chamber.

[0071] Paragraph 65: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing chamber is in fluid communication with the valve.

[0072] Paragraph 66: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing element includes a pump.

[0073] Paragraph 67: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing element includes a mechanical mixer.

[0074] Paragraph 68: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mechanical mixer is a magnetically driven stirring rod or a micro-impeller.

[0075] Paragraph 69: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing element includes a static mixer.

[0076] Paragraph 70: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing element is located in the cell culture chamber.

[0077] Paragraph 71: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes mixing the liquid upstream of the cell culture chamber.

[0078] Paragraph 72: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing is carried out in the mixing chamber.

[0079] Paragraph 73: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing is performed in the cell culture chamber.

[0080] Paragraph 74: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing includes shuttle flow through a filter that retains cells.

[0081] Paragraph 75: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing includes circulating flow via a peristaltic pump.

[0082] Paragraph 76: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing comprises alternating flow through the mixing chamber and / or through the cell culture chamber.

[0083] Paragraph 77: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the mixing includes performing mechanical mixing.

[0084] Paragraph 78: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the flow path includes the cell culture chamber.

[0085] Paragraph 79: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the flow path includes a channel in fluid communication with the cell culture chamber.

[0086] Paragraph 80: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes two mechanically stabilizing orientations, and wherein the mixing includes a flow that occurs when the system shifts from one of the mechanically stabilizing orientations to the other.

[0087] Paragraph 81: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the breathable membrane forms the wall of the cell culture chamber.

[0088] Paragraph 82: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes a breathable membrane for allowing gas to flow through the walls forming the cell culture chamber.

[0089] Paragraph 83: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cell culture chamber is enclosed in a breathable material.

[0090] Paragraph 84: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the breathable membrane comprises a silicone membrane.

[0091] Paragraph 85: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the breathable membrane includes open vents.

[0092] Paragraph 86: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the gas contains a reagent.

[0093] Paragraph 87: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the gas comprises oxygen.

[0094] Paragraph 88: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the optical detector includes a microscope.

[0095] Paragraph 89: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the optical detector includes an optical sensor.

[0096] Paragraph 90: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system further comprises a light source.

[0097] Paragraph 91: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the light source is a fluorescent light source.

[0098] Paragraph 92: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes performing optical measurements.

[0099] Paragraph 93: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the optical measurements are performed on the cell culture chamber.

[0100] Paragraph 94: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the optical measurement includes measuring the optical density of the liquid.

[0101] Paragraph 95: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes determining the amount of cells present in the liquid based on the optical measurements.

[0102] Paragraph 96: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the system includes two mechanically stabilizing orientations, one adapted for cell culture in the cell culture chamber and the other adapted for optical measurement of the cell culture chamber.

[0103] Paragraph 97: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the plurality of ports includes ports through which cells can be seeded.

[0104] Paragraph 98: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the port is sealed with a gasket.

[0105] Paragraph 99: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein cell seeding can be performed by puncturing the septum with a needle and delivering cells therethrough.

[0106] Paragraph 100: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes puncturing the septum with a needle and delivering cells therethrough.

[0107] Paragraph 101: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the port includes a cover.

[0108] Paragraph 102: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a reservoir through which cells can be seeded.

[0109] Paragraph 103: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the reservoir includes a cap.

[0110] Paragraph 104: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the cap is removed during cell seeding.

[0111] Paragraph 105: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes pumping the cells from the port or reservoir into the cell culture chamber.

[0112] Paragraph 106: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the plurality of ports includes ports through which cell recycling can be performed.

[0113] Paragraph 107: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the port is sealed with a gasket.

[0114] Paragraph 108: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein cell recovery can be performed by puncturing the septum with a needle and extracting the cells therefrom.

[0115] Paragraph 109: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes piercing the septum with a needle and extracting cells therethrough.

[0116] Paragraph 110: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the sensor is a dissolved oxygen sensor.

[0117] Paragraph 111: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes measuring the dissolved oxygen content of a liquid present in the system.

[0118] Paragraph 112: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes delivering a quantity of oxygen to the liquid based on a measurement of the dissolved oxygen content.

[0119] Paragraph 113: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes delivering a cell culture medium with a dissolved oxygen content within a range determined based on a measurement of the dissolved oxygen content.

[0120] Paragraph 114: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the sensor is a pH sensor.

[0121] Paragraph 115: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes measuring the pH of a liquid present in the system.

[0122] Paragraph 116: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes delivering a quantity of acid and / or base, the quantity being determined based on a measurement of the pH.

[0123] Paragraph 117: In some embodiments, a system or method as described in any of the paragraphs above is provided, which further includes delivering a cell culture medium with a pH content within a range determined based on the pH measurement results.

[0124] Paragraph 118: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system comprises two or more sensors.

[0125] Paragraph 119: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a sensor for a biological product.

[0126] Paragraph 120: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the biological product is IgG.

[0127] Paragraph 121: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, which further includes measuring the amount of biological products present in a liquid in the system.

[0128] Paragraph 122: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a sensor for carbon dioxide.

[0129] Paragraph 123: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the system further includes sensors for carbon dioxide, cell concentration, cell viability, nutrients, metabolites, temperature, pressure, humidity, and / or viscosity.

[0130] Paragraph 124: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the metabolites include lactate and / or glucose.

[0131] Paragraph 125: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes an optical sensor, an electrochemical sensor, and / or an acoustic sensor.

[0132] Paragraph 126: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid is present in and / or obtained from the cell culture chamber.

[0133] Paragraph 127: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the sensor includes a sensor plug.

[0134] Paragraph 128: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the sensor includes a sensor patch.

[0135] Paragraph 129: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid flows intermittently.

[0136] Paragraph 130: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid flows continuously.

[0137] Paragraph 131: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid flows as part of a batching process.

[0138] Paragraph 132: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid flows as part of a feed batching process.

[0139] Paragraph 133: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the liquid flows as part of an infusion process.

[0140] Paragraph 134: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system is located on a chip, and wherein the chip further includes one or more additional systems.

[0141] Paragraph 135: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein one or more additional systems contain the same components as the system.

[0142] Paragraph 136: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system comprises a chip having two or more modules, and wherein each module comprises the same set of components.

[0143] Paragraph 137: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the components include the pump, the valve, the cell culture chamber, and / or the sensor.

[0144] Paragraph 138: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a first chamber and a second chamber, and wherein the first and second chambers are positioned in series between a port through which a sample can be introduced and / or removed and the cell culture chamber.

[0145] Paragraph 139: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the first chamber is used as a diaphragm valve.

[0146] Paragraph 140: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the second chamber is used as a pump, the pump being configured to pump a known volume of fluid through it.

[0147] Paragraph 141: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein a membrane extends through the first chamber and the second chamber.

[0148] Paragraph 142: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the membrane can be raised and lowered to affect the pressure in the first chamber and / or the second chamber.

[0149] Paragraph 143: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the membrane can be raised to fluidly communicate the first and second chambers with each other, and can be lowered to de-fluidically communicate the first and second chambers with each other.

[0150] Paragraph 144: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes components configured to deliver gas to the cell culture chamber.

[0151] Paragraph 145: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the component configured to deliver gas to the cell culture chamber is in fluid communication with the cell culture chamber via one or more ports.

[0152] Paragraph 146: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the component configured to deliver gas to the cell culture chamber is configured to deliver the gas in a manner that mixes the contents of the cell culture chamber.

[0153] Paragraph 147: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system includes a translational sensor.

[0154] Paragraph 148: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the system comprises two translational sensors.

[0155] Paragraph 149: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the two sensors can be translated together.

[0156] Paragraph 150: In some embodiments, a system or method as described in any of the paragraphs above is provided, wherein the translational sensor includes a pH sensor and / or a dissolved oxygen sensor.

[0157] Paragraph 151: In some embodiments, a system or method as described in any of the preceding paragraphs is provided, wherein the system or method includes measuring the amount of RNA in a fluid present in the cell culture chamber and adjusting the composition of the fluid based on such measurement.

[0158] Other advantages and novel features of the invention will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying drawings. In the event of conflicting and / or inconsistent disclosures in this specification and other documents incorporated by reference, this specification shall prevail. If two or more documents incorporated by reference contain conflicting and / or inconsistent disclosures, the document with the later effective date shall prevail. Attached Figure Description

[0159] Non-limiting embodiments of the invention will be described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is generally represented by a single number. For clarity, not every component is labeled in every figure, nor is every component of every embodiment of the invention shown, unless illustrated to enable those skilled in the art to understand the invention. In the drawings: Figure 1-9 An exemplary system according to some implementation methods is described; Figure 10-53 Various methods and related structures for mixing liquids according to some embodiments are described; Figures 54-58 Exemplary methods according to some implementations are described; Figure 59 A method for introducing cells into a cell culture chamber according to some embodiments is described; Figure 60 A valve according to some embodiments is described; Figure 61-125 Exemplary systems and components thereof according to some implementations are described; Figure 126 Photographs depicting some embodiments show details related to experiments performed as described in the embodiments; Figures 127-136 These are optical micrographs showing experimental data according to some embodiments; Figure 137 It is a graph showing the degree of convergence as a function of time according to some implementation methods; Figure 138 and 139 These are optical micrographs showing experimental data according to some embodiments; Figure 140 It is a graph showing the degree of convergence as a function of time according to some implementation methods; Figure 141 It is a graph showing oxygen as a function of time according to some embodiments; Figure 142 Optical micrographs depicting experimental data according to some embodiments are shown; Figure 143 It is a graph showing the degree of convergence as a function of time elapsed after activation, according to some implementation methods; Figure 144 It is a graph showing the live cell count per image as a function of time, according to some embodiments; and Figure 145 It is a graph showing the phase object count of each image as a function of elapsed time, according to some embodiments. Detailed Implementation

[0160] Systems and methods for performing cell culture are generally described. Some such systems include one or more features that facilitate cell culture in a relatively advantageous manner. For example, such systems may include one or more features that facilitate cell culture in conjunction with components provided as part of or outside the system, such as incubators, imaging systems, and / or external reagent sources. As three further examples, such systems may include one or more features that facilitate use of the system in a flow-through manner, facilitate real-time sensing of one or more properties of the liquid in which cells are cultured, and / or allow the system (and / or one or more of its components) to have a relatively small volume. In some embodiments, the system has a combination of some or all of the advantages described above, which facilitates multiple experiments to evaluate the effects of different cell culture conditions on cell growth. Some of these experiments may ideally be performed without requiring substantial amounts of reagents and / or cells, and / or may ideally be performed in a single incubator. Some methods described herein may involve performing cell culture in one or more of the systems described herein.

[0161] In some implementations, the system described herein may have one or more advantages. Such advantages may include high throughput (which may facilitate reliable statistical analysis and / or rapid testing of different cell culture conditions), the ability to perform real-time imaging, the ability to perform uniform cell seeding, the ability to perform mixing and / or cell suspension, the ability to measure one or more properties of the liquid therein (e.g., dissolved oxygen content, pH, IgG content), the ability to control one or more properties of the liquid therein (e.g., dissolved oxygen content, pH), the ability to deliver oxygen to the liquid therein, the ability to deliver reagents to the liquid therein in an automated manner, the ability to perform perfusion, the ability to retain cells, the ability to sample the liquid therein (e.g., in a cell-free manner), and / or the ability to recover cells present therein.

[0162] Cell culture may include growing cells in the system described herein. For example, cells may be present in a cell culture medium within the system described herein, and one or more properties of the system may be selected to promote cell growth. The cultured cells may be in suspension (e.g., in a cell culture medium) and / or adherent (e.g., attached to components of the system described herein).

[0163] Figure 1 A non-limiting example of the system for cell culture described herein is shown. Figure 1 The system 100 is shown, which includes a cell culture chamber 102. Figure 1 The cell culture chamber 102 shown can be configured to allow liquid to flow through it.

[0164] In some implementations, with Figure 2 As shown in the embodiment, the cell culture chamber allows liquid to pass through two ports (e.g., Figure 2 Ports 204A and 204B in the cell culture chamber flow through it. In such cases, liquid may be able to flow into the cell culture chamber through one of the ports, through part or all of the cell culture chamber, and out from the other port. Each port may include an opening on the surface of the cell culture chamber and / or may have a different design (e.g., the port may include a connector). The cell culture chamber may also allow liquid to be introduced into and / or removed from it through components other than the ports. For example, the cell culture chamber may form part of a channel through which liquid can flow.

[0165] It should be noted that liquids can flow into and out of the cell culture chamber through various surfaces. For example, such as... Figure 1 As shown, in some embodiments, liquid can flow into and out of the cell culture chamber through opposing surfaces perpendicular to the bottom of the cell culture chamber. Liquid can also flow into and / or out of the cell culture chamber through different locations (e.g., different ports) located on a common surface. Figure 3 A non-limiting example of a cell culture chamber with this design (and also including two sensors) is shown. Furthermore, liquid can flow into, out of, and / or through the cell culture chamber horizontally, upwards, and / or downwards.

[0166] The cell culture chamber described herein can have a variety of suitable shapes. In some embodiments, the cell culture chamber includes one or more hexagonal sides. The cell culture chamber may also include sides having a basic hexagonal shape (e.g., a near-hexagonal shape, but in which one or more vertices have been replaced by shorter sides, such as shorter sides with a length of up to 10% of the length of one or more other sides present in the shape). The hexagonal sides may be opposing sides, may include a top side and / or a bottom side, and / or may include one or more sides that include the direction of liquid flow through the cell culture chamber. Without wishing to be bound by any particular theory, it is believed that cell culture chambers containing one or more hexagonal sides may exhibit a smaller dead volume compared to cell culture chambers with different geometries.

[0167] In some implementations, the cell culture chamber includes a cell filter. (Reference) Figure 2 The cell culture chamber 202 may include a cell filter. This is schematically shown by the cell filter 206 depicted therein. The cell filter allows cells to remain within it even as liquid flows through and / or is exchanged. This can be achieved in various suitable ways. In some embodiments, this is achieved by using a component near the location where the liquid exits the cell culture chamber, which prevents cells from flowing out of the cell culture chamber. For example, a membrane or other component (e.g., a spiral inertial separator, an external filter) that allows liquid to flow through but prevents cell flow can be placed in such a location. In some embodiments, such a membrane or other component can be placed on the cell culture chamber and / or a valve (e.g., a valve near the cell culture chamber). This is achieved by… Figure 2 The cell filter 206 located in the cell culture chamber 202 is schematically depicted.

[0168] Non-limiting examples of suitable membranes include microfiltration membranes (e.g., flat-sheet microfiltration membranes) and hollow fiber membranes. In some embodiments, the membrane may comprise polyethersulfone (e.g., it may be a polyethersulfone membrane comprising 0.2-micron or 5-micron pores). The aforementioned components may be flat or may include one or more bends and / or curves.

[0169] In some embodiments, the cell filter includes and / or is located within a flow path. Such a flow path (described in more detail below) may include the cell culture chamber and allow cells to remain within the flow path (and by extension, the cell culture chamber) while allowing liquid to flow in and out of the flow path. In some embodiments, the cell culture chamber may include a cell filter located outside it (e.g., a cell filter in the form of a flow path through the cell culture chamber, or a cell filter located within a flow path through the cell culture chamber).

[0170] In some implementations, cell filters are system components that retain cells through sedimentation, centrifugal force, dielectric electrophoresis, acoustic separation, and / or deterministic lateral displacement.

[0171] In some implementations, the cell culture chamber contains two or more cell filters.

[0172] In some embodiments, the system described herein includes a cell culture chamber and further includes one or more components capable of delivering to and / or configured to deliver one or more liquids and / or reagents to the cell culture chamber. Similarly, the system described herein may include a cell culture chamber and further include one or more components capable of removing from and / or configured to remove one or more liquids and / or reagents from the cell culture chamber. Such components may take the form of pumps and / or valves.

[0173] Figure 4 A non-limiting embodiment of a system 400 comprising a cell culture chamber 402 and a pump 408 is shown. In such a system, the pump may be in fluid communication with the cell culture chamber (e.g., via a conduit, such as...). Figure 4(e.g., conduit 410 in the conduit). In such cases, the pump can pump liquid into the cell culture chamber, for example, by providing positive pressure to the liquid upstream of the cell culture chamber. This may cause liquid to flow downstream into the cell culture chamber. The liquid flowing into the cell culture chamber may pressure the liquid present in the cell culture chamber, causing it to flow out of the cell culture chamber. The pump can also pump liquid out of the cell culture chamber by applying negative pressure to the liquid downstream of the cell culture chamber and / or to the components through which the liquid flows out of the cell culture chamber. In such embodiments, the liquid may be drawn out of the cell culture chamber by the negative pressure, which may cause liquid upstream of the cell culture chamber to flow into the cell culture chamber. In some embodiments, the pump may be able to pump in both directions and / or be configured to pump in both directions (e.g., applying positive pressure to the liquid to make it flow in one direction and applying negative pressure to the liquid to make it flow in the opposite direction, applying positive pressure to the liquid on one side of the liquid to make it flow in one direction and applying positive pressure to the liquid on the opposite side of the liquid to make it flow in the opposite direction). The pump may also be capable of pumping in only one direction and / or be configured to pump in only one direction.

[0174] Various suitable pumps can be used in the systems described herein. Non-limiting examples of suitable pumps include rotary membrane pumps, peristaltic pumps (e.g., tubular pumps), rotary pumps, piston pumps, diaphragm pumps, electric pumps (e.g., syringe pumps), and roller pumps. In some embodiments, the system comprises two or more pumps. In such embodiments, the two or more pumps may include two identical pumps and / or two pumps that differ from each other in one or more respects.

[0175] Figure 5 An example of a diaphragm pump and its operation are shown. Figure 5 As shown, a diaphragm pump may include multiple pump-valve units. These pump-valve units can be actuated in a sequence that promotes peristaltic flow through the diaphragm pump. For example, to drive flow from left to right, a sequence of pump-valve units can be executed. Figure 5 The steps are shown in the diagram. First, the leftmost pump valve can be opened, while the other two pump valves are closed. Then, the middle pump valve can be opened to allow liquid to flow into it. After this liquid flow, the leftmost pump valve can be closed. Then, the middle pump valve can be closed, and the rightmost pump valve can be opened. In summary, this series of steps allows liquid to flow from the leftmost pump valve to the rightmost pump valve.

[0176] As noted above, in some implementations, the system including the cell culture chamber also includes valves. Figure 6 A non-limiting embodiment of system 600 is shown, comprising a cell culture chamber 602, a pump 608, a conduit 610, and a valve 612. Also as Figure 6As shown, the valve can be in fluid communication with the pump and / or the cell culture chamber. Figure 6 This is achieved through a conduit 614 that fluidly connects valve 612 to pump 608. However, other designs are also possible. For example, the valve may be fluidly positioned between the pump and the cell culture chamber, or the cell culture chamber may be fluidly positioned between the pump and the valve.

[0177] In some implementations, the valve is in fluid communication with multiple ports. Figure 6 In some embodiments, valve 612 is in fluid communication with ports 616 and 618. In some embodiments, the valve is capable of and / or configured to allow one or more such ports to be in fluid communication with the pump and / or cell culture chamber. For example, the valve may be able to be positioned and / or configured to have two or more positions, each position allowing a unique subset of ports to be in fluid communication with the pump and / or cell culture chamber. In some embodiments, each position of the valve allows exactly one port or zero ports to be in fluid communication with the pump and / or cell culture chamber. In some embodiments, the pump includes one or more positions that allow two or more ports (e.g., each port in fluid communication with a reagent source) to be in fluid communication simultaneously with the cell culture chamber, the pump, and / or other system components (e.g., a mixing chamber).

[0178] The position of a valve may include the positioning of one or more of its components. For example, a valve may include a path that can rotate between two or more configurations that allow different combinations of ports to be fluidly connected to each other through the valve. As another example, a valve may include multiple sub-valves that can be opened or closed independently, and the position of the valve may include the position of each of the sub-valves.

[0179] A valve allows the composition of the liquid flowing into a cell culture chamber (e.g., under the action of a pump) to be altered by changing the valve's position. For example, the valve can be switched between different positions in which different port combinations are fluidly connected to each other. A first such port may be fluidly connected to a pump and / or the cell culture chamber. Other such ports may be fluidly connected to a liquid source with a different composition (e.g., containing different reagents, containing samples), to other ports (e.g., ports for cell seeding and / or recycling), and / or to other parts of the system (which can direct liquid from the cell culture chamber) (e.g., a mixing chamber, a waste chamber). In such cases, switching the valve position to allow different ports to be fluidly connected to the first port can change the composition of the liquid that can be pumped into the cell culture chamber.

[0180] In some embodiments, the system described herein includes a cell culture chamber and further includes one or more mixing components. Such components may be capable of mixing liquids flowing into, through, and / or present in the cell culture chamber, and / or may be configured to mix such liquids. The mixing may facilitate the mixing of cell suspensions and / or any reagents present in such liquids. Such components may also be provided in combination with pumps, valves, and / or other system components described herein.

[0181] Various suitable mixing components can be used. In some embodiments, the system includes a mixing component as a mixing chamber. Figure 7 A non-limiting embodiment of system 700 is shown, which includes a mixing chamber 720.

[0182] In such embodiments, the mixing chamber may be in fluid communication with the cell culture chamber. This allows liquids to mix in the mixing chamber and then, once mixed, to be transported to the cell culture chamber. Figure 7 As shown, fluid communication between the mixing chamber and the cell culture chamber can be achieved directly via a conduit (e.g., via conduit 722) (i.e., without intermediate components).

[0183] One or more of the above-described components, such as a pump and / or a valve, may also be placed between the mixing chamber and the cell culture chamber. In some embodiments, the valve is placed between the mixing chamber and the cell culture chamber, and the mixing chamber is in fluid communication with one of the ports of the valve. Figure 8 The diagram illustrates an example of a system with this design. Figure 8 The system comprises a mixing chamber and a cell culture chamber (labeled therein as a “growth chamber”), with valves and pumps placed between them. Figure 8 The valve and pump shown are a rotary diaphragm valve and a rotary diaphragm pump, respectively. Figure 8 The valve depicted includes a port in fluid communication with the mixing chamber, a port in fluid communication with the pump and the cell culture chamber, a port in fluid communication with the waste chamber, and a plurality of other ports in fluid communication with different liquids. Figure 8 Also shown are cell filters (e.g., membranes) configured to retain suspended cells in the cell culture chamber, a permeable membrane located between the cell culture chamber and the mixing chamber, and other fluid elements on opposite sides of the cell culture chamber and the growth chamber. Possible options for such other fluid elements are described in more detail elsewhere herein and should be understood to exist in some, but not all, of the embodiments described herein. Similarly, it should be understood that... Figure 8 The design shown is envisioned for use with other types of pumps, valves, components for retaining suspended cells, ports, etc., and / or the system may include Figure 8 Some, but not all, of the components are shown in the image.

