Patent Application
20250136914
There are various limitations with conventional cell therapy bioprocessing, which can be complex since they can involve (a) the extraction of cells from the patient, (b) the treatment and modification of these cells ex vivo, and then the (c) reintroduction of these cells back inside the body of the patient. High-throughput cell processing destined for cell therapy treatments can involve precise control of flow, collection, growth, and manipulation of biological cells. An aspect to high-throughput cell processing is streamlining the processes involved from seeding all the way to harvesting, with minimal invasive interventions. There is a need of a high-throughput multifunctional microfluidic chips capable of carrying out in situ cell processing without invasive interventions, which can limit the risk of cell loss and contamination that would otherwise accrue with multiple transfers.
The present disclosure provides a multifunctional microfluidic-based system that permits streamlined non-invasive in situ bioprocessing operations for adherent and suspension cells. The system can comprise one or more microfluidic chips and micro-fluidics to miniaturize culture devices, increase throughput, and couple with automation scale-ups.
The one or more microfluidic chips (solid supports) can be designed to optimize the bioprocessing operations involved in cell production for cell therapy applications. These operations aimed for cell production can include, for example, seeding, activation, viral or non-viral transduction, proliferation and/or differentiation, washing and/or purification, sampling, and harvesting—all of which can be performed within the bioprocessing chamber of the chip without the need of external transplants and/or invasive interventions.
In an aspect, provided herein is a cassette comprising: (a) a branched network of primary feeding input channels in a first plane and a branched network of primary feeding input channels in a second plane, wherein the first plane is substantially parallel to the second plane; and (b) a bioprocessing layer comprising one or more bioprocessing chambers fluidically connected to (i) the branched network of primary feeding input channels in the second plane and (ii) the branched network of primary feeding input channels in the first plane via the branched network of primary feeding input channels in the second plane, wherein the cassette permits a fluid to flow from the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane and through the one or more bioprocessing chambers.
In some cases, the one or more bioprocessing chambers comprise at least 4, 8, 16, 32, 64, 128, or 256 bioprocessing chambers.
In some cases, the one or more bioprocessing chambers are fluidically connected with one another.
In some cases, the cassette further comprises a plurality of connecting input channels connecting the branched network of primary feeding input channels in the first plane and the branched network of primary feeding input channels in the second plane.
In some cases, the cassette permits the fluid to flow from a connecting input channel of the plurality of connecting input channels to a subset of the one or more bioprocessing chambers.
In some cases, a length dimension of the connecting input channels is orthogonal to a length dimension of the branched network of primary feeding input channels in the first plane.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprises a volume of less than 15 mL, 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprise a cell culturing surface of less than 300 cm2, 200 cm2, 100 cm2, 90 cm2, 80 cm2, 70 cm2, 60 cm2, 50 cm2, 40 cm2, 30 cm2, 20 cm2, 10 cm2, 6 cm2, 5 cm2, or 1 cm2.
In some cases, the branched network of primary feeding input channels in the first plane comprises symmetrical branched channels with an axis of symmetry about an upstream-most split point.
In some cases, the branched network of primary feeding input channels in the first plane comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 split points.
In some cases, the branched network of primary feeding input channels in a second plane comprises symmetrical branched channels with an axis of symmetry about an upstream most split point.
In some cases, the branched network of primary feeding input channels in a second plane comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 split points.
In some cases, the branched network of primary feeding input channels in the first plane is above the bioprocessing layer.
In some cases, the branched network of primary feeding input channels in the first plane is below the branched network of primary feeding input channels in the second plane.
In some cases, the branched network of primary feeding input channels in the first plane is above the branched network of primary feeding input channels in the second plane.
In some cases, the cassette further comprises a branched network of primary feeding input channels in a third plane fluidically connected to the branched network of primary feeding input channels in the first plane and a branched network of primary feeding input channels in the second plane, wherein the first plane, second plane, and third plane are substantially parallel.
In some cases, the method further comprises a feeding input, and wherein the cassette permits the fluid to flow from the feeding input, through the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane and through the plurality of bioprocessing chambers.
In some cases, the branched network of primary feeding input channels in the first plane comprises binary trees.
In some cases, the branched network of primary feeding input channels in the second plane comprises binary trees.
In some cases, the one or more bioprocessing chambers are fluidically connected to each other in parallel.
In some cases, the cassette further comprises a branched network of secondary feeding input channels fluidically connected to, and between, the primary feeding input channels in the second plane and the one or more bioprocessing chambers.
In some cases, the cassette further comprises a branched network of primary feeding output channels, wherein the cassette permits the fluid to flow from the branched network of primary feeding input channels in the first plane, the branched network of primary feeding input channels in the second plane and through the one or more bioprocessing chambers, and the branched network of primary feeding output channels.
In some cases, the cassette further comprises a branched network of secondary feeding output channels fluidically connected to, and between, the one or more bioprocessing chambers and the branched network of primary feeding output channels.
In some cases, the cassette further comprises a plurality of filters, wherein the cassette permits the fluid to flow from the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane, through the plurality of bioprocessing chambers, across the plurality of filters, and through the branched network of primary feeding output channels.
In some cases, the cassette further comprises a feeding input connected to the branched network of primary feeding input channels in a first plane.
In some cases, the cassette further comprises a plurality of collection drains fluidically connected to the one or more bioprocessing chambers.
In some cases, the plurality of collection drains comprises collection channels.
In some cases, the cassette further comprises a branched network of primary collection channels in a fourth plane and a branched network of primary collection channels in a fifth plane fluidically connected to the plurality of collection drains, wherein the first plane, the second plane, the fourth plane, and the fifth plane are substantially parallel.
In some cases, a length dimension of the plurality of collection drains is not orthogonal to a length dimension of the branched network of primary collection channels in the fifth plane.
In some cases, the cassette further comprises a branched network of secondary collection channels fluidically connected to, and between, the one or more bioprocessing chambers and the branched network of primary collection channels in the fifth plane.
In some cases, the cassette further comprises a collection hole fluidically connected to the primary collection channels in a fifth plane.
In some cases, the cassette comprises a material that is transparent.
In some cases, the cassette does not comprise a valve.
In some cases, the one or more bioprocessing chambers comprises a biocompatible material.
In some cases, the biocompatible material is a U.S. Pharmacopeia Convention (USP) Class VI material.
In some cases, the cassette permits a substantially equal pressure drop across each bioprocessing chamber of the one or more bioprocessing chambers when the fluid is flowed from the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane and through the one or more bioprocessing chambers.
In some cases, the branched network of primary feeding input channels in the first plane, the branched network of primary feeding input channels in the second plane, and the one or more bioprocessing chambers comprise a fluid.
In some cases, the one or more bioprocessing chambers are sterile.
In some cases, the one or more bioprocessing chambers comprise a plurality of cells.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprise at least 0.35, 0.5, 1, 3.5, 5, 10, 15, or 20 million cells/mL.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprises at least 0.25, 0.5, or 1 million cells.
In some cases, the one or more bioprocessing chambers comprise at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, 500 million, 1 billion, or 5 billion cells.
In another aspect, provided herein is a cassette comprising: (a) a plurality of cassette structures defining a cassette flow path volume from a feeding input to a feeding output, the plurality of cassette structures comprising: the feeding input; a branched network of primary feeding input channels in a first plane; a branched network of primary feeding input channels in a second plane, wherein the first plane is substantially parallel to the second plane; one or more bioprocessing chambers, a branched network of primary feeding output channels; the feeding output; and a plurality of feeding input structures defining a feeding input flow path volume from the feeding input to the one or more bioprocessing chambers, the plurality of feeding input structures comprising: the feeding input; the branched network of primary feeding input channels in the first plane; and the branched network of primary feeding input channels in the second plane; wherein the feeding input flow path volume is 30%, 20%, or 10% or less of the cassette flow path volume, wherein the cassette flow path volume corresponds to a total volume of the feeding input channels and a total bioprocessing volume associated with the plurality of cassette structures.
In some cases, the plurality of cassette structures further comprises a branched network of primary feeding input channels in a third plane fluidically connected to the branched network of primary feeding input channels in the first plane and the branched network of primary feeding input channels in the second plane, wherein the first plane, second plane, and third plane are substantially parallel; and the plurality of feeding input structures comprises the branched comprises a branched network of primary feeding input channels in a third plane.
In some cases, the branched network of primary feeding input channels in the first plane and the branched network of primary feeding input channels in the second plane comprise microfluidic channels.
In some cases, the one or more bioprocessing chambers comprise at least 4, 8, 16, 32, 64, 128, or 256 bioprocessing chambers.
In some cases, the one or more bioprocessing chambers are fluidically connected with one another.
In another aspect, provided herein is a cassette comprising: (a) a feeding input and a branched network of primary feeding input channels; (b) a feeding output and a branched network of primary feeding output channels; and (c) a bioprocessing layer comprising one or more bioprocessing chambers, wherein the cassette permits a fluid to flow from the feeding input, through the branched network of primary feeding input channels, through the one or more bioprocessing chambers, through the branched network of primary feeding output channels, and to the feeding output, wherein the cassette provides a ΔPcassette/ΔPbioprocessing chamber that is less than or equal to (1/22)*(n)+6, wherein n is a number of the one or more bioprocessing chambers.
In some cases, n is at least 16.
In some cases, n is at least 32, 64, 128, or 256.
In some cases, a pump is fluidically connected to the cassette.
In some cases, a reagent vessel is fluidically connected to the pump and the cassette.
In some cases, an air vessel comprising CO2 is fluidically connected to the reagent vessel.
In some cases, a concentration of CO2 in the air vessel is about 5% or 8% in volume.
In some cases, a de-bubbler is fluidically connected to the reagent vessel and to the cassette.
