Bioreactors and methods for producing the same

Abstract

The present disclosure provides bioreactors and methods of producing the same. Bioreactors of the present disclosure may include a macrostructure comprising a plurality of fluidically connected minimodules that form a plurality of channels. A channel may comprise a gas permeable layer configured to provide gas diffusion to or from the channel. The macrostructure may be manufactured using three-dimensional printing of a resin. The resin may comprise a polymerizable component, photoinitiator, thermocuring agent, or any combination thereof. One or more surfaces of the microfluidic macrostructure may comprise a biocompatible coating.

Description

WSGR Ref. No.55076-707.601 BIOREACTORS AND METHODS FOR PRODUCING THE SAME CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 654,853, filed May 31, 2024, which is entirely incorporated herein by reference. BACKGROUND

[0002] Production of biological products including cells, proteins, and small and large chemical molecules has become increasingly important in manufacturing medical, food, industrial and other types of products. Consistency of the product and the ability to scale production as well as the flexibility to tailor manufacturing to different locations and for environmental conditions may be important factors for production of such products. SUMMARY

[0003] The present disclosure may provide bespoke bioreactors and systems and methods for producing the same. Bespoke bioreactors may be manufactured via three-dimensional (3D) printing. 3D printed bioreactors may permit design and manufacture of bioreactor systems with improved resolution, scalability, cost, efficiency, and consistency.

[0004] In an aspect, the present disclosure provides a bioreactor comprising: a macrostructure comprising a plurality of fluidically connected minimodules, wherein the plurality of fluidically connected minimodules form a plurality of channels, wherein a channel of the plurality of channels comprises channel wall disposed between an inner surface and an outer surface, and wherein the channel wall is configured to be: (i) permeable to gas diffusion to permit gas to flow between the inner surface and the outer surface and (ii) liquid impermeable to prevent or substantially prevent the flow of liquid between the inner surface and the outer surface.

[0005] In some embodiments, the bioreactor further comprises an inlet fluidically connected to at least one channel of the plurality if channels of the macrostructure. In some embodiments, the bioreactor further comprises an outlet fluidically connected to at least one channel of the plurality of channels of the macrostructure. In some embodiments, the channel wall has a gas permeability that is at least about 1000 barrers. In some embodiments, a composition making up the inner surface and the outer surface of the channel exhibits no more than about 30% swelling when exposed to water for about 150 hours. In some embodiments, the channel is configured to provide laminar flow of a fluid through the channel. In some embodiments, the channel is configured to provide transitional flow of a fluid through the channel. In some embodiments, theWSGR Ref. No.55076-707.601 channel is configured to provide turbulent flow of a fluid through the channel. In some embodiments, the inner surface of the channel comprises a three-dimensional (3D) structure configured to modify a fluid flow profile of a fluid flowing through the channel as compared to a channel without the 3D structure. In some embodiments, the 3D structure comprises protrusions, grooves, or a combination thereof. In some embodiments, the 3D structure is configured to generate chaotic advection in a fluid flowing through the channel. In some embodiments, the channel comprises a biocompatible material. In some embodiments, the biocompatible material is a sterilizable or autoclavable material.

[0006] In some embodiments, a diameter of the channel is from about 1 micrometer (µm) to about 10 millimeters (mm). In some embodiments, a diameter of the channel is from about 1 µm to about 50 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 100 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 500 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 1000 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 2000 µm. In some embodiments, a diameter of the channel is from about 2000 µm to about 5000 µm. In some embodiments, a diameter of the channel is from about 5000 µm to about 10 mm. In some embodiments, a diameter of the channel is at least about 100 µm.

[0007] In some embodiments, the channel wall is from about 20 µm to about 500 µm thick. In some embodiments, the channel wall is from about 500 µm to about 1000 µm thick. In some embodiments, the channel wall is at least about 10 µm thick. In some embodiments, a thickness of the channel wall varies along a length of the channel. In some embodiments, the channel comprises a first end, a center portion, and a second end and wherein the channel wall is thicker near the first end or the second end than the center portion. In some embodiments, the channel comprises a first end, a center portion, and a second end and wherein the channel wall is thicker near the center portion than the first end or the second end. In some embodiments, the channel wall is at least about 50 µm thick. In some embodiments, the channel wall is at least about 100 µm thick.

[0008] In some embodiments, the plurality of fluidically connected minimodules are disposed in at least one fluidically connected layer. In some embodiments, a layer of the at least on fluidically connected layer comprises at least one minimodule. In some embodiments, the at least one fluidically connected layer forms a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, aWSGR Ref. No.55076-707.601 toroid with a central core, or any combination thereof. In some embodiments, the macrostructure comprises a first layer comprising a first shape and a second layer comprising a second shape. In some embodiments, the first shape and the second shape are different shapes.

[0009] In some embodiments, the plurality of fluidically connected minimodules are connected at connection points. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 50 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 100 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 500 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 3000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 5000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 10 mm. In some embodiments, a diameter of a connection point of the connection points is at least about 50 µm. In some embodiments, a diameter of a connection point of the connection points is at least about 500 µm. In some embodiments, a connection point of the connection points has a modified internal diameter as compared to another connection point of the connection points. In some embodiments, an internal diameter of a connection point of the connection points in a first layer of fluidically connected minimodules is less than another internal diameter of another connection point of the connection points in a second layer of fluidically connected minimodules. In some embodiments, the modified internal diameter of the connection point is reduced compared to other connection points in the same layer. In some embodiments, the modified internal diameter of the channel is reduced compared to channels near to one or more edges of the macrostructure. In some embodiments, the modified internal diameter of the channel is reduced compared to channels adjacent to a core of the macrostructure.

[0010] In some embodiments, an internal volume of the macrostructure is from about 10 milliliters (mL) to about 50 mL. In some embodiments, an internal volume of the macrostructure is from about 10 mL to about 100 mL. In some embodiments, an internal volume of the macrostructure is from about 100 mL to about 500 mL. In some embodiments, an internal volume of the macrostructure is from about 500 mL to about 1 liter (L). In some embodiments, an internal volume of the macrostructure is from about 1 L to about 5 L. In some embodiments, an internal volume of the macrostructure is from about 5 L to about 10 L. In some embodiments, an internal volume of the macrostructure is at least about 10 mL. In some embodiments, an internal volume of the macrostructure is at least about 100 mL. In some embodiments, an internal volume of the macrostructure is at least about 1 L. In some embodiments, an internal volume of the macrostructure is at least about 5 L. In some embodiments, an internal volume ofWSGR Ref. No.55076-707.601 the macrostructure is at least about 10 L.

[0011] In some embodiments, a minimodule of the plurality of fluidically connected minimodules comprises a triply periodic minimal surface. In some embodiments, the triply periodic minimal surface comprises a gyroid structure, and wherein the gyroid structure comprises a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, of any combination thereof. In some embodiments, the minimodule comprises a single gyroid structure. In some embodiments, the macrostructure comprises layers of the one or more fluidically connected minimodules, and wherein a layer of the layers comprises from 1 to 105triply periodic minimal surfaces in a first axis and from 1 to 105triply periodic minimal surfaces in a second axis perpendicular or substantially perpendicular to the first axis.

[0012] In some embodiments, the macrostructure comprises at least one layer. In some embodiments, the macrostructure is generated using three-dimensional (3D) printing. In some embodiments, the macrostructure comprises biocompatible material, and wherein the biocompatible material comprises a three dimensional (3D) printable bioink. In some embodiments, the 3D printable bioink comprises a polymerizable component, a photoinitiatior, and a thermocuring agent. In some embodiments, the polymerizable component comprises Polyethylene Glycol Diacrylate 250 (PEGDA MW 250). In some embodiments, the PEGDA MW 250 is from about 70% to about 98% of the bioink (wt / wt). In some embodiments, the photoinitiator component comprises 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO). In some embodiments, the 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) is from about 2% to about 30 % wt / wt. In some embodiments, the thermocuring component comprises 2,2′-Azobis(2-methylpropionitrile) (AIBN). In some embodiments, the 2,2′-Azobis(2-methylpropionitrile) (AIBN) is from about 0.01% to about 10 % wt / wt. In some embodiments, the 3D printable bioink further comprises a porogen. In some embodiments, the porogen component comprises polyethylene glycol 200 (PEG-200). In some embodiments, the polyethylene glycol 200 (PEG-200) is from about 30% to about 70 % wt / wt. In some embodiments, the porogen component comprises polyethylene glycol 400 (PEG-400). In some embodiments, polyethylene glycol 400 (PEG-400) is from about 30% to about 70 % wt / wt. In some embodiments, the 3D printable bioink further comprises a light blocker. In some embodiments, the light blocker comprises Avobenzone (Avo). In some embodiments, the Avobenzone (Avo) is from about 0.001% to about 1% wt / wt. In some embodiments, the 3D printable bioink further comprises a plasticizer. In some embodiments, the plasticizer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol (n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt. In some embodiments, the porogen is about 5% wt / wt Triton X at a concentration of greater than about 10% and less thanWSGR Ref. No.55076-707.601 about 50% wt / wt. In some embodiments, the 3D printable bioink further comprises a filler. In some embodiments, the filler comprises hydroxyapatite. In some embodiments, the hydroxyapatite is from about 1% to about 10% wt / wt. In some embodiments, the filler is about 5% wt / wt hydroxyapatite. In some embodiments, the hydroxyapatite comprises modified hydroxyapatite. In some embodiments, the modification of the hydroxyapatite comprises functionalization with carbon (HApC12)-dodecanol. In some embodiments, the 3D printable bioink further comprises a rheological enhancer. In some embodiments, the rheological enhancer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol (n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt. In some embodiments, the rheological enhancer comprises SILICA. In some embodiments, the SILICA is from about15% to about 30%. In some embodiments, rheological enhancer comprises modified SILICA. In some embodiments, the modified SILICA comprises KH550 or (3- aminopropil) trietoxisilano.

[0013] In some embodiments, the 3D printable bioink further comprises a biocompatibility enhancer. In some embodiments, the biocompatible enhancer is applied in layers to at least one surface of the polymerized bioink. In some embodiments, the biocompatible enhancer comprises between 2 and 12 layers. In some embodiments, the biocompatible enhancer comprises at least 6 layers. In some embodiments, the biocompatible enhancer comprises polydimethylsiloxane (PDMS). In some embodiments, the 3D printable bioink comprises a metal. In some embodiments, the metal comprises steel, a nickel-based superalloy, aluminum, low-carbon austenitic stainless steel, or any combination thereof. In some embodiments, the 3D printing is selected from the group consisting of metal powder bed fusion (PBF), direct metal laser sintering (DMLS), selective laser melting (SLM), electron beam melting (EBM), directed energy deposition (DED), binder jetting of metallic or ceramic powders, cold spray additive manufacturing, ultraviolet vat photopolymerization, stereolithography (SLA), digital light processing (DLP), fused filament fabrication (FFF), fused deposition modeling (FDM), multi-jet printing (MJP), PolyJet printing, two-photon polymerization (2PP), electrohydrodynamic printing (EHD), inkjet bioprinting, laser-assisted bioprinting, robocasting, and material extrusion of ceramic pastes or slurries.

[0014] In some embodiments, the bioreactor comprises at least one surface compatible with establishment, maintenance, growth, differentiation, adhesion, migration, proliferation, metabolic activity, extracellular matrix deposition, signal transduction, morphogenesis, polarization, secretion of biomolecules, or viability of living cells.

[0015] In another aspect, the present disclosure provides a bioprocessor comprising a bioreactor as described elsewhere herein.WSGR Ref. No.55076-707.601

[0016] In some embodiments, the bioprocessor further comprises a housing, wherein the bioreactor is enclosed in the housing. In some embodiments, the bioprocessor further comprises a bioproduction cartridge, wherein the bioproduction cartridge comprises the bioreactor. In some embodiments, the bioproduction cartridge comprises an environmental control system. In some embodiments, the environmental control system is configured to control at least one sensor. In some embodiments, the bioprocessor further comprises at least one consumables reservoir. In some embodiments, the bioprocessor further comprises a formulator. In some embodiments, the formulator is configured to generate tailored culture media formulations. In some embodiments, the bioprocessor further comprises at least one inoculation module. In some embodiments, the bioprocessor further comprises at least one production module. In some embodiments, the bioprocessor further comprises at least one harvesting module. In some embodiments, the at least one harvesting module comprises a reservoir configured to retain a product produced by the bioreactor. In some embodiments, the harvesting module further comprises a ultrasonic harvesting system. In some embodiments, the bioprocessor further comprises a counter suspension module. In some embodiments, the counter suspension module comprises a rocking unit. In some embodiments, the counter suspension module comprises an ultrasonic unit. In some embodiments, the counter suspension module comprises a vibratory unit. In some embodiments, the counter suspension module comprises a density modified unit. In some embodiments, the counter suspension module comprises a push and pull unit.

[0017] In some embodiments, the bioprocessor further comprises at least one sensor, and wherein the at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter. In some embodiments, the biological parameter is selected from the group consisting of cell division rate, cell growth rate, a cell stress response, cell protein content, cell carbohydrate content, cell lipid content, cell viability, cell count, and cell nucleic acid content. In some embodiments, the physical parameter is selected from the group consisting of cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity, temperature, and pressure. In some embodiments, the chemical parameter is selected from the group consisting of pH, liquid media composition, concentration of individual liquid media component, gas composition, gas concentration, and dissolved gas concentration. In some embodiments, the bioprocessor further comprises at least one temperature sensor, one pH sensor, one glucose sensor, one cell production sensor, one metabolite production sensor, one ultrasound sensor, one pressure sensor, one infrared sensor, one current sensor, one gas sensor, one light sensor, one position sensor, one sound sensor, one color sensor, one hyper spectral sensor, one level sensor, one moisture sensor; and / or any combination thereof. In some embodiments, the bioprocessor further comprises at least one system to control, nutrient concentration, temperature, pH,WSGR Ref. No.55076-707.601 ultrasound, pressure, current, gas flow, gas concentration, light, position, moisture, or any combination thereof.

[0018] In another aspect, the present disclosure provides a three-dimensional (3D) printable bioink, comprising: a polymerizable component; a photoinitiatior; and a thermocuring agent, wherein the 3D printable bioink is biocompatible.

[0019] In some embodiments, the polymerizable component comprises Polyethylene Glycol Diacrylate 250 (PEGDA MW 250). In some embodiments, the PEGDA MW 250 is from about 70% to about 98% of the bioink (wt / wt). In some embodiments, the photoinitiator component comprises 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO). In some embodiments, the 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) is from about 2% to about 30 % wt / wt. In some embodiments, the thermocuring component comprises 2,2′- Azobis(2-methylpropionitrile) (AIBN). In some embodiments, the 2,2′-Azobis(2- methylpropionitrile) (AIBN) is from about 0.01% to about 10 % wt / wt.

[0020] In some embodiments, the 3D printable bioink further comprises a porogen. In some embodiments, the porogen component comprises polyethylene glycol 200 (PEG-200). In some embodiments, the polyethylene glycol 200 (PEG-200) is from about 30% to about 70 % wt / wt. In some embodiments, the porogen component comprises polyethylene glycol 400 (PEG-400). In some embodiments, polyethylene glycol 400 (PEG-400) is from about 30% to about 70 % wt / wt. In some embodiments, the 3D printable bioink further comprises a light blocker. In some embodiments, the light blocker comprises Avobenzone (Avo). In some embodiments, the Avobenzone (Avo) is from about 0.001% to about 1% wt / wt. In some embodiments, the 3D printable bioink further comprises a plasticizer. In some embodiments, the plasticizer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol (n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt. In some embodiments, the porogen is about 5% wt / wt Triton X at a concentration of from about 10% to about 50% wt / wt. In some embodiments, the 3D printable bioink further comprises a filler. In some embodiments, the filler comprises hydroxyapatite. In some embodiments, the hydroxyapatite is from about 1% to about 10% wt / wt. In some embodiments, the filler is about 5% wt / wt hydroxyapatite. In some embodiments, the hydroxyapatite comprises modified hydroxyapatite. In some embodiments, the modification of the hydroxyapatite comprises functionalization with carbon (HApC12)- dodecanol-.

[0021] In some embodiments, the 3D printable bioink further comprises a rheological enhancer. In some embodiments, the rheological enhancer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol(n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt. In some embodiments, the rheological enhancerWSGR Ref. No.55076-707.601 comprises SILICA. In some embodiments, the SILICA is from about 15% to about 30%. In some embodiments, the rheological enhancer comprises modified SILICA. In some embodiments, the modified SILICA comprises KH550 or (3-aminopropil) trietoxisilano).