[0184] Figure 8 The open paths present during the use of the mixing chamber depicted therein are shown, the process of which is described in the text below and referenced. Figure 9 A more detailed description was provided. Figure 9 It shows having with Figure 8 A system with the same design but depicted in a vertically flipped manner. For example... Figure 9 As shown, mixing can be achieved by pumping the liquids to be mixed into the mixing chamber (top), mixing them therein (not shown), and then pumping the mixed liquids into the cell culture chamber (bottom). During mixing, the position of the valve can be selected such that the port in fluid communication with the pump and the cell culture chamber is positioned to be in fluid communication with the port in fluid communication with the mixing chamber via the valve. First, liquid from the cell culture chamber can be pumped through the pump, through the valve, and into the mixing chamber using the pump. The liquids can be mixed after entering the mixing chamber. Subsequently, the liquid can be pumped back from the mixing chamber, through the valve, through the pump, and into the cell culture chamber using the pump.

[0185] In some embodiments, the system includes a mixing component as a mixing element. The system described herein may include static mixing elements and / or active mixing elements. Furthermore, the mixing element may be placed in various suitable locations within the system described herein. As two examples, the mixing element may be placed in and / or associated with a mixing chamber (e.g., the system may include a mixing chamber containing the mixing element and / or may include a mixing chamber associated with the mixing element), and / or the mixing element may be placed in and / or associated with a cell culture chamber (e.g., the system may include a cell culture chamber containing the mixing element and / or may include a cell culture chamber associated with the mixing element).

[0186] Mixing elements can mix liquids in a variety of suitable ways. In some embodiments, liquids may be mixed in chambers (e.g., mixing chambers, cell culture chambers) by pumping and / or undergoing flow. In such embodiments, the mixing element may include a pump. Non-limiting examples of flow types that liquids may undergo include oscillating flow, shuttle flow, circulating flow, and alternating flow. In some embodiments, one or more of these flow types may include flow of liquid through a cell filter that takes the form of a cell-retaining filter (e.g., shuttle flow through such a filter), flow through a pump (e.g., circulating flow through a peristaltic pump), and / or flow through one or more chambers present in the system (e.g., through a mixing chamber and / or a cell culture chamber, such as through alternating flow of either or both of these chambers).

[0187] As noted above, one example of the type of flow that can be caused by a mixing element is oscillating flow. For example, a mixing chamber may experience oscillating flow inside the chamber and / or oscillating flow into and / or out of the chamber. Figure 10 The diagram illustrates mixing via oscillating flow. As can be seen in... Figure 10 As seen in the image, the mixed liquid can flow backward and forward (central frame) within the chamber and / or flow in and / or out of the chamber. Figure 10 In this process, during oscillating flow, the liquid flowing into and out of the chamber flows along a tortuous conduit (e.g., a tortuous channel) before entering and after leaving the chamber. Flow through such conduits can further aid mixing.

[0188] The use of pumps (e.g., peristaltic pumps, piston pumps, and / or diaphragm pumps) can affect the oscillating flow in and out of chambers (e.g., mixing chambers, cell culture chambers) and / or within the chambers.

[0189] In some implementations, with Figure 10 The embodiments shown are similar, allowing oscillating flow including inflow and outflow from the chamber (e.g., a mixing chamber, a cell culture chamber) in addition to flowing in and / or out of the chamber in different directions. Similarly, the chamber may include two or more pairs of ports (and / or other components) through which liquids flow into and / or out of the chamber. One such port pair may allow one or more components that facilitate oscillating flow and / or are associated with said components, while another such port pair may allow one or more components that facilitate inflow and / or outflow from the chamber along different fluid paths (e.g., fluid paths including pumps and / or valves) and / or are associated with said components. Reference Figure 10 The chamber shown allows oscillating flow into and out of the chamber via a pair of ports and infusion through the chamber via a different pair of ports.

[0190] like Figure 10 As shown, the chamber capable of and / or configured to perform oscillating flow may also contain one or more cell filters (e.g., one or more filters and / or membranes). Such membranes may include those described elsewhere herein for retaining suspended cells, membranes capable of and / or configured to perform cross-flow filtration, membranes capable of and / or configured to perform through-flow filtration, and / or membranes capable of and / or configured to reduce fouling and / or clogging. An example of a suitable membrane is a microfiltration membrane.

[0191] Membranes capable of and / or configured to perform cross-flow filtration can be placed on surfaces through which liquid does not flow (not shown). For example, they can be placed on surfaces lacking ports or other components of the chamber through which liquid can flow in and out of the chamber, and / or can be positioned to allow liquid to flow through (e.g., rather than through) them.

[0192] In some embodiments, the membrane can be performed and / or configured to perform both cross-flow filtration and through-flow filtration (e.g., liquid can flow through the membrane and pass through the membrane, both types of flow can provide some filtration effect). Without wishing to be bound by any particular theory, such a membrane is believed to ideally exhibit reduced clogging and / or fouling.

[0193] Figure 11 Another example of a chamber in which oscillating flow can be carried out and / or configured to carry out is depicted. Figure 11 A system in which such chambers can be placed is also shown. For example... Figure 11 As shown, such a system can include air chambers that can be filled to apply pressure to the liquid present in the system and emptied to release that pressure. Filling and emptying these air chambers can cause oscillating flow. Also as... Figure 11 As shown, in some embodiments, the chamber where oscillating flow occurs may include channels (e.g., channels with microfluidic volumes). Without wishing to be bound by any particular theory, it is believed that flow through such channels can occur at relatively high velocities, which may reduce fouling of cell filters present in such systems.

[0194] Figure 12 Another example of a hybrid element applicable to some of the systems described herein is shown. Figure 12 In this design, the mixing element takes the form of two diaphragms that form two opposing walls of a chamber (e.g., a mixing chamber, a cell culture chamber). The diaphragms can be actuated (e.g., back and forth) to mechanically mix the liquids present in the chamber. The diaphragms can also be placed outside the chamber walls, for example, outside walls that can be deformed by the diaphragms, and / or the diaphragms can also be placed inside the chamber walls. Figure 12 As shown, the mixing element in the form of a diaphragm is compatible with fluid flow (e.g., perfusion) through the chamber and / or the use of cell filters (e.g., filters and / or membranes).

[0195] Figure 13-15 The text describes the... Figure 12 The slight variations in the hybrid elements shown are illustrated. Figure 13-15In the mixing element shown, two diaphragms can be placed on the same side (e.g., the upper side) of the chamber. Such diaphragms can be laterally separated. For example, one diaphragm can be located above a portion of the chamber (e.g., the upstream portion), and the other diaphragm can be located above a different portion of the chamber (e.g., the downstream portion). In some embodiments, such diaphragms can form part of the same wall of the chamber (e.g., they can form two portions of the same upper wall).

[0196] like Figure 13-15 As shown, in some embodiments, the diaphragm forming part of the mixing element may include sides with a triangular shape (e.g., sides forming part of the chamber wall). The diaphragm may also include sides with a basic triangular shape (e.g., a near-triangle shape, but where one or more vertices have been replaced by shorter sides, such as sides with a length of up to 10% of the length of one or more other sides present in the shape). Without wishing to be bound by any particular theory, it is believed that triangular and / or basically triangular diaphragms can ideally improve mixing and / or reduce cycle time compared to diaphragms with other shapes.

[0197] Figure 16 The image shows a third example of a hybrid element applicable to some of the systems described herein. Figure 16 In this context, the chamber (e.g., a mixing chamber, a cell culture chamber) contains an active mixing element located within the chamber. Non-limiting examples of suitable active mixing elements include mechanical mixers, such as stirring rods (e.g., magnetically driven stirring rods), impellers (e.g., mechanically driven impellers, micro-impellers), etc. Figure 10 and 12 The chambers shown in the image are the same. Figure 16 The chambers shown may also allow fluid flow (e.g., perfusion) and / or be used in conjunction with cell filters (e.g., filters and / or membranes).

[0198] Figure 17 An embodiment of an exemplary chamber (e.g., a mixing chamber, a cell culture chamber) containing an active mixing element and a portion of the system in which it is located is shown. As can be seen in... Figure 17 As seen in the embodiment shown therein, the chamber contains an active mixing element in the form of a magnetically driven stirring rod, which can move within the chamber to mix the liquid therein. Figure 17 The chamber shown also includes two ports located on two opposite sides of the chamber, each port being in fluid communication with a diaphragm pump. Figure 17The chamber shown (e.g., a mixing chamber, a cell culture chamber) also includes two membranes, one located at the top of the chamber and the other near one of its ports. The membrane at the top of the chamber is an elastic gas filtration membrane capable of expanding and contracting to accommodate volume loss (and configured to perform the operation described above). The membrane near one of its ports is capable of retaining and configured to retain suspended cells. This membrane is also capable of performing and configured to perform cross-flow filtration (e.g., an active mixing element such as a magnetically driven stir bar can generate flow parallel to the membrane, resulting in cross-flow filtration; in some embodiments, this cross-flow filtration occurs simultaneously with through-flow filtration). Figure 17 Each diaphragm pump shown includes multiple pump-valve components that can be used and / or configured to be actuated in a peristaltic manner to pump fluid. Furthermore, each such diaphragm pump is in fluid communication with a valve, and each such valve is in fluid communication with various ports.

[0199] Figure 18 The operation includes Figure 16 The system of the chamber shown is a non-limiting example of a method for removing waste from the chamber, delivering reagents to it, and mixing the reagents in the chamber.

[0200] Figure 18 The diagram above illustrates the use of an active mixing element (magnetically driven stirring rod) to mix the liquid in the chamber. In this diagram, the pump-valve closest to the chamber at each diaphragm port is closed. This closure restricts the liquid flow during mixing to essentially within the chamber.

[0201] exist Figure 18 As shown in the diagram, by actuating the right diaphragm pump, closing the pump-valve closest to the chamber at the left diaphragm port, and using the valve downstream of the right diaphragm port to fluidly connect the right diaphragm port to the waste chamber, waste can be pumped out of the chamber. When this is done, the liquid in the chamber can flow through the membrane located near the end of the chamber closest to the right diaphragm pump, retaining suspended cells in the chamber and allowing the liquid containing waste to flow out of the chamber. The liquid can then flow through the right diaphragm pump, the valve, and into the waste chamber.

[0202] Figure 18 The diagram below illustrates the introduction of the reagent into the chamber. This is achieved by closing the pump-valve closest to the chamber at the right diaphragm port and fluidly communicating the left diaphragm pump with the reagent source using the valve upstream of the left diaphragm pump. When this is done, the liquid containing the reagent can flow through the valve, the right diaphragm pump, and into the chamber.

[0203] Figure 18 The valve shown is also in fluid communication with other ports, the operation of which is... Figure 18Not shown, but may be present in some embodiments and / or may operate in some embodiments. For example, some valves may have more than one port in fluid communication with the reagent source (e.g., Figure 18 The valve shown in the diagram is in fluid communication with the left diaphragm pump, and the port in fluid communication with the port where cell seeding and / or cell retrieval can be performed (e.g., Figure 18 The valve shown in the diagram is in fluid communication with the left diaphragm pump, and the port in fluid communication with a component (e.g., a sensor) capable of measuring the amount of IgG in the liquid. Figure 18 The valve shown in the diagram is in fluid communication with the right diaphragm pump) and / or the port in fluid communication with the sample source (e.g., the port shown in the diagram is in fluid communication with the sample source). Figure 18 The valve shown is in fluid communication with the right diaphragm pump. The positions of these ports can be as follows: Figure 18 As shown, they can also be different (e.g., they can all be in direct fluid communication with a single valve, and / or can be in different...). Figure 18 The distribution is shown in the manner among the multiple valves.

[0204] In some embodiments, the system includes a mixing component, which is a flow path configured to facilitate mixing. The flow path may include one or more components that agitate the liquids to mix them, such as one or more pumps and / or one or more mixers (e.g., one or more active mixing elements, one or more static mixers). In some embodiments, the flow path configured to facilitate mixing includes and / or has a fluid channel (e.g., a fluid channel capable of supplying liquid (e.g., liquid flowing through the flow path) to the cell culture chamber and / or configured to do so) through and / or in fluid communication with the cell culture chamber. In such embodiments, the flow path may include components capable of retaining cells and / or configured to retain cells within the flow path while allowing liquid to enter and / or exit the flow path, such as a cell filter. A single flow path may also be configured to both facilitate mixing and take the form of a cell filter. Further details regarding exemplary flow paths configured to facilitate mixing are provided below.

[0205] Figure 19 An exemplary flow path configured to facilitate mixing is depicted. Figure 19 The flow path shown includes a peristaltic pump and a rotary valve. It should be understood that similar flow paths incorporating different types of pumps and / or valves are contemplated. Furthermore, some embodiments may include, for example... Figure 19 The flow path is shown in the diagram, but it also includes a cell culture chamber and / or may be in fluid communication with the cell culture chamber. (See diagram for details.) Figure 19 As shown, the flow path can pass through the pump and valve to form a loop through which the liquid can flow. The liquid can undergo mixing and / or become suspended during mixing. Figure 19As shown, the flow path may include a first section passing through the valve. This first section may allow fluid communication between its upstream and downstream portions and may lack a membrane or other type of cell filter.

[0206] Figure 20 It shows a way that can be obtained from Figure 19 The flow path shown depicts a method of removing some liquid and replacing it with a different liquid. More specifically, Figure 20 This illustrates a method in which liquid containing waste can be removed from the flow path and fresh culture medium can be introduced therein. These two actions can be performed simultaneously while retaining suspended cells within the flow path. In some embodiments, this can be achieved using... Figure 19 The procedure shown is used to perform the infusion.

[0207] like Figure 20 As shown in the diagram, the process can begin by selecting a second position for the valve, which modifies the flow path so that it no longer includes the initial portion (i.e., the first portion) passing through the valve, but instead includes a new, distinct portion (i.e., the second portion) (leftmost figure). This second position of the valve may result in a membrane (and / or filter) located on the valve being positioned upstream of the second portion of the flow path passing through the valve. When the valve is in this second position, the liquid pumped by the pump will encounter the membrane before flowing through the valve, thus allowing the liquid to flow through the valve, while suspended cells will be prevented from flowing through the valve by the membrane.

[0208] When the valve is in the position where the new, different flow path is filled with liquid (but not suspended cells), after sufficient flow has occurred, the valve can be positioned in a third position (middle figure). This third position allows the second portion of the flow path to be in fluid communication with a liquid source (e.g., a liquid source containing reagents, a liquid cell culture medium source) and a waste chamber. Furthermore, it can position the second portion of the flow path such that the membrane is its downstream portion (or such that the second portion of the flow path is substantially entirely upstream of the membrane). In other words, when the valve is in the third position, the orientation of the second portion relative to fluid flow may be opposite to its orientation relative to fluid flow when the valve is in the second position (i.e., the upstream and downstream directions may be opposite to those when the valve is in the second position). Therefore, flow through the second portion of the flow path can cause the liquid present therein to flow through the membrane and into the waste chamber. During this process, suspended cells present in the liquid can be retained by the membrane. The liquid flowing into the waste chamber can be replaced by liquid originating from the liquid source. This flow can be achieved by another pump (not shown).

[0209] When the valve is in the third position Figure 19 The flow path shown may no longer include any portion passing through the valve. In other words, when the valve is in the third position, it may not form a flow loop. In some such embodiments, when the valve is in the third position, the pump operation may stop, and the flow may stop (e.g., until the valve is positioned in a different position, such as the first or second position).

[0210] When the valve is in the third position, the first and second flow portions of the flow path can take the form of a “dead space” (also referred to herein as a “dead volume” and a “dead zone”). This dead space can have a known volume, which facilitates the removal of a known volume of liquid (e.g., a known volume of liquid containing waste) from the flow loop and / or the introduction of a known volume of liquid (e.g., a known volume of fresh cell culture medium) into the flow loop.

[0211] Then, Figure 20 The process shown may include positioning the valve back to the second position (rightmost figure). When the valve is in the second position, liquid present in the second portion of the flow path may flow into the flow loop (e.g., under the operation of the pump). The liquid may continue to flow until the liquid present in the second portion of the flow path is delivered to the flow loop, and / or the second portion of the flow path is filled with liquid containing waste.

[0212] Figure 20 The process shown can be repeated multiple times. Once a sufficient amount of waste has been removed from the flow path and / or a sufficient amount of liquid has been supplied to it, the valve can be repositioned to the first position.

[0213] Figure 21 It shows that it can be used Figure 19 The flow path shown is one method for cell seeding and cell recovery. For example... Figure 21 As shown, cell seeding and cell retrieval can be performed by selecting the position of the valve (e.g., the fourth position), which alters the flow path so that it is no longer a flow loop. Instead, the flow path can be in fluid communication with a portion of the system (e.g., a port) through which cell seeding and / or retrieval can be performed.

[0214] Cell seeding can be performed by actuating the pump, causing a liquid containing cells to flow from this part of the system through the valve (e.g., through a first portion of the flow path) and into the flow path. At this point, the liquid initially present in the flow path can flow out of the flow path through a different flow path present in the valve (e.g., through a second portion of the flow path). A cell filter (e.g., a membrane) may be present to prevent cells from flowing out of the flow path with the liquid.

[0215] Cell recovery can be performed by actuating the pump to reverse the flow of fluid (i.e., out of the flow path and into the cell-recoverable portion of the system). The liquid removed from the flow path by this process can be replaced by liquid flowing into the flow path (e.g., through a second portion of the flow path). The liquid flowing into the flow path may include one or more reagents and / or fresh cell culture medium.

[0216] Figure 22 Another example of a flow path configured to facilitate mixing is shown. Figure 22 The flow path shown passes through a valve, two pressurizable membranes, and two pinch valves. The two pressurizable membranes can be pressurized to induce flow through the flow path. The pinch valves allow for cell seeding and / or cell collection. Figure 22 The liquids in the path shown may undergo mixing during their flow. Figure 19 The flow path shown is the same. Figure 22 The flow path shown may include and / or be in fluid communication with a cell culture chamber. In some embodiments, as liquid flows through the flow path, the valve may be in a first position in which a first portion of the flow path passing through the valve is positioned such that liquid passes through it. This first portion may allow its upstream and downstream portions to be in fluid communication and may lack a membrane or other type of cell filter.

[0217] Figure 23 It shows that it can be obtained from Figure 23 This is a non-limiting example of a flow path shown that removes some liquid and replaces it with a different liquid (e.g., priming can be performed by the method described). Doing this can include selecting a valve that reflects... Figure 20The following are a set of positions shown and ordered. First, the valve can be positioned in the second position, and the second portion of the flow path can be filled with liquid originating from the flow path but in which suspended cells have been removed by the membrane (leftmost figure). Then, the valve can be positioned in a third position, in which the second portion of the flow path is arranged such that the membrane is its downstream portion (or such that the second portion of the flow path is substantially entirely upstream of the membrane). When in this position, liquid from the source (and may contain reagents and / or fresh cell culture medium) can be supplied to the second portion, and / or liquid initially present in the second portion can flow into the waste chamber. Finally, the valve can be positioned again in the second position, in which liquid introduced into the second portion can be supplied to the flow path, and cells present in the flow path can be blocked by the membrane from flowing into the second portion. Figure 20 As shown, these steps can be repeated multiple times, and / or the valve can be repositioned to the first position at the end of the process.

[0218] Figure 24 A third example of a flow path configured to facilitate mixing is shown. Figure 24 The flow path shown takes the form of a loop passing through the cell culture chamber, the pump, and the hollow fiber membrane. The liquid flowing through this loop may be mixed during flow. This flow can be initiated by one or more pumps. For example, a pump in fluid communication with the cell culture chamber but not part of the flow path can allow liquid (e.g., a liquid containing reagents, a liquid containing fresh cell culture medium) to flow into the cell culture chamber. An example of such a pump is... Figure 24 The leftmost pump. As another example, the pumps through which the flow path passes (e.g.) Figure 24 An intermediate pump (in the process) can cause liquid to flow through the flow loop. As a third example, a pump downstream of the hollow fiber membrane can cause liquid to flow through the hollow fiber membrane and out of the flow loop. During this flow, the hollow fiber membrane can allow liquid to flow through and out from two different outlets, but only allows cells suspended in the liquid to flow out from one such outlet. The outlet through which the suspended cells cannot flow can allow liquid flowing through to leave the flow loop, and / or the outlet through which the suspended cells can flow can supply the flow loop. The liquid flowing through the hollow fiber membrane and out of the flow loop may contain waste and / or other components that are desired to be removed from the flow loop. The suspended cells can continue to flow through the flow loop.

[0219] It should also be understood that, although Figure 24 Hollow fiber membranes were described, but in the case of… Figure 24In the flow path of the design shown, other types of cell filters (such as membranes and filters) can be used to replace and / or supplement the hollow fiber membrane.

[0220] Figure 25-27 It shows something similar to Figure 24 Other instances of flow paths configured to facilitate mixing are shown. Figure 25 This demonstrates that the hollow fiber membrane can penetrate and / or be present in the infusion chamber. Figure 25 It also shows that the cell culture chamber described herein (labeled "growth chamber") may also contain one or more sensors.

[0221] Figure 26 Showing with Figure 24 and 25 The flow path shown is a three-dimensional schematic diagram of a flow path similar to the one shown. Figure 26 The illustration also shows that liquid can flow into a perfusion chamber through which liquid is flowing out (e.g., a perfusion chamber through which a hollow fiber membrane passes). In some embodiments, this liquid may contain liquid cell culture medium (e.g., fresh liquid cell culture medium) and / or one or more reagents (e.g., one or more reagents present in the liquid and / or derived from a source). Figure 26 It is also noted that the cell culture chamber (labeled as the “growth chamber”) may also contain one or more components that allow and / or facilitate gas exchange.

[0222] Figure 27 Showing with Figure 24-26 The flow path shown is a three-dimensional schematic diagram similar to the flow path depicted. Figure 27 In this context, the infusion chamber has a cylindrical design, rather than... Figure 26 The cuboid design shown is not intended to be constrained by any particular theory; it is believed that a cylindrical infusion chamber may be able to provide similar functionality to a cuboid infusion chamber with a larger volume.

[0223] Figure 28 A fourth example of a flow path configured to facilitate mixing is shown. Figure 28 The flow path shown is similar to Figure 24-27 The flow path is shown, but includes a helical inertial separator instead of a hollow fiber membrane. Therefore, the fluid can flow in a manner similar to that described in the previous paragraph. Figure 24-27 The flow path depicted in the text describes the way the flow passes through. Figure 28 The flow path is shown in the diagram. Similar to the hollow fiber membrane described in the previous paragraph, the helical inertial separator may include two outlets: one is the outlet through which the helical inertial separator guides the suspended cells, and this outlet is also... Figure 28 One is a portion of the flow loop shown; the other is an outlet that the helical inertial separator guides the suspended cells to avoid, and this outlet leads to... Figure 28 Outside the flow path shown.

[0224] Therefore, flowing through Figure 28 The liquid in the flow path shown can be divided into two parts: one part continues to flow through the flow path (and may contain suspended cells), and the other part leaves the flow path after flowing through the helical inertial separator.