In some cases, the reagent vessel is separated from the de-bubbler by a first valve and the de-bubbler is separated from the cassette by a second valve.
In some cases, an output vessel is fluidically connected to the cassette.
In some cases, a flow-through sensor is connected to the cassette.
In some cases, a computer is electrically connected to the flow-through sensor.
In some cases, the system further comprises an imaging device.
In some cases, the system is automated.
In some cases, an agitator is coupled to the cassette.
In another aspect, provided herein is a method comprising providing a cassette; and flowing the fluid from the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane and through the one or more bioprocessing chambers.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprises a fluid inlet, and wherein a flow rate of the fluid at the inlet of each bioprocessing chamber of the one or more bioprocessing chambers is from about 10 μL/hr×a number of the one or more bioprocessing chambers to about 10 mL/min×a number of the one or more bioprocessing chambers.
In some cases, fluid comprises a plurality of solid particles.
In some cases, a flow rate of the fluid comprising a plurality of solid particles through the branched network of primary feeding input channels in the first plane and the branched network of primary feeding input channels in the second plane is greater than a settling or sedimentation velocity of the plurality of solid particles.
In some cases, the solid particles comprise cells.
In some cases, the solid particles comprise at least 20,000, 200,000, 350,000, 500,000, 1,000,000, 3,500,000, 10,000,00, 25,000,000, or 50,000,000 cells/mL.
In some cases, the cells comprise microorganisms, mammalian cells, HEK293 cells, T-cells, Jurkat cells, CHO cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells.
In some cases, the method further comprises distributing a substantially equal amount of the cells to each bioprocessing chamber of the one or more bioprocessing chambers to provide distributed cells.
In some cases, the distributing occurs within 5 minutes.
In some cases, the method further comprises washing the distributed cells.
In some cases, the method further comprises expanding the distributed cells to generate expanded cells.
In some cases, the expanding comprises expanding the distributed cells at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold.
In some cases, the expanding occurs over at least 24 hr, 48 h, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hr, or 1200 hr.
In some cases, the method further comprises imaging the distributed cells.
In some cases, the method further comprises imaging the expanded cells.
In some cases, the method further comprises, using a computer system to predict time to confluence of the expanded cells.
In some cases, the method further comprises contacting the distributed cells with a reagent after the washing.
In some cases, the method further comprises after the contacting, performing a second wash of the distributed cells.
In some cases, the method further comprises after the second wash of the distributed cells, harvesting the distributed cells.
In some cases, a pressure at a fluid input of the cassette is 3 bar or less, 2 bar or less, 1 bar or less, 0.75 bar or less, 0.5 bar or less, 0.25 bar or less, or 0.1 bar or less.
In another aspect, provided herein is a cassette, comprising: (a) a feeding input, a branched network of primary feeding input channels in a first plane, and a branched network of primary feeding input channels in a second plane; (b) a bioprocessing layer comprising one or more bioprocessing chambers; (c) a branched network of primary feeding output channels and a feeding output; (d) a plurality of filters; and (e) a collection output, a plurality of collection drains, a branched network of primary collection channels in a third plane, and a branched network of primary collection channels in a fourth plane, wherein the first plane, the second plane, the third plane, and the fourth plane are substantially parallel, wherein the cassette permits a fluid to flow in a flow path comprising, in order, the feeding input, the branched network of primary feeding input channels in the first plane, the branched network of primary feeding input channels in the second plane, the one or more bioprocessing chambers, the plurality of filters, the branched network of primary feeding output channels, and the feeding output.
In an aspect, provided herein is a method, comprising providing a cassette and flowing a fluid through the flow path.
In some cases, the fluid comprises cells.
In some cases, the method further comprises depositing the cells in the one or more bioprocessing chambers.
In some cases, the cells are not deposited in the branched network of primary feeding input channels in the first plane or the branched network of primary feeding input channels in the second plane.
In some cases, the plurality of filters prevent the cells from entering the branched network of primary feeding output channels.
In some cases, the cassette comprises a second flow path comprising, in order, the feeding input, the branched network of primary feeding input channels in the first plane, the branched network of primary feeding input channels in the second plane, the one or more bioprocessing chambers, the plurality of collection drains, the branched network of primary collection channels in the third plane, the branched network of primary collection channels in the fourth plane, and the collection output.
In some cases, the one or more bioprocessing chambers comprise cells.
In some cases, the cells comprise at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, 500 million, 1 billion, or 5 billion cells.
In some cases, the method further comprises harvesting the cells through the plurality of collection drains.
In some cases, the harvesting comprises applying a suction to the second flow path.
In some cases, the harvesting comprises harvesting at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells to provide harvested cells.
In some cases, the harvesting occurs in 5 min or less, 1 min or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less.
In some cases, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the harvested cells are viable.
In another aspect, provided herein is a method for culturing one or more cells, comprising providing a cassette, distributing the one or more cells in at least one bioprocessing chamber of the one or more bioprocessing chambers; washing the one or more cells; expanding the one or more cells; contacting the one or more cells with at least one reagent; imaging the one or more cells; and harvesting the one or more cells.
In another aspect, provided herein is a microfluidic system comprising one or more cassettes, wherein the microfluidic system is configured for i) flowing a fluid at a pressure of less than 3 bar, or less than 0.5 bar, and ii) culturing at least 50 million cells, at least 500 million cells, or at least 1 billion cells.
In some cases, the one or more cassettes comprise a branched network of primary feeding input channels in a first plane and a branched network of primary feeding input channels in a second plane, wherein the first plane is substantially parallel to the second plane.
In some cases, the one or more cassettes further comprise a bioprocessing layer comprising one or more bioprocessing chambers fluidically connected to (i) the branched network of primary feeding input channels in the second plane and (ii) the branched network of primary feeding input channels in the first plane via the branched network of primary feeding input channels in the second plane.
In some cases, the one or more cassettes permit the fluid to flow from the branched network of primary feeding input channels in the first plane, through the branched network of primary feeding input channels in the second plane and through the one or more bioprocessing chambers.
In some cases, the one or more cassettes further comprise a plurality of connecting input channels connecting the branched network of primary feeding input channels in the first plane and the branched network of primary feeding input channels in the second plane.
In some cases, the one or more cassettes permit the fluid to flow from a connecting input channel of the plurality of connecting input channels to a subset of the one or more bioprocessing chambers.
In some cases, the one or more bioprocessing chambers are separated by a liquid-permeable membrane.
In some cases, the liquid-permeable membrane is impermeable to cells.
In another aspect, provided herein is a cassette comprising (a) a branched network of feeding input channels in a first plane; (b) a bioprocessing layer in a second plane comprising one or more bioprocessing chambers fluidically connected to the branched network of feeding input channels in the first plane; and (c) a branched network of feeding output channels fluidically connected to the one or more bioprocessing chambers, wherein the cassette permits a fluid to flow from the branched network of feeding input channels in the first plane, through the one or more bioprocessing chambers in the second plane, and through the branched network of feeding output channels, and wherein the one or more bioprocessing chambers are fluidically connected with each other.
In some cases, the one or more bioprocessing chambers comprise at least 4, 8, 16, 32, 64, 128, or 256 bioprocessing chambers.
In some cases, the one or more bioprocessing chambers are separated by a liquid-permeable membrane.
In some cases, the liquid-permeable membrane is impermeable to cells.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprises a volume of less than 15 mL, 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprise a cell culturing surface of less than 300 cm2, 200 cm2, 100 cm2, 90 cm2, 80 cm2, 70 cm2, 60 cm2, 50 cm2, 40 cm2, 30 cm2, 20 cm2, 10 cm2, 6 cm2, 5 cm2, or 1 cm2.
In some cases, the branched network of feeding input channels in the first plane comprises symmetrical branched channels with an axis of symmetry about an upstream-most split point.
In some cases, the branched network of feeding input channels in the first plane comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 split points.
In some cases, the branched network of feeding input channels in the first plane is above the bioprocessing layer.
In some cases, the branched network of feeding output channels is above the bioprocessing layer.
In some cases, the branched network of feeding output channels is in the first plane.
In some cases, the branched network of feeding input channels in the first plane comprises binary trees.
In some cases, branched network of feeding output channels comprises binary trees.
In some cases, the one or more bioprocessing chambers are fluidically connected to each other in parallel.
In some cases, the cassette comprises a plurality of filters, wherein the cassette permits the fluid to flow from the branched network of feeding input channels in the first plane, through the plurality of bioprocessing chambers in the second plane, across the plurality of filters, and through the branched network of feeding output channels.
In some cases, a feeding input is connected to the branched network of feeding input channels in a first plane.
In some cases, a plurality of collection drains are fluidically connected to the one or more bioprocessing chambers.
In some cases, the plurality of collection drains comprises collection channels.
In some cases, the cassette comprises a material that is transparent.
In some cases, the cassette does not comprise a valve.
In some cases, the one or more bioprocessing chambers comprises a biocompatible material.
In some cases, the biocompatible material is a U.S. Pharmacopeia Convention (USP) Class VI material.
In some cases, the cassette permits a substantially equal pressure drop across each bioprocessing chamber of the one or more bioprocessing chambers when the fluid is flowed from the branched network of primary input channels in the first plane and through the one or more bioprocessing chambers.
In some cases, the one or more bioprocessing chambers are sterile.
In some cases, the one or more bioprocessing chambers comprise a plurality of cells.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprise at least 0.35, 0.5, 1, 3.5, 5, 10, 15, or 20 million cells/mL.
In some cases, each bioprocessing chamber of the one or more bioprocessing chambers comprises at least 0.25, 0.5, or 1 million cells.
In some cases, the one or more bioprocessing chambers comprise at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, 500 million, 1 billion, or 5 billion cells.
In some cases, the first plane is substantially parallel to the second plane.