[0022] In some embodiments, the 3D printable bioink further comprises a biocompatibility enhancer. In some embodiments, the biocompatibility enhancer is applied to a surface of a polymerized composition generated from the 3D printable bioink in layers. In some embodiments, the biocompatibility enhancer comprises from 2 to 12 layers. In some embodiments, the biocompatibility enhancer comprises at least 6 layers.

[0023] In some embodiments, the biocompatibility enhancer comprises polydimethylsiloxane (PDMS). In some embodiments, the 3D printable bioink comprises a metal. In some embodiments, the metal comprises steel, a nickel-based superalloy, aluminum, low-carbon austenitic stainless steel, or any combination thereof. In some embodiments, the 3D printing is selected from the group consisting of metal powder bed fusion (PBF), direct metal laser sintering (DMLS), selective laser melting (SLM), electron beam melting (EBM), directed energy deposition (DED), binder jetting of metallic or ceramic powders, cold spray additive manufacturing, ultraviolet vat photopolymerization, stereolithography (SLA), digital light processing (DLP), fused filament fabrication (FFF), fused deposition modeling (FDM), multi-jet printing (MJP), PolyJet printing, two-photon polymerization (2PP), electrohydrodynamic printing (EHD), inkjet bioprinting, laser-assisted bioprinting, robocasting, and material extrusion of ceramic pastes or slurries. In some embodiments, a polymerized component generated from the 3D printable bioink comprises at least one surface compatible with establishment, maintenance, growth, differentiation, adhesion, migration, proliferation, metabolic activity, extracellular matrix deposition, signal transduction, morphogenesis, polarization, secretion of biomolecules, and viability of living cells.

[0024] In another aspect, the present disclosure provides a bioreactor comprising: an input configured to receive a fluid; an output configured to output the fluid; and a macrostructure fluidically connected to the input and the output, wherein the macrostructure comprises a plurality of minimodules fluidically connected to form a plurality of channels, wherein the macrostructure comprises (i) a first equalization region in fluid communication with the input, iii) a second equalization region in fluid communication with the output, and (iii) a core region disposed between the first equalization region and the second equalization region, wherein the first equalization region comprises diverging channels of the plurality of channels and is configured to distribute fluid from the input to the core region, wherein the core region is configured to maintain uniform fluid flow through the core region, and wherein the second equalization region comprises converging channels of the plurality of channels and is configuredWSGR Ref. No.55076-707.601 to provide the fluid from the core region to the output.

[0025] In some embodiments, the macrostructure comprises a plurality of layers of fluidically connected minimodules of the plurality of fluidically connected minimodules. In some embodiments, a layer of the plurality of layers comprises at least one minimodule. In some embodiments, the plurality of layers forms a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, , cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, a toroid with a central core, or any combination thereof.

[0026] In some embodiments, the plurality of fluidically connected minimodules are connected at connection points. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 50 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 100 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 500 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 3000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 5000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 10 mm. In some embodiments, a diameter of a connection point of the connection points is at least about 50 µm. In some embodiments, a diameter of a connection point of the connection points is at least about 500 µm. In some embodiments, a connection point of the connection points has a modified internal diameter as compared to another connection point of the connection points. In some embodiments, an internal diameter of a connection point of the connection points in a first layer of fluidically connected minimodules is less than another internal diameter of another connection point of the connection points in a second layer of fluidically connected minimodules. In some embodiments, the macrostructure comprises a plurality of layers, and wherein internal diameters of the plurality of channels varies across a layer of the plurality of layers.

[0027] In some embodiments, internal diameters of channels adjacent to a center of the macrostructure are on average less than internal diameters of channels adjacent to an edge of the macrostructure. In some embodiments, internal diameters of channels adjacent to a center of the macrostructure are on average greater than internal diameters of channels adjacent to an edge of the macrostructure. In some embodiments, the core region comprises at least one layer of minimodules of the plurality of fluidically connected minimodules.WSGR Ref. No.55076-707.601

[0028] In some embodiments, an internal volume of the macrostructure is from about 10 milliliters (mL) to about 50 mL. In some embodiments, an internal volume of the macrostructure is from about 10 mL to about 100 mL. In some embodiments, an internal volume of the macrostructure is from about 100 mL to about 500 mL. In some embodiments, an internal volume of the macrostructure is from about 500 mL to about 1 liter (L). In some embodiments, an internal volume of the macrostructure is from about 1 L to about 5 L. In some embodiments, an internal volume of the macrostructure is from about 5 L to about 10 L. In some embodiments, an internal volume of the macrostructure is at least about 10 mL. In some embodiments, an internal volume of the macrostructure is at least about 100 mL. In some embodiments, an internal volume of the macrostructure is at least about 1 L. In some embodiments, an internal volume of the macrostructure is at least about 5 L. In some embodiments, an internal volume of the macrostructure is at least about 10 L.

[0029] In some embodiments, a minimodule of the plurality of fluidically connected minimodules comprises a triply periodic minimal surface. In some embodiments, a triply periodic minimal surface comprises a gyroid structure, wherein a gyroid structure comprises a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, or any combination thereof. In some embodiments, the minimodule comprises a single gyroid. In some embodiments, the layers of the core region comprise between 1 and 105triply periodic minimal surfaces in a first axis and between 1 and 105triply periodic minimal surfaces in a second axis perpendicular to or substantially perpendicular to the first axis.

[0030] In another aspect, the present disclosure provides a system for biologic production, comprising: a first module comprising a reservoir configured to contain a consumable material; a second module in fluid communication with the first module, wherein the second module comprises an inoculation device configure to (i) contain and generate a plurality of cells from at least one cell, (ii) direct cells from the plurality of cells to different segments of the inoculation device, wherein cell growth conditions in the different segments are individually configurable, and (iii) iteratively generate a set of growth conditions for the plurality of cells; a third module in fluid communication with the first module and the second module, wherein the third module comprises a bioproduction chamber enclosing a bioreactor, wherein the bioproduction chamber is configured to: (i) receive the consumable material from the reservoir, (ii) receive cell from the inoculation module, (iii) control biologic production conditions within the bioreactor, (iv) rotate, rock, vibrate or otherwise disrupt sedimentation within the bioreactor, and (v) direct a product generated in the bioreactor to a harvesting module; and a fourth module in fluid communication with the third module, wherein the fourth module comprises the harvesting module.

[0031] In some embodiments, the third module comprises a plurality of bioreactorsWSGR Ref. No.55076-707.601 comprising the bioreactor. In some embodiments, the system comprises a plurality of third modules comprising the third module. In some embodiments, the biologic production conditions are individually configurable for each bioreactor. In some embodiments, the first module, second module, third module, and fourth module are fluidically interconnected. In some embodiments, the reservoir comprises a cartridge system comprising the consumable material. In some embodiments, the inoculation module comprises a continuous microbioreactor. In some embodiments, the bioproduction chamber is a sealed chamber. In some embodiments, the harvesting module comprises a cartridge system. In some embodiments, the harvesting module comprises an ultrasonic harvesting system.

[0032] In some embodiments, the system further comprises a counter suspension system, wherein the counter suspension system is configured to rotate, rock, vibrate, or otherwise disrupt sedimentation within the bioreactor. In some embodiments, the counter suspension system comprises a rocking system. In some embodiments, the counter suspension system comprises an ultrasonic system. In some embodiments, the counter suspension system comprises a vibratory system. In some embodiments, the counter suspension system comprises a density modified system. In some embodiments, the counter suspension system comprises a push and pull system.

[0033] In some embodiments, the system further comprises at least one sensor, and wherein the at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter. In some embodiments, the biological parameter is selected from the group consisting of cell division rate, cell growth rate, a cell stress response, cell protein content, cell carbohydrate content, cell lipid content, cell viability, cell count, and cell nucleic acid content. In some embodiments, the physical parameter is selected from the group consisting of cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity, temperature, and pressure. In some embodiments, the chemical parameter is selected from the group consisting of pH, liquid media composition, concentration of individual liquid media component, gas composition, gas concentration, and dissolved gas concentration.

[0034] In some embodiments, the system further comprises at least one temperature sensor, one pH sensor, one glucose sensor, one cell production sensor, one metabolite production sensor, one ultrasound sensor, one pressure sensor, one infrared sensor, one current sensor, one gas sensor, one light sensor, one position sensor, one sound sensor, one color sensor, one hyper spectral sensor, one level sensor, one moisture sensor; and / or any combination thereof. In some embodiments, the system further comprises at least one system to control, nutrient concentration, temperature, pH, ultrasound, pressure, current, gas flow, gas concentration, light, position, moisture and / or any combination thereof. In some embodiments, the system further comprises at least one electronic circuit to control configured to control one or more of the firstWSGR Ref. No.55076-707.601 module, the second module, the third module, and the fourth module.

[0035] In some embodiments, the at least one bioreactor comprises a macrostructure. In some embodiments, the macrostructure comprises a plurality of fluidically connected minimodules, wherein the plurality of fluidically connected minimodules form a plurality of channels. In some embodiments, a channel of the plurality of channels comprises a channel wall disposed between an inner surface and an outer surface, and wherein the channel wall is permeable to gas diffusion to permit gas to flow between the inner surface and the outer surface. In some embodiments, the channel wall is liquid impermeable to prevent or substantially prevent the flow of liquid between the inner surface and the outer surface. In some embodiments, the bioreactor further comprises an inlet fluidically connected to the plurality of channels of the macrostructure. In some embodiments, the bioreactor further comprises an outlet fluidically connected to the plurality of channels of the macrostructure. In some embodiments, the channel wall has a gas permeability that is at least 1000 barrers. In some embodiments, the channel comprises a material with a composition that exhibits no more than 30% swelling when exposed to water for 150 hours.

[0036] In some embodiments, the channel is configured to provide laminar flow of a liquid through the channel. In some embodiments, the channel is configured to provide turbulent flow of a fluid through the channel. In some embodiments, the inner surface of the channel comprises a three-dimensional (3D) structure configured to modify a fluid flow profile of a fluid flowing through the channel as compared to a channel without the 3D structure. In some embodiments, the 3D structure comprises protrusions, grooves, or a combination thereof. In some embodiments, the 3D structure is configured to generate chaotic advection in a fluid flowing through the channel. In some embodiments, the channel comprises a biocompatible material.

[0037] In some embodiments, a diameter of the channel is from about 1 µm to about 50 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 100 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 500 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 1000 µm. In some embodiments, a diameter of the channel is from about 1 µm to about 2000 µm. In some embodiments, a diameter of the channel is from about 2000 µm to about 5000 µm. In some embodiments, a diameter of the channel is from about 5000 µm to about 10 mm. In some embodiments, a diameter of the channel is at least about 100 µm. In some embodiments, the channel wall is from about 20 µm to about 500 µm thick. In some embodiments, the channel wall is from about 500 µm to about 1000 µm thick. In some embodiments, the channel wall is at least about 10 µm thick.

[0038] In some embodiments, the plurality of fluidically connected minimodules is disposedWSGR Ref. No.55076-707.601 in fluidically connected layers. In some embodiments, a layer of the fluidically connected layers comprises at least one minimodule. In some embodiments, the fluidically connected layers forms a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, a toroid with a central core, or any combinations thereof.

[0039] In some embodiments, the plurality of fluidically connected minimodules are connected at connection points. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 50 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 100 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 500 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 3000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 5000 µm. In some embodiments, a diameter of a connection point of the connection points is from about 1 µm to about 10 mm. In some embodiments, a diameter of a connection point of the connection points is at least about 50 µm. In some embodiments, a diameter of a connection point of the connection points is at least about 500 µm. In some embodiments, a connection point of the connection points has a modified internal diameter as compared to another connection point of the connection points. In some embodiments, an internal diameter of a connection point of the connection points in a first layer of fluidically connected minimodules is less than another internal diameter of another connection point of the connection points in a second layer of fluidically connected minimodules. In some embodiments, the modified internal diameter of the connection point is reduced compared to other connection points in the same layer. In some embodiments, the modified internal diameter of the channel is reduced compared to channels near to one or more edges of the macrostructure. In some embodiments, the modified internal diameter of the channel is reduced compared to channels adjacent to a core of the macrostructure.

[0040] In some embodiments, an internal volume of the macrostructure is from about 10 milliliters (mL) to about 50 mL. In some embodiments, an internal volume of the macrostructure is from about 10 mL to about 100 mL. In some embodiments, an internal volume of the macrostructure is from about 100 mL to about 500 mL. In some embodiments, an internal volume of the macrostructure is from about 500 mL to about 1 liter (L). In some embodiments, an internal volume of the macrostructure is from about 1 L to about 5 L. In some embodiments,WSGR Ref. No.55076-707.601 an internal volume of the macrostructure is from about 5 L to about 10 L. In some embodiments, the internal volume of the macrostructure is between 10 mL and 5 L.

[0041] In some embodiments, a minimodule of the fluidically connected minimodules comprises a triply periodic minimal surface. In some embodiments, a triply periodic minimal surface comprises a gyroid structure, wherein a gyroid structure comprises a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, or any combinations thereof. In some embodiments, the minimodule comprises a single gyroid. In some embodiments, the bioreactor comprises a core region, and wherein the core region comprises at least one layer. In some embodiments, the layers of the core region comprise between 1 and 105triply periodic minimal surface in a first axis and between 1 and 105triply periodic minimal surface in a second axis perpendicular or substantially perpendicular to the first axis.

[0042] In some embodiments, the bioreactor comprises a biocompatible material. In some embodiments, the biocompatible material comprises a 3D printable bioink. In some embodiments, the 3D printable bioink comprises at least one polymerizable component, one photoinitiatior and one thermocuring agent. In some embodiments, the polymerizable component comprises Polyethylene Glycol Diacrylate 250 (PEGDA MW 250). In some embodiments, the PEGDA MW 250 is from about 70% to about 98% of the bioink (wt / wt). In some embodiments, the photoinitiator component comprises 2,2phenylbis(2,4,6- trimethylbenzoyl) phosphine oxide (BAPO). In some embodiments, the 2,2phenylbis(2,4,6- trimethylbenzoyl) phosphine oxide (BAPO) is from about 2% to about 30 % wt / wt. In some embodiments, the thermocuring component comprises 2,2′-Azobis(2-methylpropionitrile) (AIBN). In some embodiments, the 2,2′-Azobis(2-methylpropionitrile) (AIBN) is from about 0.01% to about 10 % wt / wt.

[0043] In some embodiments, the 3D printable bioink further comprises a porogen. In some embodiments, the porogen component comprises polyethylene glycol 200 (PEG-200). In some embodiments, the polyethylene glycol 200 (PEG-200) is between about 30% and about 70 % wt / wt. In some embodiments, the porogen component comprises polyethylene glycol 400 (PEG- 400). In some embodiments, the polyethylene glycol 400 (PEG-400) is between about 30% and about 70 % wt / wt. In some embodiments, the 3D printable bioink further comprises a light blocker. In some embodiments, the light blocker comprises Avobenzone (Avo). In some embodiments, the Avobenzone (Avo) is between about 0.001% and about 1% wt / wt. In some embodiments, the 3D printable bioink further comprises a plasticizer. In some embodiments, the plasticizer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol (n-decanol, decan-1-ol, capric alcohol) is between about 15% and about 30% wt / wt. In some embodiments, the porogen is Triton X. In some embodiments, wherein the Triton X isWSGR Ref. No.55076-707.601 at a concentration of greater than 10% and less than 50% wt / wt. In some embodiments, the Triton X is about 5% wt / wt. In some embodiments, the 3D printable bioink further comprises a filler. In some embodiments, the filler comprises hydroxyapatite. In some embodiments, the hydroxyapatite is between about 1% and about 10% wt / wt. In some embodiments, the filler is about 5% wt / wt hydroxyapatite. In some embodiments, the hydroxyapatite comprises modified hydroxyapatite. In some embodiments, the modification of the hydroxyapatite comprises functionalization with carbon (HApC12)-dodecanol-.

[0044] In some embodiments, the 3D printable bioink further comprises a rheological enhancer. In some embodiments, the rheological enhancer comprises Decanol (n-decanol, decan-1-ol, capric alcohol). In some embodiments, the Decanol (n-decanol, decan-1-ol, capric alcohol) is between about 15% and about 30% wt / wt. In some embodiments, the rheological enhancer comprises SILICA. In some embodiments, the SILICA is between about15% and about 30%. In some embodiments, the rheological enhancer comprises modified SILICA. In some embodiments, the modified SILICA comprises KH550 or (3-aminopropyl) triethoxysilane. In some embodiments, the 3D printable bioink further comprises a biocompatibility enhancer. In some embodiments, the biocompatible enhancer is applied in layers to at least one surface of the polymerized bioink. In some embodiments, the biocompatible enhancer comprises between 2 and 12 layers. In some embodiments, the biocompatible enhancer comprises at least 6 layers. In some embodiments, the biocompatible enhancer comprises polydimethylsiloxane (PDMS). In some embodiments, the bioink comprises at least one surface compatible with growth or maintenance of living cells.