[0225] Figure 29 A fifth example of a flow path configured to facilitate mixing is shown. Figure 29 The flow path shown includes both a helical inertial separator and a hollow fiber membrane. Figure 28 The use of Figure 24-28 The same symbols used to depict pumps, hollow fiber membranes, spiral inertial separators, and cell culture chambers are shown to depict these same features. Figure 29 The flow path shown is similar to Figure 28 As shown, but including another flow loop downstream of the helical inertial separator. Figure 29 As shown, the flow loop includes another pump and a hollow fiber membrane. Liquid exiting the spiral inertial separator through the outlet lacking suspended cells flows into this flow loop. Figure 29 After the second flow loop, the liquid flows through the hollow fiber membrane. Figure 29 The hollow fiber membrane shown is Figure 24 Like the hollow fiber membrane shown, it can contain two outlets: one for liquid lacking suspended cells, and another for liquid containing or lacking suspended cells. Therefore, similar to the flow through... Figure 28 Compared to the flow path depicted in the text, the liquid flowing through Figure 29 The liquid in the flow path depicted can undergo another filtration step. Any suspended cells not removed by the helical inertial separator can leave the hollow fiber membrane and be recirculated back to the cell culture chamber through the second flow loop, while the liquid can leave the second flow loop through the hollow fiber membrane.

[0226] It should also be understood that, although Figure 29 Hollow fiber membranes were described, but in the case of… Figure 29 In the flow path of the design shown, other types of cell filters (e.g., membranes and / or filters) can be used to replace and / or supplement the hollow fiber membrane.

[0227] Figure 30 A sixth example of a flow path configured to facilitate mixing is shown. Figure 30 The flow path shown passes through the cell culture chamber (in Figure 30 The middle is labeled "growth chamber"), and the spiral inertial separator (in Figure 30 The upper left portion is shown as a spiral, a hollow fiber membrane or a channel parallel to it, a pump, and a chamber containing a static mixing element. Liquid flowing through this flow path may mix and / or be mixed by the static mixing element during flow. Figure 29 As shown in the flow path, the liquid exiting the helical inertial separator can be separated into a liquid relatively rich in suspended cells and a liquid relatively poor in suspended cells. The former liquid can flow directly into the pump, which causes the fluid to flow along the flow path. The latter liquid can pass through a hollow fiber membrane, where it is further separated into a liquid even more poor in suspended cells and a liquid containing some or all of the suspended cells from the latter liquid. The liquid even more poor in suspended cells can flow out of the flow path and into the waste chamber. Another liquid can flow into the pump.

[0228] In some implementations, with Figure 30 As in the illustrated implementation, the flow path is divided into one or more branches, each containing a component that guides a larger volume of fluid along that branch and / or a component that guides a larger volume of fluid along another branch. As an example, Figure 30 A flow restrictor downstream of the hollow fiber membrane is depicted (represented by the narrowing of the channel). The flow restrictor increases the resistance of this branch along the flow path, thereby directing a higher percentage of liquid flow through the branch parallel to it (i.e., the branch accessed by the outlet of the helical inertial separator, which outputs a liquid quite rich in suspended cells).

[0229] like Figure 30 As shown, liquid may also flow into the flow path depicted therein, for example through a port and / or connector located between the pump and the chamber containing the static mixing element.

[0230] Also Figure 30 As shown, the flow path can pass through the pump. Figure 30 The flow path shown passes through a peristaltic pump. In some such embodiments, one or more portions of the flow path (e.g., the portion of the flow path passing through the pump) are surrounded by a material (e.g., tubing) that allows gas exchange and / or is configured to facilitate gas exchange. For example, the flow path may be surrounded by a permeable membrane (e.g., a semi-permeable membrane that allows one or more gases to pass through but not liquids). In some such embodiments, the material that allows gas exchange and / or is configured to facilitate gas exchange and surrounds a portion of the flow path is further surrounded by a chamber that controls gas exchange (e.g., a sealed pressure chamber).

[0231] It should also be understood that, although Figure 30 Hollow fiber membranes were described, but in the case of… Figure 30In the flow path design shown, other types of cell filters (e.g., membranes and / or filters) can be used to replace and / or supplement the hollow fiber membrane. Similarly, although Figure 30 A static mixing element is described, but the flow path can also have... Figure 30 A similar design to the one shown, but which may alternatively include an active mixing element. Similarly, in a design with... Figure 30 In some aspects of the flow path shown in the design, cell filters other than helical inertial separators can be used.

[0232] Figure 31 and 32 Depicting Figure 30 Other possible aspects of the flow path shown. Figure 31 Further evidence suggests that IgG pipelines may exist (e.g., for recovering liquids to analyze their IgG content via an IgG sensor). Figure 32 The other components of the flow path are described in more detail. It should be understood that some flow paths may have one or more characteristics similar to... Figure 31 The features shown, but in one or more aspects similar to Figure 32 different.

[0233] Figure 33 The seventh example of a flow path configured to facilitate mixing is shown. Figure 33 The flow path shown passes through the cell culture chamber (in Figure 33 The cell culture chamber is labeled "growth chamber" and contains cell filters (e.g., membrane filters, such as microfiltration membranes) near two points where it is in fluid communication with the flow path. As can be seen in... Figure 33 As seen in the diagram, liquid can flow through the flow path in two directions (e.g., from left to right through the cell culture chamber and from right to left through the cell culture chamber). The flow direction can be selected by choosing the position of a valve in the flow path. For example, liquid can enter the flow path and be guided by a valve to flow on a left or right conduit originating from a branch point upstream of the cell culture chamber. The liquid can then flow into the cell culture chamber through a cell filter fed by an associated conduit, pass through the cell culture chamber, and exit through another membrane. The liquid can then continue flowing to a valve that guides the liquid to continue circulating in the flow path (e.g., back to the first cell filter) or to exit the flow path (e.g., to waste for IgG analysis). Advantageously, this allows liquids present in and / or flowing through the cell culture chamber (e.g., liquid cell culture medium) to be backwashed through each cell filter, which may help reduce fouling on these filters.

[0234] In some implementations, by means of, Figure 33 The flow in the flow path shown can be driven by a pump, for example... Figure 33 The piston pump shown.

[0235] like Figure 33 As shown, it is able to supply liquid from the temporary storage orifice to the flow path. Figure 34 A non-limiting example of a suitable temporary storage hole is shown. As can be seen in... Figure 34 As seen in the diagram, the temporary storage orifice may include an outlet for supplying the flow path. The temporary storage orifice may also include one or more inlets. The inlets may include one or more inlets suitable for receiving liquids (e.g., liquids containing reagents, liquid cell culture media) and / or one or more inlets suitable for receiving gases (e.g., gases to be dissolved in the liquid and / or exchanged with the liquid). In some embodiments, one or more gases and one or more liquids may undergo an exchange in the temporary storage orifice, which may allow the introduction of a liquid containing a desired amount of gas into the flow path. This exchange may occur across a gas-liquid interface (e.g., the gas inlet may be located above the liquid inlet, which may result in the formation of a gas-liquid interface where the gas is above the liquid).

[0236] In some implementations, the temporary storage orifice also includes a mixing element. (See reference) Figure 34 The temporary storage hole depicted therein contains a magnetically driven stirring rod.

[0237] Figure 35 An eighth example of a flow path configured to facilitate mixing is shown. Figure 34 Like the seventh flow path shown, this flow path allows liquid to flow in the opposite direction. Figure 35 The left side shows the liquid flow through the flow path in the first direction. As can be seen therein, liquid can enter the flow path from the right side, flow into the cell culture chamber, and pass through the cell filter located therein (e.g., Figure 35 The liquid enters the pump (as shown in the membrane filter) and then flows out of the flow path. Alternatively, the liquid can enter the flow path from the left, flow through the pump, through the cell filter, and out of the flow path. This flow direction is shown in... Figure 35 On the right side. In some embodiments, the flow direction can be determined by actuating the pump. The pump can be actuated to cause the liquid present in the flow path to flow back and forth (e.g., through a cell filter), which can promote mixing of the liquid (and possibly promote the co-mixing of any components therein, such as reagents and / or the suspension of cells therein). This flow may also result in backflushing of the cell filter, which may help prevent and / or reduce scaling.

[0238] like Figure 35As shown, a flow path with this design may include a pump as a piston pump and / or a pump containing an air bladder. In some embodiments, an air bladder exists between one or more parts of the pump (e.g., the piston in a piston pump) and the liquid flowing in the flow path, which can ideally reduce and / or prevent contamination of these parts of the pump. When present, the piston pump can influence the pressure in the flow path by actuating the piston up and down, thereby causing the liquid to flow.

[0239] Also Figure 35 As shown, the flow path depicted may include pinch valves (e.g., on the left side of the pump and the right side of the cell culture chamber). When present, pinch valves may be able to open and close and / or be configured to open and close. An open valve may allow liquid to flow through (e.g., into or out of the flow path), while a closed valve may prevent liquid from flowing through (e.g., when it is desired to retain liquid present in the flow path).

[0240] Figure 35 It is also shown that the cell cultures depicted therein may be covered by a gas exchange membrane (e.g., a semi-permeable membrane), and / or may contain a cell filter to retain the cells in the cell culture chamber. The former may facilitate and / or allow gas exchange.

[0241] Liquid can be supplied from a source (e.g., liquid cell culture medium source, reagent source) to... Figure 35 The flow path is shown in the figure.

[0242] Figure 36 It shows the way Figure 35 Another example of flow in the flow path shown is where liquid flows from right to left, and liquid is introduced into the flow path. As can be seen in... Figure 36 As seen in the diagram, liquid can be introduced into the flow path through a dispensing orifice located near the right side of the flow path. During this process, a right-hand clamp valve can be opened to allow flow from the dispensing orifice into the cell culture chamber. Furthermore, the pump can be used to reduce the pressure in the flow path (e.g., by raising the piston of a piston pump). Liquid can be introduced into the dispensing orifice through a reagent dispensing head or by any other suitable method. In some embodiments, the dispensing orifice may be designed such that the liquid does not completely fill the orifice during and / or after its introduction. This may result in an air gap between the reagent dispensing head and the liquid in the dispensing orifice during liquid introduction. The liquid may contain reagents, cell culture medium, and / or cells.

[0243] Figure 37 It shows the way Figure 35Another example of the flow path shown is where liquid flows from right to left and is removed from the flow path. Figure 37 As shown, the liquid can be removed from the flow path by flowing through the left pinch valve (which can open in the process). This liquid can then flow to the waste chamber and / or another system component for analyzing IgG levels (e.g., using an IgG sensor). Figure 37 During the process described, the right-hand pinch valve can be closed, and the pressure in the flow path can be increased (e.g., by raising the piston of the piston pump). This may apply positive pressure to the liquid, causing it to flow out as... Figure 37 The flow path is shown in the figure.

[0244] Figure 38 The ninth example of a flow path configured to facilitate mixing is shown. Figure 38 The flow path shown passes beneath the path under which a roller can roll at two locations (e.g., the flow path may have a U-shape, with opposite ends of the U-shape located beneath this path). The roller can roll at one of these locations, compressing the conduit surrounding the flow path and thus applying pressure to the liquid present in the flow path. This pressure can cause the liquid in the flow path to flow away from the roller across the flow path (e.g., toward another location beneath the path under which the roller can roll). In this way, flow through the flow path can be initiated by rolling the roller, and the direction of flow can be selected by the position where the roller rolls. Flow through the flow path induced in this way can result in mixing of the flowing liquid and / or suspension of cells in the flowing liquid. In some embodiments, the liquid mixed in this way can take the form of a slug, occupying a space smaller than the entire spatial extent of the flow path.

[0245] As pointed out above, Figure 38 The flow path shown can be surrounded by a conduit that can deform upon pressure applied by rollers. Figure 38 As shown, this can take the form of an elastic material (e.g., a silicone membrane). In some such embodiments, the elastic material (and / or another material) can allow gas exchange as described elsewhere herein. Also as Figure 38As shown, the flow path can pass through the growth chamber. The flow path may also include one or more ports through which one or more liquids (e.g., liquid containing reagents, liquid cell culture medium) can be delivered; one or more ports through which gases (e.g., air) can be removed from the flow path; and / or one or more ports through which waste (e.g., one or more ports in fluid communication with a conduit in fluid communication with a waste chamber) can be removed.

[0246] Some flow paths are configured to facilitate mixing (e.g.) Figure 19-33 The flow path depicted has a fixed volume. In such embodiments, liquid removed therefrom (e.g., to a waste chamber) must be replaced by another fluid (e.g., another liquid). In some embodiments, such liquid is replaced by liquid cell culture medium (e.g., fresh liquid cell culture medium). Such liquid may also be replaced by a liquid containing one or more reagents. In some embodiments, it may be desirable that the amount of liquid entering and leaving the flow path is relatively small compared to the total amount of liquid in the flow path. For example, in some embodiments, the amount of liquid entering and leaving the flow path is less than 10%, less than 7.5%, less than 5%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, or less than 0.2% of the total amount of liquid in the flow path (and, for example, greater than 0.1%, greater than 0.2%, greater than 0.5%, greater than 0.75%, greater than 1%, greater than 2%, greater than 5%, or greater than 7.5% of the total amount of liquid in the flow path).

[0247] Some flow paths are configured to facilitate mixing (e.g.) Figure 19-38 The flow path depicted may undergo one or more processes before cells are introduced into it. As an example, in some implementations, the flow path may be cleaned, pre-filled, and / or degassed before cells are introduced into it.

[0248] In some embodiments, the system includes two or more flow paths configured to facilitate mixing. In such embodiments, each of the two or more flow paths may be in fluid communication with one or more common system components. For example, the system may include two or more flow paths in fluid communication with a common component that helps retain cells and / or a common component that can deliver one or more liquids and / or reagents (e.g., fresh cell culture medium) to the flow path. In some such embodiments, one or more flow paths may lack such a component. Advantageously, designs that include two or more flow paths configured to facilitate mixing can facilitate multiplexing and / or reduce the need for multiple repetitive components in multiplexed designs.

[0249] Figure 39 A non-limiting example of a system comprising two flow paths is depicted, each of which is configured to facilitate mixing. These two flow paths are depicted in... Figure 39 On the left side. As can be seen in this figure, each flow path includes a cell culture chamber (labeled "growth chamber") and a pump (e.g., a peristaltic pump). The operation of the pump causes the liquid present in the flow path to flow around the flow path through the cell culture chamber and the pump.

[0250] Just as it can also be in Figure 39 As seen in the diagram, each flow path is fluidly connected to two ports, one port connecting the relevant flow path to its upstream hollow fiber membrane and the other port connecting the relevant flow path to its downstream hollow fiber membrane. (As can be seen from...) Figure 39 Understood, the liquid flowing through the two flow paths depicted herein can therefore be removed from the respective flow path via the first port, pass through the hollow fiber membrane, and then be resupplyed to the respective flow path (or the other flow path). Various suitable ports can be employed; a non-limiting example is a rotary shear valve.

[0251] When the liquid passes through Figure 39 When a hollow fiber membrane is depicted, cells suspended within it can be retained in a liquid that is resupplying to one of two flow paths, while simultaneously being removed by a portion of the liquid through the other portion of the hollow fiber membrane. This separation allows for the removal of liquid waste, as this liquid can be directed to a waste chamber.

[0252] exist Figure 39 As can also be seen, the ports depicted that connect the flow path to the liquid exiting the hollow fiber membrane are also in fluid communication with other flow paths present in the system. These flow paths may be in fluid communication with a liquid source (e.g., a reagent source and / or a cell culture medium source). Therefore, these ports can also be used to supply... Figure 39 The flow path depicted in the diagram supplies this type of liquid.

[0253] Liquid inflow and outflow Figure 39 The flow path depicted can be achieved by using one or more pumps. Figure 39 An exemplary piston pump suitable for this purpose is shown. Other types of pumps (such as those described herein) may also exist in place of and / or as a supplement to piston pumps. Similarly, although Figure 39 Hollow fiber membranes are depicted, but other types of cell filters (such as membranes and / or filters) may be used in place of hollow fiber membranes or as a supplement to hollow fiber membranes.

[0254] like Figure 40As shown, having Figure 39 The design shown (and / or similar) Figure 39 The system (as shown in the design) can be divided into two or more modules. Reference Figure 40 The system described includes a filtration module comprising a hollow fiber membrane and a piston pump, and two modules, each containing a cell culture chamber (labeled therein as a "growth chamber"). As can also be seen from... Figure 40 It is understood that a system comprising multiple modules may be able to allow liquid to flow between and / or through the different modules therein, and / or be configured to perform any or both of these functions.

[0255] When a system comprises two or more flow paths and / or modules through which liquids can be transferred, the system may further comprise one or more components that reduce cross-contamination between different liquids flowing primarily through different flow paths, but also at least partially through a common module and / or system component. As an example, the common module and / or system component (and / or the entire system) may be capable of being sterilized and / or configured to undergo sterilization treatment. Sterilization may occur after the liquid has flowed through the relevant module and / or system component from one flow path, and before the liquid has flowed through that module and / or system component from a different flow path.

[0256] Figure 41 Depicting Figure 39 and 40 This is a non-limiting instance of a module present in the system shown. As can be seen in... Figure 41 As seen in the image, the module may contain one or more ports through which liquid can be introduced into the module. For example, see reference... Figure 41 The module may include inoculation ports (e.g., pores and / or reservoirs) through which cells can be inoculated and / or one or more ports that are in fluid communication with another module (e.g., a filtration module). Figure 41 (As depicted in the image, there are two ports). The ports, which are in fluid communication with other modules, allow liquid to be transferred between modules. Figure 41 A pump that can be used to induce flow through the module is also described. For example... Figure 41 As shown, this flow may lead to the circulation of liquids, such as the circulation of liquid cell culture medium present in the module. Figure 41 A peristaltic pump is described, but other pumps may be used alternatively or as alternatives. Finally, Figure 41 A breathable membrane (referred to herein as a semi-permeable membrane) is shown. The breathable membrane allows any gas dissolved in the liquid present in the module to be removed therefrom and / or allows gases present in the environment outside the module to diffuse through the breathable membrane and into the liquid.

[0257] Figures 42-43 This describes a non-limiting example of a way in which liquid can flow through a cell culture chamber while retaining cells within it. (Reference) Figure 42 The old liquid cell culture medium is drained from the cell culture chamber and replaced with fresh liquid cell culture medium, while the cells remain in the chamber. Figure 42 As shown, old liquid cell culture medium can flow out of the growth chamber, through a port exiting a module therein, and into the hollow fiber membrane (labeled therein as "cross-flow filter" and "filter"). Cells in the old liquid cell culture medium can be retained by the hollow fiber membrane and subsequently returned to the module from which they originated (and the growth chamber therein). Some or all of the old liquid cell culture medium can exit the hollow fiber membrane via different pathways and through a pump (e.g., Figure 42 The piston pump depicted in the diagram pumps out of the system and / or into the waste chamber. During and / or after this process, it can be added to the module by allowing fresh liquid cell culture medium to flow from its source into the module (e.g., in some embodiments, such as...). Figure 42 The embodiment shown is via a port and / or via a valve that fluidly connects the module to the liquid cell culture medium source. Also as... Figure 42 As shown, the fresh liquid cell culture medium may contain one or more reagents.

[0258] In some embodiments, the system may have one or more features that facilitate the exchange of the aforementioned liquid cell culture medium. For example, such as... Figure 42 As shown, the system may include one or more components capable of containing different volumes of liquid and / or undergoing volume changes in response to changes in the volume of the liquid therein. (Reference) Figure 42 The cell culture chamber possesses this characteristic. In some embodiments, such as Figure 42 In the embodiments shown, the system may include one or more components that can perform this function because they are formed of a flexible material (and / or include one or more portions formed of such material).

[0259] like Figure 43 As shown, in some implementations, when executing Figure 42 During the procedure shown, some cells may initially be retained in the cell culture chamber (in...) Figure 43 The cells are located outside the hollow fiber membrane (labeled "growth chamber") but still within the flow path containing the cell culture chamber. These cells can be reintroduced into the cell culture chamber by allowing liquid to flow through the hollow fiber membrane and into the cell culture chamber (e.g., through a port). In some embodiments, such as Figure 43In the embodiment shown, the liquid used for this purpose is old liquid cell culture medium. For example, some old liquid cell culture medium may be pumped back by the pump to which it was originally pumped, through the hollow fiber membrane, and into the cell culture chamber.

[0260] Figure 44 Depicting having such Figure 39 The system design shown is a non-limiting example of a method for sterilization and cleaning. For example... Figure 44 As shown, sterilization can be performed by flowing one or more fluids (e.g., one or more cleaning fluids, such as one or more cleaning solvents) through one or more components of the system, which may undergo flow originating from and / or terminating at more than one module. As an example, sterilization can be performed by flowing one or more fluids through a hollow fiber membrane and / or one or more conduits and / or valves. The fluids can sterilize and / or assist in sterilization of the components through which they flow. In some embodiments, after sterilization, and as... Figure 44 As shown, another fluid may flow through one or more components of the system (e.g., one or more sterilized components). For example, and as... Figure 44 As shown, liquid growth media (e.g., fresh liquid growth broth), also referred to herein as cell culture media, can flow through some or all of these components.

[0261] Figure 45 and 46 Other instances of systems comprising multiple flow paths configured to facilitate mixing are described. More specifically, Figure 45 An example of a system comprising multiple flow paths is described, each of which has Figure 41 The design shown. As can be seen from... Figure 45 and Figure 41 As can be seen, each such flow path can have two locations where liquid can be supplied to and / or removed from the flow path: upstream and downstream of the pinch valve. Figure 45 This demonstrates that, if such flow paths are properly arranged, they can be positioned such that these positions in each flow path are aligned (i.e., for each flow path, the pinch valves near the pump are aligned, and the pinch valves near the cell culture chamber are also aligned). In such cases, they can be easily connected as needed to a liquid source (e.g., a liquid cell culture medium source, a reagent source) and / or a conduit in fluid communication with the waste chamber. This connection can be provided as needed when it is necessary to supply a specific liquid to a specific flow path or remove waste from it. This connection can be further facilitated by one or more components that allow easy movement of the liquid source and the conduit in fluid communication with the waste chamber, such as an electrically powered component.

[0262] In some implementations, liquid can be dispensed into the flow path via a liquid dispensing head (e.g., a reagent dispensing head). It is not desired to be bound by any particular theory, but it is believed that an air gap between the reagent dispensing head and any liquid in the dispensing orifice can facilitate the dispensing of reagent from a single reagent dispensing head across multiple flow paths without causing significant contamination.

[0263] Figure 46 Depicting and Figure 45 Examples of systems related to the system are shown. It shows that, in some embodiments, a bracket may be used to hold and / or seal one or more system components (e.g., pumps in multiple flow paths). It also shows that, in addition to or instead of a conduit in fluid communication with the waste chamber, the system may include conduits in fluid communication with system components (e.g., IgG sensors) capable of determining the IgG content of liquid recovered from the flow paths and / or configured to determine such IgG content.