In some cases, a cassette further comprises: (a) a branched network of gas input channels in a third plane; (b) a gas layer in a fourth plane fluidically connected to the branched network of as input channels in the third plane; and (c) a branched network of gas output channels in the third plane fluidically connected to the one or more bioprocessing chambers, wherein the cassette permits a gas to flow from the branched network of gas input channels in the third plane, through the gas layer in the fourth plane, and through the branched network of gas output channels in the third plane.
In some cases, the cassette permits the gas, while in the gas layer, to dissolve directly into the fluid in the one or more bioprocessing chambers.
In some cases, the third plane is substantially parallel to the fourth plane.
In some cases, the first plane, second plane, third plane, and fourth plane are all substantially parallel.
In some cases, the third plane is above the fourth plane.
In some cases, the fourth plane is above the first plane, and wherein the first plane is above the second plane.
In some cases, the gas layer and the one or more bioprocessing chambers are not separated by a gas permeable membrane.
Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.
Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
While various embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed.
Whenever the term “about,” “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “about,” “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.
Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.
The present disclosure provides a multifunctional microfluidic-based system that can permit streamlined non-invasive in situ bioprocessing operations for adherent and suspension cells. The system can comprise a microfluidic chip, or a cassette comprising more than one microfluidic chip as described in further detail below.
The one or more microfluidic chips can be designed to optimize the operations involved in cell production for cell therapy applications. The operations for cell production can include, for example, seeding, treatment, proliferation and/or differentiation, washing and/or purification, sampling, and harvesting—all of which can be performed within the bioprocessing chamber (e.g., on a culture surface or within a volume of the bioprocessing chamber) of the microfluidic chip or cassette without the need of external transplants and/or invasive interventions.
The systems and methods of the present disclosure can be used for cell therapy applications. Cell therapy can be a treatment approach in which functional and healthy cells, cultured ex vivo, are administered into or to a subject (e.g., a patient).
The systems of the present disclosure are designed to improve the cell culture process. In one aspect, the present disclosure provides a miniaturized chip comprising a bioprocessing chamber that is capable of performing bioprocessing operations involved in cell culture. The chip can utilize microfluidics, which can involve manipulating fluids inside channel dimensions of the micrometer range. In some embodiments, the channels described herein (including, for instance, feeding input channels, feeding output channels, input harvest channels, output harvest channels, etc.) can have one or more channel dimensions. The one or more channel dimensions can correspond to one or more of a channel width, a channel length, a channel height, or a channel diameter. The channel dimensions can range from about 1 micrometer to about 10 centimeters. In some cases, the channel dimensions can be less than 1 micrometer. In some cases, the chip design extends to the principles of mesofluidics or macrofluidics because certain \channel dimensions can be greater than 1 centimeter and sometimes greater than 10 centimeters. In some cases, the channels described herein (including, for instance, feeding input channels, feeding output channels, input harvest channels, output harvest channels, etc.) can have a channel volume. The channel volume can range from 10% of the total chip volume to 90% of the total chip volume. In some cases, the channel volume can be less than 10% of the total chip volume. In some cases, the channel volume can be greater than 90% of the total chip volume.
Microfluidic cell culture can provide several advantages, including, for instance: (1) better control of process parameters: cells can have equal access to molecules present in the surrounding fluid due to homogenous cell distribution and fluid circulation in microenvironments, which can result in a more homogeneous end product and less process failure (e.g. cell death); (2) better overall cell health: e.g., paracrine effects can be amplified in a small environment (i.e. microscale), which can lead to better cell expansion and phenotype control; (3) a reduction in reactant volume: can be 10-20 fold reduction compared to other conventional bioprocessing systems, due to smaller volumes of fluid used in microfluidic chips as well as the ability to recirculate unspent reactant (e.g., growth media (fluid) can be re-enriched and recirculated at defined intervals, e.g., due to rapid oxygen or glucose depletion inside the chip). As used herein, homogenous cell distribution may refer to the distribution of cells such that a density of cells is approximately uniform across a target area.
In some cases, the chip can comprise a microfluidic chip. The chip or microfluidic chip can comprise a bioprocessing chamber. The bioprocessing capabilities of the chip can be scaled up via parallelization to achieve high throughput bioprocessing. A plurality of chips can be parallelized in a cassette. In some cases, a plurality of cassettes can be placed in a machine for parallelized bioprocessing. The plurality of cassettes can be stackable.
These input channels 101 can feed into a bioprocessing chamber 105, which can comprise a recess in fluid communication with the one or more feeding input channels 101. The recess can comprise a vertical depth perpendicular to the flow direction where cells can settle. The recess can be protected from damaging shear stress because of minimal fluid velocity acting on the recess. The bioprocessing chamber 105 can be elongated in the primary direction of seeding and perfusion flow, such that length>>width. In some cases, the microfluidic chip or the bioprocessing chamber of the microfluidic chip can have a length that is about, at least, or at most 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 times its width. In some embodiments, the edges of the bioprocessing chamber 105 (e.g., at the ends) can be curved to minimize culture dead zones. Having curved edges can also facilitate initial chip wetting as opposed to rigid, sharp edges. In some cases, the curved edges may comprise a fillet or a rounded section that provides a transitional surface between two adjacent edges of the bioprocessing chamber.
In some cases, the bioprocessing chamber can comprise a recess with one or more walls that are angled relative to the feeding input channels and/or the feeding output channels. In some non-limiting embodiments, the angle can range from about 45 degrees to about 90 degrees. In some cases, the bioprocessing chamber can have one or more dimensions. The one or more dimensions can comprise, for example, a length, a width, a height, or a depth. The one or more dimensions of the bioprocessing chamber can range from about 1 millimeter to about 60 centimeters. In some cases, the dimensions of the bioprocessing chamber can be less than 1 millimeter. In some cases, the dimensions of the bioprocessing chamber can be greater than 60 centimeters. The bioprocessing chamber can comprise a volume of less than 15 mL, 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL.
In some cases, the bioprocessing chamber can have a bottom surface, as described elsewhere herein. The bottom surface can be used for cell culturing. The bottom surface can have a surface area ranging from about 1 mm2 to about 300 cm2. In some cases, the surface area can be less than 1 mm2. In some cases, the surface area can be greater than 300 cm2. The bottom surface can have a surface area of less than 300 cm2, 200 cm2, 100 cm2, 90 cm2, 80 cm2, 70 cm2, 60 cm2, 50 cm2, 40 cm2, 30 cm2, 20 cm2, 10 cm2, 6 cm2, 5 cm2, or 1 cm2.
In some cases, a length dimension of the bioprocessing chamber can be at least 2x, 3x, 4x, 5x, 10x, 15x, or 20x a width dimension of the bioprocessing chamber. In some cases, the bioprocessing chamber has a height of at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, or 0.5 mm.
The bioprocessing chamber can comprise a cross-sectional shape. The cross-sectional shape can be a circle, an oval, an ellipse, a triangle, a square, a rectangle, or any other polygon having three or more sides. The cross-sectional shape can correspond to a horizontal cross-section and/or a vertical cross-section of the bioprocessing chamber.
Towards the downstream end of the bioprocessing chamber 105, there can be two outlets positioned above and below the bioprocessing chamber 105. The upper outlet can connect to a filter 104 (e.g., a filter membrane) and/or a feeding output channel 103. In some cases, the chip may comprise a plurality of upper outlets connected to one or more filters. The one or more filters may be positioned at or near one or more upper outlets. In some cases, the one or more filters may be positioned in front of or within the one or more upper outlets. In some cases, the chip may comprise a plurality of filters that are stacked on top of each other, or arranged side-by side or in series relative to each other. The plurality of filters may have different shapes, sizes, and/or filtering capabilities. In some cases, one or more filters may be provided in each of a plurality of upper outlets. The filter 104 can serve as a barrier to prevent cells from exiting the chamber prematurely, hence increasing seeding efficiency. During perfusion via the feeding input channel, while growth medium can be replenished, e.g., gradually replenished, by a fresh batch of fluid, the cells can still be retained in the bioprocessing chamber 105 while the fluid exits through the feeding output channel 103. In some cases, the filter 104 can comprise a filter membrane, and the filter membrane can comprise filter materials such as polyethersulfone (PES), polyester track-etched (PETE), cellulose nitrate, polytetafluororthethylene (PTFE) e.g., with a pore size structure of about 5 micrometers. In some cases, the filter 104 comprises a pore size of less than 10 μm, less than 7.5 μm, less than 5 μm, less than 2.5 μm, or less than 1 μm, or less than 0.45 μm. The shape of the filter can be rectangular, oval, elliptical, or circular. In some cases, the shape can comprise any regular or irregular shape. In some cases, the shape can comprise any shape having three or more sides. In some cases, a dimension of the filter can range from about 1 μm to about 50 cm. The dimension can correspond to a length, a width, or a thickness of the filter. In some cases, a single filter strip can be used across the entire bioprocessing chamber. The lower outlet of the chip can be fluidically connected to a drain 108 for harvesting or collection purposes. In some cases, the chip may comprise a plurality of drains disposed at or near a bottom surface of the bioprocessing chamber. In some cases, the chip may comprise a plurality of lower outlets fluidically connected to the plurality of drains. When the outlet is open, fluid containing cells can be drawn out of the bioprocessing chamber 105. To improve harvest efficiency, fluid can be pulled via the lower outlet, e.g., via a syringe pump or a suction generated by a negative pressure source. Alternatively, fluid can be introduced via the feeding input and/or feeding output to help “push” the fluid out. Alternatively, collecting can also be done via a combination of push and pull, where fluid is simultaneously pulled from the lower outlet and pushed via the feeding input and/or output. The collection drain 108 can be positioned directly below the filter 104 or at a position away from the filter 104. In some non-limiting embodiments, an inclined/sloped structure can be provided to facilitate the fluid's exit. In some cases, the inclined/sloped structure can be integrated with the bottom surface of the bioprocessing chamber 105 and can connect the bioprocessing chamber 105 to the drain 108. Alternatively, the inclined/sloped structure can be formed as part of the drain 108. In some cases, the drain may be positioned at or near a bottom portion of one or more walls of the bioprocessing chamber. In some cases, the drain may be located upstream of a feeding output and/or downstream of a feeding input. In some cases, the drain may be positioned to the left of the feeding input or feeding output. In other cases, the drain may be positioned to the right of the feeding input or feeding output.