[0045] 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. INCORPORATION BY REFERENCE

[0046] 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 supersedeWSGR Ref. No.55076-707.601 and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] 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:

[0048] FIG.1 schematically illustrates an example polymerization reaction;

[0049] FIG.2 shows an example coated three dimensional (3D) printed well-type material;

[0050] FIG.3 schematically illustrates an example coating process;

[0051] FIG.4 shows example permeability of an example resin;

[0052] FIG.5 shows example results of biocompatibility assay for an example resin;

[0053] FIGs.6A and 6B show example results of another biocompatibility assay for an example resin;

[0054] FIG.7 shows example biocompatibility assay results for another example resin;

[0055] FIG.8A shows example microfluidic bioreactors produces using an example resin; FIG.8B shows scale of example microfluidic macrostructure devices;

[0056] FIG.9 schematically illustrates an example microfluidic macrostructure device;

[0057] FIG.10A shows an example andromeda shaped microfluidic macrostructure device; FIG.10B shows an example trunk shaped microfluidic macrostructure device;

[0058] FIG.11A shows another example andromeda shaped microfluidic macrostructure device; FIG.11B shows an example spherical microfluidic macrostructure device;

[0059] FIG.12 shows an example system for coating a microfluidic macrostructure device;

[0060] FIG.13 shows an example configuration for removing excess coating material from a microfluidic macrostructure device;

[0061] FIG.14 shows an example configuration for providing fluid to multiple inlets;

[0062] FIG.15 schematically illustrates another coating process;

[0063] FIG.16A schematically illustrates a system for producing biological, biochemical, or chemical products; FIG.16B schematically illustrates another system for producing biological, biochemical, or chemical products;

[0064] FIG.17 schematically illustrates an example bioproduction chamber;

[0065] FIG.18 shows an example counter sedimentation system;

[0066] FIG.19 shows an example inoculation module;

[0067] FIG.20 shows example 3D printed bioreactor test configuration;WSGR Ref. No.55076-707.601

[0068] FIG.21 shows an example follow-up test configuration;

[0069] FIG.22 shows another example follow-up test configuration;

[0070] FIG.23 shows another example follow-up test configuration;

[0071] FIG.24 schematically illustrates an example bioproduction diagram;

[0072] FIG.25 shows example inoculum concentration as a function of outlet volume for an example culture system;

[0073] FIG.26 shows example cell recovery as a function of time for an example culture system;

[0074] FIG.27 shows example total cell concentration and viability as a function of time for an example culture system;

[0075] FIG.28 shows example cell concentration prior to and after culture in an example continuous flow bioreactor;

[0076] FIG.29 show example cell recovery and percent viability a function of time for an example culture system;

[0077] FIG.30 shows example nitrogen metabolism as a function of time for an example culture system;

[0078] FIG.31 shows example ion concentration as a function of time for an example culture system;

[0079] FIG.32 shows an example specific productivity and titer as a function of time for an example culture system;

[0080] FIG.33 shows example harvest and total cells as a function of time for an example culture system;

[0081] FIG.34 shows example carbon metabolism as a function of time for an example culture system;

[0082] FIG.35 shows example cellular viability and monoclonal antibody production as a function of time for an example culture system;

[0083] FIG.36A schematically illustrates a microfluidic macrostructure device without equalization layers; FIG.36B schematically illustrates a microfluidic macrostructure device comprising equalization layers;

[0084] FIG.37A schematically illustrates an example gyroid structure;

[0085] FIG.37B schematically illustrates an example gyroid structure comprising gas permeable channels for gas exchange;

[0086] FIG.38A schematically illustrates an example of a Schwarz P crystal when the diameter is 0; FIG.38B schematically illustrates an example of a Schwarz P crystal when the diameter is 1;WSGR Ref. No.55076-707.601

[0087] FIG.39 shows example fluid flow through mesh equalization layers;

[0088] FIG.40 shows example fluid flow uniformity as a function of central diameter for example microfluidic devices various numbers of equalization layers;

[0089] FIG.41 shows example fluid flow uniformity in an example microfluidic device without equalization layers;

[0090] FIG.42 shows example fluid flow uniformity in an example microfluidic device with three equalization layers;

[0091] FIG.43 shows example diameter variation in an example equalization layer;

[0092] FIG.44 shows example flow uniformity as a function of gyroid width and number of layers;

[0093] FIG.45 schematically illustrates an example biologic production workflow;

[0094] FIG.46 shows a computer system that is programmed or otherwise configured to implement methods provided herein;

[0095] FIG.47 schematically illustrates a bioprocessor comprising an example bioproduction cartridge;

[0096] FIG.48 schematically illustrates an example bioprocessor configuration;

[0097] FIG.49 schematically illustrates an example bioproduction chamber;

[0098] FIG.50 schematically illustrates an example bioproduction chamber comprising an example bioreactor;

[0099] FIG.51 schematically illustrates another example bioproduction chamber comprising an example cartridge;

[0100] FIG.52 schematically illustrates an example cartridge disposed in a bioproduction chamber; and

[0101] FIG.53 schematically illustrates an example bioproduction chamber and cartridge. DETAILED DESCRIPTION

[0102] While various embodiments of the invention 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 may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0103] Whenever the term “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 “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 orWSGR Ref. No.55076-707.601 equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0104] 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.

[0105] The terms “production bioreactor”, “bioreactor”, “microbioreactor”, “3D printed bioreactor,” as used herein, generally refers to a bioreactor device suitable for scaling production of cells and / or products produced by cells. A production bioreactor may include one or more channels or other openings for inputting cells, for providing liquid media, gas composition and other cell environment factors and one or more channels for harvesting cells or products produced by cells.

[0106] The term “minimodule,” as used herein, generally refers to a segment of a production bioreactor that may be interconnected and assembled into a larger structure (e.g., macrostructure or macroshape) to constitute at least a portion or an entirety of the production bioreactor.

[0107] The term “gyroid,” as used herein, generally refers to a connected periodic minimal surface containing no straight lines. Such surface may have a mathematically infinite number of connections. In some examples, a gyroid is a unique non-trivial embedded member of the associate family of the Schwarz P and D surfaces with angle of association approximately 38.01°. A gyroid may be configured as a single gyroid or a double gyroid. A double gyroid may be oriented and configured for a particular application in a microfluidic device. The double gyroid may be configured by balancing geometric aspects related to fluid dynamic performances observed in minimodules and macrostructures (e.g., macroshapes), such as the double gyroids crystallographic structure and space group. The gyroid (e.g., single or double gyroid) may be implemented in a variety of macrostructures.

[0108] The term “chaotic advection,” as used herein, generally refers to a process by which fluid particles are transported through a flow path in a deterministic, highly sensitive, and non- linear manner. Chaotic advection may provide complex, seemingly random flow trajectories that enhance mixing.

[0109] The term “advecting design,” as used herein, generally refers to any intentionally formed features, for example, protrusions, grooves, or patterns, that modify the surface relief of a channel’s internal geometry to promote, generated, or enhance chaotic advection.

[0110] The term “selective” or “chemo selective,” as used herein, generally refers to a reactivity that is based on the chemical composition of a polymer. Where used in an expression such as “… to selectively polymerize a polymerizable material…” or “…to induce selectiveWSGR Ref. No.55076-707.601 polymerization…”, the term generally refers to polymerization of a predetermined type of the material for which the initiator is specifically intended or selected.

[0111] The “light-sensitive material” or “light-curable material,” as used interchangeably herein, generally refer to any material that upon irradiation with a light source of a given wavelength is susceptible to undergoing photo, thermal, or pH-induced transformation into a reactive species (e.g., a radical or an ion) to initiate polymerization or material differentiation.

[0112] The term “bioink” or “resin” or “blend”, as used herein, generally refers to a “homogeneous solution” or a “dispersion” or an “emulsion” which comprises reaction precursors “polymerizable material”, initiators “photoinitiatior” or “thermoinitiator” or catalysts and potentially other agents and fillers such as polymers, waxes, ceramic materials, inorganic, sand, metal particles, or liquids, such glycols, oils and others, which may or may not be soluble in the resin composition. A resin may or may not comprise an inactive carrier medium, such as a solvent (e.g., water). A vat reaction system holding the resin may comprise a built platform and one or a plurality of light sources. The resin may be provided into a single reaction mixture, forming the resin. In an example, a resin or bioink may be a the thermo-sensitive or photo- sensitive composition prepared according to the present invention. A bioink may be in the form of a bulk, aggregate, or a hydrogel product.

[0113] As stated herein, “PHR” or “phr”, as used herein, generally refers to “per hundred resin”. Where phr units are in grams and can be described as the amounts of the additive per 100 g of resin.

[0114] The term “base resin”, as used herein, generally refers to a homogeneous solution that serves as base preparation for the bioinks.

[0115] The term “coating” or “coating material” or “rheologic enhancer” or “biocompatibility enhancer”, as used here, generally refer to products that are used to enhance the characteristics of 3D printed objects that include, but are not limited to rheological, biocompatibility and porosity characteristics.

[0116] The term “equalization layer”, as used herein, generally refers to implementing variations in the diameters of structures within the same layer or across a small number of layers (e.g., less than 10, 5, 4, 3, or 2 layers) to ensure uniform flow distribution by changing the size of a connection point.

[0117] The term “connection point”, as used herein, generally refers to the minimum passage between two interconnected pores. The connection points may be disposed on the external surface(s) or inner surface(s) of the minimodules. A plurality of minimodules may be connected via the connection points. In an example, a minimodule may be connected to 4, 6, 8, 10, 12, or more other minimodules. As the number of minimodules connected via connectionWSGR Ref. No.55076-707.601 points increases so does the length of the channel formed by the minimodules and the length between the connection points. In some embodiments, the connection point may provide a location to fluidically connect a minimodule with another minimodule. The minimodules may be coupled to one another via the connection points.

[0118] The term “macrostructures”, as used herein, generally refers to a variety of structures made up of a plurality of minimodules. A macrostructure may be a microfluidic macrostructure. Alternatively, the macrostructure may provide fluidic channels with dimensions larger than microfluidic dimensions (e.g., channel diameters of greater than 1 millimeters). Minimodules may be fluidically connected to generate the macrostructures. Such structures may include, but are not limited to lamella, pyramid, hollow pyramid, spheric and cubic.

[0119] The terms “complex geometric structures” or “intricate geometries”, as used herein, generally refer to connected periodic minimal surfaces that may be hollow mathematically infinite number of connections that can be used to flow a fluid through it.

[0120] The term “bioprocessor”, as used herein, generally refers to the systems and components for controlling various aspects of producing and maintaining cells, isolating cells, producing and isolating products made by cells, or any combination thereof.

[0121] Bioreactors may provide an environment for production of cells and for producing proteins and other molecules from such cells. Many bioreactor systems involve a large capital investment as well as a large physical space. Additionally, the environment of a large bioreactors can differ in environment from smaller scale growth chambers and thereby result in suboptimal growth and production conditions. The larger scale of bioreactors can also make it difficult to investigate growth conditions on an individual cell level. This can result in population heterogeneity of cells as well as affect the quality, purity, and yield of bioproducts produced by the cells.

[0122] Included herein in the system and components are one or more bioreactors for growing cells. The bioreactors are on a microbioreactor scale, such that the system can be constructed as a benchtop bioreactor with a capacity to grow and produce cells and / or cell products in both small and large amounts. This system and methods of use are advantageous in their scalability, flexibility, and conservation of resources.

[0123] Provided herein are processes and methods that, among other implementations, provide components and a method that facilitates an effective chemical or physical production of light curable, durable, porous, hydrophobic, biocompatible, sterilizable, high resolution, and gas- permeable three dimensional (3D) printed bespoke bioreactors.

[0124] Provided herein are systems, components, and methods for producing and maintaining cells and for producing and isolating cells and products made by cells. TheWSGR Ref. No.55076-707.601 systems, components and methods herein provide flexibility to tailor production for different types of cells, types of cellular environments, and types of molecules produced. The systems, components and methods also provide flexibility of scale. For example, the systems, components, and methods described herein may provide for production scale-up without the altering or significantly altering bench-scale growth conditions.

[0125] Provided herein are methods and components for preparing, producing, or otherwise manufacturing mixture(s) that may serve as raw material for producing bioreactors, e.g., 3D printed bioreactors for growing cells. Also provided herein are methods and systems for coating a bioreactor, e.g., the 3D printed bioreactors for growing cells. The bioreactors may be any scale. In an example, a bioreactor is a microbioreactor scale, such that the objects can be constructed as a bench-top bioreactor with a capacity to grow and produce cells, cell products, or both in small and large amounts. These systems and methods may be advantageous in scalability, flexibility, and conservation of resources.

[0126] The present disclosure further provides complex three dimensional (3D)-printed objects. Particularly, to the production of bioreactors that is suitable for cell culturing made of bioinks comprised of light-activated resins comprising a multifunctional acrylate or methacrylate monomers, a curing agent (e.g., a thermal curing agent or thermocurator, or a light curing agent), a light absorbent, a photo-initiator, or any combination thereof. The disclosures further relate to the usage of silicone coatings configured to enhance lifespan, rheologic, biologic, and mechanical characteristics of 3D printed objects.

[0127] The systems and methods described herein may be usable for the production of biological, chemical, or biochemical products. Biological, chemical, or biochemical products may comprise, but are not limited to, cells, proteins, antibodies, biochemical substances or molecules, chemical substances or molecules, or any combination thereof. The systems and methods described herein may provide production of biological, chemical, or biochemical products with improved scalability, cost, efficiency, and consistency. Bioreactors and methods of use

[0128] In an aspect, the present disclosure provides bioreactors comprising a macrostructure. The macrostructure may, or may not be, a microfluidic macrostructure. The microfluidic macrostructure may comprise a plurality of fluidically connected minimodules. The plurality of fluidically connected minimodules may form a plurality of channels. The plurality of channels may comprise a channel wall that is gas permeable such that gas may diffusion between an inner surface and an outer surface of the channels. The inner surface and outer surface of the channels may be impermeable or substantially impermeable to a liquid.WSGR Ref. No.55076-707.601

[0129] In another aspect, the present disclosure provides a bioreactor comprising a macrostructure comprising a plurality of fluidically connected minimodules. The fluidically connected minimodules may form at least one channel. A channel of the at least one channel may comprise at least one connection point.

[0130] In another aspect, the present disclosure provides bioreactors comprising an input, an output, and a macrostructure. The input may be configured to receive a fluid. The output may be configured to output the fluid, cells, biological products produced in the bioreactor, or any combination thereof. The macrostructure may be fluidically connected to the input and the output. The macrostructure may include (i) a first equalization region in fluid communication with the input, (ii) a second equalization region in fluid communication with the output, and (iii) a core region disposed between the first equalization region and the second equalization region. The first equalization region may include diverging channels. The first equalization region may be configured to distribute or may distribute fluid from the input to the core region. The core region may be configured to maintain uniform fluid flow through the plurality of channels in the core region. The second equalization region may include converging channels. The second equalization region may be configured to provide the fluid from the core region to the output.

[0131] A bioreactor may include one or more inlets fluidically connected to the channel of the macrostructure. A bioreactor may include at least 1, 2, 3, 4, 5, 6, 8, 10, or more inlets. In an example, the bioreactor includes at least one inlet. A bioreactor may include one or more outlets fluidically connected to the channel of the macrostructure. The bioreactor may include at least 1, 2, 3, 4, 5, 6, 8, 10, or more outlets. In an example, the bioreactor comprises at least one outlet. A bioreactor may have an equal number of inlets and outlets, more inlets than outlets, or more outlets than inlets.

[0132] In some embodiments, the bioreactor comprises a plurality of minimodules. The bioreactor may provide an environment for scaled-up growth and production of cells and / or bioproduct from cells. The production bioreactor may provide a 3-D structure comprised a multiple minimodules. The production bioreactor may include greater than or equal to 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 80, 100, or more minimodules. The minimodules may assemble to generate a plurality of fluidically connected channels. The minimodules can include shapes such as gyroid, single gyroid, modified single gyroid, double gyroid, modified double gyroid, or any shapes that may be described as a triply periodic minimal surface (TPMS). This type of surface may form a lattice system that can grow on all three axes (X, Y, Z) periodically. TPMS may be free of self-intersections and divide a given volume into two (or more) independent sub volumes. A self-intersection may comprise a surface with a single normal vector per point defining the surface. If the surface divides the volume in which it is circumscribed into twoWSGR Ref. No.55076-707.601 independent and congruent sub volumes, this surface is called a balanced surface. TPMS are described in terms of a fundamental patch or asymmetric unit from which the entire surface may be built up by its symmetry elements. The minimodules may be fluidically connected (e.g., interconnected) with one another such that gasses, media, cells, byproduct, or any combination thereof can flow from one minimodule to another minimodule.