[0264] In some embodiments, the system includes a mixing component, which is a component capable of rotation and / or configured to rotate (in other words, a rotatable component). This rotation can be performed by another component of the system (e.g., a component capable of rotating the rotatable component and / or configured to rotate the rotatable component). The rotation may result in mixing of liquids present in the rotatable component. In some embodiments, the rotatable component includes one or more other system components (e.g., cell culture chambers, cell filters, flow paths) described elsewhere herein. In some embodiments, the cell culture chamber is rotatable, which allows it to be both a mixing component (i.e., a rotatable component) and a cell culture chamber. The system as a whole may also be rotatable (i.e., the system consists of rotatable components) and / or substantially composed of rotatable components.

[0265] In some embodiments, the rotatable component may include two or more mechanically stable orientations. In other words, the rotatable component may include two or more orientations in which it may remain stably stationary indefinitely unless an external force (e.g., a rotational force) is applied. This mechanical stability may be due to the weight distribution and / or shape of the rotatable component (e.g., individually), and / or may be facilitated by the presence of one or more system components also present in the system (e.g., one or more grooves, tabs, and / or other features that enhance the mechanical stability of the rotatable component when properly positioned). Rotating the rotatable component may include rotating it between mechanically stable orientations. In some embodiments, rotating the rotatable component may cause liquids therein to flow within and / or through the rotatable component. This flow may cause the liquids to mix. In such embodiments, mixing may include flow that occurs when the rotatable component is transferred between mechanically stable orientations (e.g., from one mechanically stable orientation to another).

[0266] In some embodiments, the rotatable component includes different mechanically stable orientations that may be adapted to perform different functions. As an example, the rotatable component may include one mechanically stable orientation suitable for cell culture in a cell culture chamber therein, and another mechanically stable orientation suitable for optical measurements on the cell culture chamber. In such embodiments, the rotatable component can be positioned in the first mechanically stable orientation during cell culture and in the second mechanically stable orientation during optical measurements. The rotatable component can rotate therein during the transition from cell culture to optical measurements, and vice versa. Without wishing to be bound by any particular theory, it is believed that this may facilitate cell culture when the rotatable component is in a mechanically stable orientation that facilitates cell culture (e.g., where liquid flows relatively easily through the cell culture chamber), while also allowing the cell culture chamber to be easily used for optical measurements, which may require different geometries (e.g., geometries more compatible with optical detectors but less suitable for cell culture) and / or be facilitated by different geometries.

[0267] Figure 47 and 48 A non-limiting example of a rotatable assembly comprising two mechanically stabilizing orientations is shown. As can be seen in these figures, the rotatable assembly may include an orientation suitable for flow-through and / or infusion (e.g., Figure 47 The orientation shown) and another orientation (e.g. Figure 48 (The orientation shown). The other orientation may only be suitable for mixing liquids present in the rotatable component, or it may be applicable to one or more other purposes. As an example, and as... Figure 48As shown, the other orientation may be suitable for imaging the liquid present in the rotatable component.

[0268] Figure 47 An example of liquid flow through an exemplary rotatable assembly is illustrated. As can be seen therein, liquid can be introduced into the rotatable assembly through one or more ports and can flow through it after pressure is applied (e.g., supplied by another port, such as a port supplying pressurized gas or liquid). The flow rate can be adjusted by regulating the amount of pressure applied. The flow of liquid through the rotatable assembly can include flow in multiple directions, including upward, downward, and / or lateral. After flowing out of the rotatable assembly, the liquid can be directed to a waste chamber (e.g., Figure 47 (as shown in the diagram) and / or components used to determine the IgG content of the liquid (e.g., an IgG sensor).

[0269] Figure 48 An example of a rotatable assembly through which no liquid flows is shown. In some cases, such as during imaging of a chamber present in a rotatable assembly, it may be desirable that no liquid flows through it.

[0270] like Figure 47 and 48 As shown, the rotatable component may also include additional hybrid components. For example, such as... Figure 47 and 48 As shown, the rotatable component may also include a mixing element, such as a magnetically driven stirring rod. As another example, and as... Figure 49 As further shown, the rotatable component may also have one or more structural features that promote mixing. (Reference) Figure 49 As can be seen, the rotatable component includes baffles that separate the liquid flowing into it. When liquid flows into it and / or the rotatable component rotates, the liquid can flow upward through a central conduit defined by the baffles and / or downward through one or both side conduits defined by the baffles. This can promote convection and / or swirling flow in the rotatable component, thereby promoting mixing.

[0271] Figure 49 A port (labeled "control port") through which liquid can be removed from the rotatable assembly is also depicted. In some embodiments, the port may be associated with a valve that can be opened and closed to allow or prevent such liquid removal.

[0272] Figure 50 A second example of a rotatable component is shown. It should be noted that this component is also depicted in... Figure 38 In China, and also as a reference. Figure 38 The described flow path. For example... Figure 50As shown, in some embodiments, the rotatable component is capable of rotating after a centrifugal force is applied and / or configured to rotate after a centrifugal force is applied. The application of this force may cause liquid therein to migrate away from the axis around which the rotatable component rotates. When this force is combined with the opening of a port available for removing liquid from the rotatable component, the liquid present in the rotatable component can be removed in this way. This is illustrated in [illustration]. Figure 50 On the right side, it shows liquid being removed from a port in fluid communication with the waste chamber during the application of centrifugal force. In some embodiments, this force can be applied to remove old liquid cell culture medium from the rotatable assembly. During this removal process, cells may also remain in the rotatable assembly. This can be facilitated by a cell filter that retains cells in the rotatable assembly and / or by the formation of cell clumps that are too large to be removed from the rotatable assembly. Such cell clumps can form after the application of centrifugal force if the centrifugal force is high enough.

[0273] Alternatively, centrifugal force can be applied to create cell clumps without the accompanying removal of liquid from the rotatable assembly. This centrifugal force can be applied with all ports of the rotatable assembly closed, and / or to push liquid away from ports from which liquid can be removed from the rotatable assembly, creating cell clumps away from such ports. After the application of centrifugal force, some or all of the liquid not present in the cell clumps can then be removed through such ports (e.g., by suction). During this removal process, cells present in the cell clumps can remain in the rotatable assembly. In some such embodiments, a second port can also be opened during liquid removal (e.g., a port allowing air to escape). In such embodiments, air can flow into the rotatable assembly through this second port as liquid is removed.

[0274] In some implementations, two or more rotatable components may be positioned such that they can rotate after a single force (e.g., a single centrifugal force) is applied. Figure 51 The diagram illustrates an example of a system containing such a rotatable component. For example... Figure 51 As shown, multiple rotatable components are arranged on a single substrate that is rotatable as a whole. This ideally allows for multiplexing, facilitating the easy delivery of liquids (e.g., liquids containing one or more reagents, fresh liquid cell culture media) to and / or simultaneously facilitating the removal of liquids from the rotatable components. Figure 51 As shown, the rotation can be achieved by a motor (e.g., a spindle motor). In some embodiments, the substrate can be in the form of a CD.

[0275] Also Figure 51As shown, a system with the above design may also include additional features, such as one or more reagent dispensing heads (which can be translated to access different rotatable components). Figure 51 The rotatable assembly may contain a conduit (labeled "reagent station") and / or a conduit supplying liquid to it, in fluid communication with a waste chamber (not shown), and / or a conduit in fluid communication with a system component (e.g., an IgG sensor (not shown)) capable of determining the IgG content of the liquid recovered from the rotatable assembly. In some such embodiments, the rate and / or amount of liquid supplied from the reagent dispensing head may be controlled by an actuator, and / or the translation of the reagent dispensing head may be controlled by an actuator.

[0276] Similarly, and as Figure 51 As shown, a system with the above design may also include one or more components that allow monitoring of the liquid present in the rotatable component, such as one or more fiber optic and / or optical sensors (not shown).

[0277] In some implementations, when having Figure 51 A single component used in the system of the design shown may be capable of performing more than one function and / or configured to perform more than one function. For example, in some embodiments, the system may include a single motor that can rotate the substrate, translate one or more rollers (thereby mixing liquids in one or more flow paths), translate one or more reagent dispensing heads and / or dispense liquids, and / or translate one or more optical fibers.

[0278] In some embodiments where the rotatable component is configured to rotate under high-amplitude centrifugal forces, it may be desirable to seal the rotatable component so that liquid cannot be removed from it through any leakage. For example, in some embodiments, the rotatable component may be fabricated in a sealing chip. When the rotatable component is sealed, it may be necessary to break the seal to supply and / or remove liquid from it. This can be achieved by using a seal that can be opened and then resealed to allow liquid introduction and / or removal, followed by subsequent sealing. Figure 52 The image shows an example of a seal design that can be opened and then resealed. In some implementations, such as... Figure 52 The flow path shown may include ports sealed in this manner, such as the “growth chamber access port” marked therein.

[0279] exist Figure 52In this assembly, the port is sealed by two components: an upper component (labeled therein as the "dispensing needle seal") and a lower component mounted on a spring. The upper component may be made of an elastic material that can be punctured by a needle and / or pipette but can be resealed (e.g., a rubber septum). Applying a force to the lower component (e.g., through a needle and / or pipette containing fluid to be introduced into the rotatable assembly) compresses the spring and opens the port. Removing the force from the component allows the spring to return to its original position, causing the assembly to reseal the port.

[0280] In some embodiments, the liquid can be passed through a series of steps, such as... Figure 52 The design shown illustrates the sealing distribution. First, the upper assembly can be punctured with a needle and / or pipette. Then, a small amount of fluid can be dispensed into the space between the upper and lower assemblies (e.g., approximately a volume sufficient to fill the space between these assemblies) using the needle and / or pipette. Subsequently, the lower assembly can be depressed, allowing the dispensed fluid to be drawn into the rotatable assembly. Introducing fluid into the rotatable assembly in this manner advantageously allows for the introduction of fluid in a way that results in low dead volume and / or no displacement interface.

[0281] Figure 53 The filling is shown to have the following characteristics: Figure 52 The sealing assembly of the seal shown is a non-limiting example. As can be seen in... Figure 53 As seen in the diagram, the sealing assembly can initially be started in a sealed and liquid-free state. When liquid is introduced into it through an open seal, it fills the assembly. During this process, other ports (e.g., all other ports) can be sealed. In some such embodiments, the additional volume introduced into the sealing assembly is accommodated by the expansion of a membrane (e.g., the membrane shown at the bottom of the sealing assembly). Liquid can also be introduced into a sealing assembly that already contains liquid (e.g., liquid containing one or more reagents and / or fresh liquid cell culture medium can be introduced into a sealing assembly that already contains liquid).

[0282] Some implementations involve methods. These methods can be performed on the systems described herein and / or on systems that include one or more of the components described herein. Figure 54 A non-limiting implementation of this method is described. It should be noted that... Figure 54 The methods shown herein, as well as those shown in other accompanying figures, depict one possible order of performing the steps illustrated therein. However, it should also be understood that the steps may be performed in other orders, and / or two or more steps may be performed within partially or completely overlapping time periods.

[0283] Figure 54The method shown includes step 5424, which involves flowing a liquid (e.g., cell culture medium) through a cell culture chamber. It also includes step 5426, which involves retaining the cells in the cell culture chamber. Figure 54 In the method illustrated, the cell culture chamber may contain cells (e.g., during step 5424). Steps 5424 and 5426 may be performed simultaneously (e.g., liquid may flow through the cell culture chamber while cells remain therein). In some embodiments, the method includes causing liquid to flow through the cell culture chamber in a manner such that after the flow stops, at least some cells remain in the cell culture chamber (possibly after being removed from the cell culture chamber and returned to the cell culture chamber during liquid flow).

[0284] Figure 54 The method shown may also include one or more optional steps, some of which are in Figure 54 As shown in the image.

[0285] As an example, such as Figure 54 The method shown may include an optional step of using a pump to flow liquid through the cell culture chamber. This step is... Figure 54 The process is schematically depicted as step 5428. Alternatively, another system component may be employed to flow the liquid and / or force may be applied to flow the liquid by components other than the pump (e.g., system components and / or forces described elsewhere herein).

[0286] As described herein, flow through a cell culture chamber may include flow from various system components (such as one or more system components described elsewhere herein) into the cell culture chamber. As an example, and as... Figure 54 As shown in optional step 5430, the method may include allowing liquid to flow into a cell culture chamber. In some embodiments, the liquid flowing into the cell culture chamber enters from a port. For example, the liquid may flow through the port and into the cell culture chamber. In such embodiments, the liquid may originate from its source (e.g., a reagent source, a cell culture medium source). In some embodiments, the liquid flows out of the cell culture chamber and into (e.g., through) the port. The liquid may also flow through a valve (e.g., a valve located upstream of the port, or a valve fluidically located between the port and the cell culture chamber).

[0287] As described herein, flow through the cell culture chamber may include flow from the cell culture chamber to various system components (such as one or more system components described elsewhere herein). As an example, and as... Figure 54As shown in optional step 5432, the method may include allowing liquid to flow out of the cell culture chamber. Subsequently, the liquid may flow into a waste chamber and / or into a component containing a sensor for a biological product (e.g., a component capable of determining the amount of IgG in the liquid, such as an IgG sensor). The liquid flowing out of the cell culture chamber may flow through a port and / or a valve (e.g., a valve located between the cell culture chamber and the port, or a valve located downstream of the port).

[0288] In some embodiments where the method includes allowing liquid to flow into and out of a cell culture chamber, a single valve may be used to guide the liquid into the cell culture chamber through a first port and out of the cell culture chamber through a second port (e.g., a port different from the first port). For example, the valve may allow a reagent source to be in fluid communication with the cell culture chamber through the first valve, while simultaneously allowing a waste chamber and / or a component that can determine the amount of IgG in the liquid to be in fluid communication with the second port.

[0289] Can be used as Figure 54 Another example of an optional step performed as part of the method shown is optional step 5434. Optional step 5434 includes mixing the liquid. This mixing can occur before the liquid flows into the cell culture chamber (e.g., it can occur upstream of the cell culture chamber). This mixing can be performed as described elsewhere herein. For example, it can be performed in a mixing chamber and / or in a flow path configured to facilitate mixing. Similarly, it can be performed by mixing elements described herein (e.g., active mixing elements, static mixers), and / or can include mechanical mixing via one or more such mixing elements. Optional step 5434 or similar steps can also be performed in the cell culture chamber (e.g., after the liquid flows into the cell culture chamber and / or before the liquid flows out of the cell culture chamber). In some embodiments, optional step 5434 may be omitted. Figure 54 Perform these steps within a time period (e.g., during periods when liquid is not pumped into, out of, and / or through the cell culture chamber). It is not intended to be bound by any particular theory, but it is believed that performing these steps separately can improve the accuracy of the other steps.

[0290] Can be used as Figure 54 Another example of an optional step performed as part of the method shown is optional step 5436. Optional step 5436 includes allowing gas to flow through a permeable membrane. The permeable membrane may be located within the cell culture chamber (e.g., it may form its walls). Advantageously, allowing gas to flow through the permeable membrane allows for gas exchange between the liquid present in the cell culture chamber and its external environment. The gas may flow into (e.g., from) the cell culture chamber and / or out of (e.g., into) the cell culture chamber. The gas may contain reagents (e.g., gaseous reagents) and / or oxygen.

[0291] Can be used as Figure 54 A sixth example of an optional step performed as part of the method shown is optional step 5438. Optional step 5438 includes performing one or more measurements on the liquid. Such measurements can be performed while the liquid is present in the cell culture chamber, before the liquid enters the cell culture chamber, and / or after the liquid leaves the cell culture chamber. Such measurements can be performed using one or more sensors and / or optical detectors.

[0292] Can be used as Figure 54 Two additional examples of optional steps performed as part of the method shown are optional steps 5440 and 5442. Optional step 5440 includes introducing cells into a cell culture chamber, and optional step 5442 includes removing cells from the cell culture chamber. Cells may be introduced into the cell culture chamber for culture and / or removed at the end of culture and / or to evaluate one or more of their properties.

[0293] Some implementations involve methods of pumping one or more fluids (e.g., one or more liquids and / or one or more gases) into a mixing chamber. Figure 55 A non-limiting example of this method is shown. Figure 55 The method shown can be used as Figure 54 To perform a part of the method shown (e.g., Figure 55 Some or all of the steps shown can be used as a component Figure 54 Performed as part of some or all of the steps shown, and / or Figure 54 Step 5434 may include Figure 55 Some or all of the steps shown in the diagram), or they can be performed individually (e.g. Figure 55 Some or all of the steps shown can be performed without execution. Figure 54 (Execute in the case of any of the steps shown).

[0294] Figure 55 The method shown includes optional step 5544, in which fluid is pumped into the mixing chamber. In such embodiments, the fluid may be pumped into the mixing chamber via a port and / or valve (and / or from a cell culture chamber and / or reagent source), as described elsewhere herein.

[0295] Figure 55The method shown also includes optional step 5546. Optional step 5546 includes pumping a second fluid into the mixing chamber. When performing both optional steps 5544 and 5546, they can be performed simultaneously and / or sequentially. In other words, two or more fluids can be pumped into a single mixing chamber simultaneously and / or sequentially. In such cases, the fluids can be pumped into the mixing chamber through a single port (e.g., the fluids can be pumped sequentially through the port, or they can be mixed upstream of the port and then pumped into the mixing chamber) and / or through two different ports (e.g., a first fluid can be pumped into the mixing chamber through a first port, and a second fluid can be pumped into the mixing chamber through a second port). In some such embodiments, both the first and second fluids can flow through a common valve (e.g., via different paths through the valve, originating from different locations and / or exiting the valve to flow into different ports before entering the valve).

[0296] Figure 55 The method shown also includes an optional step 5548. Optional step 5548 includes pumping a third fluid into the second mixing chamber. This fluid may flow through a valve (e.g., a valve through which the first and / or second fluids flow into the first mixing chamber) and / or through a port. In some embodiments, the third fluid is a mixture of the first and second fluids (e.g., after mixing in the first mixing chamber). The third fluid may also be a different fluid (e.g., a fluid flowing from a reagent source). In such embodiments, two mixing chambers may be used simultaneously (and possibly with a common valve) to mix different fluids.

[0297] As noted above, some implementations involve methods for performing one or more measurements on a liquid. Figure 56 A non-limiting example of such a method is shown. Figure 56 The method shown can be used as Figure 54 To perform a part of the method shown (e.g., Figure 56 Some or all of the steps shown can be used as a component Figure 54 Performed as part of some or all of the steps shown, and / or Figure 54 Step 5438 may include Figure 56 Some or all of the steps shown in the diagram), or they can be performed separately (e.g. Figure 56 Some or all of the steps shown can be performed without execution. Figure 54 (Execute in the case of any of the steps shown).

[0298] Figure 56The method shown includes optional step 5650. Optional step 5650 includes performing an optical measurement. The optical measurement may be performed using an optical detector (possibly including an optical sensor) and / or a light source, as described elsewhere herein. In some embodiments, the optical measurement is performed on the cell culture chamber and / or the liquid present therein. The optical measurement may also be performed on a system component outside the cell culture chamber (e.g., a component configured to determine the amount of IgG in liquid flowing out of the cell culture chamber) and / or liquid in another such system component. Various suitable optical measurements can be performed; a non-limiting example is the measurement of the optical density of a liquid (e.g., liquid present in the cell culture chamber, liquid flowing out of the cell culture chamber).

[0299] Relatedly, it can be used as Figure 56 Another optional step performed as part of the method shown is optional step 5652. Optional step 5652 includes determining the amount of cells present in the liquid based on the optical measurement. For example, if the optical measurement is a measurement of the optical density of the liquid, the amount of cells present in the liquid can be determined from the optical density.

[0300] Figure 57 Another non-limiting example of a method for performing one or more measurements on a liquid is shown. Figure 57 The method shown can be used as Figure 54 To perform a part of the method shown (e.g., Figure 57 Some or all of the steps shown can be used as including Figure 54 Performed as part of some or all of the steps shown, and / or Figure 54 Step 5438 may include Figure 57 Some or all of the steps shown), or they can be performed separately (e.g. Figure 57 Some or all of the steps shown can be performed without execution. Figure 54 (Execute in the case of any of the steps shown).

[0301] Figure 57The method shown includes optional step 5754. Optional step 5754 includes measuring the properties of the liquid. In some embodiments, the measurement is performed on the liquid present in the cell culture chamber. The measurement may also be performed on liquids present in system components other than the cell culture chamber (e.g., liquids present in components configured to determine the amount of IgG in liquid flowing out of the cell culture chamber, liquids obtained from the cell culture chamber). Non-limiting examples of measurable liquid properties include dissolved oxygen content, pH, the amount of biological products (e.g., IgG) and / or other substances (e.g., carbon dioxide, nutrients, metabolites such as lactate and / or glucose) present in the liquid, cell concentration, cell viability, temperature, pressure, and / or viscosity. Liquid properties can be measured using sensors described elsewhere herein.

[0302] Figure 57 The method shown also includes an optional step 5756. Optional step 5756 includes delivering a reagent to the liquid based on measurements of the liquid's properties. Non-limiting examples of reagents that can be delivered include oxygen, acids (e.g., hydrochloric acid), bases (e.g., sodium bicarbonate), and cell culture media. The reagent can be delivered to the liquid at the location where the measurement is performed (e.g., a cell culture chamber) and / or to a location upstream or downstream of it (e.g., to the liquid flowing out of the cell culture chamber, delivered to the cell culture chamber based on measurements of the liquid flowing out of the cell culture chamber).

[0303] In some implementations, optional steps 5754 and 5756 may be performed together to adjust one or more properties of the liquid and / or maintain one or more properties of the liquid within a desired range.

[0304] As an example, in some implementations, the method includes measuring the dissolved oxygen content of a liquid and, based on the measurement, supplying the liquid with an amount of oxygen (e.g., an amount sufficient to bring the dissolved oxygen content of the liquid within a desired range). The oxygen can be supplied as dissolved oxygen in the liquid to which the dissolved oxygen content is measured. This liquid may also have one or more properties required for use in a cell culture chamber. For example, the method may include supplying a cell culture medium (e.g., a pre-prepared cell culture medium) with a dissolved oxygen content within a range determined based on the measurement of the dissolved oxygen content.

[0305] As another example, in some implementations, the method includes measuring the pH of a liquid and delivering a quantity of acid and / or base, the quantity determined based on the pH measurement (e.g., an amount sufficient to bring the pH of the liquid within a desired range). The acid and / or base may be in the liquid, which may also have one or more properties required for inclusion in a cell culture chamber. For example, the method may include delivering a cell culture medium with a pH content within a range determined based on the pH measurement (e.g., containing a sufficient quantity of acid and / or base to have such a pH).

[0306] Figure 58 Non-limiting examples of methods in which cells can be introduced into and / or recovered from a cell culture chamber are shown. Figure 58 The method shown in the figure can be used as Figure 58 To perform a part of the method shown (e.g., Figure 58 Some or all of the steps shown can be used as including Figure 54 Performed as part of some or all of the steps shown, and / or Figure 54 Steps 5440 and / or 5442 may include Figure 58 Some or all of the steps shown), or they can be performed separately (e.g. Figure 58 Some or all of the steps shown can be performed without execution. Figure 54 (Performed in any of the steps shown). In some implementations, Figure 58 The methods shown can be used in whole or in part as Figure 54 The first step of the method shown is to be executed, or in the execution Figure 54 The method shown is performed prior to this. For example, cells may be introduced into the cell culture chamber when the liquid begins to flow through the cell culture chamber and / or before the liquid flows through the cell culture chamber.