In some embodiments, the filter can comprise a pore size. The pore size can range from about 1 nanometer to about 1 millimeter. In some cases, the filter can comprise a plurality of different pore sizes ranging from about 1 nanometer to about 1 millimeter.
In some cases, the filter can comprise a membrane. The membrane can be permeable or semi-permeable. The membrane can comprise, for example, Polytetrafluoroethylene (PTFE) or expanded polytetrafluoroethylene (ePTFE), polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone (PS), modified polysulfone (mPS), ceramics, polypropylene (PP), cellulose, regenerated cellulose or a cellulose derivative (e.g., cellulose acetate or combinations thereof), polyolefin, polypropylene, polytetrafluoroethylene, polyvinyl chloride, polyester, or any other type of polymer. In some non-limiting embodiments, the membrane can comprise a biomedical polymer, e.g., polyurethane, polyethylene, polypropylene, polyester, poly tetra fluoro-ethylene, polyamides, polycarbonate, or polyethylene-terephthalate.
In some embodiments, the chip can comprise a middle layer 106 comprising a bioprocessing chamber 105 that is carved out of the middle layer 106. In some embodiments, the chip can further comprise a bottom layer 107. The bottom layer can comprise a collection/harvest drain 108. In one embodiment, the drain 108 can comprise a circular structure. In some cases, the drain may have a cross-sectional shape that is circular, oval, elliptical, square, or rectangular. In some cases, the drain may have a cross-sectional shape having three or more sides. The cross-sectional shape may correspond to a regular shape or an irregular shape. In some cases, the bottom layer 107 can have an inclined or sloped structure leading to the drain 108 to help facilitate the fluid's exit. In other cases, the inclined or sloped structure can be formed as part of the drain 108 to help facilitate the fluid's exit. In some embodiments, the collection drain 108 can be fluidically connected to the bioprocessing chamber via the bottom surface of the bioprocessing chamber. In some cases, the collection drain 108 may be fluidically connected to the bioprocessing chamber via one or more holes, apertures, channels, or passageways in or through at least a portion of the bottom surface of the bioprocessing chamber.
The cells described herein can comprise a range of sizes. In some cases, the cells can have a size of at least about 1 micrometer, 5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, or any size that is between any of the preceding values. In some cases, the cells can have a size that is less than about 1 micrometer. In some cases, the cells can have a size that is at most about 1 micrometer, 900 nanometers, 800 nanometers, 700 nanometers, 600 nanometers, 500 nanometers, 400 nanometers, 300 nanometers, 200 nanometers, 100 nanometers, 90 nanometers, 80 nanometers, 70 nanometers, 60 nanometers, 50 nanometers, 40 nanometers, 30 nanometers, 20 nanometers, 10 nanometers, or less.
The microfluidic device can be fabricated in optically transparent material or a combination of different types of materials. The bioprocessing chamber that can be used for cell culture can be made of a USP (United States Pharmacopoeia) Class VI Material. Such materials can be transparent so that imaging technology can be coupled. The device can also possess tolerances on the design requirements (e.g., channels) not lower than 5 micrometers in absolute value for the smallest feature and 5% for larger dimensions. This can ensure that fabrication of these devices can be suitable with standard manufacturing processes (e.g., sheet or roll processing). The device can also comprise usable surface culture space (for the individual chip) that is potentially capable of handling up to at least about 10 million cells, 20 million cells, 30 million cells, 40 million cells, 50 million cells, 60 million cells, 70 million cells, 80 million cells, 90 million cells, 100 million cells, or more.
The device can have certain favorable properties. For example, the device can favor homogenous distribution or collection of solids (i.e., cells) and prevent premature collection of seeded cells. In some embodiments, seeding efficiency (i.e., the number of cells actually seeded relative to the number of cells provided) can be greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The device can also be versatile for adherent and suspension solids (i.e., cell culture). The fluid flow coming from perfusion can avoid generating high shear stress that would potentially damage the cells. For suspension cells, the flow can circulate and exit the chip without flushing the cells. The device can also favor cell growth with minimal invasion and favor sampling procedures without invasive procedures. In some cases, the device can favor cell/particle extraction from the device at >90% efficiency with very minimal cells still stuck in the system. In some cases, the device can favor cell/particle extraction from the device at about 50%, 60%, 70%, 80%, or 90% efficiency with very minimal cells still remaining in the system.
The chip can possess usable surface culture area (for the network) of at least 1 cm2, which can represent at least about 70% of the total chip footprint.
The chips of the present disclosure can be designed in a way such that scale-up does not require a massive overhaul of design, i.e., the chip can retain its original features even when parallelized and multiplexed in a cassette.
First, an individual microfluidic chip can be fabricated. Then, the individual chip can be used as a building block towards a more sophisticated parallel array network for high-throughput cell processing. In some cases, the network array can handle up to 3 bars of pressure without leakage. In some cases, the network array can handle pressure of up to about 1 bar, 2 bars, 3, bars, 4 bars or more without leakage. Through parallelization, there can be no significant change in the cell's microenvironment, since in some cases, at every point in the scale-up, the dimensions of the individual chips and the bioprocessing chambers do not change. Rather, a greater number of chips can be added to the array in a controlled manner to manage hydrodynamic resistances.
In some embodiments, the chip configuration can further comprise one or more primary feeding output channels 203. The one or more primary feeding output channels 203 can function as the output of the entire array of parallelized chips.
In some embodiments, the chip configuration can further comprise one or more primary feeding input channels 204. The one or more primary feeding input channels 204 can connect to the feeding input hole 201 and feed the entire array network. In some embodiments, the chip configuration can further comprise one or more secondary feeding input channels 205. The secondary feeding input channels 205 can feed one or more individual chips in the array.
In some embodiments, the chip configuration can further comprise one or more bioprocessing chambers 207. In some embodiments, the chip configuration can further comprise one or more collection or harvest drains 208. The one or more collection or harvest drains 208 can comprise a circular structure. In some cases, an inclined or sloped structure leading to the drain can be utilized to help facilitate the fluid's exit.
In some embodiments, the chip configuration can further comprise one or more secondary collection channels 209. The one or more secondary collection channels 209 can be configured to collect fluid or cells from each individual chip.
In some embodiments, the chip configuration can further comprise one or more primary collection channels 210. The one or more primary collection channels 210 can be configured to collect fluid or cells from the entire array network and can be fluidically connected to at least one collection or harvest hole 211. The one or more primary collection channels 210 can comprise one or more primary collection channels in a first plane and one or more primary collection channels in a second plane. The first plane can be different than the second plane.
In some cases, the one or more secondary feeding output channels 202 and the one or more primary feeding output channels 203 can be located on a same layer of the parallelized chip configuration. In some cases, the one or more secondary feeding output channels 202 and the one or more primary feeding output channels 203 can be on different layers than the one or more filters 206 (e.g., filter membranes). In some cases, the one or more secondary feeding output channels 202, the one or more primary feeding output channels 203, and the one or more filters 206 can be on different layers than the one or more primary feeding input channels 204 and the one or more secondary feeding input channels 205. In some cases, the one or more bioprocessing chambers 207 and the one or more collection/harvest drains 208 can be on different layer than any of the other components or features of the parallelized chip configuration. In some embodiments, the one or more secondary collection channels 209, the one or more primary collection channels 210, and the one or more collection/harvest holes 211 can be provided on a same layer, which layer can be a different layer than the other previously described layers.
Referring to
In some cases, the fluid distribution network can comprise one or more primary input channels and one or more secondary input channels which feed into one or more compartments comprising 4 chips. The fluid distribution network can further comprise one or more primary output channels and one or more secondary output channels.
In any of the embodiments described herein, the chip arrays may comprise input channels that feed into the bioprocessing chambers. The input channels may be configured to ensure that the cells in solution do not settle in the input channels, and instead reach the bioprocessing chambers in fluid communication with the input channels.
The total area occupied by the feeding input channels can be optimized to reduce the likelihood of cells settling in the feeding input channels. In some cases, the size, shape, and/or spatial configuration of the feeding input channels may be adjusted to minimize the amount of space (e.g., area or volume) occupied by the channels, thereby reducing the amount of dead space or dead volume available for the settling of cells. Minimizing the footprint of the channels may facilitate the settling of cells in the bioprocessing chambers of the chip arrays. The percentage of dead volume for a chip array may be computed as a ratio between (i) the total volume of the input channels and (ii) the total volume of the input channels plus the total bioprocessing volume associated with the chips in the chip array. In some cases, the percentage of dead volume for a chip array may be computed as a ratio between (i) the total volume of the input channels and (ii) the total volume of the input channels plus the total bioprocessing volume associated with the chips in the chip array plus the volume of the outlet and/or collection channels.
The chip array shown in
In one instance, a 0.5 mL volume of cells was collected at each port. The above procedure was repeated and the chip array was further flushed with cell media, and another 0.5 mL of volume was collected at each port. The chip array was flushed again with cell media and another 0.5 mL of volume was collected. The cells from every port were counted to determine the total number of cells passing through the chip array and the total number of cells collected.