[0133] In some embodiments, the minimodules of the production bioreactor may include a single gyroid, modified single gyroid, double-gyroid, or modified double-gyroid shape. In an example, one or more of the minimodules comprises a triply periodic minimal surface. In another example, the TPMS comprises a gyroid. In another example, the gyroid is a single gyroid. In another example, the gyroid is a double gyroid. A modified gyroid shape may include internal diameters, wall thicknesses, or both that vary across the minimodule or from one minimodule to another. In some embodiments, modifications may include blocking of a portion of the connections or intersections (e.g., ‘mouths’), modifying the diameter of one or both phase channels of the structure, or complete or partial elimination of any of the phase channels present in a gyroid structure. In some embodiments, the channel may provide a surface on which certain cell types can adhere and grow. In some embodiments, the diameter of the channel of the minimodules can vary as required for specific cell types, production requirements and the like.

[0134] The minimodules may be fitted together into a macrostructure or macroshape that makes up the production bioreactor. In an example, the fluidically connected minimodule layers may be assembled into a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, a toroid with a central core, or combinations thereof. In some embodiments, the macrostructure is a pyramid. In some embodiments, the macrostructure is hollow pyramid, a lamella pyramid, a chessboard arrangement or a log. The macrostructure and number of minimodules within the production bioreactor can be tailored to cell division rate of the cells to be grown, as well as to regulate the velocities of liquid media, gas exchange and cell movement through the bioreactor. Each macrostructure can provide different possibilities to interact with cells, and it may be chosen given the specific process the production bioreactor is intended to stimulate. Pyramid and hollow pyramid macrostructure enable a suitable environment for growth while keeping constant velocities and cell density. More sensitive strains may require more interventions over time, in which case hollow pyramids may provide that capacity. Lamella pyramids enable a suitableWSGR Ref. No.55076-707.601 environment for growth and development, by keeping both velocity and density constant while providing complete access to each cell at each moment in time, enabling direct intervention and treatment. The chessboard and log arrangements may also provide complete access to every cell at every point of the process while permitting control over homogenous velocity and density. In some embodiments, cells are inputted at the top of the macrostructure and a cell collection device at the base of the macrostructure.

[0135] FIG.9 shows an example microfluidic macrostructure. The bioreactor may comprise one or more fluidically connected minimodules 910. In some embodiments, the minimodules can form a plurality of different macrostructures comprising a plurality of channels (FIGS.8- 11).

[0136] In some embodiments, the channel can comprise an inner surface and an outer surface. In some embodiments, the channels are permeable to gas diffusion between the inner surface and the outer surface. In some embodiments, the inner surface and / or the outer surface is impermeable to liquid. In some embodiments, the bioreactor may further comprise at least one inlet 920 fluidically connected to at least one channel of the microfluidic macrostructure. In some embodiments, the bioreactor may further comprise at least one outlet 930 fluidically connected to at least one channel of the microfluidic macrostructure. As shown in FIG.9, the at least one minimodule can comprise a triply periodic minimal surface. In some embodiments, the triply periodic minimal surface can comprise a gyroid structure. In some embodiments, the gyroid structure may comprise a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, or / and any combinations thereof. In some embodiments, the at least one minimodule can comprise a single gyroid 940.

[0137] FIG.10A shows an example andromeda shaped microfluidic macrostructure device. FIG.10B shows an example trunk shaped microfluidic macrostructure device. In some embodiments, the device can have one inlet and one outlet (FIG.10A). In some embodiments, the device can have more than one inlet and more than one outlet (FIG.10B). In some embodiments, the device can have a height from about 100 mm to about 200 mm, from about 100 mm to about 300 mm, from about 100 mm to about 400 mm, from about 100 mm to about 500 mm, from about 100 mm to about 1000 mm, from about 200 mm to about 300 mm, from about 200 mm to about 400 mm, from about 200 mm to about 500 mm, from about 200 mm to about 1000 mm, from about 300 mm to about 400 mm, from about 300 mm to about 500 mm, from about 300 mm to about 1000 mm, from about 400 mm to about 500 mm, from about 400 mm to about 1000 mm, or from about 500 mm to about 1000 mm.

[0138] FIG.11A shows another example andromeda shaped microfluidic macrostructure device. FIG.11B shows an example spherical microfluidic macrostructure device. In someWSGR Ref. No.55076-707.601 embodiments, the spherical device may have a diameter from about 50 mm to about 100 mm, from about 50 mm to about 200 mm, from about 50 mm to about 300 mm, from about 50 mm to about 400 mm, from about 50 mm to about 500 mm, from about 50 mm to about 1000 mm, from about 100 mm to about 200 mm, from about 100 mm to about 300 mm, from about 100 mm to about 400 mm, from about 100 mm to about 500 mm, from about 100 mm to about 1000 mm, from about 200 mm to about 300 mm, from about 200 mm to about 400 mm, from about 200 mm to about 500 mm, from about 200 mm to about 1000 mm, from about 300 mm to about 400 mm, from about 300 mm to about 500 mm, from about 300 mm to about 1000 mm, from about 400 mm to about 500 mm, from about 400 mm to about 1000 mm, or from about 500 mm to about 1000 mm.

[0139] In an example, a bioreactor is a continuous flow bioreactor. The continuous flow bioreactor may comprise a plurality of minimodules fluidically connected together at connection points (see, for example, 3710 of FIG.37A) to form a macrostructure. The minimodules may comprise a single gyroid shape (see, for example, 940 of FIG.9). The minimodules may not comprise a double gyroid shape. The single gyroid shape may permit the plurality of minimodules to assemble into a single fluidic channel, as shown in FIG.37B. The single fluidic channel may provide a tortuous fluidic pathway with a plurality of branches and tributaries, as shown in FIG.37B. The single fluidic pathway may be configured to flow, or may flow, a liquid media comprising cells. The single fluidic pathway may be configured to provide laminar flow such that the cells in suspension or adhered to the wall of the channel are subjected to little or no shear stress. The wall of the channel may be formed of a polymerized 3D printable bioink. The polymerized bioink may or may not be biocompatible. The polymerized bioink may or may not be coated or otherwise treated to increase the biocompatibility of the surface(s) of the channel. The wall of the channel may be at least partially gas permeable. The wall of the channel may be impermeable to liquid. In an example, the wall is impermeable to aqueous liquids such as culture media. The wall of the channel may be configured to be sufficiently gas permeable to permit gas to diffuse from an environment external to the channel into the liquid flowing within the channel. The liquid impermeability and gas permeability of the channel wall may be sufficient gas to enter the channel to support cell culture while preventing evaporative or liquid loss from the channel. In some embodiments, the bioreactor may comprise a first channel configured to flow a liquid media and a second channel configured to flow a gas such that gas may exchange from the gas channel to the liquid channel. Alternatively, forming the channel of a gas permeable material may permit a single channel flowing liquid media to be used (e.g., without using a second or separate channel to flow gas). This may reduce the complexity of the printed structure and the amount of material used during manufacturing of the bioreactor.WSGR Ref. No.55076-707.601

[0140] In an example, the one or more surfaces of the bioreactor may include functionalization groups. The functionalization groups may be distributed through the channel walls of the bioreactor or may be disposed on an inner surface of the bioreactor channels. The bioreactor may be printed with surface functionalization groups. Alternatively, surface functionalization groups may be added subsequent to printing. In an example, functionalization groups may be incorporated in the bioink prior to printing. Alternatively, or in addition to, functionalization groups may be incorporated and / or added to the channel wall after generation or printing of the bioreactor. Functionalization groups may be used in bioreactors for adherent cell processing, suspension cell processing, or both. Functionalization groups may be configured to permit cell differentiation, cell adhesion, cell detachment, activation of metabolic pathways, membrane permeabilization, pH control, or any combination thereof. Functionalization groups may include peptides, proteins, functional polymers, metabolites, or any combination thereof. Functionalization groups may include growth factors (e.g., bone morphogenetic protein-2, transforming growth factor-beta, vascular endothelial growth factor, nerve growth factor, etc.), cell differentiation molecules (e.g., retinoic acid, dexamethasone, beta-glycerophosphate, etc.), short peptides (e.g., derived from extracellular matrix proteins), proteins, activation molecules (e.g., forskolin, resveratrol, insulin, insulin-like growth factor 1, etc.), membrane permeabilization molecules (e.g., saponins, cell-penetrating peptides, cytolytic, etc.), buffers for pH control (e.g., Tris buffer, HEPES buffer, magnesium hydroxide, calcium carbonate, bioactive glass, etc.), or any combinations thereof. In an example, proteins and larger molecules may be encapsulated in microspheres or nanoparticles prior to incorporation into the bioink or resin to protect the proteins and larger molecules from degradation during photo- or thermos-curing of the bioink or resin. Alternatively, the proteins or larger molecules may be coupled to or associated with a surface of the channel wall after forming of the bioreactor. The bioink, resin, or surface of the channel wall may comprise one or more reactive groups configured to permit or that permit immobilization of the functional groups to the bioreactor (e.g., channel wall). The reactive groups may include maleimide groups, thiol groups, N- hydroxysuccinimide esters, alkyne groups, azide groups, imidazole groups, catechol groups, carboxyl groups, or any combinations thereof.

[0141] In an example, the bioreactor is configured for adherent cell processing. The cells may be cultured and, during culturing, the cells may adhere to the inner surfaces of the bioreactor (e.g., via surface functionalization groups). The channel walls may permit gasses, such as oxygen, to contact the adhered cells via diffusion through the wall. The channel walls may permit other gasses, such as carbon dioxide, to diffuse away from the adhered cells. The channels may be configured to continuously flow culture media past the adhered cells. The cellsWSGR Ref. No.55076-707.601 may be detached from the channel walls via enzymatic, chemical, or physical detachment in order to permit harvesting of the cells. Enzymes for detaching the cells may include trypsin, TrypLETM, Accutase®, collagenase, proteases (e.g., dispase), ReLeSRTM, or any combination thereof. Chemicals for detaching cells may include ethylenediaminetetraacetic acid (EDTA). Physical detachment may include subjecting the bioreactor and cells to ultrasonic, acoustic, vibration, or other energy. In an example, the channels of the bioreactor are washed or flushed with buffer (e.g., HEPES, Tris, phosphate-buffered saline, etc.) and EDTA to remove cellular debris from the bioreactor. In another example, the channels may be washed to remove cellular debris and, subsequently or simultaneously, cells may be detached via enzymatic harvesting (e.g., contacting the adhered cells with an enzyme configured to detach the cells from the channel walls). The bioreactor may comprise a material configured to facilitate cell detachment. The materials may comprise thermoresponsive polymers (e.g., poly(N-isopropylacrylamide), competitive inhibition compounds (e.g., peptides, chelators, etc.) in solution within the channel(s), light-cleavable adhesion peptides, or any combinations thereof. Harvesting the adherent cells may include washing culture medium with a buffer (e.g., phosphate-buffered saline), EDTA, or both to remove cellular debris. The bioreactor may be subjected to sonication using an ultrasonic source. Sonication may promote cell detachment. The ultrasonic frequency may be greater than to equal to about 10 kilohertz (kHz), 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 80 kHz, 100 kHz, or greater. In an example the ultrasonic frequency is greater than or equal to 40 kHz. The bioreactor may be subjected to the ultrasonic frequency for a time period of greater than or equal to about 30 seconds, 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, 30 min, or more. The bioreactor may be subjected to the ultrasonic frequency for a time period of less than or equal to about 30 min, 20 min, 15 min, 10 min, 8 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, 30 seconds, or less. The bioreactor may be subjected to enzymatic detachment prior to, during, or subsequent to subjecting the bioreactor to the ultrasonic frequency. In an example, the bioreactor is subjected to enzymatic detachment subsequent to subjecting the bioreactor to the ultrasonic frequency.