[0307] Figure 58The method shown includes optional step 5858. Optional step 5858 includes delivering cells to a port and / or reservoir. In some embodiments, cells are delivered in the presence of a liquid (e.g., they may be suspended in the liquid delivered to the port and / or reservoir). The port and / or reservoir may be a port and / or reservoir suitable for cell seeding. In some embodiments, the port and / or reservoir is sealed (e.g., by a septum, such as a rubber septum and / or a silicone septum). In such embodiments, delivering cells may include piercing the septum (e.g., with a needle and / or pipette) and delivering cells through the piercing (e.g., through the needle and / or pipette). The port and / or reservoir may also be covered with a cap (and may be sealed or unsealed). In such embodiments, delivering cells may include removing the cap. In some embodiments, cells may be delivered automatically (e.g., without needing to pierce the septum or remove the cap). Such automated delivery may include the use of one or more pumps.

[0308] Figure 58 The method shown also includes an optional step 5860. Optional step 5860 includes allowing cells to flow from the port and / or reservoir into the cell culture chamber. This can be achieved by pumping the cells (and / or the liquid in which they reside) along a fluid path connecting the port and / or reservoir to the cell culture chamber.

[0309] Figure 58 The method shown also includes an optional step 5862. Optional step 5862 includes allowing cells to flow from the cell culture chamber to a second port. This step can also be achieved by pumping cells (and / or the liquid in which they reside) along a fluid path connecting the second port and / or reservoir to the cell culture chamber. The second port and / or reservoir may be a different port and / or reservoir from which cells flow into the cell culture chamber. For example, it may be a port and / or reservoir from which cell recovery can be performed.

[0310] Figure 58The method shown also includes an optional step 5864. Optional step 5864 includes removing cells from the second port and / or reservoir. In some embodiments, the cells are recovered in the presence of liquid (e.g., they may be suspended in liquid recovered from the port and / or reservoir). The port and / or reservoir may be a port and / or reservoir capable of cell recovery. In some embodiments, the port and / or reservoir is sealed (e.g., by a septum). In such embodiments, cell recovery may include piercing the septum (e.g., with a needle and / or pipette) and recovering the cells through the piercing (e.g., aspirating cells through the needle and / or pipette). The port and / or reservoir may also be covered by a cap (and may be sealed or unsealed). In such embodiments, cell recovery may include removing the cap. In some embodiments, cells may be recovered automatically (e.g., without needing to pierce the septum or remove the cap). Such automated delivery may include the use of one or more pumps.

[0311] Figure 59 The illustration depicts a method for introducing cells into a cell culture chamber (e.g., a method for cell seeding). The introduction of cells can be performed manually and / or automatically. In some embodiments, this introduction can result in a relatively uniform distribution of cells within the cell culture chamber.

[0312] As can be Figure 59 As seen in the diagram, cells can be introduced into the cell culture chamber from the inoculation port. During this introduction, a pump can be used to flow liquid on demand. Prior to this introduction, the cell culture chamber can be pre-filled by flowing liquid (e.g., liquid supplied by the pump and / or from a reagent source, such as cell culture medium) through the cell culture chamber and / or other system components. During this process, valves (e.g., ...) can be opened. Figure 59 The inoculation port valve and rotary valve shown (to allow air and / or liquid to escape from the cell culture chamber) are used to allow air and / or liquid to escape. After this introduction, one or more valves (e.g., including...) can be closed. Figure 59 (See the inoculation port valve shown). This can seal the cell culture chamber and / or system, which can be beneficial during incubation.

[0313] In some implementations, it can be achieved by... Figure 59 The inoculation port shown retrieves cells and / or removes cells from the cell culture chamber by retrieving cells from different inoculation ports (e.g., inoculation ports located on opposite sides of different inoculation port valves). Cells can be removed from the cell culture chamber for further analysis, on which one or more assays are performed, and / or for further cell culture (e.g., in a larger chamber and / or container).

[0314] Cell culture chambers can have a variety of suitable designs. In some embodiments, the cell culture chamber includes, is substantially composed of, and / or consists of channels. In some embodiments, such channels may be microfluidic channels.

[0315] The cell culture chambers described herein can have various suitable volumes. In some embodiments, the cell culture chamber has a microfluidic volume. For example, the cell culture chamber can have a volume of less than or equal to 500 μL, less than or equal to 400 μL, less than or equal to 300 μL, less than or equal to 200 μL, less than or equal to 100 μL, less than or equal to 75 μL, or less than or equal to 50 μL. In some embodiments, the cell culture chamber has a volume greater than or equal to 30 μL, greater than or equal to 50 μL, greater than or equal to 75 μL, greater than or equal to 100 μL, greater than or equal to 200 μL, greater than or equal to 300 μL, or greater than or equal to 400 μL. Combinations of the above ranges are also possible (e.g., less than or equal to 500 μL and greater than or equal to 30 μL). Other ranges are also possible.

[0316] Some cell culture chambers exhibit relatively small dead volumes. In some embodiments, the dead zone comprises less than or equal to 20% or 15% or 10% or 7.5% or 5% or 2% or 1% of the cell culture chamber. In some embodiments, the dead zone comprises more than or equal to 0% or 1% or 2% or 5% or 7.5% or 10% or 15% of the cell culture chamber. Combinations of the above ranges are also possible (e.g., less than or equal to 20% and greater than or equal to 10%). Other ranges are also possible. In some embodiments, the dead zone comprises exactly 0% of the cell culture chamber.

[0317] In some implementations, mixing is minimal or nonexistent in the “dead zone” (e.g., a portion of a cell culture chamber present in the system).

[0318] In some embodiments, the cell culture chamber may have one or more features that facilitate the measurement of the amount of cells present in the liquid therein. For example, the cell culture chamber may include a surface that is transparent to light of at least one wavelength (e.g., a transparent path may exist between the outer surface of the system and the interior of the cell culture chamber). When present, this surface may extend through the entire cell culture chamber, or only through one or more portions thereof. In some embodiments, the cell culture chamber as a whole is transparent to light of at least one wavelength (e.g., a transparent path through the interior of the cell culture chamber may exist between two outer surfaces of the system (e.g., two opposing outer surfaces)). In some embodiments, the entire cell culture chamber may be transparent to light of at least one wavelength. It is also possible that only one or more portions thereof are transparent to light of at least one wavelength.

[0319] In some embodiments, light that can pass through the cell culture chamber surface and / or the cell culture chamber has a wavelength greater than or equal to 400 nm, greater than or equal to 450 nm, greater than or equal to 500 nm, greater than or equal to 550 nm, greater than or equal to 600 nm, or greater than or equal to 650 nm. In some embodiments, light that can pass through the cell culture chamber surface and / or the cell culture chamber has a wavelength less than or equal to 700 nm, less than or equal to 650 nm, less than or equal to 600 nm, less than or equal to 550 nm, less than or equal to 500 nm, or less than or equal to 450 nm. Combinations of the above ranges are also possible (e.g., greater than or equal to 400 nm and less than or equal to 700 nm). Other ranges are also possible. In some embodiments, the cell culture chamber surface and / or the cell culture chamber is transparent to light of all wavelengths within one or more of the above ranges.

[0320] In some embodiments, the cell culture chamber surface and / or the cell culture chamber itself is transparent to light within one or more wavelengths in the aforementioned ranges, with a transparency greater than or equal to 70%, 80%, 90%, 95%, 99%, or 99.9%. In some embodiments, the cell culture chamber surface and / or the cell culture chamber itself is transparent to light within one or more wavelengths in the aforementioned ranges, with a transparency less than or equal to 100%, 99.9%, 99%, 95%, 90%, or 80%. Combinations of the aforementioned ranges are also possible (e.g., greater than or equal to 70% and less than or equal to 100%). Other ranges are also possible. In some embodiments, the cell culture chamber surface and / or the cell culture chamber itself is 100% transparent to light within one or more wavelengths in one or more of the ranges mentioned above.

[0321] Transparency can be assessed using ultraviolet-visible spectroscopy.

[0322] Cells present in the cell culture chamber can be retained with relatively high efficiency. In some embodiments, cells are retained in the cell culture chamber (e.g., during fluid flow) with an efficiency greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 99%. In some embodiments, cells are retained in the cell culture chamber (e.g., during fluid flow) with an efficiency less than or equal to 100%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, or less than or equal to 75%. Combinations of the above ranges are also possible (e.g., greater than or equal to 75% and less than or equal to 100%). Other ranges are also possible. In some embodiments, exactly 100% of the cells are retained in the cell culture chamber (e.g., during fluid flow).

[0323] In some embodiments, the cell culture chamber has one or more features that facilitate cell culture therein. For example, in some embodiments, the cell culture chamber may be biocompatible. For example, cells may be cultured in the cell culture chamber. Such cells may have relatively high viability as measured by a dye (e.g., trypan blue), may exhibit adhesion and / or proliferation in the cell culture chamber, and / or exhibit cellular markers and / or proteins related to cell health and function upon immunofluorescence staining.

[0324] In some embodiments, the cell culture chamber is sterilizable (e.g., by autoclave, exposure to gamma radiation, exposure to X-ray radiation, exposure to ethylene oxide, dry heat sterilization, and / or chemical sterilization, such as by exposure to hydrogen peroxide vapor, bleach, and / or alcohol). Such a cell culture chamber may discolor and / or deform during sterilization (or it may remain unchanged).

[0325] The cell culture chamber described herein can comprise and / or be formed from a variety of suitable materials. In some embodiments, the cell culture chamber is formed of and / or comprises plastics such as polystyrene, PMMA, cyclic olefin copolymers, polycarbonate, polysulfone, polyetherimide (e.g., Ultem), polyurethane, and / or 3D printing resin. In some embodiments, the cell culture chamber is formed of and / or comprises PDMS. In some embodiments, the substrate is formed of and / or comprises glass.

[0326] In some embodiments, the system for cell culture includes a permeable membrane. The permeable membrane may allow one or more gases to pass through but prevent the passage of one or more liquids. In some embodiments, the permeable membrane present in the systems described herein does not allow the passage of liquids present in the system and / or flowing through the system. For example, the permeable membrane may be impermeable to water and / or aqueous liquids. The permeable membrane may be permeable to one or more gases that are desired to be exchanged therethrough, such as gaseous reagents and / or oxygen. Some suitable permeable membranes include open pores, which are believed to facilitate such exchange.

[0327] The breathable membrane can be placed in various suitable locations within the system described herein. In some embodiments, the breathable membrane forms the wall of the cell culture chamber, and / or the cell culture chamber is surrounded by a breathable material (e.g., it can be surrounded by a breathable membrane). In some such embodiments, the breathable membrane can provide transport of gases in and / or into the cell culture chamber and its external environment (e.g., the surrounding environment where the cell culture chamber and / or system are located). Without wishing to be bound by any particular theory, it is believed that placing the breathable membrane near the cell culture chamber facilitates the necessary gas exchange within it because it shortens the distance between the membrane and the cell culture chamber, the distance that gases must diffuse to for exchange to occur.

[0328] Various suitable breathable membranes can be used. A non-limiting example of a suitable breathable membrane is a silicone membrane. Advantageously, some silicone membranes can allow ideally high oxygen diffusion (which can ideally limit oxygen depletion near the membrane surface) and / or withstand relatively high oxygen pressure (which can allow relatively rapid diffusion).

[0329] In some implementations, the breathable membrane may be relatively flexible. For example, some breathable membranes may be able to undergo and / or be configured to undergo reversible deformation under pressure applied to them during flow through the system.

[0330] In some embodiments, the system for performing cell culture includes one or more sensors (e.g., two or more sensors). Such sensors can be placed in one or more locations where it may be necessary to sense the characteristics of the system and / or the characteristics of the liquids present in the system. For example, sensors may be present in a cell culture chamber, a mixing chamber, and / or in a flow path configured to facilitate mixing. Sensors may also be located in a position fluidly in communication with one or more such locations. In such embodiments, liquid can be removed from the relevant portion of the system and supplied to the location containing the sensors.

[0331] Non-limiting examples of sensors that may be included in the systems described herein include dissolved oxygen sensors, pH sensors, bioproduct sensors (e.g., IgG sensors, monoclonal antibody sensors), carbon dioxide sensors, cell concentration sensors, cell viability sensors, nutrient sensors, metabolite sensors (e.g., lactate sensors, glucose sensors), temperature sensors, pressure sensors, humidity sensors, and / or viscosity sensors.

[0332] The sensors described herein can be used to perform sensing using various techniques. For example, the system may include optical sensors (e.g., optical detectors and / or portions of optical detectors), electrochemical sensors, electrical sensors, and / or acoustic sensors. In some embodiments, one or more of these types of sensors may be employed to sense one or more substances described in the preceding paragraph. For example, optical sensors may be used to perform optical measurements to determine the amount of a biological product. The substances described in the preceding paragraph may also be sensed directly (e.g., by a sensor that detects the binding between the relevant substance and sensor components). Further examples of sensor types that can be used to sense different properties are described below.

[0333] pH sensors can be optical sensors and / or electrochemical sensors (e.g., ion-sensitive field-effect transistors, solid polymer films, capacitive sensors, sensors containing carbon nanotubes, and / or enzyme sensors). Optical sensors measure pH by measuring the optical properties of a pH-sensitive indicator dye and thereby calculating the pH. Electrochemical sensors measure pH by measuring the potential, current, and / or capacitance of a liquid and thereby calculating the pH. In some embodiments, pH sensors are capable of and / or configured to measure pH values ​​within a specific range (e.g., between 5.5 and 8.5).

[0334] Dissolved oxygen sensors may include optical sensors (e.g., fluorescence sensors, colorimetric sensors, and / or surface plasmon resonance sensors) and / or electrochemical sensors (e.g., microfabricated Clarke electrodes, thin-film platinum electrodes, screen-printed electrodes, and / or polymer electrolyte-based electrodes). Optical sensors can measure dissolved oxygen content by measuring the absorption, fluorescence, and / or refractive index of a liquid and thereby calculating the dissolved oxygen content. Electrochemical sensors can measure dissolved oxygen content by measuring the potential, current, and / or capacitance of a liquid and thereby calculating the dissolved oxygen content. In some embodiments, dissolved oxygen sensors are capable of and / or configured to measure dissolved oxygen values ​​within a specific range (e.g., between 0% and 100%).

[0335] Sensors for carbon dioxide can include optical and / or electrochemical sensors (e.g., potentiometric and / or amphoteric sensors, ion-sensitive field-effect transistors, solid-state electrolyte sensors, and / or enzyme sensors). Optical sensors can measure carbon dioxide content by measuring the absorption, fluorescence, and / or refractive index of a liquid and thereby calculating the carbon dioxide content. Electrochemical sensors can measure carbon dioxide content by measuring the potential, current, and / or capacitance of a liquid and thereby calculating the carbon dioxide content.

[0336] Sensors used for cell concentration can include optical (e.g., optical density sensors, bioreflectance sensors, fluorescence sensors, and / or near-infrared spectroscopy sensors), electrical (e.g., electrical impedance sensors, dielectric spectroscopy sensors, RF impedance sensors, and / or capacitance-based sensors), and / or acoustic sensors (e.g., surface acoustic wave sensors, bulk acoustic wave sensors). Such sensors can measure the optical, electrical, and / or acoustic properties of a liquid and thereby calculate cell concentration.

[0337] Sensors used for cell viability can include optical (e.g., fluorescence, reflectivity) and / or electrical sensors (e.g., impedance sensors, capacitance-based sensors, potential sensors, and / or conductivity sensors). Such sensors can measure the optical and / or electrical properties of a liquid and thereby calculate cell concentration.

[0338] Sensors for nutrients and metabolites can include optical (e.g., fluorescent NADH sensors and / or fluorescent NADPH sensors) and / or electrochemical sensors (e.g., ion-selective electrodes, conductivity sensors, current-type glucose sensors, and / or current-type and / or potential-type lactate sensors). Such sensors can measure the optical and / or electrochemical properties of liquids and thereby calculate the amount of nutrients and / or metabolites contained therein.

[0339] Sensors for biological products can include optical (e.g., surface plasmon resonance sensors, fluorescence sensors, surface-enhanced Raman scattering sensors, interferometric sensors, colorimetric sensors, and / or luminescent sensors) and / or electrical (e.g., field-effect transistors, electrochemical impedance spectroscopy sensors, dielectric spectroscopy sensors, capacitive sensors, impedance sensors, and / or sensors incorporating carbon nanotubes) sensors. Such sensors can measure the optical and / or electrical properties of liquids and thereby calculate the amount of cellular products contained therein.

[0340] Temperature sensors can include optical (e.g., fluorescent and / or luminescent sensors) and / or electrical (e.g., resistance temperature detectors) sensors. Such sensors can measure the optical and / or electrical properties of a liquid and thereby calculate its temperature.

[0341] Pressure sensors may include optical (e.g., optical pressure sensors) and / or electrical (e.g., capacitive pressure sensors) sensors.

[0342] Humidity sensors may include optical (e.g., absorbance sensors and / or reflection-based sensors) and / or electrical sensors (e.g., capacitive sensors, resistive sensors, conductivity sensors and / or dielectric sensors).

[0343] Viscosity sensors may include electrical sensors (such as capacitance-based sensors and / or impedance sensors).

[0344] Other examples of sensors that can be used to sense one or more properties of a liquid medium include optical sensors (e.g., transmittance sensors and / or reflectance sensors) and electrical sensors (e.g., conductivity sensors and / or capacitance-based sensors).

[0345] Optical detectors can contain various components that facilitate optical detection. Non-limiting examples of such components include optical sensors, light sources, and components that facilitate imaging (e.g., microscopes). The light source present in an optical detector can provide light in various suitable ways; a non-limiting example is fluorescence (e.g., the light source could be a fluorescence source). In some embodiments, the optical detector takes the form of and / or includes a reader.

[0346] The sensors described herein can have a variety of suitable designs; two non-limiting examples include sensor plugs and sensor patches. In some embodiments, the sensor includes a sensor plug capable of receiving optical fiber (e.g., polymer fiber, 1 mm diameter fiber). The optical fiber may be capable of and / or configured to transmit an optical signal to a location where it can be detected.

[0347] As noted above, some systems may include valves. A variety of suitable valves can be used in the systems described herein. Non-limiting examples of suitable valves include pneumatic valves, rotary valves, rotary valves (e.g., rotary diaphragm valves), pinch valves, solenoid valves, shape memory alloy valves, and diaphragm valves.

[0348] In some embodiments, the valve used in the system described herein is designed such that it fluidly communicates with one port of the pump inlet and is located upstream of it, and fluidly communicates with another port of the pump outlet and is located downstream of it. In some such embodiments, these ports can be arbitrarily selected (i.e., any port fluidly communicating with the valve can be configured to fluidly communicate with the pump inlet and be located upstream of it, and any port fluidly communicating with the valve can be configured to fluidly communicate with the pump outlet and be located downstream of it, and / or any pair of ports can be selected for these two locations). It is not desirable to be bound by any particular theory; this can facilitate fluid flow through the system between various components located within the system, the flow being driven by a pump that can only pump in a single direction. In other words, the direction in which the pumped fluid flows through the system may be selectable by the valve (e.g., even if the pump can only pump in a single direction).

[0349] Figures 60-66 A non-limiting example of a valve having the above design and the associated system in which the valve is located is shown. Figure 60 The valve push rod is shown. From Figure 60 As can be seen, the valve push rod has two open positions, which allows selection of the port in fluid communication with the pump (e.g., fluid communication can occur through the open positions). Figure 61 A non-limiting example is shown as part of a system that includes such a valve and also includes a pump. As can be seen from... Figure 61 As can be seen, the push rod is rotatable, allowing various ports to be in fluid communication with the pump and located upstream or downstream of the pump.

[0350] Figure 62 An example of the valve position described above is shown, in which a port (fluidly connected to the reagent source) is fluidly connected to and upstream of the pump inlet, and a port (upstream of and fluidly connected to the mixing chamber) is fluidly connected to and downstream of the pump outlet. Figure 62 The arrows in the diagram illustrate the flow of fluid from a port in fluid communication with the reagent source through a valve, through a pump, through the valve again, and then to a chamber (e.g., a cell culture chamber).

[0351] Figures 63-65 Other examples of how fluid can flow through the valve depicted herein are shown. Figure 63 and 64 The valve is described as being used to induce a flow that promotes mixing (i.e., by allowing the fluid to flow back and forth between the two mixing chambers). Figure 65 The flow of fluid being removed from the mixing chamber to the waste chamber using the valve is illustrated. As can be seen from... Figures 62-65As can be seen, the flow direction through the valve and / or pump can be adjusted to flow in multiple different directions, to multiple different locations, and / or out of multiple different locations.

[0352] Figure 66 The base of the system in which the aforementioned valve is positioned is depicted. As can be seen from... Figure 62 As can be seen, the various components of the system may have static positions (e.g., they may not move and / or they may be mechanically coupled to a substrate present in the system), although flow may be selected to flow to them, through them, and / or out of them as needed.

[0353] The valves described herein can be in fluid communication with multiple ports. In some embodiments, some or all of the ports are themselves in fluid communication with a reagent source. For example, each port may be in fluid communication with a different source of a different reagent, and / or each port may be fluidly isolated from each other without being in fluid communication with a valve to which it is in fluid communication. Such reagents may be those described elsewhere herein with respect to cell culture.

[0354] The valve may also be in fluid communication with certain ports, which are in fluid communication with fluid sources other than reagents (e.g., sample sources) and / or system components other than sources (e.g., mixing chambers, pumps, waste chambers), and / or system components other than ports (e.g., mixing chambers, pumps, and waste chambers).

[0355] As noted above, some of the valves described herein are in fluid communication with multiple ports. Such ports may include ports in fluid communication with various chambers (e.g., cell culture chambers, mixing chambers), pumps, reagent sources (e.g., sources of oxygen, acid, alkali, and / or cell culture media), sample sources, gas sources, and / or waste chambers. As described elsewhere herein, multiple ports may include ports for cell seeding and / or ports for cell retrieval. As also described elsewhere herein, such ports may be sealed with septa. In such cases, cell seeding and cell retrieval can be performed by puncturing the septa with a needle and / or pipette and delivering and / or withdrawing cells through it, respectively. As discussed above, Figure 52 A non-limiting example of a port sealed by a septum is shown. Alternatively, ports for cell seeding and / or cell retrieval may include caps that can be opened and / or removed to allow delivery and / or withdrawal of cells.

[0356] In some embodiments, the system includes a reservoir through which cell seeding and / or cell retrieval can be performed. When present, such reservoirs may be sealed with septa and / or may include caps, which can be punctured and / or removed, respectively, to allow cell seeding and / or retrieval.