The results shown in
In some cases, a detachable filter unit can be added to the input or output channel of any of the cassettes described herein. As shown in
In some embodiments, bioprocessing chambers can be connected or “fused”, while still maintaining the same number of bifurcations as other chip configurations with bioprocessing chambers that have not been connected or “fused.” These fused bioprocessing chambers can further facilitate communication between cells in a much larger space without having to separately seed multiple chambers. This is because in a classic 2×2 array, the cell numbers in each of the four chambers are not significantly different, while in the fused array, only one or two chambers can have approximately equal numbers (instead of all four chamber). The same level of bifurcation can be maintained from the primary input to the bioprocessing chambers since this bifurcation can optimize fluid distribution while minimizing fluid pressure drops. In some cases, an advantage of a fused chamber is that it is not necessary to rely on the exact same number of cells reaching each chamber (when seeding) and the exact same flow rate in each chamber (e.g., when perfusing). If there is any imbalance in one of the channels (e.g., because of imperfect fabrication, bubble, debris), the fact that the chambers are fused can compensate for this through agitation of the fluid in the fused chamber, therefore homogenizing the cells and the fluid across the fused chamber.
In some cases, a cassette comprises (a) a branched network of feeding input channels in a first plane, (b) a bioprocessing layer in a second plane comprising a fused bioprocessing chamber fluidically connected to the branched network of feeding input channels in the first plane; and (c) a branched network of feeding output channels fluidically connected to the fused bioprocessing chamber. In some cases, the cassette permits a fluid to flow from the branched network of feeding input channels in the first plane, through the fused bioprocessing chamber in the second plane, and through the branched network of feeding output channels. The fused bioprocessing chamber may have one or more sub-chambers that are separated by a liquid permeable membrane. The liquid permeable membrane may be impermeable to cells or other biological samples. In some cases, the input and output channels are located in the same plane. This plane may be located above the bioprocessing chamber.
Fluid channels may be designed and optimized to achieve certain characteristics, such as uniform seeding or injection rates.
In some embodiments, the harvested cells from the presently disclosed chips can be used for various applications. The applications can include, for example, regenerative medicine, treatment of diabetes, cancer, and/or treatment of cardiac-related diseases or neurogenerative diseases. In some cases, the application can include autologous cell transplantation, allogenic cell transplantation, or reinfusion of cells in a patient.
The chips and cassettes disclosed herein can yield a large number of cells after cell expansion occurs. In some cases, at least about 1 million, 10 million, 100 million, 150 million, 500 million, 1 billion, 5 billion, or more cells can be harvested from the chips disclosed herein. The cells can comprise, for example, human cells (e.g., stem cells, bone cells, blood cells (e.g., white blood cells (monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages), red blood cells (erythrocytes), or platelets), muscle cells, fat cells, skin cells, nerve cells, immune cells (e.g., T-cells, B-cells or NK cells, lymphocytes, neutrophils, or monocytes/macrophages), cancer cells (e.g., cells associated with carcinoma, sarcoma, melanoma, lymphoma, or leukemia), or non-human cells (including, for instance, animal cells, plant cells, bacterial cells, fungal cells, etc.). Plant cells may include, for example, collenchyma, sclerenchyma, parenchyma, xylem or phloem. Bacterial cells may include, for example, spherical bacterial cells (cocci), rod-shaped bacterial cells (bacilli), spiral bacterial cells (spirilla), comma bacterial cells (Vibrios), or corkscrew bacterial cells (Spirochaetes). Fungal cells may include, for example, hyphae, yeast cells, spores, Chytridiomycota (chytrids), Zygomycota (bread molds), Ascomycota (yeasts and sac fungi), and the Basidiomycota (club fungi). In some cases, the cells may comprise chimeric antigen receptor T-cells.
The systems of the present disclosure can provide a multi-functional design with numerous advantages over other systems. The systems referred to herein can comprise any of the chips, chip arrays, cassettes, and other devices, hardware, or apparatuses described herein.
A device provided herein can be closed at all times, i.e., operations can be carried out in a closed environment (no opening of the chip at any time). The presently disclosed chips, chip arrays, and cassettes can be configured to carry out a plurality of functions in situ (cell seeding, perfusion, sampling, transduction, differentiation, purification, cell harvest) without opening the chip or cassette.
When cells are provided to the chips described herein, the cells can settle on or come in contact with cyclic olefin copolymer (COC), which can possess glasslike clarity that can exceed thermoplastic substitutes such as polycarbonate. COC can be sterilized using standard methods (e.g., steam, ethylene oxide, gamma irradiation, and hydrogen peroxide) without altering its properties. It can also permit UV transmission, which can be best suited for diagnostic analysis. COC can also have low leachables and extractables, making it best suited for direct drug contact. It can be classified USP Class VI and is ISO 10993 compliant including biocompatibility, USP 661.1 and FDA drug and device master files.
In some cases, the chip or cassette can comprise, for example, a polydimethylsiloxane (PDMS) component, which can form the wall of the bioprocessing chambers, as well as the top layer, which can form part of its ceiling. PDMS can have gas permeability, which can be advantageous for cell growth. PDMS can permit gas equilibration between the bioprocessing chamber and that of the surrounding controlled environment (e.g., incubator), and can withstand autoclave conditions. In some cases, the PDMS component can be replaced with another gas permeable polymer. Cells can settle on the COC portion of the bioprocessing chamber.
In some cases, the chip or cassette can comprise a gas input line. In some gases, a chip or cassette with a gas input line does not have a gas permeable membrane. A gas input line can allow for gas to be injected into the chip or cassette, thereby dissolving directly onto the fluid within the chip or cassette. In some cases, a gas input is separate from a fluid input. The chip or cassette may also comprise a gas outlet. In some gases, the gas outlet contains a filter to reduce convection and increase gas residence time above the fluid in the chip or cassette, thereby favoring gas diffusion into the fluid.
In some cases, the chip or cassette can comprise a plurality of components or layer comprising a plurality of materials. The plurality of materials can comprise different materials. In some cases, the plurality of materials can comprise a cyclic olefin polymer (COP), a cyclic olefin copolymer (COC), or a polydimethylsiloxane (PDMS) material. In some cases, the plurality of materials can comprise a USP Class VI material such as other medical-grade thermoplastics In some cases, the plurality of materials can comprise any type of material that is biocompatible and/or biostable. In some cases, the materials for the various components or layers of the chip can have a high permeability (e.g., liquid or gas permeability) to permit a flow of fluid and/or cells into, out of, or through the chip (and any components or layers thereof).
The presently disclosed chips can also contain a filter, e.g., filter membrane made of polyethersulfone (PES). The filter can have one or more pores. The pore size can be about 5 μm or less, which can be used to retain cells of 10-μm diameter in the bioprocessing chamber.
In some cases, systems provided herein can be primed with fluid, e.g., in order to facilitate injection of the growth media. This priming can reduce the interfacial tension effects that can be associated with flowing liquid in an initially gas-filled chamber. Interfacial tension between gas-liquid can contribute to hydrodynamic resistance. This can be true of microfluidic devices with relatively small dimensions, whose inherent resistance can be high.
In some cases, the chips and chip arrays (e.g., cassettes) disclosed herein can have a height of the bioprocessing chamber of around 3-5 mm, such that priming is no longer needed, as the fluid does not experience significant resistance when injected into the bioprocessing chamber.
Due to the fact that PDMS is gas permeable, evaporation can happen, resulting in bubbles in the bioprocessing chamber. In a bioprocessing chamber of small heights, the bubbles can be detrimental to cell growth.
In the presently disclosed chips and chip arrays (e.g., cassettes), the height of the bioprocessing area can be, e.g., around 3-5 mm, which can permit natural “separation” of the cells and occurring bubbles. While the cells settle at the bottom surface, bubbles can be naturally buoyant and thus float towards the top part of the bioprocessing chamber, away from the cells. The environment can be controlled to minimize evaporation and mitigate impacts on the cell growth.
The chips and cassettes provided herein can permit high efficiency cell seeding, while minimizing loss. Such cell seeding may be determined by monitoring the number of cells that are provided to a bioprocessing chamber and the number of cells that adhere to a portion or a surface of the bioprocessing chamber. The cells can be spread homogeneously throughout the bioprocessing chamber to enable optimal growth. In some cases, confluent growth can be prematurely reached in some areas and therefore decrease cell culture efficiency.
The presence of the filter (e.g., filter membrane) can help in blocking cells from prematurely exiting the bioprocessing chamber, ensuring that they stay detained inside the bioprocessing chamber. Mass transport or advection can be a phenomenon due in large part that cells can be relatively large (>10 μm). Their large size can help them sediment into the recess. To help in homogenous distribution, the chip can be attached to a mechanical agitation device, which can facilitate re-distribution of the seeded cells all throughout the bioprocessing chambers. In some cases, the mechanical agitation device can be used with a single, chip, a plurality of chips, a chip array, multiple chip arrays, or any cassettes or compartments of such cassettes.
In some cases, the cells can comprise at least 20,000, 200,000, 350,000, 500,000, 1,000,000, 3,500,000, 10,000,00, 25,000,000, or 50,000,000 cells/mL. In any of the embodiments described herein, the cells can comprise microorganisms, mammalian cells, HEK293 cells, T-cells, Jurkat cells, CHO cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. The bioprocessing chambers can comprise at least 0.35, 0.5, 1, 3.5, 5, 10, 15, or 20 million cells/mL.
Cells can deplete surrounding media from nutrients in static conditions. The rate of media flow in the microfluidic chip can be carefully regulated. The chip designs disclosed herein can balance perfusion flow rate with shear stress so that cells get access to enough nutrients while at the same time, the flow is low enough so as not to remove the cells from their substrate.
In the microscale, numerous parameters can be involved in ensuring cell growth, including temperature gradients, oxygen levels, chemical gradients, cell-to-cell interactions, cell-to-molecule interactions, CO2 level, shear stress, and cell-substrate interactions.