[0142] The minimodules may be connected to one another at connection points. The average diameter (e.g., internal diameter) of a connection point may be greater than or equal to about 0.1 micrometer (µm), 0.5 µm, 1 µm, 5 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 75 µm, 100 µm, 150 µm, 200 µm, 300 µm, 400 µm, 500 µm, 750 µm, 1000 µm, 2000 µm, 3000 µm, 4000 µm, 5000 µm, 6000 µm, 7000 µm, 8000 µm, 9000 µm, 10 millimeters (mm), or more. The average diameter of a connection point may be less than or equal to about 10 mm, 9000 µm, 8000 µm, 7000 µm, 6000 µm, 5000 µm, 4000 µm, 3000 µm, 2000 µm, 1000 µm, 750 µm, 500 µm, 400 µm, 300 µm, 200 µm, 150 µm, 100 µm, 75 µm, 50 µm, 40 µm, 30 µm, 20 µm, 10 µm,WSGR Ref. No.55076-707.601 5 µm, 1 µm, 0.5 µm, 0.1 µm, or less. A connection point may have an average diameter from about 0.1 µm to 0.5 µm, 0.1 µm to 1 µm, 0.1 µm to 5 µm, 0.1 µm to 10 µm, 0.1 µm to 20 µm, 0.1 µm to 30 µm, 0.1 µm to 40 µm, 0.1 µm to 50 µm, 0.1 µm to 60 µm, 0.1 µm to 75 µm, 0.1 µm to 100 µm, 0.1 µm to 150 µm, 0.1 µm to 200 µm, 0.1 µm to 300 µm, 0.1 µm to 400 µm, 0.1 µm to 500 µm, 0.1 µm to 750 µm, 0.1 µm to 1000 µm, 0.1 µm to 2000 µm, 0.1 µm to 3000 µm, 0.1 µm to 4000 µm, 0.1 µm to 5000 µm, 0.1 µm to 6000 µm, 0.1 µm to 7000 µm, 0.1 µm to 8000 µm, 0.1 µm to 9000 µm, 0.1 µm to 10 mm, 0.5 µm to 1 µm, 0.5 µm to 5 µm, 0.5 µm to 10 µm, 0.5 µm to 20 µm, 0.5 µm to 30 µm, 0.5 µm to 40 µm, 0.5 µm to 50 µm, 0.5 µm to 60 µm, 0.5 µm to 75 µm, 0.5 µm to 100 µm, 0.5 µm to 150 µm, 0.5 µm to 200 µm, 0.5 µm to 300 µm, 0.5 µm to 400 µm, 0.5 µm to 500 µm, 0.5 µm to 750 µm, 0.5 µm to 1000 µm, 0.5 µm to 2000 µm, 0.5 µm to 3000 µm, 0.5 µm to 4000 µm, 0.5 µm to 5000 µm, 0.5 µm to 6000 µm, 0.5 µm to 7000 µm, 0.5 µm to 8000 µm, 0.5 µm to 9000 µm, 0.5 µm to 10 mm, 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to 30 µm, 1 µm to 40 µm, 1 µm to 50 µm, 1 µm to 60 µm, 1 µm to 75 µm, 1 µm to 100 µm, 1 µm to 150 µm, 1 µm to 200 µm, 1 µm to 300 µm, 1 µm to 400 µm, 1 µm to 500 µm, 1 µm to 750 µm, 1 µm to 1000 µm, 1 µm to 2000 µm, 1 µm to 3000 µm, 1 µm to 4000 µm, 1 µm to 5000 µm, 1 µm to 6000 µm, 1 µm to 7000 µm, 1 µm to 8000 µm, 1 µm to 9000 µm, 1 µm to 10 mm, 5 µm to 10 µm, 5 µm to 20 µm, 5 µm to 30 µm, 5 µm to 40 µm, 5 µm to 50 µm, 5 µm to 60 µm, 5 µm to 75 µm, 5 µm to 100 µm, 5 µm to 150 µm, 5 µm to 200 µm, 5 µm to 300 µm, 5 µm to 400 µm, 5 µm to 500 µm, 5 µm to 750 µm, 5 µm to 1000 µm, 5 µm to 2000 µm, 5 µm to 3000 µm, 5 µm to 4000 µm, 5 µm to 5000 µm, 5 µm to 6000 µm, 5 µm to 7000 µm, 5 µm to 8000 µm, 5 µm to 9000 µm, 5 µm to 10 mm, 10 µm to 20 µm, 10 µm to 30 µm, 10 µm to 40 µm, 10 µm to 50 µm, 10 µm to 60 µm, 10 µm to 75 µm, 10 µm to 100 µm, 10 µm to 150 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 750 µm, 10 µm to 1000 µm, 10 µm to 2000 µm, 10 µm to 3000 µm, 10 µm to 4000 µm, 10 µm to 5000 µm, 10 µm to 6000 µm, 10 µm to 7000 µm, 10 µm to 8000 µm, 10 µm to 9000 µm, 10 µm to 10 mm, 20 µm to 30 µm, 20 µm to 40 µm, 20 µm to 50 µm, 20 µm to 60 µm, 20 µm to 75 µm, 20 µm to 100 µm, 20 µm to 150 µm, 20 µm to 200 µm, 20 µm to 300 µm, 20 µm to 400 µm, 20 µm to 500 µm, 20 µm to 750 µm, 20 µm to 1000 µm, 20 µm to 2000 µm, 20 µm to 3000 µm, 20 µm to 4000 µm, 20 µm to 5000 µm, 20 µm to 6000 µm, 20 µm to 7000 µm, 20 µm to 8000 µm, 20 µm to 9000 µm, 20 µm to 10 mm, 30 µm to 40 µm, 30 µm to 50 µm, 30 µm to 60 µm, 30 µm to 75 µm, 30 µm to 100 µm, 30 µm to 150 µm, 30 µm to 200 µm, 30 µm to 300 µm, 30 µm to 400 µm, 30 µm to 500 µm, 30 µm to 750 µm, 30 µm to 1000 µm, 30 µm to 2000 µm, 30 µm to 3000 µm, 30 µm to 4000 µm, 30 µm to 5000 µm, 30 µm to 6000 µm, 30 µm to 7000 µm, 30 µm to 8000 µm, 30 µm to 9000 µm, 30 µm to 10 mm, 40 µm to 50 µm, 40 µm to 60 µm, 40 µm to 75 µm, 40 µm to 100 µm, 40 µm to 150 µm, 40 µm to 200 µm, 40 µm to 300 µm, 40WSGR Ref. No.55076-707.601 µm to 400 µm, 40 µm to 500 µm, 40 µm to 750 µm, 40 µm to 1000 µm, 40 µm to 2000 µm, 40 µm to 3000 µm, 40 µm to 4000 µm, 40 µm to 5000 µm, 40 µm to 6000 µm, 40 µm to 7000 µm, 40 µm to 8000 µm, 40 µm to 9000 µm, 40 µm to 10 mm, 50 µm to 60 µm, 50 µm to 75 µm, 50 µm to 100 µm, 50 µm to 150 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 750 µm, 50 µm to 1000 µm, 50 µm to 2000 µm, 50 µm to 3000 µm, 50 µm to 4000 µm, 50 µm to 5000 µm, 50 µm to 6000 µm, 50 µm to 7000 µm, 50 µm to 8000 µm, 50 µm to 9000 µm, 50 µm to 10 mm, 60 µm to 75 µm, 60 µm to 100 µm, 60 µm to 150 µm, 60 µm to 200 µm, 60 µm to 300 µm, 60 µm to 400 µm, 60 µm to 500 µm, 60 µm to 750 µm, 60 µm to 1000 µm, 60 µm to 2000 µm, 60 µm to 3000 µm, 60 µm to 4000 µm, 60 µm to 5000 µm, 60 µm to 6000 µm, 60 µm to 7000 µm, 60 µm to 8000 µm, 60 µm to 9000 µm, 60 µm to 10 mm, 75 µm to 100 µm, 75 µm to 150 µm, 75 µm to 200 µm, 75 µm to 300 µm, 75 µm to 400 µm, 75 µm to 500 µm, 75 µm to 750 µm, 75 µm to 1000 µm, 75 µm to 2000 µm, 75 µm to 3000 µm, 75 µm to 4000 µm, 75 µm to 5000 µm, 75 µm to 6000 µm, 75 µm to 7000 µm, 75 µm to 8000 µm, 75 µm to 9000 µm, 75 µm to 10 mm, 100 µm to 150 µm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 750 µm, 100 µm to 1000 µm, 100 µm to 2000 µm, 100 µm to 3000 µm, 100 µm to 4000 µm, 100 µm to 5000 µm, 100 µm to 6000 µm, 100 µm to 7000 µm, 100 µm to 8000 µm, 100 µm to 9000 µm, 100 µm to 10 mm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 750 µm, 200 µm to 1000 µm, 200 µm to 2000 µm, 200 µm to 3000 µm, 200 µm to 4000 µm, 200 µm to 5000 µm, 200 µm to 6000 µm, 200 µm to 7000 µm, 200 µm to 8000 µm, 200 µm to 9000 µm, 200 µm to 10 mm, 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 750 µm, 300 µm to 1000 µm, 300 µm to 2000 µm, 300 µm to 3000 µm, 300 µm to 4000 µm, 300 µm to 5000 µm, 300 µm to 6000 µm, 300 µm to 7000 µm, 300 µm to 8000 µm, 300 µm to 9000 µm, 300 µm to 10 mm, 400 µm to 500 µm, 400 µm to 750 µm, 400 µm to 1000 µm, 400 µm to 2000 µm, 400 µm to 3000 µm, 400 µm to 4000 µm, 400 µm to 5000 µm, 400 µm to 6000 µm, 400 µm to 7000 µm, 400 µm to 8000 µm, 400 µm to 9000 µm, 400 µm to 10 mm, 500 µm to 750 µm, 500 µm to 1000 µm, 500 µm to 2000 µm, 500 µm to 3000 µm, 500 µm to 4000 µm, 500 µm to 5000 µm, 500 µm to 6000 µm, 500 µm to 7000 µm, 500 µm to 8000 µm, 500 µm to 9000 µm, 500 µm to 10 mm, 750 µm to 1000 µm, 750 µm to 2000 µm, 750 µm to 3000 µm, 750 µm to 4000 µm, 750 µm to 5000 µm, 750 µm to 6000 µm, 750 µm to 7000 µm, 750 µm to 8000 µm, 750 µm to 9000 µm, 750 µm to 10 mm, 1000 µm to 2000 µm, 1000 µm to 3000 µm, 1000 µm to 4000 µm, 1000 µm to 5000 µm, 1000 µm to 6000 µm, 1000 µm to 7000 µm, 1000 µm to 8000 µm, 1000 µm to 9000 µm, 1000 µm to 10 mm, 2000 µm to 3000 µm, 2000 µm to 4000 µm, 2000 µm to 5000 µm, 2000 µm to 6000 µm, 2000 µm to 7000 µm, 2000 µm to 8000 µm, 2000 µm to 9000 µm, 2000 µm to 10 mm, 3000 µm to 4000 µm, 3000 µm to 5000 µm, 3000 µm to 6000 µm, 3000 µm to 7000 µm,WSGR Ref. No.55076-707.601 3000 µm to 8000 µm, 3000 µm to 9000 µm, 3000 µm to 10 mm, 4000 µm to 5000 µm, 4000 µm to 6000 µm, 4000 µm to 7000 µm, 4000 µm to 8000 µm, 4000 µm to 9000 µm, 4000 µm to 10 mm, 5000 µm to 6000 µm, 5000 µm to 7000 µm, 5000 µm to 8000 µm, 5000 µm to 9000 µm, 5000 µm to 10 mm, 6000 µm to 7000 µm, 6000 µm to 8000 µm, 6000 µm to 9000 µm, 6000 µm to 10 mm, 7000 µm to 8000 µm, 7000 µm to 9000 µm, 7000 µm to 10 mm, 8000 µm to 9000 µm, 8000 µm to 10 mm, or 9000 µm to 10 mm.

[0143] In an example, a connection point has an average diameter from about 1 µm to 50 µm. In another example, a connection point has an average diameter from about 1 µm to 100 µm. In another example, a connection point has an average diameter from about 1 µm to 500 µm. In another example, a connection point has an average diameter from 1 µm to 3000 µm. In another example, a connection point has an average diameter from about 1 µm to 10 mm. In another example, a connection point has an average diameter of at least about 50 µm. In another example, at least one connection point of a minimodule may have a modified internal diameter.

[0144] The microfluidic macrostructure may have a plurality of channels. A channel of the plurality of channels may have a volume. The volume of the channels may be constant throughout the macrostructure. Alternatively, the volume of the channels may vary across the macrostructure. The volume of the channels may be added to generate an internal volume of the macrostructure. The internal volume of the macrostructure may be to total volume of liquid that the macrostructure may be able to contain. The internal volume of the macrostructure may be greater than or equal to 10 mL, greater than or equal to 50 mL, greater than or equal to 100 mL, greater than or equal to 1000 mL, greater than or equal to 5 L, or greater than or equal to 10 L.

[0145] The channel may comprise a gas permeable material. The channel wall may be comprised of a gas permeable material. The gas permeable material may permit gas to diffuse through a wall of the channel while preventing or substantially preventing flow of liquid through a wall of the channel. Gas permeability of the channel may be determined via wall material, coatings, thickness, or any combination thereof. The channel may have a gas permeability of greater than or equal to about 50 barrers, 100 barrers, 150 barrers, 200 barrers, 300 barrers, 400 barrers, 500 barrers, 600 barrers, 700 barrers, 800 barrers, 1000 barrers, 1250 barrers, 1500 barrers, 1750 barrers, 2000 barrers, or greater. In an example, the channel wall may have a gas permeability of at least about 1000 barrers.

[0146] The channel may comprise a liquid impermeable material. The channel wall may be comprised of a liquid impermeable material. Liquid permeability of the channel wall may be less than or equal to about 10 microDarcy (µD), 5 µD, 4 µD, 3 µD, 2 µD, 1 µD, 900 nanoDarcy (nD), 800 nD, 700 nD, 600 nD, 500 nD, 300 nD, 200 dD, 100 nD, 50 nD, 25 nD, 20 nD, 15 nD, 10 nD, 8 nD, 6 nD, 4 nD, 2 nD, 1 nD, or less. In an example, the liquid permeability of theWSGR Ref. No.55076-707.601 channel wall is less than or equal to about 1 µD. In another example, the liquid permeability of the channel wall is less than or equal to about 100 nD. In another example, the liquid permeability of the channel wall is less than or equal to about 10 nD. In another example, the liquid permeability of the channel wall is less than or equal to about 1 nD.

[0147] The gas permeable material may be formed of a polymer, hydrogel, resin, other printable materials, or combinations thereof. The gas permeable material may be a 3D printable material. The 3D printable material may be a liquid material that, upon being subjected to or contacted with a stimulus, undergoes a phase change to a semi-solid or solid material. The semi- solid or solid material may form the channels. The material(s) forming the channels may or may not swell in water. In an example, the material(s) forming the channel swells in water. The material(s) forming the channels may increase in volume by less than or equal to 75%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1%, or less when exposed to room temperature (e.g., 20-22 °C) water for 150 hours. In an example, the material(s) forming the channel increase in volume by less than or equal to 30% when exposed to room temperature water for 150 hours.

[0148] The channels may be configured to provide laminar flow, transitional flow, or turbulent flow of a liquid through the channels or substantially laminar flow of a liquid. The diameter of the channel, fluid properties (e.g., density and viscosity), and flow rate may affect the flow regime of fluid moving through the channel. The channels may be configured to provide or may provide laminar flow of a fluid with a viscosity of equal to or larger than about 1.4, 1.5, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0, 10, 50, 60, or more of a viscosity of water. In some embodiments, the viscosity of the liquid may increase as the cell concentration increases. In some embodiments, the denser the culture, the more laminar the flow may be. In some embodiments, the Reynolds number of the flow may be less than 1000, less than 100, less than 10, less than 1, less than 0.1, less than 0.01, less than 0.001. In some embodiments, the flow rate may be from 0.1 µm / s to 10 mm / s. In some embodiments, the flow rate may be 2 µm / s.

[0149] An inner surface of the channel(s) may include a 3D structure. The 3D structure may include protrusions, grooves, or a combination thereof. The 3D structure may be a patterned 3D structure or may be a random 3D structure. The 3D structure may be small enough such that it does not interfere with the flow of cells through the channels. For example, individual structures of the 3D structure may have a dimension (e.g., length, width, height, or depth) that is less than the average diameter of a cell being processed in the bioreactor. The 3D structure may generate chaotic advection in the fluid flowing through the channel. The chaotic advection may enhance mixing within the channels without adversely affecting the cells.

[0150] In some embodiments, a residence time for the cells may be 1 hr to 7 days. In someWSGR Ref. No.55076-707.601 embodiments, a residence time for the cells may be 24 hr. In some embodiments, a volume may be processed per day due to the laminar flow and the positioning of the cells within the channel. This depends on the cell doubling time. With human cells, given that a volume is processed each day, the residence time could be around 4 days.