[0357] The system may also contain two valves, each in fluid communication with one or more ports. Figure 67 A non-limiting example of a system with this design is described. Figure 67 The system shown includes a first valve (rotary valve) in fluid communication with a port that allows for cell seeding and / or cell recovery. The system also includes a second valve (rotary valve) in fluid communication with multiple ports, including a port in fluid communication with a reagent source and a port in fluid communication with a waste chamber. Figure 67 The system shown also includes a cell culture chamber, an oxygen sensor, a filter, and a rotary pump, with a breathable membrane forming one wall of the cell culture chamber (which is labeled "growth chamber" and "gas passage," respectively).

[0358] The volume of each pumping cycle of the pumps described herein can be greater than or equal to 100 nL, greater than or equal to 200 nL, greater than or equal to 500 nL, greater than or equal to 750 nL, greater than or equal to 1 μL, greater than or equal to 2 μL, or greater than or equal to 5 μL. The volume of each pumping cycle of the pumps described herein can be less than or equal to 10 μL, less than or equal to 5 μL, less than or equal to 2 μL, less than or equal to 1 μL, less than or equal to 750 nL, less than or equal to 500 nL, or less than or equal to 200 nL. Combinations of the above ranges are also possible (e.g., greater than or equal to 100 nL and less than or equal to 10 μL, greater than or equal to 100 nL and less than or equal to 1 μL, greater than or equal to 5 μL and less than or equal to 10 μL). Other ranges are also possible.

[0359] When the system described herein contains two or more pumps, each pump may independently have a volume for each pumping cycle within one or more of the ranges described above.

[0360] The maximum flow rate of the pumps described herein can be greater than or equal to 10 nL / s, greater than or equal to 20 nL / s, greater than or equal to 50 nL / s, greater than or equal to 75 nL / s, greater than or equal to 100 nL / s, greater than or equal to 150 nL / s, greater than or equal to 200 nL / s, greater than or equal to 300 nL / s, or greater than or equal to 400 nL / s. The maximum flow rate of the pumps described herein can be less than or equal to 500 nL / s, less than or equal to 400 nL / s, less than or equal to 300 nL / s, less than or equal to 200 nL / s, less than or equal to 150 nL / s, less than or equal to 100 nL / s, less than or equal to 75 nL / s, less than or equal to 50 nL / s, or less than or equal to 20 nL / s. Combinations of the above ranges are also possible (e.g., greater than or equal to 10 nL / s and less than or equal to 500 nL / s). Other ranges are also possible.

[0361] When the system described herein contains two or more pumps, each pump may independently have a maximum flow rate within one or more of the ranges mentioned above.

[0362] Some of the pumps described herein may be able to withstand, or be configured to withstand, and / or withstand high negative pressures. Similarly, some such pumps may be able to deliver, or be configured to deliver, and / or may deliver fluids of predictable volumes at high pressures (e.g., high negative pressures). Without wishing to be bound by any particular theory, it is believed that pumps may experience relatively high negative pressures when used to pump fluids through a permeable membrane (e.g., from a cell culture chamber through a permeable membrane forming part or all of its wall).

[0363] In some implementations, the pump includes one or more valves. Fluid flow through such a pump may include flow through one or more valves. Figure 68 Examples of pumps and associated valves that can be applied to the systems described herein are shown.

[0364] In some embodiments, the pumped liquid can flow into the pump through an inlet valve and out through an outlet valve. Between these two passages, the liquid can flow through a diaphragm valve. In some such embodiments, the pump may also include one or more air passages associated with its valves. Such air passages can facilitate pumping action.

[0365] As can be Figure 68 As seen in some embodiments, the pump may include more than one inlet valve and / or may include one or more routing valves. The routing valves may be adapted to route liquid to diaphragm valves within the pump and / or different locations within the system. The presence of more than one routing valve and / or more than one inlet valve allows a single pump to supply liquid from multiple sources and / or locations within the system (e.g., one or more reagent sources, one or more mixing chambers, one or more cell culture chambers) to a single outlet valve (e.g., supplying a mixing chamber, cell culture chamber, and / or waste chamber).

[0366] Just as it can also be in Figure 68 As seen in some embodiments, more than one pump (and / or more than one valve present in the system and / or on the substrate) may be associated with a common air passage. For example, see reference... Figure 68The diaphragm valve and outlet valve share an air passage with other pumps. When the common air passage is associated with more than one passage, a common air pressure clock can be used to drive the actuation of all pumps associated with the common air pressure passage. In such embodiments, if it is desired to flow through one but not all such pumps (e.g., if it is desired to add reagents (e.g., cell culture medium) to one but not all cell culture chambers supplied by these pumps), the routing valve on the selected pump can be closed to prevent pumping by those pumps (and the addition of the relevant reagents to the relevant cell culture chamber). In such embodiments, the routing valve may be able to be controlled independently.

[0367] In some implementations, the liquid can flow through the pump in multiple directions (e.g., in opposite directions). In such implementations, flowing through the pump in one direction may result in the liquid flowing into the cell culture chamber, while flowing through the pump in another direction may result in the liquid flowing into the waste chamber.

[0368] In some implementations, the system includes more than one pump. Figure 69 A non-limiting example of such a system is shown. Figure 69 The system shown also includes other exemplary components discussed elsewhere in this document (such as two sets of multiple ports, a waste chamber, and a cell culture chamber). Figure 70 and 71 Another non-limiting example of such a system is shown. Figure 70 and 71 The system shown includes two pumps, each driven by a manifold that can also be used to drive pumps associated with other systems, such as other systems located on a common substrate, as described in further detail below.

[0369] In some embodiments, the system described herein is located on a substrate. Some (but not all) components of the system described herein may also be placed on a substrate. For example, the system may include a cell culture chamber, one or more mixing components and / or valves located on the substrate, and also include a pump located outside the substrate. As a second example, the system may include one or more cell culture chambers and / or mixing components located on the substrate, and also include valves and a pump located outside the substrate. Other system components described herein, such as sensors (e.g., optical detectors), may be placed on and / or outside such a substrate. Non-limiting examples of suitable such substrates include chips, such as microfluidic chips.

[0370] In some implementations, one or more system components include volumes that have been removed (e.g., etched away) from the substrate. Some or all of these system components may be located on a single substrate, and / or the system may include a single substrate from which one or more portions have been removed to form the system components.

[0371] In some embodiments, the system includes a substrate from which various volumes have been removed, and two components are disposed thereon. In some such embodiments, various channels and ports take the form of volumes removed from the substrate. Valve pins and pump pins are disposed on the substrate, and / or may position a membrane between the substrate and these components. Several volumes may also be removed from the membrane. These volumes may take the form of through-holes (forming membrane valves) disposed below valve pins and channels (forming membrane pumps) disposed below pump pins. The valve pins are rotatable to allow various ports on the substrate to fluidly communicate with channels taking the form of volumes removed from the substrate. The pump pins are rotatable to allow the pump to deliver fluid to and / or remove fluid from the channels.

[0372] Some systems can be thermally bonded together. This bonding can be advantageously performed in the absence of solvents and / or laser welding.

[0373] A portion of the substrate can be removed, through which one or more sensors can be inserted.

[0374] The substrate can be assembled in a variety of suitable ways. In some embodiments, one or more materials can be machined to form the substrate and / or one or more layers thereof. As another example, one or more materials can be laser-cut to form the substrate and / or one or more layers thereof.

[0375] In some embodiments, the substrate (e.g., a chip, a microfluidic chip) may include a surface that is transparent to light of at least one wavelength. This surface may extend throughout the entire substrate or only through a portion of it. In some embodiments, the substrate may include one or more portions that, as a whole, are transparent to light of at least one wavelength. The entire substrate may also be transparent to light of at least one wavelength. The wavelength of light that the substrate can transmit and the level of transparency may fall within one or more of the ranges provided above regarding the transparency of the channel to light wavelengths.

[0376] In some embodiments, the substrate on which the system is placed has one or more features that facilitate cell culture therein. For example, in some embodiments, the substrate may be biocompatible and / or sterilizable (e.g., as described elsewhere herein with respect to cell culture chambers). The substrate may also be disposable. In such cases, some system components (e.g., components located outside the substrate and / or components removable from the substrate, such as valves and / or pumps) may be reused.

[0377] The substrates described herein can comprise and / or be formed from a variety of suitable materials. In some embodiments, the substrate is formed from and / or comprises plastics such as polystyrene, PMMA, cyclic olefin copolymers, polycarbonate, polysulfone, polyetherimide (e.g., Ultem), polyurethane, and / or 3D printing resin. In some embodiments, the substrate is formed from and / or comprises PDMS. In some embodiments, the substrate is formed from and / or comprises glass.

[0378] In some embodiments, the system includes a substrate and one or more additional components. These components may be located above or below the substrate. For example, the system may also include a bottom cover disposed below the substrate and / or a cover layer disposed on the substrate. In some such embodiments, the substrate may be attached to these components using adhesives. Figure 72 A non-limiting example of a system having this design is shown. In some such embodiments, the cover layer disposed on the substrate comprises a membrane valve and pump and / or silicone.

[0379] The system may also include one or more components that can be housed in a volume that has been removed from the substrate (e.g., a pump that can be housed in such a volume, a sensor that can be housed in such a volume, an optical cable that can be housed in such a volume, a light emitter that can be housed in such a volume, and / or a valve that can be housed in such a volume).

[0380] The system may also include one or more additional components not placed on and / or under the substrate. For example, the system may include one or more motors (e.g., for one or more pumps, one or more sensors, one or more optical cables, one or more optical transmitters, and / or one or more valves) placed in these locations.

[0381] Figure 73 and 74 An example of a system is shown, comprising components placed on a substrate and components not placed on the substrate (e.g., these may be part of a manifold that can be integrated with a first substrate). Figure 73 and 74 In this process, these components are placed on manifolds (e.g., manifolds placed on different second substrates). Figure 73 and 74 The system shown includes a substrate A66 and a manifold A68. The substrate A66 is equipped with a cell culture chamber, a dissolved oxygen sensor, a pH sensor and a cell filter. The manifold A68 is equipped with multiple valves, pumps, reagent sources (in the form of capped or uncapped reservoirs) and related conduits. Figure 73 and 74 The substrate A66 shown also includes an inoculation port. Figure 73 and 74The manifold A68 shown also includes ports configured to connect to and / or be able to connect to an external air supply, ports configured to connect to and / or be able to connect to a waste chamber and / or a conduit in fluid communication therewith, and ports configured to connect to and / or be able to connect to a liquid source that can be used to clean a cell culture chamber and / or samples therein. Ports present on the substrate and / or manifold may include fittings (e.g., Luer barb connectors, threaded connectors).

[0382] Figure 73 This illustrates a configuration where the substrate and manifold are separate. Figure 74 The diagram illustrates a configuration in which a substrate is nested within a manifold. In some embodiments, systems with a design containing two substrates (e.g., having a...) Figure 73 and 74 The design shown (of which some systems are similar) may include one or more features that facilitate this nesting. For example, the first manifold may include one or more components (such as one or more tabs, fasteners, washers, etc.) that are mechanically coupled together when the substrate is nested in the manifold. Figure 75 It shows Figure 73 and 74 The image shows an example of another view of the system, which is arranged such that the substrate is nested within the manifold.

[0383] for Figures 73-75 In the system illustrated, components located on the manifold can be connected to and / or configured to be connected to components on the substrate via multiple ports provided on the manifold and / or multiple ports provided on the substrate. Liquids (e.g., liquids containing reagents, culture media) and / or pressures (e.g., positive pressure, negative pressure) can be supplied to these ports. The pressure supplied to the ports may cause liquid to flow in, out, and / or flow through the ports and any system components in fluid communication with them (e.g., cell culture chambers provided on the substrate). The substrate may also be electrically connected to the manifold (e.g., sensors placed on the substrate may be controllable and / or able to provide data to one or more components placed on the substrate via electrical communication).

[0384] In some implementations, a system comprising components disposed on two substrates (e.g.) Figures 73-75The system shown can include a reusable substrate and a disposable substrate. For example, referring to these figures, the substrate on which the cell culture chamber is placed can be disposable, while the other substrate can be reusable. This allows cell culture to be performed in a disposable, replaceable system without the need to discard other, more expensive, and / or difficult-to-replace system components (e.g., pumps, valves, and / or reagent sources). At the end of cell culture in the cell culture chamber, the appropriate substrate can be discarded, and cell culture can then be performed in a new cell culture chamber placed on a new substrate using the same components on the manifold.

[0385] Figure 76 An example of a disposable substrate (e.g., one that can be used in combination with a second non-disposable substrate) is shown. As can be seen in... Figure 76 As seen in the image, the disposable substrate may include a cell culture chamber in direct fluid communication with two conduits, and wherein a dissolved oxygen sensor and a pH sensor are disposed. The cell culture chamber may also be in direct fluid communication with other conduits (not shown). The disposable substrate may also include multiple ports that are in fluid communication with and / or configured to be in fluid communication with a second non-disposable substrate (e.g., through multiple ports on the second non-disposable substrate). Also as... Figure 76 As shown, the disposable substrate may include a breathable membrane (in which it is labeled “silicone membrane”), an inoculation port (in which it is labeled “sample well”), and an air chamber that can be used to induce mixing within the cell culture chamber (described in further detail elsewhere herein). Figure 76 The disposable substrate shown also includes a conduit that provides fluid communication between the inoculation port and one of a plurality of ports, through which it can be supplied to a conduit in direct fluid communication with (and possibly to) the cell culture chamber. The flow path of the liquid from the inoculation port to the cell culture chamber may pass through the manifold (e.g., from one of the plurality of ports on the disposable substrate, through a portion of the manifold, to a second of the plurality of ports on the disposable substrate).

[0386] and Figures 73-75 The system shown is similar to a system (which may contain a disposable substrate, and...) Figure 76(Similar to the one shown) can be assembled by filling a reagent reservoir, which serves as the reagent source, with reagents and / or a liquid containing the reagents. This can be done in a controlled environment (e.g., in a fume hood) if the reagents and / or liquids are toxic and / or require special handling. Use of the system can begin with a pre-filling operation (e.g., controlled by a controller, such as a controller to which the system is inserted). Cell culture medium and / or cells can then be introduced into the cell culture chamber (e.g., via an inoculation process). The system can then be sealed and / or placed in the instrument (as described further below). Sealing the system allows it to be transported to the instrument without excessive spillage and / or flow through different system components. After introducing the system into the instrument, the inoculation port can be cleaned (e.g., manually) and / or cells can be recovered through the inoculation port and / or through different ports. The recovered cells can be further analyzed (e.g., through one or more components on a manifold, via the instrument).

[0387] Figure 77 This demonstrates the process of pre-charging. Figures 73-75 This is an example of liquid flow in a system illustrated. Pre-charging can include allowing liquid to flow through some or all of the system components (e.g., through one or more conduits). Pre-charging prepares the system for subsequent flow of other liquids through it. As can be seen in... Figure 77 As seen in the diagram, liquid flowing through the manifold depicted therein may flow into, out of, and / or through one or more reservoirs (e.g., one or more reservoirs used as a reagent source) and / or through one or more valves.

[0388] Figure 78 This demonstrates the process after prefilling but before introducing cells into the cell culture chamber, via... Figures 73-75 An example of liquid flow in the system shown is illustrated. This may include initial filling of the cell culture chamber with liquid before introducing cells. During this process, liquid may flow from its source (e.g., located on a manifold) into the cell culture chamber (e.g., located on a substrate). The liquid may flow therebetween via one or more ports and / or one or more conduits. Liquid may also flow through and / or out of the cell culture chamber (e.g., into a waste chamber and / or container). The liquid flowing through the cell culture chamber may contain cell culture medium. In some embodiments, a port for supplying cleaning fluid may be provided during this process (in... Figure 78 The port shown as "sample cleaning port" is not in fluid communication with the rest of the system (e.g., it is isolated from the rest of the system via a valve).

[0389] Figure 79 This illustrates how, during the introduction of cells into the system (e.g., during cell seeding), [the process is achieved] through [method / method / etc.]. Figures 73-75 An example of liquid flow in the system shown. As can be seen in... Figure 79As seen in the diagram, the cell-containing liquid can be introduced from the inoculation port, flow through one of the multiple ports on the substrate into a manifold, flow through the manifold and back to the substrate through another of the multiple ports on the substrate, and then flow into the cell culture chamber. The liquid can then flow out of the cell culture chamber and into the manifold (and possibly out of the manifold and into a waste chamber and / or container). In some embodiments, a port for supplying cleaning fluid can be provided during this process (in...). Figure 79 The port shown as the "sample cleaning port" is not in fluid communication with the rest of the system (e.g., it is isolated from the rest of the system via a valve). In some embodiments, a cell filter located in the cell culture chamber allows liquid to flow out of the cell culture chamber while retaining the cells within it.

[0390] Figure 80 It shows the flow of liquid during infusion. Figures 73-75 An example of liquid flow in the system shown. As can be seen in... Figure 80 As seen in the diagram, fresh liquid culture medium can flow into the cell culture chamber, while old liquid culture medium can flow out of the cell culture chamber, while cells are retained in it (e.g., through a cell filter).

[0391] Figure 81 This illustrates the process of cleaning the catheter that fluidly connects the inoculation port, located on the manifold, to the cell culture chamber, via... Figures 73-75 An example of liquid flow in the system shown. Figure 81 As shown, liquid can flow into open (e.g., uninserted) ports that can supply cleaning fluid and through these conduits. The liquid flowing in this process may contain, consist of, or be substantially composed of cleaning fluid and / or liquids containing one or more cleaning agents.

[0392] Figure 82 This demonstrates the process of recovering cells from the system via... Figures 73-75 An example of liquid flow in the system shown. Figure 82As shown, cells can be recovered from the inoculation port. During this process, liquid can flow into and through the cell culture chamber (e.g., from a liquid culture medium source located on a manifold). The liquid can flow out of the cell culture chamber through a conduit in fluid communication with the inoculation port (e.g., through a port and / or conduit located on the substrate and / or manifold). In some such embodiments, the cell culture chamber may include a cell filter positioned to allow cells to flow through this path (e.g., it may be positioned close to and / or across a conduit that fluidly communicates the cell culture chamber with different ports on the substrate (e.g., ports where perfusion can occur)). Thus, cells can flow with the liquid to the inoculation port, from where they can be recovered. During this process, the port where cleaning fluid can be supplied may not be in fluid communication with the rest of the system (e.g., it is isolated from the rest of the system via a valve).

[0393] In some implementations, the system resides on a substrate (e.g., a chip), which also includes one or more additional systems. In such implementations, the additional systems may include some or all of the same components present in the system (e.g., the substrate may include multiple systems, each having at least one identical component). The additional systems may also differ from the system (e.g., the substrate may include multiple systems, and some or all of the systems may differ from each other in one or more ways).

[0394] Similarly, in some embodiments, the substrate comprises two or more modules. Each such module may be the system described herein, and / or two or more modules together may be part of a common system. In some such embodiments, each module may contain the same set of components. Such identical components may include pumps, valves, cell culture chambers, and / or sensors. The two or more modules present in the substrate may also differ in one or more respects and / or contain different components. In some embodiments, two or more modules on the substrate share one or more components (e.g., they may share pumps, valves, mixing chambers, and / or sensors). In such embodiments, liquid may flow out of the module as needed, flow through some or all of the common components, and / or flow back to the module.

[0395] In some implementations, the substrate comprising two or more systems and / or two or more modules has dimensions similar to standard laboratory consumables (e.g., 96-well plates). Without wishing to be bound by any particular theory, it is believed this may facilitate operation of the substrate with existing laboratory instruments.

[0396] Figure 83 and 84 Two non-limiting examples of a substrate comprising multiple modules are shown. Figure 83In the process, the protruding component may contain a motor (e.g., a motor capable of driving and / or configured to drive valves and / or pumps). Figure 85-91 It shows Figure 83 Other views of the substrate shown. (See also...) Figure 91 As shown in the figures, in some embodiments, such modules may be in fluid communication with a common chip manifold. As illustrated in these figures, in some embodiments, multiple systems and / or modules are horizontally arranged on a substrate (e.g., they pass through and / or are disposed on a single substrate). Without wishing to be bound by any particular theory, it is believed that such an arrangement may contribute to high throughput.

[0397] In some embodiments, a substrate comprising multiple modules may be equipped with a manifold (e.g., a second substrate) on which control valves and / or other components are disposed (e.g., such as...). Figure 73 and 74 (A manifold similar to the second substrate shown). In some such embodiments, both substrates can be placed, configured to be placed, and / or placed within an instrument. Without being bound by any particular theory, this design ideally reduces the number of lines passing through the instrument, which facilitates operation of the instrument in combination with such substrates and requires relatively few modifications. Furthermore, the manifold and substrates can be placed relatively close together, which may result in shorter lines. Without being bound by any particular theory, it is believed that shorter lines (e.g., shorter air lines) can improve pumping speed and / or accuracy by reducing response time. When a manifold is used, in some embodiments, one or more sensors can be placed on the manifold (e.g., an IgG sensor can be placed in this way). Figure 92 A non-limiting example is shown of a first substrate comprising multiple modules and a manifold (e.g., a manifold placed on a second substrate) on which control valves are placed. Figure 93 Another non-limiting example of a manifold and substrate is shown.

[0398] In some implementations, a single second substrate may be equipped with multiple substrates (e.g., multiple substrates, each in the form of a module). Figure 94 A non-limiting example of a system having this design is shown. In some embodiments, the system may include multiple similar and / or identical components placed on a second substrate (e.g., the second substrate may include multiple modules, each configured to be in contact with and / or capable of being in contact with the substrate). The second substrate may also include a set of common components configured to be in contact with and / or capable of being in contact with multiple modules (e.g., including a single common pump that can supply pressure to two or more modules; a single common reagent source that can supply reagents to two or more modules, etc.).

[0399] As noted above, some embodiments involve instruments that incorporate the systems described herein and / or are otherwise associated with the instruments described herein. In some such embodiments, the system may fit components present in the instrument (e.g., chip racks and / or trays). A non-limiting example of such an instrument is an incubator (e.g., a carbon dioxide incubator). In some embodiments, part or all of the system described herein is located inside the instrument that serves as an incubator. For example, in some embodiments, a cell culture chamber is located inside the incubator. In some embodiments, the system is located within the incubator, and the incubator itself is located within an optical detector (e.g., an optical microscope).

[0400] The system can also be mechanically and / or electronically connected to an instrument. For example, in some embodiments, the system is capable of being "plugged" into the instrument electronically and / or mechanically. In such embodiments, the instrument is capable of and / or configured to provide pressure (e.g., positive and / or negative pressure) to one or more components of the system, causing liquid to flow in, out, and / or through the system. In some embodiments, pressure is provided by infusing air (e.g., compressed air) into the components to pressurize them. In such embodiments, the instrument may include one or more air lines that can be mechanically coupled to the system. The instrument can also serve as a source and / or a source of one or more liquids (e.g., liquids containing reagents, liquid culture media) and / or a container to which one or more liquids can be delivered (e.g., as waste, as liquids to be analyzed by the instrument). In some embodiments, the instrument includes a controller that provides signals (e.g., electrical signals) to the system and / or receives signals from the system.