The fact that the perfusion input channels can be on a different plane than the cells (settling at the bottom of the bioprocessing chamber), means that they can be protected from damaging shear stress induced by the fluid streamlines during perfusion. Shear stress can decrease with depth. Thus, the cells can avoid significant shear stress in the chip/chip array/cassette configurations described herein.
In some cases, the perfusion can come from the width-wise side because one or more fluid inlets are positioned at a higher plane than the bottom of the bioprocessing chamber, which can cause the reduction in shear stress. When injected from the lengthwise side, the input channels can be positioned at a higher plane than the bottom surface of the bioprocessing chamber.
Nutrient and gas diffusion as well as cell consumption can also be optimized. When cells are not homogeneously distributed during seeding, a consumption rate that follows a Poisson distribution can be expected where there is a higher consumption rate near the position of the feeding input channels (since it can be in first contact with the nutrients). Mechanical agitation can be employed during seeding can be beneficial for perfusion to ensure that nutrients are distributed all throughout the bioprocessing chamber. In some embodiments, the system can comprise an agitation device comprising one or more motors configured to produce vibration waveforms. The vibration waveforms may or may not be periodic. The vibration waveforms can agitate at least a portion of one or more chips in a parallelized array, which may aid in homogenously distributing cells or other solid particles in one or more bioprocessing chambers of a parallelized chip array. In some embodiments, a human operator may manually agitate at least a portion of the one or more chips in the parallelized array to aid in homogenously distributing cells or other solid particles in the one or more bioprocessing chambers of the parallelized chip array.
In some embodiments, the methods described herein can further comprise expanding the distributed cells to generate expanded cells. In some cases, the expanding comprises expanding the distributed cells about, or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold. In some cases, the expanding occurs over about, or at least 24 hr, 48 h, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hr, or 1200 hr.
Appropriate surface treatment can also be performed inside the bioprocessing chamber of the chip or cassette depending on the experimental conditions. Such surface treatments can allow adherent particles or cells to stick unto the surface such that particle-wall adhesion takes place. Additional coating methods can be used to facilitate cell attachment or detachment on the COC substrate. In some cases, the coating can comprise one or more polymeric surfactants. In some cases, the coating can comprise any type of biocompatible or biostable material that facilitates cell adhesion or growth. In some cases, the coating may comprise, for example, biological materials such as extracellular matrix, attachment and adhesion proteins, collagen, laminin, fibronectin, mucopolysaccharides, heparin sulfate, hyaluronidase, or chondroitin sulfate. In some cases, the coating may comprise a non-biological material.
Positioning the harvest/collection channels on a different plane can optimize space by effectively reducing the dead space between chips on a cassette. This can also reduce to constraint of adding lateral channels on either side of the chip in the lengthwise direction, which can make the chip design cumbersome and at the same time, generally adds chip footprint.
In some cases, harvesting can comprise harvesting at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells to provide harvested cells. Harvesting may begin when the seeded or expanded cells are collected by any manual or automatic operation or process, and may end when at least a portion of the seeded or expanded cells are collected by a human or a machine. In some cases, the harvesting occurs in 5 min or less, 1 min or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less. In some cases, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the harvested cells can be viable. In some cases, the harvesting of cells from the presently disclosed chips can result in about, or least, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% cell recovery with about or at least 95%, 94%, 93%, 92%, 91%, or 90% viability for the recovered cells.
Harvesting cells through the bottom surface of the bioprocessing chamber can take advantage of gravity, facilitating the exit of the fluid and cells. Drawing the fluid out (via a syringe pump or negative pressure) while pushing fluid from the perfusion side (via a second syringe pump or positive pressure) can also help complete removal of the fluid and cells to increase harvest efficiency.
Sampling can involve taking representative samples of cells from inside the bioprocessing chamber without interfering with cell growth and without opening the chip. Varying the drawing flow rate at the harvest or collection drain can control the amount of fluid (and cells) collected.
The parallelization systems and methods disclosed herein can exhibit the following characteristics:
As more chips are parallelized, resistances can increase, which can require large pressures to inject fluids into the parallelized chip arrays disclosed herein.
As more chips are parallelized on a cassette, the entire array can be compartmentalized into smaller arrays. This can make the cross-section that the fluid passes through more manageable because the fluid is no longer required to feed all of the chips on a cassette but only a portion of them. As a consequence of compartmentalization, feeding inputs can be separated into different layers, which can then permit feeding into smaller arrays.
As more chips are being parallelized or multiplexed on a cassette, more feeding input layers can be added to manage the global pressure across the network. In the examples illustrated in
In some embodiments, the 16-chip array on a cassette can also be separated into smaller compartments of four separate 4-chip arrays on a cassette. A 16-chip array that has one input and output fluidically connected in the same layer as the chambers can have a large resistance because the fluid needs to travel greater lengths to reach all the chips in the array.
Referring back to
In one aspect, the present disclosure provides a cassette for bioprocessing. The cassette can comprise a feeding input (201), a branched network of primary feeding input channels in a first plane (204a), and a branched network of primary feeding input channels in a second plane (204b).
In some cases, the cassette can further comprise a bioprocessing layer comprising a plurality of bioprocessing chambers (207). In some embodiments, the plurality of bioprocessing chambers may be fluidically connected to (i) the branched network of primary feeding input channels in the first plane and (ii) the branched network of primary feeding input channels in the second plane. In some cases, the plurality of bioprocessing chambers may be fluidically connected to the branched network of primary feeding input channels in the first plane via the branched network of primary feeding input channels in the second plane.
In some cases, the cassette can further comprise a branched network of primary feeding output channels (203) and a feeding output (214).
In some cases, the cassette can further comprise a plurality of filters (206) (e.g., filter membranes).
In some cases, the cassette can further comprise a collection output (215), a plurality of collection drains (208), a branched network of primary collection channels in a third plane (210a), and a branched network of primary collection channels in a fourth plane (210b). In some embodiments, the first plane, the second plane, the third plane, and/or the fourth plane are substantially parallel.
In some cases, the cassette permits a fluid to flow in a flow path comprising, in order, the feeding input (201), the branched network of primary feeding in-put channels in the first plane (204a), the branched network of primary feeding input channels in the second plane (204b), the plurality of bioprocessing chambers (207), the plurality of filters (e.g., filter membranes), the branched network of primary feeding output channels (203), and the feeding output (214).
In some cases, any one or more of the cassettes disclosed herein can be provided, and a fluid can be flowed along a flow path as described elsewhere herein. In some cases, the fluid can comprise one or more cells.
In some cases, the cells can be deposited into the plurality of bioprocessing chambers (207). In other cases, the cells may not or need not be deposited in the branched network of primary feeding input channels in a first plane (204a) and branched network of primary feeding input channels in a second plane (204b).
In some cases, one or more filters (206) (e.g., filter membranes) can be used to prevent the cells from entering the branched network of primary feeding output channels (203).
In some cases, the cassette can comprise a second flow path comprising, in order, the feeding input (201), the branched network of primary feeding input channels in the first plane (204a), the branched network of primary feeding input channels in the second plane (204b), the plurality of bioprocessing chambers (207), the plurality of collection drains (208), the branched network of primary collection channels in the third plane (210a), the branched network of primary collection channels in the fourth plane (210b), and the collection output (215). In some cases, the plurality of bioprocessing chambers (207) can comprise a plurality of cells. The chambers can comprise at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, 500 million, 1 billion, or 5 billion cells.
In some cases, the cells can be harvested through the plurality of collection drains. In some cases, the harvesting can comprise applying a suction to the second flow path. In some cases, harvesting can comprise harvesting at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells to provide harvested cells. In some cases, the harvesting occurs in 5 min or less, 1 min or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less. In some cases, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the harvested cells can be viable.
In one aspect, the present disclosure provides a multiple fluid layer cassette with a bioprocessing layer. The cassette can comprise a branched network of primary feeding input channels in a first plane (204a) and a branched network of primary feeding input channels in a second plane (204b). In some cases, the first plane can be substantially parallel to the second plane.
The cassette can further comprise a bioprocessing layer comprising a plurality of bioprocessing chambers (207) fluidically connected to the branched network of primary feeding input channels in the first plane (204a) and the branched network of primary feeding input channels in the second plane (204b). In some cases, the cassette permits a fluid to flow from the branched network of primary feeding input channels in the first plane (204a), through the branched network of primary feeding input channels in the second plane (204b) and into or through the plurality of bioprocessing chambers (207). In some embodiments, the plurality of bioprocessing chambers (207) can comprise at least 4, 8, 16, 32, 64, 128, or 256 bioprocessing chambers. The plurality of bioprocessing chambers (207) can comprise 2n bioprocessing chambers, where n is any integer greater than zero. In some embodiments, the plurality of bioprocessing chambers (207) can be fluidically connected to each other in series or in parallel. In some cases, the plurality of bioprocessing chambers (207) can be sterile.
In some cases, each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) comprises a volume of less than 15mL, 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL. In some cases, each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) comprises a cell culturing surface of less than about 60 cm2, 50 cm2, 40 cm2, 30 cm2, 20 cm2, 10 cm2, 6 cm2, 5 cm2, or 1 cm2.
In some embodiments, the plurality of bioprocessing chambers (207) comprises cells. In some cases, each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) can comprise at least 0.35, 0.5, 1, 3.5, 5, 10, 15, or 20 million cells/mL. In some embodiments, each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) comprises at least 0.25, 0.5, or 1 million cells. In some cases, the plurality of bioprocessing chambers (207) comprises at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, 500 million, 1 billion, or 5 billion cells.
In some cases, the plurality of bioprocessing chambers (207) can comprise a biocompatible material. In some cases, the biocompatible material can be a U.S. Pharmacopeia Convention (USP) Class VI material.