[0151] The channels of the macrostructure may have the same diameter or may vary in diameter across the macrostructure. A channel may have a constant diameter along the length of the channel. Alternatively, the channel diameter may vary along the length of the channel. A channel may have an average diameter (e.g., internal diameter) of greater than or equal to about 0.1 micrometer (µm), 0.5 µm, 1 µm, 5 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 75 µm, 100 µm, 150 µm, 200 µm, 300 µm, 400 µm, 500 µm, 750 µm, 1000 µm, 2000 µm, 3000 µm, 4000 µm, 5000 µm, 6000 µm, 7000 µm, 8000µm, 9000 µm, 10 mm or more. A channel may have an average diameter (e.g., internal diameter) of less than or equal to about 10 mm, 9000 µm, 8000 µm, 7000 µm, 6000 µm, 5000 µm, 4000 µm, 3000 µm, 2000 µm, 1000 µm, 750 µm, 500 µm, 400 µm, 300 µm, 200 µm, 150 µm, 100 µm, 75 µm, 50 µm, 40 µm, 30 µm, 20 µm, 10 µm, 5 µm, 1 µm, 0.5 µm, 0.1 µm, or less. The channel may have an average diameter (e.g., internal diameter) from about 0.1 µm to 0.5 µm, 0.1 µm to 1 µm, 0.1 µm to 5 µm, 0.1 µm to 10 µm, 0.1 µm to 20 µm, 0.1 µm to 30 µm, 0.1 µm to 40 µm, 0.1 µm to 50 µm, 0.1 µm to 60 µm, 0.1 µm to 75 µm, 0.1 µm to 100 µm, 0.1 µm to 150 µm, 0.1 µm to 200 µm, , 0.1 µm to 300 µm, 0.1 µm to 400 µm, 0.1 µm to 500 µm, 0.1 µm to 750 µm, 0.1 µm to 1000 µm, 0.1 µm to 2000 µm, 0.1 µm to 3000 µm, 0.1 µm to 4000 µm, 0.1 µm to 5000 µm, 0.1 µm to 6000 µm, 0.1 µm to 7000 µm, 0.1 µm to 8000 µm, 0.1 µm to 9000 µm, 0.1 µm to 10 mm, 0.5 µm to 1 µm, 0.5 µm to 5 µm, 0.5 µm to 10 µm, 0.5 µm to 20 µm, 0.5 µm to 30 µm, 0.5 µm to 40 µm, 0.5 µm to 50 µm, 0.5 µm to 60 µm, 0.5 µm to 75 µm, 0.5 µm to 100 µm, 0.5 µm to 150 µm, 0.5 µm to 200 µm, 0.5 µm to 300 µm, 0.5 µm to 400 µm, 0.5 µm to 500 µm, 0.5 µm to 750 µm, 0.5 µm to 1000 µm, 0.5 µm to 2000 µm, 0.5 µm to 3000 µm, 0.5 µm to 4000 µm, 0.5 µm to 5000 µm, 0.5 µm to 6000 µm, 0.5 µm to 7000 µm, 0.5 µm to 8000 µm, 0.5 µm to 9000 µm, 0.5 µm to 10 mm, 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to 30 µm, 1 µm to 40 µm, 1 µm to 50 µm, 1 µm to 60 µm, 1 µm to 75 µm, 1 µm to 100 µm, 1 µm to 150 µm, 1 µm to 200 µm, 1 µm to 300 µm, 1 µm to 400 µm, 1 µm to 500 µm, 1 µm to 750 µm, 1 µm to 1000 µm, 1 µm to 2000 µm, 1 µm to 3000 µm, 1 µm to 4000 µm, 1 µm to 5000 µm, 1 µm to 6000 µm, 1 µm to 7000 µm, 1 µm to 8000 µm, 1 µm to 9000 µm, 1 µm to 10 mm, 5 µm to 10 µm, 5 µm to 20 µm, 5 µm to 30 µm, 5 µm to 40 µm, 5 µm to 50 µm, 5 µm to 60 µm, 5 µm to 75 µm, 5 µm to 100 µm, 5 µm to 150 µm, 5 µm to 200 µm, 5 µm to 300 µm, 5 µm to 400 µm, 5 µm to 500 µm, 5 µm to 750 µm, 5 µm to 1000 µm, 5 µm to 2000 µm, 5 µm to 3000 µm, 5 µm to 4000 µm, 5 µm to 5000 µm, 5 µm to 6000 µm, 5 µm to 7000 µm, 5 µm to 8000 µm, 5 µm to 9000 µm, 5 µm to 10 mm, 10 µm to 20 µm, 10 µm to 30WSGR Ref. No.55076-707.601 µm, 10 µm to 40 µm, 10 µm to 50 µm, 10 µm to 60 µm, 10 µm to 75 µm, 10 µm to 100 µm, 10 µm to 150 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 750 µm, 10 µm to 1000 µm, 10 µm to 2000 µm, 10 µm to 3000 µm, 10 µm to 4000 µm, 10 µm to 5000 µm, 10 µm to 6000 µm, 10 µm to 7000 µm, 10 µm to 8000 µm, 10 µm to 9000 µm, 10 µm to 10 mm, 20 µm to 30 µm, 20 µm to 40 µm, 20 µm to 50 µm, 20 µm to 60 µm, 20 µm to 75 µm, 20 µm to 100 µm, 20 µm to 150 µm, 20 µm to 200 µm, 20 µm to 300 µm, 20 µm to 400 µm, 20 µm to 500 µm, 20 µm to 750 µm, 20 µm to 1000 µm, 20 µm to 2000 µm, 20 µm to 3000 µm, 20 µm to 4000 µm, 20 µm to 5000 µm, 20 µm to 6000 µm, 20 µm to 7000 µm, 20 µm to 8000 µm, 20 µm to 9000 µm, 20 µm to 10 mm, 30 µm to 40 µm, 30 µm to 50 µm, 30 µm to 60 µm, 30 µm to 75 µm, 30 µm to 100 µm, 30 µm to 150 µm, 30 µm to 200 µm, 30 µm to 300 µm, 30 µm to 400 µm, 30 µm to 500 µm, 30 µm to 750 µm, 30 µm to 1000 µm, 30 µm to 2000 µm, 30 µm to 3000 µm, 30 µm to 4000 µm, 30 µm to 5000 µm, 30 µm to 6000 µm, 30 µm to 7000 µm, 30 µm to 8000 µm, 30 µm to 9000 µm, 30 µm to 10 mm, 40 µm to 50 µm, 40 µm to 60 µm, 40 µm to 75 µm, 40 µm to 100 µm, 40 µm to 150 µm, 40 µm to 200 µm, 40 µm to 300 µm, 40 µm to 400 µm, 40 µm to 500 µm, 40 µm to 750 µm, 40 µm to 1000 µm, 40 µm to 2000 µm, 40 µm to 3000 µm, 40 µm to 4000 µm, 40 µm to 5000 µm, 40 µm to 6000 µm, 40 µm to 7000 µm, 40 µm to 8000 µm, 40 µm to 9000 µm, 40 µm to 10 mm, 50 µm to 60 µm, 50 µm to 75 µm, 50 µm to 100 µm, 50 µm to 150 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 750 µm, 50 µm to 1000 µm, 50 µm to 2000 µm, 50 µm to 3000 µm, 50 µm to 4000 µm, 50 µm to 5000 µm, 50 µm to 6000 µm, 50 µm to 7000 µm, 50 µm to 8000 µm, 50 µm to 9000 µm, 50 µm to 10 mm, 60 µm to 75 µm, 60 µm to 100 µm, 60 µm to 150 µm, 60 µm to 200 µm, 60 µm to 300 µm, 60 µm to 400 µm, 60 µm to 500 µm, 60 µm to 750 µm, 60 µm to 1000 µm, 60 µm to 2000 µm, 60 µm to 3000 µm, 60 µm to 4000 µm, 60 µm to 5000 µm, 60 µm to 6000 µm, 60 µm to 7000 µm, 60 µm to 8000 µm, 60 µm to 9000 µm, 60 µm to 10 mm, 75 µm to 100 µm, 75 µm to 150 µm, 75 µm to 200 µm, 75 µm to 300 µm, 75 µm to 400 µm, 75 µm to 500 µm, 75 µm to 750 µm, 75 µm to 1000 µm, 75 µm to 2000 µm, 75 µm to 3000 µm, 75 µm to 4000 µm, 75 µm to 5000 µm, 75 µm to 6000 µm, 75 µm to 7000 µm, 75 µm to 8000 µm, 75 µm to 9000 µm, 75 µm to 10 mm, 100 µm to 150 µm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 750 µm, 100 µm to 1000 µm, 100 µm to 2000 µm, 100 µm to 3000 µm, 100 µm to 4000 µm, 100 µm to 5000 µm, 100 µm to 6000 µm, 100 µm to 7000 µm, 100 µm to 8000 µm, 100 µm to 9000 µm, 100 µm to 10 mm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 750 µm, 200 µm to 1000 µm, 200 µm to 2000 µm, 200 µm to 3000 µm, 200 µm to 4000 µm, 200 µm to 5000 µm, 200 µm to 6000 µm, 200 µm to 7000 µm, 200 µm to 8000 µm, 200 µm to 9000 µm, 200 µm to 10 mm, 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 750 µm, 300 µm to 1000 µm, 300 µm to 2000 µm,WSGR Ref. No.55076-707.601 300 µm to 3000 µm, 300 µm to 4000 µm, 300 µm to 5000 µm, 300 µm to 6000 µm, 300 µm to 7000 µm, 300 µm to 8000 µm, 300 µm to 9000 µm, 300 µm to 10 mm, 400 µm to 500 µm, 400 µm to 750 µm, 400 µm to 1000 µm, 400 µm to 2000 µm, 400 µm to 3000 µm, 400 µm to 4000 µm, 400 µm to 5000 µm, 400 µm to 6000 µm, 400 µm to 7000 µm, 400 µm to 8000 µm, 400 µm to 9000 µm, 400 µm to 10 mm, 500 µm to 750 µm, 500 µm to 1000 µm, 500 µm to 2000 µm, 500 µm to 3000 µm, 500 µm to 4000 µm, 500 µm to 5000 µm, 500 µm to 6000 µm, 500 µm to 7000 µm, 500 µm to 8000 µm, 500 µm to 9000 µm, 500 µm to 10 mm, 750 µm to 1000 µm, 750 µm to 2000 µm, 750 µm to 3000 µm, 750 µm to 4000 µm, 750 µm to 5000 µm, 750 µm to 6000 µm, 750 µm to 7000 µm, 750 µm to 8000 µm, 750 µm to 9000 µm, 750 µm to 10 mm, 1000 µm to 2000 µm, 1000 µm to 3000 µm, 1000 µm to 4000 µm, 1000 µm to 5000 µm, 1000 µm to 6000 µm, 1000 µm to 7000 µm, 1000 µm to 8000 µm, 1000 µm to 9000 µm, 1000 µm to 10 mm, 2000 µm to 3000 µm, 2000 µm to 4000 µm, 2000 µm to 5000 µm, 2000 µm to 6000 µm, 2000 µm to 7000 µm, 2000 µm to 8000 µm, 2000 µm to 9000 µm, 2000 µm to 10 mm, 3000 µm to 4000 µm, 3000 µm to 5000 µm, 3000 µm to 6000 µm, 3000 µm to 7000 µm, 3000 µm to 8000 µm, 3000 µm to 9000 µm, 3000 µm to 10 mm, 4000 µm to 5000 µm, 4000 µm to 6000 µm, 4000 µm to 7000 µm, 4000 µm to 8000 µm, 4000 µm to 9000 µm, 4000 µm to 10 mm, 5000 µm to 6000 µm, 5000 µm to 7000 µm, 5000 µm to 8000 µm, 5000 µm to 9000 µm, 5000 µm to 10 mm, 6000 µm to 7000 µm, 6000 µm to 8000 µm, 6000 µm to 9000 µm, 6000 µm to 10 mm, 7000 µm to 8000 µm, 7000 µm to 9000 µm, 7000 µm to 10 mm, 8000 µm to 9000 µm, 8000 µm to 10 mm, or 9000 µm to 10 mm.

[0152] In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 3000 µm. In another example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 50 µm. In another example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 100 µm. In another example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 500 µm. In another example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 1000 µm. In another example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 2000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 3000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 4000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 5000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 6000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 7000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 8000 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 9000WSGR Ref. No.55076-707.601 µm. In an example, the average diameter (e.g., internal diameter) of a channel is from about 1 µm to 10 mm.

[0153] A wall of the channel may have a thickness. The thickness may be a distance between the inner surface and the outer surface of a channel. The wall thickness (e.g., distance between the inner surface and outer surface) may be less than or equal to about 1000 µm, 800 µm, 600 µm, 500 µm, 400 µm, 300 µm, 200 µm, 150 µm, 100 µm, 75 µm, 50 µm, 40 µm, 30 µm, 20 µm, 10 µm, 5 µm, 1 µm, or less. The wall thickness (e.g., distance between the inner surface and outer surface) may be greater than or equal to about 1 µm, 5 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 75 µm, 100 µm, 150 µm, 200 µm, 300 µm, 400 µm, 500 µm, 600 µm, 800 µm, 1000 µm, or more. The wall thickness (e.g., distance between the inner surface and outer surface) may be from 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to 30 µm, 1 µm to 40 µm, 1 µm to 50 µm, 1 µm to 75 µm, 1 µm to 100 µm, 1 µm to 150 µm, 1 µm to 200 µm, 1 µm to 300 µm, 1 µm to 400 µm, 1 µm to 500 µm, 1 µm to 600 µm, 1 µm to 800 µm, 1 µm to 1000 µm, 5 µm to 10 µm, 5 µm to 20 µm, 5 µm to 30 µm, 5 µm to 40 µm, 5 µm to 50 µm, 5 µm to 75 µm, 5 µm to 100 µm, 5 µm to 150 µm, 5 µm to 200 µm, 5 µm to 300 µm, 5 µm to 400 µm, 5 µm to 500 µm, 5 µm to 600 µm, 5 µm to 800 µm, 5 µm to 1000 µm, 10 µm to 20 µm, 10 µm to 30 µm, 10 µm to 40 µm, 10 µm to 50 µm, 10 µm to 75 µm, 10 µm to 100 µm, 10 µm to 150 µm, 10 µm to 200 µm, 10 µm to 300 µm, 10 µm to 400 µm, 10 µm to 500 µm, 10 µm to 600 µm, 10 µm to 800 µm, 10 µm to 1000 µm, 20 µm to 30 µm, 20 µm to 40 µm, 20 µm to 50 µm, 20 µm to 75 µm, 20 µm to 100 µm, 20 µm to 150 µm, 20 µm to 200 µm, 20 µm to 300 µm, 20 µm to 400 µm, 20 µm to 500 µm, 20 µm to 600 µm, 20 µm to 800 µm, 20 µm to 1000 µm, 30 µm to 40 µm, 30 µm to 50 µm, 30 µm to 75 µm, 30 µm to 100 µm, 30 µm to 150 µm, 30 µm to 200 µm, 30 µm to 300 µm, 30 µm to 400 µm, 30 µm to 500 µm, 30 µm to 600 µm, 30 µm to 800 µm, 30 µm to 1000 µm, 40 µm to 50 µm, 40 µm to 75 µm, 40 µm to 100 µm, 40 µm to 150 µm, 40 µm to 200 µm, 40 µm to 300 µm, 40 µm to 400 µm, 40 µm to 500 µm, 40 µm to 600 µm, 40 µm to 800 µm, 40 µm to 1000 µm, 50 µm to 75 µm, 50 µm to 100 µm, 50 µm to 150 µm, 50 µm to 200 µm, 50 µm to 300 µm, 50 µm to 400 µm, 50 µm to 500 µm, 50 µm to 600 µm, 50 µm to 800 µm, 50 µm to 1000 µm, 75 µm to 100 µm, 75 µm to 150 µm, 75 µm to 200 µm, 75 µm to 300 µm, 75 µm to 400 µm, 75 µm to 500 µm, 75 µm to 600 µm, 75 µm to 800 µm, 75 µm to 1000 µm, 100 µm to 150 µm, 100 µm to 200 µm, 100 µm to 300 µm, 100 µm to 400 µm, 100 µm to 500 µm, 100 µm to 600 µm, 100 µm to 800 µm, 100 µm to 1000 µm, 150 µm to 200 µm, 150 µm to 300 µm, 150 µm to 400 µm, 150 µm to 500 µm, 150 µm to 600 µm, 150 µm to 800 µm, 150 µm to 1000 µm, 200 µm to 300 µm, 200 µm to 400 µm, 200 µm to 500 µm, 200 µm to 600 µm, 200 µm to 800 µm, 200 µm to 1000 µm, 300 µm to 400 µm, 300 µm to 500 µm, 300 µm to 600 µm, 300 µm to 800 µm, 300 µm to 1000 µm, 400 µm to 500 µm, 400 µm to 600 µm,WSGR Ref. No.55076-707.601 400 µm to 800 µm, 400 µm to 1000 µm, 500 µm to 600 µm, 500 µm to 800 µm, 500 µm to 1000 µm, 600 µm to 800 µm, 600 µm to 1000 µm, or 800 µm to 1000 µm. In an example, the wall thickness (e.g., distance between the inner and outer surface) is from about 20 µm to 500 µm. In another example, the wall thickness (e.g., distance between the inner and outer surface) is at least about 50 µm.

[0154] In some embodiments, the minimodules are arranged in levels or layers. The layers may be fluidically connected to one another. A layer may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minimodules. Channels within a layer may have the same internal diameter. Alternatively, internal diameter of channels within a layer may vary within the layer. Channels in one layer may have the same internal diameter as channels in another layer. Alternatively, or in addition to, channels in one layer may have different internal diameters as channels in other layers. In an example, a layer comprises channels with varying internal dimensions. In an example, levels or layers of minimodules may be assembled into a macroshape or macrostructure. In another example, internal diameters of channels may be reduced near the edges of the macrostructure. The internal diameters of the channels near an edge of the macrostructure may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or more as compared to average internal dimensions near the center of the macrostructure. Alternatively, internal diameters of channels may be reduced near the center of the macrostructure as compared to internal diameters of channels near edges of the macrostructure. The internal diameters of the channels near the center of the macrostructure may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or more as compared to average internal dimensions near the edge(s) of the macrostructure.

[0155] In some embodiments, the bioreactor can comprise a microfluidic macrostructure 3710, as shown in FIG.37A. In some embodiments, the microfluidic macrostructure may or may not comprise minimodules. In some embodiments, the minimodules can comprise channels. In some embodiments the channels may or may not be connected at connection points. In some embodiments, the connection points are the minimum passage between two interconnected pores e.g., 3710, as shown in FIG.37A. FIG.36A shows the flow velocity profile when the bioreactor does not have equalization layers. Arrows in FIG.36A left panel shows the different flow velocity of the fluid in the macrostructure or macroshape 3610. With no equalization layer, the velocity in the center of the bioreactor may be higher than the perimeter of the bioreactor. FIG. 36A right panel shows the cross-section view of the two layers wherein the diameter of the channels is substantially similar. The circles 3620 demonstrate the channels. An equalization layer may be configured equalize the flow through the macrostructure such that the average velocity across the macrostructure is constant or substantially constant, as shown in FIG.36BWSGR Ref. No.55076-707.601 left panel. FIG.36B right panel shows the cross-section view of an equalization layer 3631 wherein the diameter of the channel at the center is different than at the perimeter and a normal layer 3632 wherein the diameter of the channels is substantially similar. In some embodiments, the diameter increases from the center to the corner. In some embodiments, the diameter decreases from the center to the corner. The diameter and variation of the diameter may correlate to the number and size of inlets, the shape of the macro structure, the number of gyroids, and / or the size of the device, the flow rate. The equalization layer can be at any suitable location of the bioreactor. The macrostructure may be configured such that flow across the macrostructure varies by less than or equal to about 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1%, or less.

[0156] In some embodiments, at least one minimodule can comprise a triply periodic minimal surface. In some embodiments, the triply periodic minimal surface can comprise a gyroid structure. In some embodiments, the gyroid structure can comprise a single gyroid; a modified single gyroid; a double gyroid; a modified double gyroid; or / and any combinations thereof. In some embodiments, the at least one minimodule comprises a single gyroid. FIG.37B schematically illustrates an example macrostructure (or bioreactor) formed of single gyroid minimodules. A fluid (including cells and culture medium) can be fed into the bioreactor through inlet 3770 and the fluid (including cells, products made by cells such as proteins, monoclonal antibodies, etc.) can exit the bioreactor through outlet 3760. The fluid may be in a laminar flow 3740 inside the channel. The bioreactor is gas permeable or partially permeable. A gas 3730 can penetrate or diffuse through the wall of the channel into the channel and mix with the fluid inside the channel.

[0157] The velocity of liquid media and cells in each level may be the same or may vary across the levels. In an example, the velocity of liquid media and cells is substantially the same in each level. Alternatively, or in addition to, the velocity of the liquid media in each level or layer may vary. For example, the velocity of the liquid media may increase or decrease between levels or layers. The velocity of the liquid media may vary from minimodule to minimodule or may be substantially the same between minimodules.