[0401] As noted above, liquids can flow between the system components described herein via conduits. In some embodiments, the system includes one or more conduits in the form of tubing (e.g., PEEK tubing).

[0402] As noted above, some embodiments relate to systems and / or methods for performing cell culture. Therefore, cells may be present in some systems and / or during some methods described herein. As an example, in some embodiments, the cell culture chamber contains cells. Such cells may include adherent cells and / or suspension cells. The suspension cells may be suspended in a liquid also contained in the cell culture chamber. Non-limiting examples of suitable adherent cell types include HeLa cells, HEK293 cells, iPSC cells, and hMSC cells (human mesenchymal stem cells). Non-limiting examples of suitable suspension cell types include CHO cells, iPSC aggregates, T cells, HEK293S cells, and Jurkat cells.

[0403] Various suitable liquids may be contained in and / or flow through the cell culture chamber described herein (and / or contained in and / or flow through other system components described herein). As mentioned elsewhere herein, some of these liquids may contain cells (e.g., suspended cells) and / or reagents. Such liquids may be aqueous.

[0404] Liquid flow through the cell culture chamber and other system components can be intermittent and / or continuous. It can flow as part of a batch process, a fed-batch process, and / or a perfusion process (e.g., using continuous and / or push-feed addition, and / or allowing reagent delivery between additions).

[0405] In some embodiments, the method includes flowing a liquid through one or more system components to clean the components. For example, in some embodiments, 10% bleach and / or 70% alcohol may be flowed through the system component (e.g., sequentially) to clean it. After cleaning, the system component can be prepared for cell culture by rinsing with a cleaning solution (e.g., PBS cleaning solution).

[0406] As mentioned elsewhere herein, in some embodiments, one or more method steps described herein can be automated. For example, the flow through a cell culture chamber, the introduction of reagents and / or cells into the cell culture chamber, the removal of waste from the cell culture chamber, and / or the removal of samples and / or cells from the cell culture chamber can be automated. Automation may include performing relevant method steps in response to specific time points and / or specific sensing conditions (e.g., pH, oxygen content).

[0407] Figure 95 Another exemplary system is shown. Figure 95 The system shown includes a cell culture chamber (labeled "Growth Chamber") containing a cell filter, ports through which samples can be introduced and / or removed (e.g., via cell seeding and / or recycling), two air sources (labeled "L Air" and "R Air"), a waste chamber (labeled "Waste"), three reagent sources (labeled "L 5 mL", "R 5 mL", and "1 mL"), multiple pumps, multiple valves, and multiple ports. Figure 95 In the system shown, one of the plurality of ports is used to allow fluid communication between the cell culture chamber and its upstream reagent source, and between the filter and its downstream waste chamber. Pumps and valves are also provided in the fluid path between each port and each reagent source or waste chamber. These pumps are used to pump fluid to and / or pump fluid out of the ports in fluid communication with them. These valves allow such pumping to occur when open and prevent such pumping when closed. Also as Figure 95 As shown in the diagram, in the system described therein, a port for introducing and / or removing samples is in fluid communication with a cell culture chamber.

[0408] Figure 96 Another exemplary system is shown. Figure 97 The system shown is Figure 95 The system shown is similar, except that the multiple ports comprise only one port that allows fluid communication between the cell culture chamber and two reagent sources (labeled "L 5mL" and "R mL"), and it also includes additional fluid components associated with the reagent source labeled "1mL". Regarding the former, the reagent source is separated from the single port by a partitioned valve and a single pump in fluid communication with it. When pumping, the pump can pump reagent from the reagent source in fluid communication with it through the open valve. Regarding the latter, a first valve (labeled "Valve 5") is located between the port and a branch flow path. The branch flow path includes a first branch in fluid communication with the pump and the reagent source labeled "1mL", and a second branch in fluid communication with the waste chamber through a second valve (labeled "Valve 4"). This branch flow path allows reagent to be pumped from the reagent source labeled "1mL" into the cell culture chamber (if Valve 5 is open and Valve 4 is closed).

[0409] Figure 97 Yet another exemplary system is shown. Figure 97 The system shown comprises two modules, each including a cell culture chamber containing a cell filter (labeled "Growth Chamber"), a port through which samples can be introduced and / or removed (labeled "S"; e.g., via cell seeding and / or recovery), and multiple ports. The system also includes two different types of reagent sources: a non-temperature-controlled reagent source (labeled "#1 5mL" and "#2 5mL") and a cooled reagent source (labeled "#3" and "#4"). Figure 97 The system shown further includes a waste chamber (labeled "waste"), multiple pumps, and multiple valves. Figure 97 In the system shown, a pump is located between each reagent source and the connector, and can pump reagents from these reagent sources to the connector. On the other side of the connector are two valves (labeled "Valve 1" and "Valve 3"), each in fluid communication with a port on the module. When the valve of the module is open, reagents pumped into the connector from one or more reagent sources can flow further into the cell culture chamber through the port. Waste flows out of each module through a filter, through the port, and into the waste chamber. In this system, there are also two pumps (labeled "Pump 1" and "Pump 6") that can pump waste accordingly. Another valve (labeled "Valve 2") is also located between the connector and the waste chamber.

[0410] Figure 97 The uncontrolled reagent source shown can be present in the manifold. Figure 97The temperature-controlled reagent shown can be stored separately (e.g., in a cold storage container). The reagent source may be purgeable.

[0411] Figure 98 Yet another exemplary system is shown. Figure 98 The system shown is Figure 97 The system shown is similar, except that it includes a different number of reagent tanks, two waste chambers (each labeled "waste orifice"), and a flow pool located between the waste chambers and a pump capable of pumping waste. A similar system could also be conceived. Figure 98 As shown in the diagram, but including the valve instead of pump 5. Figure 98 The system shown can be used to perform perfusion by pumping pump 5 at twice the speed of pumps 1 and 4. Perfusion can be paused to supply one or more reagents to the cell culture chamber from one or more reagent sources.

[0412] Figure 99 Yet another exemplary system is shown. Figure 99 The system shown illustrates the relationship with Figure 98 A similar system is shown, but many of its components are mounted on a substrate (labeled "Incucyte tray") suitable for inclusion in an incubator. The waste chamber is not mounted on this substrate.

[0413] Figure 100 Another exemplary system is shown. In this system, various components are mounted on a substrate, including three manifolds (one for a fluid pump, one for a fluid valve, and one for an air mixer), various ports, two consumables each containing a cell culture chamber (labeled "growth chamber consumable"), and one consumable containing a reagent source (labeled "reagent container consumable"). The manifolds may be integrally connected to the substrate, the consumable containing the cell culture chamber may be removable, and / or the consumable containing the reagent source may be removable.

[0414] The system described in this article, such as Figure 95-100 The systems shown and described elsewhere herein can include temperature sensors in a variety of suitable locations. Non-limiting examples of such locations include on and / or inside a manifold, on and / or inside a port in fluid communication with a reagent source (which may be particularly suitable for measuring the temperature of reagents supplied through such ports), and on and / or inside a reagent source. For the latter, for temperature-controlled reagents, the temperature sensor can monitor the temperature at the location where temperature control is being applied (e.g., a refrigerator, a heated receiver in fluid communication with a refrigerator).

[0415] In some embodiments, the system described herein may include one or more translational sensors (e.g., temperature sensors, other sensors such as pH and / or dissolved oxygen sensors) relative to one or more other components of the system, such as cell culture chambers therein. Such sensors may advantageously be capable of measuring, configured to measure, and / or measure characteristics at one or more locations within the system (e.g., one or more locations within a cell culture chamber, two or more different cell culture chambers). The translation can be achieved using a motor. The motor may be controlled automatically (e.g., by software and / or electrical signals) and / or manually (e.g., by an operator). In some embodiments, the sensors are designed such that they can measure system characteristics without contact (e.g., they may be optical sensors). Translational sensors may be particularly well-suited for performing multiplexing.

[0416] In some implementations, the system includes a component comprising two sensors that can be translated together (e.g., mechanically coupled, integrally connected, inseparable without special tools, mounted on the same component and / or bracket). Figure 101 A non-limiting example of a block diagram depicting an exemplary translational component comprising two sensors is shown. Figure 101 The two sensors shown are a pH patch sensor and a dissolved oxygen patch sensor (labeled "DO patch sensor"), both of which are optical sensors containing optical fibers. These sensors are mounted on a single bracket and controlled by a separate electro-optical OEM module board, which itself is electrically connected to a computer.

[0417] Figure 102 It shows Figure 101 The image shows the location of the component within an exemplary system. In this image, it is labeled "PreSens pH Patch Sensor" and is positioned for measurement in a cell culture chamber.

[0418] Figure 103 Depicting Figure 101 and 102 The second block diagram of the components is shown. As can be seen from this block diagram, the pH and dissolved oxygen sensors are electrically connected to a light emitter, which itself is electrically connected to a plate that can be powered and communicate with the sensors. The light emitter is also electrically connected to a PC, which can be used to trigger cell imaging and / or analysis, provide information, and / or detect and / or control the position and / or movement of the components (e.g., position, whether the component is moving, whether the component is idle). The PC is electrically connected to a computer (labeled "Incucyte Control Computer") that controls the incubator in which the components can be placed.

[0419] Figure 104A cross-sectional view of a portion of an exemplary system is shown. Figure 104 In the diagram, the system includes a silicone gasket (identified by an arrow). This silicone gasket seals the components located therebetween (in...). Figure 104 In this context, the component is formed from polycarbonate. Figure 104 The system shown further depicts the screw, but it can also be considered in other aspects with... Figure 104 The system shown is similar to a system without screws (e.g., a system thermally riveted together). Figure 104 Some of the systems shown can be easily injection molded and / or can be sealed, thereby limiting or preventing leakage, contamination, and / or bubbles. Some of such systems may ideally exhibit a relatively low dead volume.

[0420] In some embodiments, the system described herein includes components capable of delivering gases (e.g., air, oxygen, nitrogen) and configured to deliver gases and / or deliver gases to a cell culture chamber. Such components are referred to herein elsewhere as an "air mixer manifold". Figure 105 An exemplary air mixer manifold is shown in the figure. Figure 106 A system diagram of an exemplary air mixer manifold is depicted.

[0421] An air mixer manifold may be fluidly connected to the cell culture chamber described herein through one or more ports. This component may also be a mixing element described elsewhere herein. In such cases, the air mixer manifold is configured to deliver gas to the cell culture chamber in a manner that mixes the contents of the cell culture chamber, and / or to deliver gas in this manner. The air mixer manifold may also supply fresh gas to the cell culture chamber while causing minimal or no mixing. Gas supplied from the air mixer manifold may diffuse through a permeable membrane forming part (or all) of the cell culture chamber wall.

[0422] In some embodiments, the amount of dissolved oxygen in the cell culture chamber supplied with gas by the air mixer manifold may be controlled independently of the mixing amount achieved by the air mixer manifold, and may be configured to be controlled in this way, and / or may be controlled in this way. This can be achieved by selecting the gas composition to deliver the desired amount of oxygen, selecting the gas quantity (e.g., at a specific pressure and / or flow rate) to deliver the desired mixing amount, and / or applying positive pressure at two or more ports (e.g., both at the port supplied by the air mixer manifold and the exhaust port).

[0423] In some implementations, the air mixing manifold may be present in a system containing a cell culture chamber with adherent cells. In such cases, it may be desirable for the air mixing manifold to supply gas with a smooth flow so that the membrane through which the gas is supplied deforms minimally or not at all.

[0424] In some embodiments, the air mixer manifold includes a pump, such as a piezoelectric pump. The pump can draw fresh air from the atmosphere and / or discharge exhaust gases back into the atmosphere (e.g., through the aforementioned permeable membrane). In some embodiments, the pump maintains a high-pressure reservoir. In such embodiments, fluidly communicating the air mixer manifold with the cell culture chamber (e.g., by opening a valve located therebetween) may result in a rapid increase in pressure applied to the fluidically oriented permeable membrane located therebetween, which may enhance mixing.

[0425] In some implementations, a single air mixer manifold may be configured to cause mixing and / or supply air to two or more cell culture chambers. Fluid communication between the cell culture chambers and the air mixer manifold can be initiated by opening a valve located therebetween and stopped by closing such a valve.

[0426] The air mixer manifold described herein can be operated automatically (e.g., via software and / or electrical signals) and / or manually (e.g., by an operator).

[0427] The air mixer manifolds described herein may have a certain size and / or shape, so that they can be placed in the systems and / or incubators described herein.

[0428] In some implementations, the system described herein is designed to facilitate cell sampling. Figure 107 and 108 A portion of such a system is shown. As illustrated in these figures, the system may include a bracket that, in a recessed state, seals the membrane to other system components. Figure 107 However, in the convex state, the membrane is allowed to deform upwards ( Figure 108 When the membrane deforms upward, fluid can flow past its previous location and to a location where it can be removed from the system (e.g., from a cell culture chamber). This location can be a port for cell retrieval and / or allow the insertion of capillaries, pipettes, and / or collection containers (in which fluid can enter via capillary action and / or aspiration). The bracket can function as a valve that is normally locked in a closed position (i.e., recessed) but can be opened (i.e., allows for a raised position). Opening can be achieved by pressing a snap fastener. Advantageously, Figure 107 and 108 The system shown prevents direct fluid communication between the cell culture chamber and the external environment, which reduces contamination of the cell culture chamber. Alternatively, this system may also exhibit reduced dead volume.

[0429] Figure 109and 110 This describes another example of a design that facilitates cell sampling. Figure 109 It is a top view. Figure 110 It is a side view. Figure 109 and 110 The design shown includes two chambers arranged in series between a port for introducing and / or removing samples (e.g., via cell seeding and / or recycling) and a cell culture chamber: a first chamber serving as a membrane valve (e.g., exhibiting zero dead volume), and a second chamber serving as a pump capable of pumping, configured to pump, and / or pump a known volume of sample (e.g., from the port to the cell culture chamber, and from the cell culture chamber to the port). A membrane (e.g., a silicone membrane) runs through these chambers. This membrane can be raised and lowered to affect the pressure within these chambers and their fluid communication with each other, with the cell culture chamber, and with the port. Figure 109 and 110 The system shown also includes a passageway that runs through the second chamber and into the cell culture chamber.

[0430] like Figure 109 and 110 The diaphragm valve shown may benefit from the application of positive pressure to maintain the required seal.

[0431] Figure 111 Depicting what can be done Figure 109 and 110 The system shown employs a method that allows samples to be transferred from the port to the cell culture chamber. For example... Figure 111 As shown, the portion of the membrane spanning the two chambers can be raised and lowered to allow fluid to flow out of the port, through the two chambers, into the channel, and into the cell culture chamber. The channel may also contain a buffer solution with which the sample can be mixed before flowing into the cell culture chamber. During execution... Figure 111 Following the method shown, the port can be flushed and / or cleaned with liquid directed to the waste chamber.

[0432] Figure 112 This describes another example of a design that facilitates cell sampling. Figure 112 The system shown includes a cell culture chamber containing a filter (labeled "Growth Chamber"), a port for introducing and / or removing samples (labeled "Inoculation"), a waste chamber (labeled "Waste"), various reagent sources (labeled "L 5mL", "R 5mL", and "1mL"), and various pumps and valves. The valves allow fluid flow when open and prevent fluid flow when closed. The pumps may be able to... Figure 112The arrows shown indicate the direction of pumping, and the system is configured to pump fluid in such a way as to pump and / or pump fluid through the system. As can be seen from the accompanying drawings, the waste chamber can receive waste from the ports (via valve 1, valve 8, and pump 1) and / or the cell culture chamber (via filter, valve 2, and pump 1). Figure 112 In some of the systems shown, the port through which samples can be introduced and / or removed can be used, configured to be used, and / or used as an exhaust port (e.g., after pre-filling and / or by keeping it open). In some such embodiments, when the system is used in this manner, the valve located between the cell culture chamber and the port can then be closed, and the port and any associated flow paths can be cleaned by a sterilization cycle.

[0433] Figures 113-114 This describes another example of a design that facilitates cell sampling. Figure 113 The majority of the system with this design is described. Figure 114 The capillary and its associated components within the system are depicted. These figures illustrate spring-loaded mechanical parts configured together to actuate, and to actuate, the loading and removal of the capillary. The top figure in each depicts the presence of a plug at the end of the capillary. As shown in the middle figure, this plug can be removed (e.g., by a first pusher), allowing the capillary to be filled to a specified volume with fluid originating from a system component (e.g., a cell culture chamber) to its left. Following this filling, as shown in the bottom figure, a second pusher is actuated, causing the capillary to move so that it is no longer positioned to receive fluid from the previously receiving location. This can be achieved, for example, by removing the capillary from that location, such as by pulling it out of the septum.

[0434] In some implementations, having as Figures 113-114 The system shown in the illustration may be able to store multiple capillaries, be configured to store multiple capillaries, and / or store multiple capillaries. An example of such a system is shown in... Figure 115 The image shows a cell culture chamber (labeled "growth chamber") containing multiple capillaries (shown as lines extending downwards from them). In some embodiments, such as Figure 115 The system shown can be pre-loaded with multiple capillaries that can be used to sample cells as needed during cell culture. Pre-loading all the capillaries expected to be needed in a specific cell culture process ideally allows the entire system to be sterilized together.

[0435] Figure 115 The source of cleaning fluid (labeled "clean") is further described, which can be supplied to ports and / or cell culture chambers where samples (labeled "inoculation") can be introduced and / or removed.

[0436] In some implementations, the system described herein is designed to facilitate cell counting. Figure 116 A non-limiting example of such a system is described. The system includes a flow pool whose certain dimension is small enough that cells cannot flow through it in an overlapping manner along that dimension (e.g., such that the cells form a monolayer, such that if said dimension is height, the cells cannot flow above or below each other, such that the projection of the flow pool onto a plane perpendicular to said direction does not include any spatially overlapping cells). This ideally allows for easy cell counting, which can be used to determine cell density.

[0437] Figure 116 The process for retrieving samples from a cell culture chamber (labeled "growth chamber") is also described. In this process, pump 1 can be used to extract the sample from the cell culture chamber via valve 3. Then, pump 2 can be used to pump fresh cell culture medium into the cell culture chamber via valve 3, thereby flushing any cells located between pump 2 and valve 3 (e.g., cells upstream of the connector between valve 3 and valve 4, and cells on the valve 3 branch of that connector) into the cell culture chamber. Any remaining cells between pump 2 and valve 4 (e.g., cells on the valve 4 branch of that connector) can be pumped through a flow cell by pump 1 and / or pump 2.

[0438] By opening valves 2 and 3 and using pump 1, the sample can be introduced from the port where the sample can be introduced and / or removed (labeled "inoculated") into... Figure 116 In the cell culture chamber shown, fluid is made to flow from the port through valve 1, valve 3, cell culture chamber, filter, valve 2, flow cell and pump 1 to the waste chamber (labeled "waste").

[0439] Figure 117 and 118 Two different designs are depicted that can seal a valve. Both designs include a push rod (which may be plastic) that can be pushed down to apply force to the valve components, resulting in a seal. Figure 117 A valve is depicted that includes sealing ribs set in hard plastic and diagonal channels. Figure 118 A valve comprising two silicone components with a diaphragm disposed between them is depicted. In some embodiments, the valve includes a spring-loaded push rod such that the push rod closes unless a force is applied to compress the spring. Figure 117 and 118 The valve shown can be opened and / or closed manually (e.g. by an operator) and / or automatically (e.g., in response to electrical signals sent by software and / or electrical controllers).

[0440] Without wanting to be bound by any particular theory, various designs for cell sampling and counting may have different advantages. A rotary valve located between the cell culture chamber and a port where samples can be introduced and / or removed allows for sample injection in precise volumes and exhibits a simple design that can be operated by an electromagnetic actuator. Membrane valves (e.g.) Figure 109-111 The membrane valve shown can exhibit reduced dead volume, a linear fluid element with reduced contamination risk, a compact structure, and allows for easy sample recovery from the port. Components configured to actuate, capable of actuating, and / or actuating capillaries (e.g., Figure 113 and 114 The components shown can be mechanically simple and can be used with existing fluid elements without modification. For example... Figure 116 The flow cell shown allows for rapid and / or accurate cell counting, and the likelihood of introducing contaminants into the system is very low.

[0441] Figure 119 This is a photograph of an exemplary cross-slit valve through which a capillary tube can be inserted. Figure 120 Mechanical diagrams are included, showing valves with and without inserted capillary tubes. The capillary tube inserted through the cross-slit valve can form a radial seal with the silicone gasket present in the system described herein.

[0442] Some of the systems described herein include chips having a substrate formed from a relatively small number of components. As an example, some systems include chips having a substrate formed from a silicone layer (e.g., a cast silicone layer) located between two plastic sheets. Figure 121 A top view of a chip with this design is shown. Figure 122 A side view of the chip is shown. This chip can be sealed by a compression seal along the edge of any open portion (e.g., any chamber and / or channel). In some embodiments, one or more such open portions (e.g., one or more chambers and / or channels) may be surrounded by a silicone layer on three sides and by a plastic sheet on one side. In some embodiments, either or both of the plastic sheets include one or more positioning ribs that facilitate the formation of a seal.

[0443] In some embodiments, in a chip having a design that includes a silicone layer located between two plastic sheets, the silicone layer may also include one or more relatively thin portions that form one or more films described elsewhere herein. Figure 123 This part is depicted with an arrow.

[0444] Figure 124 and 125 Another example of a chip with a substrate formed by a silicone layer located between two plastic sheets is depicted. Figure 124 and 125 The chip shown comprises a cell culture chamber with hexagonal walls at the top and bottom. One such hexagonal wall comprises a portion formed of cast silicone and includes multiple protrusions extending toward the opposing hexagonal wall. These protrusions can form a seal with and / or apply compressive force to the opposing hexagonal wall. In some embodiments, the space between such protrusions can take the form of channels through which fluid can flow. This flow may include flow through a filter located within the cell culture chamber and / or flow through one or more sensors (e.g., a pH sensor and / or a dissolved oxygen sensor).

[0445] In some embodiments, the system described herein may be suitable for performing RNA transcription, and / or a method may include performing RNA transcription within the system described herein. In some embodiments, RNA transcription may be performed in the cell culture chamber described herein.

[0446] Without being bound by any particular theory, RNA transcription is a process of copying a DNA sequence into a messenger RNA strand. The process comprises three steps: initiation, elongation, and termination. Initiation involves binding RNA polymerase to a DNA promoter sequence on the double-stranded DNA, which then opens the DNA. Elongation involves growing an RNA strand with the aid of RNA polymerase. In this step, one strand of the opened double-stranded DNA serves as a template for the growing RNA strand. Furthermore, nucleotides may be added to the RNA strand complementary to the template DNA strand. Termination occurs at the end of RNA transcription. Termination may be triggered by the formation of a termination sequence in the grown RNA strand. This results in the release of RNA from the RNA polymerase.