In some embodiments, the cassette can further comprise a plurality of connecting input channels (212) connecting the branched network of primary feeding input channels in the first plane (204a) and the branched network of primary feeding input channels in the second plane (204b). In some embodiments, the cassette permits the fluid to flow from a connecting input channel (212) of the plurality of connecting input channels (212) to a subset of the plurality of the bioprocessing chambers (207).
In some embodiments, a length dimension of the connecting input channels (212) can be disposed at an angle (e.g., orthogonal) to a length dimension of the branched network of primary feeding input channels in the first plane (204a).
In some embodiments, the branched network of primary feeding input channels in the first plane (204a) comprises symmetrical branched channels with an axis of symmetry about an upstream-most split point in the branched net-work of primary feeding input channels in the first plane (204a). In some cases, the branched network of primary feeding input channels in the first plane (204a) can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 split points (213).
In some embodiments, the branched network of primary feeding input channels in a second plane (204b) comprises symmetrical branched channels with an axis of symmetry about an upstream most split point in the branched network of primary feeding input channels in a second plane (204b). In some cases, the branched network of primary feeding input channels in a second plane (204b) can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 split points (213).
In some cases, the branched network of primary feeding input channels in the first plane (204a) can be positioned above the bioprocessing layer of the cassette. In some cases, the branched network of primary feeding input channels in the first plane (204a) can be positioned below the branched network of primary feeding input channels in the second plane (204b). In some cases, the branched network of primary feeding input channels in the first plane (204a) is above the branched network of primary feeding input channels in the second plane (204b).
In any of the embodiments described herein, the cassette can comprise a branched network of primary feeding in-put channels in a third plane fluidically connected to a branched network of primary feeding input channels in the first plane (204a) and a branched network of primary feeding input channels in the second plane (204b). In some cases, first plane, second plane, and third plane can be substantially parallel. In some cases, the cassette comprises a branched network of primary feeding input channels in at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more planes, wherein the planes are substantially parallel, and the branched network of primary feeding input channels are fluidically connected.
In any of the embodiments described herein, the feeding layer can comprise a feeding input (201). In some cases, the cassette can permit the fluid to flow from the feeding input (201), through the branched network of primary feeding input channels in the first plane (204a), through the branched network of primary feeding input channels in the second plane (204b) and into or through the plurality of bioprocessing chambers (207).
In some cases, the branched network of primary feeding input channels in the first plane (204a) can comprise binary trees. In some cases, the branched network of primary feeding input channels in the second plane (204b) can comprise binary trees.
In some cases, the cassette further comprises a branched network of secondary feeding input channels (205) fluidically connected to, and between, the primary feeding input channels in the second plane (204b) and the plurality of bioprocessing chambers (207).
In any of the embodiments described herein, the cassette can further comprise a branched network of primary feeding output channels (203). In some cases, the cassette permits the fluid to flow from the branched network of primary feeding input channels in the first plane (204a), the branched network of primary feeding input channels in the second plane (204b) and through the plurality of bioprocessing chambers (207), and the branched network of primary feeding out-put channels (203). In some cases, the cassette comprises a branched network of secondary feeding output channels (202) fluidically connected to, and between, the plurality of bioprocessing chambers (207) and the branched network of primary feeding output channels (203). In some cases, the cassette comprises a branched network of feeding output channels (e.g., primary feeding output channels) in at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more planes, wherein the planes are substantially parallel, and the branched network of feeding output channels (e.g., primary feeding output channels) are fluidically connected.
In any of the embodiments described herein, the cassette can comprise a plurality of filters (206) (e.g., filter membranes). The cassette can permit the fluid to flow from the branched network of primary feeding input channels in the first plane (204a), through the branched network of primary feeding input channels in the second plane (204b), through the plurality of bioprocessing chambers (207), across the plurality of filters (206), and through the branched network of primary feeding output channels (203). In some embodiments, the cassette comprises a feeding input (201).
In any of the embodiments described herein, the cassette can further comprise a plurality of collection drains (208) fluidically connected to the plurality of bioprocessing chambers (207). The plurality of collection drains can be located below the bioprocessing layer (e.g., when fluid is flowed through the cassette). In some cases, the plurality of collection drains (208) can comprise one or more collection channels. In some cases, the collection layer can further comprise a branched network of primary collection channels in a fourth plane (210a) and a branched net-work of primary collection channels in a fifth plane (210b) fluidically connected to the plurality of collection drains (208). The first plane, the second plane, the third plane, the fourth plane, and/or the fifth plane can be substantially parallel. In some cases, a length dimension of the plurality of collection drains (208) may not or need not be orthogonal to a length dimension of the branched network of primary collection channels in the fifth plane (210b).
In some embodiments, the collection layer can further comprise a branched network of secondary collection channels (209) fluidically connected to, and between, the plurality of bioprocessing chambers (207) and the branched network of primary collection channels in the fifth plane (210b). In some cases, the cassette further comprises a collection hole (211) fluidically connected to the primary collection channels in a fifth plane (210b).
In some cases, the cassette can comprise a material that is transparent or translucent. In some cases, the cassette may not or need not comprise one or more valves.
In some embodiments, the cassette permits a substantially equal pressure drop across each bioprocessing chamber (207) of the plurality of bio-processing chambers (207) when the fluid is flowed from the branched network of primary feeding input channels in the first plane (204a), through the branched net-work of primary feeding input channels in the second plane (204b) and through the plurality of bioprocessing chambers (207). The branched network of primary feeding input channels in the first plane (204a), the branched network of primary feeding in-put channels in the second plane (204b) and the plurality of bioprocessing chambers (207) can be configured to permit a flow of fluid therethrough.
In another aspect, the present disclosure provides a cassette comprising a plurality of cassette structures defining a cassette flow path volume from a feeding input (201) to a feeding output (214). The plurality of cassette structures can comprise the feeding input (201), a branched network of primary feeding input channels in a first plane (204a), a branched network of primary feeding input channels in a second plane (204b), which first plane can be substantially parallel to the second plane, a plurality of bioprocessing chambers (207), a branched network of primary feeding output channels (203), a feeding output (214), and a plurality of feeding input structures defining a feeding input flow path volume from the feeding input (201) to the plurality of bioprocessing chambers (207). In some cases, the plurality of feeding input structures can comprise the feeding input (201), the branched network of primary feeding input channels in the first plane (204a), and the branched network of primary feeding input channels in the second plane (204b). In some embodiments, the feeding input flow path volume can be 30%, 20%, or 10% or less of the cassette flow path volume.
In some cases, the plurality of bioprocessing chambers (207) can comprise at least 4, 8, 16, 32, 64, 128, or 256 bioprocessing chambers (207).
In some embodiments, the plurality of cassette structures can further comprise a branched network of primary feeding input channels in a third plane fluidically connected to the branched network of primary feeding input channels in the first plane (204a) and the branched network of primary feeding input channels in the second plane (204b). In some cases, the branched network of primary feeding input channels in the first plane (204a) and the branched network of primary feeding input channels in the second plane (204b) can comprise one or more microfluidic channels. In some cases, the first plane, second plane, and third plane can be substantially parallel.
In another aspect, the present disclosure provides a cassette with an optimal pressure drop. The cassette can comprise a feeding input (201) and a branched network of primary feeding input channels (204a); a feeding output (214) and a branched network of primary feeding output channels (203); and a bioprocessing layer comprising a plurality of bioprocessing chambers (207). In some cases, the cassette permits a fluid to flow from the feeding input (201), through the branched network of primary feeding input channels (204a), through the plurality of bioprocessing chambers (207), through the branched network of primary feeding output channels (203), and to the feeding output. In some cases, the plurality of bioprocessing chambers (207) comprises at least 32, 64, 128, or 256 bioprocessing chambers (207).
In some cases, the cassette provides a [ΔPcassette/ΔPbioprocessing chamber]/n of less than 0.3, wherein n is greater than 20. In some cases, the cassette provides a ΔPcassette/ΔPbioprocessing chamber with a value equal or lower than the result of (1/22)*n+6, wherein n is the number of bioprocessing chambers. In some cases, n is at least 16, 32, 64, 128, or 256. In some cases, the cassette may provide a ΔPcassette/ΔPbioprocessing chamber with a value equal or lower than the result of (1/a)*n+b, where n is the number of bioprocessing chambers.
In another aspect, the present disclosure provides a system comprising any one or more of the cassettes described herein and a pump fluidically connected to the one or more cassettes. In some cases, the system can further comprise a reagent vessel fluidically connected to the pump and the cassette. In some cases, the system can further comprise an air vessel comprising CO2 fluidically connected to the reagent vessel. In some cases, a concentration of CO2 in the air vessel can be about 5% or 8% in volume.
In some cases, the system can further comprise a de-bubbler fluidically connected to the reagent vessel and/or to the cassette. In some cases, the reagent vessel can be separated from the de-bubbler by a first valve and the de-bubbler can be separated from the cassette by a second valve.
In some cases, the system can further comprise an output vessel fluidically connected to the one or more cassettes. In some cases, the system can further comprise a flow-through sensor connected to the one or more cassettes. In some cases, the system can further comprise a computer electrically connected to the flow-through sensor. In some cases, the system can further comprise an imaging device. In some cases, the system can further comprise an agitator coupled to the one or more cassettes. In any of the embodiments described herein, the system can be automated (e.g., using on one or more computer processors). In some non-limiting embodiments, the system can be operated manually (e.g., either locally or remotely).
In another aspect, the present disclosure provides a method for bioprocessing. The method can comprise providing any one or more of the cassettes described herein. The method can further comprise flowing fluid from the branched network of primary feeding input channels in the first plane (204a), through the branched network of primary feeding input channels in the second plane (204b) and through the plurality of bioprocessing chambers (207). The fluid can comprise a plurality of solid particles. In some cases, a pressure at a fluid input of the cassette can be 3 bar or less, 2 bar or less, 1 bar or less, 0.75 bar or less, 0.5 bar or less, 0.25 bar or less, or 0.1 bar or less.