[0158] In some embodiments, the production bioreactor further comprises a liquid media input device. The liquid media device may be structured to provide liquid media to each level of minimodules within the macrostructure. In some embodiments, the volume of liquid media provided to each level maintains a substantially constant cell density in each of the levels. Channel radius may be linked to the radius of the cells, cell density or to other parameters (e.g., filamentous arrangements, chain arrange, etc.). In some embodiments, cell density can vary from 1 x 106cells / ml to 1 x 1012cells / ml. In some embodiments, the velocity of the liquid mediaWSGR Ref. No.55076-707.601 through each minimodule is determined by the cell division rate such that the time for a cell to traverse a single minimodule or level of minimodules is substantially the same as the cell division rate or is proportional to the cell division rate so that the cell divides greater than or equal to 1, 2, 3, 4, 5, or more than 5 times during the transit.

[0159] An inner surface of the channel may contact media, cells, or both media and cells. The channel may be formed of a biocompatible material. Alternatively, the channel may be formed of a bioincompatible material. The inner surface, outer surface, or both surfaces may be coated in a biocompatible material to permit the use of biologically incompatible materials for cell culture applications. In an example, the channel is formed of a biocompatible material. The biocompatible material may be configured to be or may be sterilizable, autoclavable, or both. Sterilizable or autoclavable materials may permit the bioreactor cartridge to be reused, increasing the efficiency of the bioproduction system. The biocompatible material may be a 3D printable bioink as described elsewhere herein. Cell culture systems and methods of use

[0160] In another aspect, the present disclosure provides a system for biological production. The system may comprise a first module, a second module, a third module, and a fourth module. The first module may comprise a reservoir configured to contain or that contains at least one consumable material. The second module may be in fluid communication with the first module. The second module may comprise at least one inoculation device. The inoculation device may be configured to, or may, (i) contain and generate a plurality of cells from at least one cell, (ii) direct a cells of the plurality of cells to different segments of the inoculation device, and (iii) iteratively generate a set of growth conditions for the plurality of cells. Cell growth conditions in the different segments may be individually configurable. The third module may be in fluid communication with the first module and the second module. The third module may comprise at least one bioproduction chamber enclosing at least one bioreactor. The at least one bioreactor may be configured to, or may, (i) interface with the reservoir, (ii) interface with the inoculation module, (iii) control biologic production conditions that may or may not be individually configurable, (iv) rotate, rock, vibrate, or otherwise disrupt sedimentation within the bioreactor, and (v) direct a plurality of biological production to a fourth module. The fourth module may be in fluid communication with the third module. The fourth module may comprise a harvesting module. In an example, the first, second, third, and fourth modules are fluidically interconnected.

[0161] Provided herein are systems and components for producing biological products, biochemical, or chemical substances. FIGS.16A and 16B schematically illustrate a system forWSGR Ref. No.55076-707.601 producing biological, biochemical, or chemical products. FIG.16A shows an example production system. The system may comprise a production module 1630 configured to hold the bioreactor macrostructure under select environmental conditions. The system may comprise a local user interface 1600 configured to provide the user with key information about the ongoing bioprocess or maintenance routine. The system may comprise an inoculation module 1620 configured to retain a cell chip (e.g., cell-line-on-a-chip) which may be configured to provide a continuous flow of fresh cells lines to the production module. The system may comprise a harvest module 1680 with a replaceable cartridge configured to maintain the output of the bioreactor (e.g., production module) for use or further downstream processing. In an example, the harvesting module may include an ultrasonic harvesting system. The ultrasonic harvesting system may use high-frequency sound waves to separate or extract cells, biologic products produced from the cells, or both from the liquid media.

[0162] FIG.16B schematically illustrates example internal components for an example system. The systems and components can be modular (e.g., comprising or involving a module or a plurality of modules as the basis of design or construction) and interconnectable. In some embodiments, the system comprises one or more modules. In some embodiments, the system comprises at least one, at least two, at least three, at least four, at least five, at least six, or more modules. In some embodiments, the system comprises more than three modules. In some embodiments, one or more modules are configured for laminar flow of liquid (e.g., media, media and cells, and / or solvents). In some embodiments, the one or more modules in the system can be interconnected. In some embodiments, the system can comprise a first module 1610. In some embodiments, the first module 1610 can comprise a reservoir 1611 configured to contain at least one consumable material. In some embodiments, the reservoir can comprise a cartridge system. In some embodiments, the system can comprise a second module. In some embodiments, the second module may or may not be in fluid communication with the first module. In some embodiments, the second module can comprise at least one inoculation device 1620 configured to contain at least one cell and to direct a subset of cells from the plurality of cells to different segments. In some embodiments, the cell growth conditions in the different segments can be individually configurable. In some embodiments, the cell growth conditions can iteratively generate a set of growth conditions for the plurality of cells. In some embodiments, the second module can comprise an inoculation chamber, as shown in FIG.19. In some embodiments, the second module can comprise at least one inlet 1920, a culture chamber 1910, at least one outlet 1930, or any combination thereof.

[0163] In some embodiments, the system can comprise a third module. In some embodiments, the third module may or may not be in fluid communication with the first moduleWSGR Ref. No.55076-707.601 and the second module. In some embodiments, the third module can comprise at least one bioproduction chamber 1630, as shown in FIG.17. In some embodiments, the bioproduction chamber can comprise at least one bioreactor 1640 and 1705. In some embodiments, the bioreactor may or may not be configured to interface with the reservoir; may or may not be configured to interface with the inoculation module. In some embodiments, the biologic production conditions may or may not be individually configurable. In some embodiments, the third module can be configured to direct a plurality of biologic production to a fourth module. In some embodiments, the bioreactor can comprise one or more fluidically connected minimodules 1640 and 1705. In some embodiments, the minimodules can form a plurality of channels. The channels can comprise an inner surface and an outer surface. In some embodiments, the channels are permeable to gas diffusion between the inner surface and the outer surface. In some embodiments, the inner surface and / or the outer surface is impermeable to liquid. In some embodiments, the bioreactor may further comprise at least one inlet 1710 fluidically connected to at least one channel of the microfluidic macrostructure. In some embodiments, the bioreactor may further comprise at least one outlet 1735 fluidically connected to at least one channel of the microfluidic macrostructure. In some embodiments, the gyroid structure may comprise a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, or any combinations thereof. In some embodiments, the at least one minimodule can comprise a single gyroid. In some embodiments, the bioreactor can comprise a counter sedimentation system 1660, as shown in FIG.18. In some embodiments, the bioproduction chamber can comprise a heating system 1650 and 1720. In some embodiments, the heating system can comprise a heating mesh 1720. In some embodiments, the bioproduction chamber can comprise a counter sedimentation system 1660, 1730, 2060, and 2430, and as shown in FIGS.18, 20, and 23. In some embodiments, the system can comprise a counter sedimentation system. In some embodiments, the counter sedimentation system can comprise a rocking system; an ultrasonic system; a vibratory system; a density modified system and / or a push and pull system. In some embodiments, and as shown in FIG.21, the bioproduction chamber can comprise a gas supplier system 2110. FIG.22 shows another example follow-up test configuration. In some embodiments, the bioproduction chamber can comprise inlets 2210 (can comprise one inlet for water to humify the environment and one inlet to add CO2), peristaltic pumps 2220, and a temperature probe 2230. FIG.23 shows another example follow-up test configuration. In some embodiments, the bioproduction chamber 2420 can comprise inlets 2410 (can comprise one inlet for water to humify the environment and one inlet to add CO2), roller / rocking system 2430, heating mesh 2470, and peristaltic pumps 2440. The bioproduction chamber 2420 may be connected to a culture medium reservoir 2450 and an inoculum reservoir 2460.WSGR Ref. No.55076-707.601

[0164] In some embodiments, the bioproduction chamber can comprise a moisturizing system 2120. In some embodiments, the bioproduction chamber can comprise at least one inlet 1710, 2010, 2130, 2210, and 2410. In some embodiments, the bioproduction chamber can comprise at least one outlet 1723 and 2020. FIG.24 schematically illustrates an example bioproduction diagram. The bioprocessor can comprise an inoculation module and a production module that are interconnected. The inoculation module can comprise a supply module to supply medium and cells. After inoculation, the cells can be supplied to the production module. The production module can comprise a gyroid structure. In some embodiments, the production module can comprise a single gyroid structure. In some embodiments, the single gyroid structure can comprise at least one channels for the liquid (medium, or medium and cells) to pass through. In some embodiments, the production module may work without a gas channel. In some embodiments, the production module may comprise gas permeable and liquid impermeable channel walls that allow the gas to penetrate through to enter the inner space of the production module. In some embodiments, the production module may be encased in a chamber. In some embodiments, the chamber may comprise an inlet to supply gas to the chamber, and the gas may permeate to the inside of the production module. The production module can comprise a supply module to supply medium to the production module. In some embodiments, the supply module may be encased in the same chamber. In some embodiments, the supply module may be independent of the chamber.

[0165] In some embodiments, the system can comprise a fourth module. In some embodiments, the system may or may not be in fluid communication with a fourth module. In some embodiments the fourth module may comprise a sampling platform 1670. In some embodiments, the sampling platform can be configured to automatically take samples from at least one bioreactor of the third module. In some embodiments, the fourth module may comprise a peristaltic pump 1690.

[0166] In some embodiments, the system can comprise a fifth module. In some embodiments the fifth module may or may not be in fluid communication with the third and the fourth module. In some embodiments, the fifth module can comprise a harvesting module 1680. In some embodiments, the harvesting module can comprise a cartridge system.

[0167] The system for biological production may comprise at least one reservoir. The reservoir may comprise a cartridge system configured to receive a cartridge. The cartridge may be a consumable cartridge.

[0168] The system for biological production may comprise at least on inoculation module. The inoculation module may comprise a continuous microbioreactor configured to provide a constant source of cells to the bioreactor.WSGR Ref. No.55076-707.601

[0169] The system for biological production may comprise at least one harvest module. A harvest module may comprise a cartridge system configured to receive or that receives a cartridge. A harvest module may comprise an ultrasonic harvesting system. The harvesting module may initiate harvesting procedures when a given cell concentration is achieved. Harvesting procedures may include washing culture medium with a buffer (e.g., phosphate- buffered saline), EDTA, or both to remove cellular debris. The bioreactor may be subjected to sonication using an ultrasonic source. Sonication may promote cell detachment. The ultrasonic frequency may be greater than to equal to about 10 kilohertz (kHz), 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 80 kHz, 100 kHz, or greater. In an example the ultrasonic frequency is greater than or equal to 40 kHz. The bioreactor may be subjected to the ultrasonic frequency for a time period of greater than or equal to about 30 seconds, 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, 30 min, or more. The bioreactor may be subjected to the ultrasonic frequency for a time period of less than or equal to about 30 min, 20 min, 15 min, 10 min, 8 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, 30 seconds, or less. The bioreactor may be subjected to enzymatic detachment prior to, during, or subsequent to subjecting the bioreactor to the ultrasonic frequency. In an example, the bioreactor is subjected to enzymatic detachment subsequent to subjecting the bioreactor to the ultrasonic frequency.

[0170] The system for biological production may comprise at least one production module. The production module may comprise a chamber. The chamber may be configured to retain (or contain) or may retain the bioreactor. The chamber may be configured to retain or may retain at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, or more bioreactor macrostructures. During operation, the chamber may be maintained at a pressure configured to provide gaseous nutrients through the wall(s) of the channels of the bioreactor. The chamber may be operated at a pressure of greater than or equal to about 0.1 atmospheres (atm), 0.5 atm, 1 atm, 5 atm, 10 atm, or more. The chamber may be operated at a pressure of less than or equal to about 10 atm, 5 atm, 1 atm, 0.5 atm, 0.1 atm, or less. The chamber may be operated at a pressure from about 0.1 atm to 0.5 atm, 0.1 atm to 1 atm, 0.1 atm to 5 atm, 0.1 atm to 10 atm, 0.5 atm to 1 atm, 0.5 atm to 5 atm, 0.5 atm to 10 atm, 1 atm to 5 atm, 1 atm to 10 atm, or 5 atm to 10 atm.

[0171] The system for biological production may comprise at least one counter suspension module. The counter suspension module may comprise a rocking unit. The rocking unit may comprise a sedimentation unit. The counter suspension unit may further comprise an ultrasonic unit. The counter suspension unit may comprise a vibratory unit. The counter suspension unit may comprise a density modified unit. The counter suspension unit may comprise a push and pull unit.

[0172] The system may further comprise at least one sensor. The system may comprise atWSGR Ref. No.55076-707.601 least 1, 2, 3, 4, 6, 8, 10, 15, 20, or more sensors. The sensors may measure one or more biological, physical, or chemical parameters. The one or more biological parameters may include, but are not limited to, cell division rate, cell growth rate, a cell stress response, cell protein content, cell carbohydrate content, cell lipid content, cell nucleic acid content, or any combination thereof. The one or more physical parameters may include, but are not limited to, cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity, temperature, pressure, or any combination thereof. The one or more chemical parameters may include, but are not limited to, pH, liquid media composition, concentration of individual liquid media component, gas composition, gas concentration, dissolved gas concentration, or any combination thereof. The system may include one or more temperature sensors, pH sensors, glucose sensors, cell production sensors, metabolite production sensors, ultrasound sensors, pressure sensors, infrared sensors, current sensors, gas sensors, light sensors, position sensors, sound sensors, color sensors, hyper spectral sensors, level sensors, moisture sensors, or any combinations thereof.

[0173] The system may comprise one or more control units. The one or more control units may monitor, control, or monitor and control nutrient concentration, temperature, pH, ultrasound, pressure, current, gas flow, gas concentration, light, position, moisture, or any combination thereof in the system.

[0174] The system may be configured to be used or may be used at zero gravity (e.g., in space). The laminar flow configuration, channel configuration, and production chamber may permit zero gravity cell culture without development of phase separation due to gas bubbles within the media that may interfere with cell culture at zero gravity.

[0175] The system may comprise one or more bioprocessors. A bioprocessor may be configured to retain a bioproduction chamber. The bioproduction chamber may be configured to retain a bioreactor cartridge. The bioreactor cartridge may comprise the bioreactor macrostructure. In an example, the system comprises a plurality of bioprocessors (e.g., stacked on top of one another). A bioprocessor of the plurality of bioprocessors may comprise one bioproduction chamber encapsulating one bioreactor cartridge. Alternatively, or in addition to, a bioprocessor may comprise a plurality of bioproduction chambers each comprising a bioreactor cartridge. A bioprocessor may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more bioproduction chambers. A bioproduction chamber may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more bioreactor cartridges. As shown in FIGs.47 and 48, the bioprocessor may comprise an environmental control system 4705, 4805 that houses the bioproduction chamber 4730. The environmental control system 4705, 4805 may be configured to control or may control the temperature, oxygen content, etc. of within and around the bioreactor. The bioprocessor mayWSGR Ref. No.55076-707.601 have a user interface 4710, 4810 that permits a user to control bioprocessing, monitor bioprocessing, or both. The bioprocessor may have one or more fluidic systems 4715, 4815 that control fluid flow through the channels of the bioreactor. The bioprocessor may be coupled to a media formulation system 4720, 4820 that is configured to generate tailored media formulations. The bioprocessor may be coupled to a harvesting system 4725, 4825 that is configured to collect cells or cell products from the bioreactor. As shown in FIG.48, the bioprocessing system may further comprise a gas control system 4840 that controls pressure, flow, and composition of gas provided to the bioreactor. The bioprocessing system may further include a heating and humidity system 4830 that controls the temperature and humidity within the bioproduction chamber and bioreactor. The system may further comprise a counter sedimentation system 4835 that is configured to rotate, rock, vibrate, oscillate, or otherwise disrupt sedimentation within the bioreactor. The counter sedimentation system may rotate, rock, vibrate, or oscillate the bioreactor, the bioproduction chamber, or both.

[0176] FIG.49 shows an example bioprocessor with the bioproduction chamber removed. The heating and humidity system 4930 may be disposed on the bottom, side, or both of the inside of the bioprocessor. The counter sedimentation system 4935 may be disposed in a side wall of the bioprocessor system such that it can couple or otherwise interface with an end of the bioproduction chamber, bioreactor cartridge, or both. The gas control system 4940 may comprise an interface panel disposed on an interior wall of the bioprocessor to permit coupling with the bioproduction chamber. FIG.50 shows an example of a bioprocessor with bioproduction chamber and bioreactor cartridge 5000 in place. FIG.51 shows another example of a bioprocessor with bioproduction chamber and bioreactor cartridge 5100 in place. As shown in FIG.51, both the user interface 5110 and fluidic system 5115 controls may be external facing to permit user access.