[0447] The RNA transcription process may require the presence of one or more reagents. Such reagents can be introduced from their sources into the systems and / or instruments described herein (and / or one or more components thereof). The systems and / or instruments described herein may also contain reagent sources. Non-limiting examples of such reagents include DNA templates (i.e., double-stranded DNA used as a template for RNA growth), RNA polymerase, nucleoside triphosphates (also known as “NTPs”), cofactors, Mg... 2+ Capping reagents, acids and bases, RNase inhibitors, and pyrophosphatase. Not wishing to be bound by any particular theory, NTPs can act as precursors undergoing a reaction in which they are incorporated into the growing RNA chain. Cofactors are proteins that can bind to the transcribed DNA and control the rate of RNA transcription. Mg 2+(These can be provided as salts such as MgCl2) and can help stabilize the structure of RNA polymerase during transcription. Capping reagents can help form cap structures on growing RNA. Acids and bases can be used for pH control. RNase inhibitors can reduce RNA degradation by inhibiting RNases. Pyrophosphatases can remove pyrophosphate formed during RNA synthesis by catalyzing the hydrolysis of pyrophosphate to orthophosphate. In some embodiments, one or more reagents are provided in a buffer and / or added to a fluid containing a buffer (e.g., transcription buffer).

[0448] A non-restricted example of RNA polymerase is T7.

[0449] Non-restrictive examples of suitable NTPs include adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0450] Non-restricted examples of suitable cofactors include universal transcription factors (GTFs), mediator complexes, chromatin-modifying enzymes, RNA-binding proteins (RBPs), transcription activators, transcription repressors, and transcription elongation factors.

[0451] Non-limiting examples of suitable capping reagents include reagents suitable for enzymatic capping and / or post-transcriptional capping (e.g., vaccinia virus capping enzyme (VCE), Forstovirus capping enzyme (FCE)) and co-transcriptional capping enzymes (e.g., cap analogs).

[0452] Non-limiting examples of suitable acids include acetic acid and citric acid.

[0453] Non-limiting examples of suitable bases include sodium hydroxide (NaOH), potassium hydroxide (KOH), and sodium bicarbonate (NaHCO3).

[0454] In some embodiments, the method of RNA transcription includes performing one or more steps involving allowing RNA to be synthesized from a DNA template, monitoring the progress of RNA synthesis, and / or collecting the RNA synthesized during RNA transcription.

[0455] Such methods may include measuring the amount of RNA in a fluid (e.g., a fluid present in a cell culture chamber as described herein) and / or adjusting the fluid composition based on such measurement. For example, if the measured amount of RNA indicates that RNA transcription is proceeding in an appropriate and / or expected manner, the fluid composition may be maintained. As another example, if the measured amount of RNA indicates that the rate of RNA transcription is too fast or too slow, the fluid composition may be adjusted.

[0456] In some embodiments, in addition to measuring the amount of RNA in the fluid and adjusting the fluid composition based on the measured amount of RNA, or as an alternative, the method may include measuring the amount of reagents present in the fluid and / or adjusting the fluid composition based on the measured amount of reagents. For example, the method may include measuring NTP, Mg... 2+ The amount of capping reagent and / or the amount of such reagent in the fluid can be adjusted, and the fluid composition can be maintained or adjusted based on the measurements. For example, if measurements indicate that the amount of such reagent in the fluid is too little or too much for RNA synthesis at the desired rate, the fluid composition can be adjusted.

[0457] When performed, adjusting the composition of the fluid may include adding reagents to the fluid, such as from a source. For example, adjustment may include adding NTP, Mg, etc. 2+ And / or capping reagents. Adjusting the composition of the fluid may also include adding a buffer solution (e.g., transcription buffer) that does not contain any reagents to the fluid to dilute the reagent concentration in the fluid.

[0458] Example 1

[0459] This embodiment describes an exemplary system and its use in an incubator.

[0460] Figure 126 An exemplary system described herein and various photographs of its placement in an incubator are shown.

[0461] Example 2

[0462] This embodiment describes the execution of HeLa cell culture in the system described herein.

[0463] Figures 127-129 The images show photomicrographs taken at various time points during the culture of HeLa cells in a cell culture chamber within an exemplary system set up in a humidified chamber. As can be seen from these photomicrographs, cell proliferation is possible in such a cell culture chamber.

[0464] Figures 130-133 The image shows the cells before perfusion on the second day of cell culture. Figure 130 Immediately after infusion Figure 131 ) and a few days later ( Figure 132 and 133 The obtained micrographs. Cell culture is basically performed as described above. Perfusion involves removing the cell culture chamber from the humidification chamber, then removing waste into the waste chamber, adding fresh liquid culture medium, and mixing. As can be seen from these figures, it is possible to perform perfusion and continue cell proliferation while still in the cell culture chamber.

[0465] Figure 134 and 135The images show micrographs taken on day eight of cell culture after perfusion on days two and seven, as well as micrographs taken on day eight of cell culture without perfusion. Cell culture and perfusion were performed essentially as described above. As can be seen from these figures, perfusion resulted in a higher degree of cell proliferation.

[0466] Example 3

[0467] This embodiment describes the execution of iPSC cell culture in the system described herein.

[0468] Cell culture was performed substantially as described in Example 2. Perfusion was performed on day 1, substantially as described in Example 2. Figure 136 The images shown are photomicrographs taken at various different time points. As can be seen from these photomicrographs, iPSC cells were able to be cultured under these conditions.

[0469] Example 4

[0470] This embodiment describes the execution of CHO cell culture in the system described herein.

[0471] Cell culture was performed substantially as described in Example 2. Perfusion was also performed substantially as described in Example 2. Perfusion consisted of removing waste for 20 seconds, followed by adding fresh liquid culture medium for 20 seconds. Perfusion was performed every 8 hours for the first 6.5 days of cell culture, and then every 4 hours thereafter. Furthermore, during perfusion, the pump was run at 22 rpm for the first two days of cell culture, and then at 11 rpm thereafter.

[0472] Figure 137 The diagram shows how the convergence degree changes over time (in hours). Figure 138 The image shown is a photomicrograph obtained on the first day of cell culture. Figure 139 The images shown are photomicrographs obtained on day nine of cell culture. As can be seen from these figures, CHO cells were able to be cultured under the conditions described above.

[0473] Example 5

[0474] This embodiment describes the execution of HeLa cell culture in the system described herein.

[0475] The sample, containing HeLa cells, is introduced into a system comprising the cell culture chamber described herein by means of a port pumped into the cell culture chamber. Air is then distributed to opposite sides of the cell culture chamber using an air-filled syringe, inducing flow and mixing of the sample within the cell culture chamber. Cell culture is then performed. At certain points during the cell culture process, the cell culture medium is removed from the cell culture chamber and replaced with fresh cell culture medium.

[0476] During cell culture, changes in confluence and dissolved oxygen over time were measured. Figure 143 The diagram shows the change in convergence degree over time. Figure 141 The change in dissolved oxygen over time is shown. Figure 140 The arrows indicate the points in time when cells were accidentally removed from the cell culture chamber. Figure 141 The arrows indicate the points in time when fresh culture medium was added. As can be seen from... Figure 140 and 141 The observed confluence increases over time, while dissolved oxygen content decreases, indicating cell proliferation. Furthermore, the introduction of fresh culture medium leads to an increase in dissolved oxygen.

[0477] In addition, optical micrographs were taken showing the changes over time at various locations within the cell culture chamber during cell culture. These optical micrographs demonstrate that the cells were well mixed and relatively uniformly dispersed throughout the cell culture chamber.

[0478] Example 6

[0479] This example describes the execution of HeLa cell culture after transfection in the system described herein.

[0480] As described in Example 5, a sample containing HeLa cells was introduced into a system containing a cell culture chamber and cultured for 24 hours. Then, the HeLa cells were transfected with a mixture of GFP mRNA and jetRNA+. Cell culture continued after transfection, and fluorescence images of the cell culture chamber were obtained at various time points. Figure 142 Two sets of these images are shown in the image. As can be seen from... Figure 142 As you can see, the transfection was successful.

[0481] Furthermore, the average intensity of green fluorescence present in the live cell count and fluorescence images was determined and compared with the average intensity of live cell count and green fluorescence present in HeLa cells transfected in the same manner but cultured in the wells of a microplate. The average intensity of live cell count and green fluorescence was comparable for both fluorescence images obtained from the cell culture chamber and those obtained from the microplate, further demonstrating the suitability of the system for culturing HeLa cells.

[0482] Example 7

[0483] This embodiment describes the execution of T cell culture in the system described herein.

[0484] PBMC cells were thawed, concentrated, and then introduced into the cell culture chamber of the system described herein. After introduction into the cell culture chamber, these cells were mixed and cultured as described in Example 5. Alternatively, PBMC cells were prepared in the same manner, but instead of being introduced into the cell culture chamber, they were cultured in the wells of a microplate. Optical micrographs of the cell culture chamber and microplate were obtained at various time points, and cell samples were removed from the cell culture chamber and microplate at various time points.

[0485] The optical micrographs show that the confluence of PBMCs cultured in the cell culture chamber is comparable to that of PBMCs cultured in the wells of the microplate. This is illustrated in Figure 146, where the arrow indicates the introduction of an air bubble that artificially reduces further confluence measurements. Furthermore, the visually observed color of the cell culture chamber is comparable to that of the wells in the microplate, indicating comparable T cell expansion.

[0486] At 140 hours after introduction into the cell culture chamber or microplate, the cell viability in cell samples taken from the cell culture chamber (82-83%) was comparable to that taken from the wells of the microplate (80-85%).

[0487] Example 8

[0488] This embodiment describes the execution of iPSC cell culture in the system described herein.

[0489] A sample containing iPSC cells was introduced into the humidified cell culture chamber of the system as described in Example 5. After introduction, the sample was mixed and then cultured as described in Example 5. Optical micrographs were obtained at various time points during the cell culture process, from which viable cell counts were obtained. These viable cell counts are shown in... Figure 144 The results show that the cell culture was successfully carried out.

[0490] Further experiments were conducted in which fresh cell culture medium was supplied via manifold. Figure 145 The image shows the phase object count for each image of a cell culture chamber where cell culture medium is supplied in this manner. As can be seen from... Figure 145 As observed, using the manifold in this manner does not produce significant cytotoxicity.

[0491] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the invention. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications using the teachings of the invention. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using no more than conventional experimentation. Therefore, it should be understood that the above embodiments are presented by way of example only, and the invention can be practiced in ways different from those specifically described and claimed within the scope of the appended claims and their equivalents. The invention relates to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention if such features, systems, articles, materials, kits, and / or methods do not contradict each other.

[0492] All definitions used in this document should be understood to have a superseding power over dictionary definitions, definitions in referenced literature, and / or the general meaning of the defined terms.

[0493] Unless explicitly stated otherwise, references to numbers used herein in the specification and claims without a specific number shall be understood to mean “at least one”.

[0494] The phrase “and / or” as used herein in the specification and claims should be understood to mean “any one or both” of the elements so connected, i.e., elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., “one or more” of the elements so connected. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language (e.g., “comprising”), the reference to “A and / or B” may in one embodiment refer to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so on.

[0495] The word “or” as used herein in the specification and claims should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, meaning it includes multiple elements or at least one of the elements in the list, but also includes more than one element, as well as other optional unlisted items. Only terms that explicitly indicate the opposite, such as “only one” or “exactly one” or “consisting of” as used in the claims, refer to multiple elements or exactly one of the elements in the list. Generally, the term “or” as used herein should only be interpreted as indicating an exclusive option (i.e., “one or the other, but not both”), provided that it is preceded by an exclusive term such as “either of,” “one of,” “only one of,” or “exact one of.” When used in the claims, “consisting substantially of” should have its ordinary meaning as used in the field of patent law.

[0496] The phrase “at least one” as used herein in the specification and claims, when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether or not they are related to those specifically identified elements. Thus, as a non-limiting example, “at least one of A and B” (or equivalents “at least one of A or B” or equivalents “at least one of A and / or B”) in one embodiment may mean at least one (optionally including more than one) A without B (and optionally including elements other than B), in another embodiment at least one (optionally including more than one) B without A (and optionally including elements other than A), in yet another embodiment at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements), and so on.

[0497] It should also be understood that, unless expressly indicated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0498] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “possessing,” “composed of,” etc., should be understood as open-ended, meaning including but not limited to. As set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures, only the transitional phrases “consisting of” and “substantially composed of” should be closed or semi-closed transitional phrases, respectively.

Claims

1. A system for performing cell culture, comprising: A valve, wherein the valve is in fluid communication with a plurality of ports; A cell culture chamber, wherein the cell culture chamber includes a cell filter, wherein at least a portion of the surface of the cell culture chamber is transparent to light of at least one wavelength, and wherein the cell culture chamber has a volume of less than or equal to 500 microliters; and sensor, The system further includes a mixing chamber, mixing elements, and / or flow paths configured to facilitate mixing.

2. A method for performing cell culture, the method comprising: Liquid is passed through a cell culture chamber containing cells, and the cell culture chamber has a volume of less than or equal to 500 microliters. and As the liquid flows through the cell culture chamber, the cells are retained within the cell culture chamber, wherein: The cell culture chamber is present in a system used for cell culture. The system also includes pumps, valves, and sensors. The system also includes a mixing chamber, mixing elements, and / or flow paths configured to facilitate mixing. The valve is in fluid communication with multiple ports, and At least a portion of the surface of the cell culture chamber is transparent to light of at least one wavelength.

3. A system for performing cell culture, comprising: Pump; A valve, wherein the valve is in fluid communication with a plurality of ports; A cell culture chamber, wherein the cell culture chamber includes a cell filter, and wherein the cell culture chamber has a volume of less than or equal to 500 microliters; sensor; Optical detectors; and Breathable membrane, The system further includes a mixing chamber, mixing elements, and / or flow paths configured to facilitate mixing.

4. The system of claim 1, wherein the system is located on a substrate.

5. The system according to claim 4, wherein the substrate is a chip.

6. The system of claim 5, wherein the surface of the chip is transparent to light of at least one wavelength.

7. The system of claim 5, wherein the chip is formed of a biocompatible and / or sterilizable material.

8. The system of claim 1, wherein the system comprises an incubator.

9. The system of claim 1, wherein the cell culture chamber is located inside the incubator.

10. The system of claim 1, wherein the system comprises a pump.

11. The system of claim 11, wherein the pump is a rotary membrane pump, peristaltic pump, rotary pump, piston pump, diaphragm pump, or roller pump.

12. The system according to claim 1, wherein the valve is a pneumatic valve, a rotary valve, a rotary diaphragm valve, a shape memory alloy valve, and / or a solenoid valve.

13. The system of claim 1, wherein at least one port is in fluid communication with a reagent source.

14. The system of claim 1, wherein at least one port is in fluid communication with the waste chamber.

15. The method of claim 2, wherein flowing the liquid through the cell culture chamber comprises flowing the liquid from the reagent source through a port and then into the cell culture chamber.

16. The method of claim 2, wherein flowing the liquid through the cell culture chamber comprises flowing the liquid out of the cell culture chamber, through a port, and then into the waste chamber.

17. The method of claim 2, wherein the fluid comprises a cell culture medium.

18. The system of claim 1, wherein the valve enables two or more ports to be in simultaneous fluid communication with the first mixing chamber.

19. The system of claim 1, wherein the valve enables two or more ports to be in simultaneous fluid communication with the second mixing chamber.

20. The system of claim 1, wherein the cell culture chamber contains cells.

21. The system of claim 20, wherein the cells comprise adherent cells.

22. The system of claim 20, wherein the cells comprise suspended cells.

23. The system of claim 1, wherein the cell culture chamber is substantially composed of channels.

24. The system of claim 23, wherein the channel is a microfluidic channel.

25. The method of claim 2, wherein the surface of the cell culture chamber is transparent to light of at least one wavelength.

26. The system of claim 1, wherein the cell culture chamber is formed of a biocompatible and / or sterilizable material.

27. The system of claim 1, wherein the fluid dead zone occupies less than or equal to 20% of the volume of the cell culture chamber.

28. The system of claim 1, wherein the system comprises a cell filter, the cell filter being a filter, a helical inertial separator, a hollow fiber membrane, for retaining cells by sedimentation, by centrifugal force, by dielectric electrophoresis, by acoustic separation, and / or by deterministic lateral displacement.

29. The system of claim 28, wherein the filter is flat.

30. The system of claim 28, wherein the filter is located on the valve.

31. The method of claim 2, wherein the cells are retained with an efficiency of 70% or higher.

32. The system of claim 1, wherein the system includes the mixing chamber.

33. The system of claim 32, wherein the mixing chamber is in fluid communication with the cell culture chamber.

34. The system of claim 32, wherein the mixing chamber is in fluid communication with the valve.

35. The system of claim 1, wherein the system includes the mixing element, and wherein the mixing element includes a pump.

36. The system of claim 1, wherein the mixing element comprises a mechanical mixer.

37. The system of claim 1, wherein the mixing element comprises a static mixer.

38. The system of claim 1, wherein the mixing element is located in the cell culture chamber.

39. The method of claim 2, further comprising mixing the liquid in the mixing chamber.

40. The method of claim 2, further comprising mixing the liquid in the cell culture chamber.

41. The method of claim 2, further comprising mixing the liquid, wherein the mixing comprises shuttle flow through a filter that retains cells.

42. The method of claim 2, further comprising mixing the liquid, wherein the mixing comprises circulating flow via a peristaltic pump.

43. The method of claim 2, further comprising mixing the liquid, wherein the mixing comprises alternating flow through the mixing chamber and / or through the cell culture chamber.

44. The method of claim 2, further comprising mixing the liquid, wherein the mixing comprises mechanical mixing.

45. The system of claim 1, wherein the flow path includes the cell culture chamber.

46. ​​The system of claim 1, wherein the flow path includes a channel in fluid communication with the cell culture chamber.

47. The system of claim 1, wherein the system comprises two mechanically stabilizing orientations, and wherein the mixing comprises a flow that occurs when the system shifts from one of the mechanically stabilizing orientations to the other.

48. The system of claim 3, wherein the breathable membrane forms the wall of the cell culture chamber.

49. The method of claim 2, further comprising a breathable membrane through which gas flows through the wall forming the cell culture chamber.

50. The system of claim 3, wherein the breathable membrane comprises a silicone membrane.

51. The system of claim 49, wherein the gas comprises a reagent.

52. The system of claim 49, wherein the gas comprises oxygen.

53. The system of claim 3, wherein the optical detector comprises a microscope.

54. The system of claim 1, wherein the system further comprises a light source, and wherein the light source is a fluorescent light source.

55. The system of claim 1, wherein the system comprises two mechanically stabilizing orientations, one adapted for performing cell culture in the cell culture chamber and the other adapted for performing optical measurements of the cell culture chamber.

56. The method of claim 2, wherein the plurality of ports includes a port through which cells can be seeded.

57. The method of claim 56, wherein the port is sealed with a gasket.

58. The method of claim 57, wherein cell seeding can be performed by puncturing the septum with a needle and delivering cells therethrough.

59. The method of claim 56, wherein the port includes a cover.

60. The method of claim 2, wherein the system comprises a reservoir through which cell seeding can be performed.

61. The method of claim 60, wherein the reservoir includes a cap.

62. The method of claim 61, wherein the cap is removed during cell seeding.

63. The method of claim 2, wherein the plurality of ports includes a port through which cell recycling can be performed.

64. The method of claim 2, wherein the port is sealed with a gasket.

65. The method of claim 64, wherein cell recovery can be performed by puncturing the septum with a needle and extracting the cells therefrom.

66. The method according to claim 2, wherein the sensor is a dissolved oxygen sensor.

67. The method of claim 66, further comprising: (i) Measure the dissolved oxygen content of the liquid present in the system, (ii) deliver a certain amount of oxygen to the liquid based on the measurement of the dissolved oxygen content, and / or (iii) deliver a cell culture medium with a dissolved oxygen content within a range determined based on the measurement of the dissolved oxygen content.

68. The method of claim 2, wherein the sensor is a pH sensor.

69. The method of claim 68, further comprising: (i) measuring the pH of the liquid present in the system, (ii) delivering a certain amount of acid and / or base, the amount being determined based on the pH measurement, and / or (iii) delivering cell culture medium with a pH content within a range determined based on the pH measurement.

70. The system of claim 1, wherein the system comprises two or more sensors.

71. The system of claim 1, wherein the system comprises a sensor for a biological product, and wherein the biological product is IgG.

72. The system of claim 71, wherein the biological product is IgG.

73. The method of claim 2, further comprising measuring the amount of biological products in the liquid present in the system.

74. The system of claim 1, wherein the system further comprises sensors for carbon dioxide, cell concentration, cell viability, nutrients, metabolites, temperature, pressure, humidity and / or viscosity.

75. The system of claim 74, wherein the metabolite comprises lactate and / or glucose.

76. The system of claim 1, wherein the system comprises an optical sensor, an electrochemical sensor, and / or an acoustic sensor.

77. The method of claim 2, wherein the liquid is present in and / or obtained from the cell culture chamber.

78. The system of claim 1, wherein the sensor comprises a sensor plug and / or a sensor patch.

79. The method of claim 2, wherein the liquid flows intermittently.

80. The method of claim 2, wherein the liquid flows continuously.

81. The method of claim 2, wherein the liquid flows as part of a batching process.

82. The method of claim 2, wherein the liquid flows as part of a feed batching process.

83. The method of claim 2, wherein the liquid flows as part of the infusion process.

84. The system of claim 1, wherein the system is located on a chip, and wherein the chip further comprises one or more additional systems.

85. The system of claim 84, wherein the one or more additional systems comprise the same components as the system.

86. The system of claim 1, wherein the system comprises a chip having two or more modules, and wherein each module comprises the same set of components.

87. The system of claim 86, wherein the components include the pump, the valve, the cell culture chamber, and / or the sensor.

88. The system of claim 1, wherein the system comprises a first chamber and a second chamber, and wherein the first chamber and the second chamber are positioned in series between a port capable of introducing and / or removing samples and the cell culture chamber.

89. The system of claim 88, wherein the first chamber is used as a diaphragm valve.

90. The system of claim 88, wherein the second chamber serves as a pump, the pump being configured to pump a known volume of fluid through the pump.

91. The system of claim 88, wherein the membrane extends through the first chamber and the second chamber.

92. The system of claim 91, wherein the membrane can be raised and lowered to affect the pressure in the first chamber and / or the second chamber.

93. The system of claim 91, wherein the membrane can be raised to fluidly communicate with the first chamber and the second chamber, and can be lowered to de-fluidically communicate with each other.

94. The system of claim 1, wherein the system includes a component configured to deliver gas to the cell culture chamber, and wherein the component configured to deliver gas to the cell culture chamber is in fluid communication with the cell culture chamber via one or more ports.

95. The system of claim 1, wherein the system comprises a translational sensor.

96. The system of claim 95, wherein the translational sensor comprises a pH sensor or a dissolved oxygen sensor.

97. A method for performing RNA transcription in the system of claim 1, the method comprising measuring the amount of RNA in a fluid present in the cell culture chamber and adjusting the composition of the fluid based on such measurement.

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