In some cases, each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) can comprise a fluid inlet. A flow rate of the fluid at the inlet of each bioprocessing chamber of the plurality of bioprocessing chambers (207) can range from about 10 μL/hr×a number of the plurality of bioprocessing chambers (207) to about 10 mL/min×a number of the plurality of bioprocessing chambers (207).
In some cases, a flow rate of the fluid comprising the plurality of solid particles through the branched network of primary feeding input channels in the first plane (204a) and the branched network of primary feeding input channels in the second plane (204b) can be greater than a settling or sedimentation velocity of the plurality of solid particles.
In some embodiments, the solid particles can comprise cells. In some cases, the solid particles can comprise at least 20,000, 200,000, 350,000, 500,000, 1,000,000, 3,500,000, 10,000,00, 25,000,000, or 50,000,000 cells/mL. In any of the embodiments described herein, the cells can comprise microorganisms, mammalian cells, HEK293 cells, T-cells, Jurkat cells, CHO cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells.
In some embodiments, the method can further comprise distributing a substantially equal amount of the cells to each bioprocessing chamber (207) of the plurality of bioprocessing chambers (207) to provide distributed cells. In some cases, the distributing can occur within 5 minutes.
In some embodiments, the method can further comprise washing the distributed cells. In some embodiments, the method can further comprise expanding the distributed cells to generate expanded cells. In some cases, the expanding comprises expanding the distributed cells about, or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold. In some cases, the expanding occurs over about, or at least 24 hr, 48 h, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hr, or 1200 hr.
In some embodiments, the method can further comprise imaging the distributed cells. In some embodiments, the method can further comprise imaging the expanded cells. In some embodiments, the method can further comprise using a computer system to predict time to confluence of the expanded cells.
In some embodiments, the method can further comprise contacting the distributed cells with a reagent after the washing. In some embodiments, the method can further comprise, after the contacting, performing a second wash of the distributed cells. In some embodiments, the method can further comprise, after the second wash of the distributed cells, harvesting the distributed cells.
The reagents can comprise, for example, balanced salt solutions, buffers, detergents, chelators, or any materials or substances that promote or facilitate cell adhesion.
In some cases, the cells can be seeded at a flow rate ranging from 0.1 microliters per second (μL/s) to 10 μL/s or more. In some cases, the cells can be seeded at a flow rate of at least about 0.17 μL/s.
In some cases, the cells can be harvested at a flow rate ranging from about 0.1 microliters per second (μL/s) to about 10 μL/s or more. In some cases, the cells can be harvest at a flow rate ranging from about 0.17 μL/s to about 1.59 μL/s.
In another aspect, the present disclosure provides a method for culturing one or more cells. The method may comprise distributing the one or more cells in one or more bioprocessing chambers. In some embodiments, one or more bottom surfaces of the bioprocessing chambers may be used for cell culturing. The one or more bioprocessing chambers may be associated with one or more cassettes and/or one or more chips within a parallelized array of chips. In some cases, the one or more cells may be cultured using a microfluidic system comprising a plurality of chips arranged in a parallelized configuration. In some cases, the microfluidic system may comprise a plurality of components that are disposed on different layers or planes to enhance fluid flow characteristics through the microfluidic system, perfusion of growth media through the microfluidic system, cell seeding efficiency, and cell harvesting efficiency.
In some embodiments, the method may comprise washing the one or more cells. In some cases, the one or more cells may be washed a plurality of times. In some cases, the one or more cells may be washed using a buffer (e.g., a PBS buffer). The buffer may be provided via one or more perfusion inlets or feeding inputs of the microfluidic system.
In some embodiments, the method may comprise expanding the one or more cells. In some cases, the cells may undergo an expansion that is about, or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold. In some cases, the expansion may occur within a time period of at least about 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hr, or 1200 hr.
In some embodiments, the method may comprise contacting the one or more cells with at least one reagent. The at least one reagent may comprise, for example, a balanced salt solution, a buffer, a detergent, a chelator, or any materials or substances that promotes or facilitates cell adhesion. In some cases, the reagent may be provided to the one or more cells after the cells have been washed.
In some embodiments, the method may comprise imaging the one or more cells. In some cases, the one or more cells may be imaged using an imaging sensor or a camera. The imaging sensor or camera may be positioned and/or oriented such that an optical/imaging axis of the imaging sensor or camera is aligned with a portion of the microfluidic system comprising the one or more cells. In some cases, the portion of the microfluidic system may be transparent or semi-transparent to facilitate the imaging of the one or more cells.
In some embodiments, the method may comprise harvesting the one or more cells. In some cases, the cells may be harvested via one or more drains. In some cases, the one or more drains may be located on a bottom surface of the plurality of bioprocessing chambers of the microfluidic system.
In another aspect, the present disclosure provides a microfluidic system. The microfluidic system may be configured for cell culturing. The microfluidic system may comprise one or more cassettes or one or more chips arranged in a parallelized configuration as described elsewhere herein.
In some embodiments, the microfluidic system may be configured for flowing a fluid through a fluid input of the microfluidic system at a target pressure. In some cases, the target pressure may be less than 3 bars. In some cases, the target pressure may be less than 2 bars. In some cases, the target pressure may be less than 1 bar. In some cases, the target pressure may be less than 0.5 bars.
In some embodiments, the microfluidic system may be configured for culturing a plurality of cells. In some cases, the plurality of cells may comprise at least about 50 million cells. In some cases, the plurality of cells may comprise at least about 500 million cells. In some cases, the plurality of cells may comprise at least about 1 billion cells.
In an aspect, the present disclosure provides computer systems that are programmed or otherwise configured to implement methods of the disclosure, e.g., any of the subject methods for bioprocessing.
The computer system 2001 can include a central processing unit (CPU, also “processor” and “computer processor” herein) 2005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 2001 also includes memory or memory location 2010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 2015 (e.g., hard disk), communication interface 2020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2025, such as cache, other memory, data storage and/or electronic display adapters. The memory 2010, storage unit 2015, interface 2020 and peripheral devices 2025 are in communication with the CPU 2005 through a communication bus (solid lines), such as a motherboard. The storage unit 2015 can be a data storage unit (or data repository) for storing data. The computer system 2001 can be operatively coupled to a computer network (“network”) 2030 with the aid of the communication interface 2020. The network 2030 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 2030 in some cases is a telecommunication and/or data network. The network 2030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 2030, in some cases with the aid of the computer system 2001, can implement a peer-to-peer network, which can enable devices coupled to the computer system 2001 to behave as a client or a server.
The CPU 2005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as the memory 2010. The instructions can be directed to the CPU 2005, which can subsequently program or otherwise configure the CPU 2005 to implement methods of the present disclosure. Examples of operations performed by the CPU 2005 can include fetch, decode, execute, and writeback.
The CPU 2005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 2001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
The storage unit 2015 can store files, such as drivers, libraries and saved programs. The storage unit 2015 can store user data, e.g., user preferences and user programs. The computer system 2001 in some cases can include one or more additional data storage units that are located external to the computer system 2001 (e.g., on a remote server that is in communication with the computer system 2001 through an intranet or the Internet).
The computer system 2001 can communicate with one or more remote computer systems through the network 2030. For instance, the computer system 2001 can communicate with a remote computer system of a user (e.g., an operator managing or monitoring the bioprocessing). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iphone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 2001 via the network 2030.
Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 2001, such as, for example, on the memory 2010 or electronic storage unit 2015. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 2005. In some cases, the code can be retrieved from the storage unit 2015 and stored on the memory 2010 for ready access by the processor 2005. In some situations, the electronic storage unit 2015 can be precluded, and machine-executable instructions are stored on memory 2010.
The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
Aspects of the systems and methods provided herein, such as the computer system 2001, can be embodied in programming. Various aspects of the technology can be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which can provide non-transitory storage at any time for the software programming. All or portions of the software can at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, can enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that can bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also can be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.
Hence, a machine readable medium, such as computer-executable code, can take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media including, for example, optical or magnetic disks, or any storage devices in any computer(s) or the like, can be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media can be involved in carrying one or more sequences of one or more instructions to a processor for execution.
The computer system 2001 can include or be in communication with an electronic display 2035 that comprises a user interface (UI) 2040 for providing, for example, a portal for an operator to monitor or track one or more steps or operations of the bioprocessing methods and systems described herein. The portal can be provided through an application programming interface (API). A user or entity can also interact with various elements in the portal via the UI. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 2005. For example, the algorithm can be configured to adjust a flow rate or an amount of fluid flow into or through the one or more chips or cassettes, based on one or more sensor readings. In some embodiments, the algorithm can be further configured to adjust the flow rate or an amount of fluid flow into or through the one or more chips or cassettes in order to optimize (i.e., decrease) an amount of pressure drop across the system.
A sample of T-cells is taken from a cancer patient. The T-cells are modified by transduction in a chip to produce chimeric antigen receptors that target the patient's cancer. The cassette of
Perfusion is performed to introduce media; the perfusion does not disturb the seeded modified T-cells because they are in the recess and on the bottom surface (1001) (see
This application is a continuation of International Patent Application No. PCT/GB2022/053051, filed Dec. 1, 2022, which claims priority to 63/285,066, filed Dec. 1, 2021, U.S. Provisional Patent Application No. 63/285,062, filed on Dec. 1, 2021, and U.S. Provisional Patent Application No. 63/409,983, filed on Sep. 26, 2022, all of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63285066 | Dec 2021 | US | |
| 63285062 | Dec 2021 | US | |
| 63409983 | Sep 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/GB2022/053051 | Dec 2022 | WO |
| Child | 18679196 | US |