[0177] FIGs.52 and 53 show example bioproduction chambers. As shown in FIG.52, the bioproduction chamber may comprise a cylindrical structure that opens at one end. The bioproduction chamber may be configured to seal the bioreactor cartridge 5200 within the chamber. As shown in FIG.53, the bioproduction chamber may comprise a removable door or hatch that permits the bioreactor cartridge 5300 to be accessed and removed from the bioproduction chamber. As described elsewhere herein, the bioreactor cartridge may be a single use consumable. Alternatively, the bioreactor cartridge may be sterilizable or autoclavable to permit repeat usage. The bioreactor cartridge 5300 may be disposed in a cartridge holder 5305 that secures the cartridge within the bioproduction chamber. The bioproduction chamber may comprise an aseptic container 5310 that maintains the bioreactor in an aseptic environment. The bioproduction chamber may comprise one or more sensors 5315 for monitoring bioprocessingWSGR Ref. No.55076-707.601 and an actuator 5320 to permit movement of the bioproduction chamber or the bioreactor cartridge.

[0178] The system may comprise one or more consumables. For example, media reservoirs, inoculation components, harvesting components, the bioproduction chamber, bioreactor, or any combination thereof may be a single use consumable. In an example, the bioproduction chamber is integrated with the bioreactor such that the bioreactor cannot be removed and the entire bioproduction chamber is a consumable. In another example, the bioreactor cartridge is removable from the bioproduction chamber and the bioreactor cartridge is a consumable and the bioproduction chamber is reusable. Methods of forming bioreactor structures

[0179] In another aspect, the present disclosure provides a three dimensional (3D) printable bioink (e.g., bioink or resin). The 3D printable bioink (e.g., bioink or resin) may comprise at least one polymerizable component, at least one photoinitiator, at least one thermocuring agent, or combinations thereof. In an example, the bioink comprises at least one polymerizable component, at least one photoinitiator, and at least one thermocuring agent.

[0180] Provide herein are processes and methods that, among other implementations, provides components and a method that facilitates an effective chemical or physical production of light-curable, durable, porous, hydrophobic, biocompatible, sterilizable, high resolution, and gas-permeable 3D printed bespoke bioreactors. In some embodiments, each material set comprises (i) one or more polymerizable materials, (ii) one or more light sensitive materials, wherein the polymerizable material may be in a form of monomers and / or oligomers; (iii) one or more initiators or catalysts capable of causing polymerization of the polymerizable monomers / oligomers; or transformation of the light-sensitive materials in the set. In some embodiments, the polymerizable materials comprise a corresponding initiator capable of inducing polymerization by light of a particular wavelength. In an example, the polymerization wavelength may be about 405 nanometers (nm).

[0181] The bioinks or resins described herein may be configured for printing intricate 3D geometries for use in, for example, bioreactors for the production of biologics. The bioinks and resins described herein may be configured for high resolution light-polymerization. Upon polymerization, the bioinks or resins may be biologically compatible or may not be biologically compatible. In an example, a bioink or resin may have low biological compatibility and the polymerized structure may be coated with a biologically compatible layer.

[0182] The bioink, or resin, may comprise at least 1, 2, 3,4, 5, 6, 8, 10, or more polymerizable components. In an example, the polymerizable component is polyethylene glycolWSGR Ref. No.55076-707.601 diacrylate 250 (PEGDA MW 250). The bioink may comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more polymerizable component by weight percent. The bioink may comprise less than or equal to about 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or less polymerizable component by weight percent. The bioink may comprise from about 50% to 55%, 50% to 60%, 50% to 65%, 50% to 70%, 50% to 75%, 50% to 80%, 50% to 85%, 50% to 90%, 55% to 95%, 55% to 98%, 55% to 60%, 55% to 65%, 55% to 70%, 55% to 75%, 55% to 80%, 55% to 85%, 55% to 90%, 55% to 95%, 55% to 98%, 60% to 65%, 60% to 70%, 60% to 75%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 98%, 65% to 70%, 65% to 75%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 98%, 70% to 75%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 98%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 98%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 98%, 85% to 90%, 85% to 95%, 85% to 98%, 90% to 95%, 90% to 98%, or 90% to 98% polymerizable component by weight percent. In an example, the bioink comprises from about 70% to 98% by weight polymerizable component. In another example, the bioink comprises from about 70% to 98%, weight by weight, PEGDA MW 250.

[0183] The bioink, or resin, may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more photoinitiators. In an example, the photoinitiator comprises 2,2phenylbis(2,4,6- trimethylbenzoyl) phosphine oxide (BAPO). The bioink may comprise great than or equal to about 1%, 2%, 4%, 6%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, or more photoinitiator by weight. The bioink may comprise less than or equal to about 50%, 40%, 30%, 20%, 15%, 10%, 8%, 6%, 4%, 2%, 1%, or less photoinitiator by weight. The bioink may comprise from about 1% to 2%, 1% to 4%, 1% to 6%, 1% to 8%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 2% to 4%, 2% to 6%, 2% to 8%, 2% to 10%, 2% to 15%, 2% to 20%, 2% to 30%, 2% to 40%, 2% to 50%, 4% to 6%, 4% to 8%, 4% to 10%, 4% to 15%, 4% to 20%, 4% to 30%, 4% to 40%, 4% to 50%, 6% to 8%, 6% to 10%, 6% to 15%, 6% to 20%, 6% to 30%, 6% to 40%, 6% to 50%, 8% to 10%, 8% to 15%, 8% to 20%, 8% to 30%, 8% to 40%, 8% to 50%, 10% to 15%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 15% to 20%, 15% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 40%, 30% to 50%, or 40% to 50% photoinitiator by weight. In an example, the bioink comprises from about 2% to 30% by weight photoinitiator. In another example, the bioink comprises from about 2% to 30%, weight by weight, BAPO.

[0184] The bioink, or resin, may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more thermocuring agents. In an example, the thermocuring agent comprises 2,2′-Azobis(2- methylpropionitrile) (AIBN). The bioink may comprise greater than or equal to about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, or more thermocuring agent by weight. TheWSGR Ref. No.55076-707.601 bioink may comprise less than or equal to about 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less thermocuring agent by weight. The bioink may comprise from about 0.01% to 0.05%, 0.01% to 0.1%, 0.01% to 0.5%, 0.01% to 1%, 0.01% to 5%, 0.01% to 10%, 0.01% to 20%, 0.05% to 0.1%, 0.05% to 0.5%, 0.05% to 1%, 0.05% to 5%, 0.05% to 10%, 0.05% to 20%, 0.1% to 0.5%, 0.1% to 1%, 0.1% to 5%, 0.1% to 10%, 0.1% to 20%, 0.5% to 1%, 0.5% to 5%, 0.5% to 10%, 0.5% to 20%, 1% to 5%, 1% to 10%, 1% to 20%, 5% to 10%, 5% to 20%, or 10% to 20% thermocuring agent by weight. In an example, the bioink comprises from about 0.01% to 10% thermocuring agent by weight. In another example, the bioink comprises from about 0.01% to 10%, weight by weight, AIBN.

[0185] The bioink, or resin, may further comprise a porogen. The porogen may comprise polyethylene glycol 200 (PEG-200), PEG-400, Triton X, or any combination thereof. The bioink may comprise greater than or equal to about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more by weight porogen. The bioink may compress less than or equal to about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less porogen by weight. The bioink may comprise from about 1% to 5%, 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 80%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 50% to 60%, 50% to 70%, 50% to 80%, 60% to 70%, 60% to 80%, or 70% to 80% porogen by weight. In an example, the bioink comprises from about 30% to 70% by weight PEG-200. In another example, the bioink comprises from about 30% to 70% by weight PEG-400. In another example, the bioink comprises from about 10% to 50% by weight Triton X.

[0186] The bioink, or resin, may further comprise a light blocker. In an example, the light blocker comprises Avobenzone (Avo). The bioink may comprise greater than or equal to about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, or greater light blocker by weight. The bioink may comprise less than or equal to about 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, or less light blocker by weight. The bioink may comprise from about 0.0001% to 0.001%, 0.0001% to 0.01%, 0.0001% to 0.1%, 0.0001% to 1%, 0.0001% to 10%, 0.001% to 0.01%, 0.001% to 0.1%, 0.001% to 1%, 0.001% to 10%, 0.01% to 0.1%, 0.01% to 1%, 0.01% to 10%, 0.1% to 1%, 0.1% to 10%, or 1% to 10% light blocker by weight. Light blocker concentration may be configured for printing resolution and bioink transmittance. In an example, the bioink comprises from about 0.001% to 1% light blocker by weight. In another example, the bioink comprises from about 0.001% to 1%, weight by weight, Avo.WSGR Ref. No.55076-707.601

[0187] The bioink, or resin, may further comprise a plasticizer. In an example, the plasticizer comprises Decanol (e.g., n-decanol, decan-1-ol, capric alcohol, etc.). The bioink may comprise greater than or equal to about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or more plasticizer by weight. The bioink may comprise less than or equal toa bout 40%, 30%, 25%, 20%, 15%, 10%, 5%, or less plasticizer by weight. The bioink may comprise from about 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 40%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 40%, 20% to 25%, 20% to 30%, 20% to 40%, 25% to 30%, 25% to 40%, or 30% to 40% plasticizer by weight. In an example, the bioink comprises from about 15% to 30% plasticizer by weight. In another example, the bioink comprises from about 15% to 30%, weight by weight, decanol.

[0188] The bioink, or resin, may further comprise a filler. In an example, the filler comprises hydroxyapatite. The bioink may comprise greater than or equal to about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, or more filler by weight. The bioink may comprise less than or equal to about 20%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less filler by weight. The bioink may comprise from about 0.01% to 0.05%, 0.01% to 0.1%, 0.01% to 0.5%, 0.01% to 1%, 0.01% to 5%, 0.01% to 10%, 0.01% to 20%, 0.05% to 0.1%, 0.05% to 0.5%, 0.05% to 1%, 0.05% to 5%, 0.05% to 10%, 0.05% to 20%, 0.1% to 0.5%, 0.1% to 1%, 0.1% to 5%, 0.1% to 10%, 0.1% to 20%, 0.5% to 1%, 0.5% to 5%, 0.5% to 10%, 0.5% to 20%, 1% to 5%, 1% to 10%, 1% to 20%, 5% to 10%, 5% to 20%, or 10% to 20% filler by weight. In an example, the bioink comprises from about 1% to 10% filler by weight. In another example, the bioink comprises from about 1% to 10%, weight by weight, hydroxyapatite. In another example, the bioink comprises greater than or equal to about 5%, weight by weight, hydroxyapatite. In another example, the hydroxyapatite comprises modified hydroxyapatite. The modified hydroxyapatite is functionalized with carbon (HApC12)-dodecanol-.

[0189] The bioink, or resin, may further comprise a rheological enhancer. The rheological enhancer and the biocompatibility coating may be the same or different coatings. The rheological enhancer may enhance biocompatibility and improve rheological characteristics by preventing or slowing swelling In some embodiments, the rheological enhancer can comprise a coating material. In some embodiments, the coating material can comprise a material that is not reactive to the resin. In some embodiments, the coating may comprise a dilution of the coating material. In some embodiments, the coating process can further comprise a stage of heat treating the object in oxygen, air, or in an inert gas environment. In some embodiments, the heat treatment can comprise thermal radiation, microwave radiation, pressure, or chemical treatment, to curate the coating. The rheological enhancer may comprise Decanol (e.g., n-decanol, decan- 1-ol, capric acid, etc.) or silica (e.g., KH550, (3-aminoprpil) trietoxisilano), etc.). The bioinkWSGR Ref. No.55076-707.601 may comprise greater than or equal to about 5%, 10%, 15%, 20%, 25%, 30%, 40%, or more rheological enhancer by weight. The bioink may comprise less than or equal to about 40%, 30%, 25%, 20%, 15%, 10%, 5%, or less rheological enhancer by weight. The bioink may comprise from about 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 40%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 40%, 20% to 25%, 20% to 30%, 20% to 40%, 25% to 30%, 25% to 40%, or 30% to 40% rheological enhancer by weight. In an example, the bioink comprises from about 15% to 30% rheological enhancer by weight. In another example, the bioink comprises from about 15% to 30%, weight by weight, decanol or silica.

[0190] The bioink may comprise one or more non-organic materials. Non-organic materials may include metals, ceramics, composites, or any combination thereof. The non-organic materials may be particulates within the bioink or may be dissolved within the bioink. The non- organic materials may be homogenously dispersed within the bioink or heterogeneously dispersed within the bioink. Alternatively, or in addition to, the non-organic material may be a powder or other solid that is sintered to generate a 3D structure. The sintered powder may be deposited layer by layer to generate the 3D structure. In an example, the bioink comprises a metal. The metal may be one or more of steel, a nickel-based superalloy, aluminum, low-carbon austenitic stainless steel, or any combination thereof. In another example, the bioink comprises a ceramic. The ceramic may be one or more of alumina, zirconia, silicon carbide, porcelain, titania, borosilicate, carbon, tricalcium phosphate, hydroxyapatite, lithium silicate glass ceramics, bioactive glass, aluminosilicate glass ceramics, phosphate-based glass ceramics, clay, or any combination thereof.

[0191] The bioink may be compatible with any 3D printing technique. For example, the bioink may be compatible with metal powder bed fusion (PBF), direct metal laser sintering (DMLS), selective laser melting (SLM), electron beam melting (EBM), directed energy deposition (DED), binder jetting of metallic or ceramic powders, cold spray additive manufacturing, ultraviolet vat photopolymerization, stereolithography (SLA), digital light processing (DLP), fused filament fabrication (FFF), fused deposition modeling (FDM), multi-jet printing (MJP), PolyJet printing, two-photon polymerization (2PP), electrohydrodynamic printing (EHD), inkjet bioprinting, laser-assisted bioprinting, robocasting, and / or material extrusion of ceramic pastes or slurries. Any one of these techniques may be used to generate the bioreactor from the bioink.

[0192] The bioink may comprise at least one surface that is compatible with growing or maintaining viability of living cells. In an example, the bioink is compatible with growing or maintaining viability of living cells. The bioink, or resin, may further comprise aWSGR Ref. No.55076-707.601 biocompatibility enhancer. Alternatively, the polymerized bioink may be coated or layered with a biocompatibility enhancer. The biocompatibility enhancer may be applied in layers or coatings to at least one surface of the polymerized bioink. The at least one surface of the polymerized bioink may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or more layers of biocompatibility enhancer. The at least one surface of the polymerized bioink may have less than or equal to 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less layers of biocompatibility enhancer. The at least one surface of the polymerized bioink may have from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 12, 2 to 15, 2 to 20, 2 to 25, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 12, 3 to 15, 3 to 20, 3 to 25, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 12, 4 to 15, 4 to 20, 4 to 25, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 12, 5 to 15, 5 to 20, 5 to 25, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 12, 6 to 15, 6 to 20, 6 to 25, 7 to 8, 7 to 9, 7 to 10, 7 to 12, 7 to 15, 7 to 20, 7 to 25, 8 to 9, 8 to 10, 8 to 12, 8 to 15, 8 to 20, 8 to 25, 9 to 10, 9 to 12, 9 to 15, 9 to 20, 9 to 25, 10 to 12, 10 to 15, 10 to 20, 10 to 25, 12 to 15, 12 to 20, 12 to 25, 15 to 20, 15 to 25, or 20 to 25. In an example, the at least one surface comprises between 2 and 12 layers of biocompatibility enhancer. In another example, the at least one surface comprises at least 6 layers of biocompatibility enhancer. The biocompatibility enhancer may comprise polydimethylsiloxane (PDMS).

[0193] The biocompatibility coating or layer may be hydrophilic, hydrophobic, or neutral. In an example, the coating or layer is hydrophobic. In another example, the coating or layer is neutral. In another example, the coating or layer is hydrophilic. The coating or layer may have a low swelling ratio. The coating or layer may be configured to be robust (e.g., not susceptible to degradability) and have robust mechanical properties. The biocompatibility coating may enhance biocompatibility and improve rheological characteristics by preventing or slowing swelling.

[0194] In an example, the biocompatibility coating or layer may be applied by dip coating. For example, the polymerized macrostructure may be submerged in a solution comprising the biocompatibility coating. After submersion, the solution may be drained from the macrostructure channels. In another example, the biocompatibility coating or layer may be applied by pumping the coating material inside the polymerized macrostructure.

[0195] In some embodiments, the bioreactor disclosed herein can be made from the resin. FIG.1 shows an example polymerization reaction. To provide initiation 110, the initiator 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide can generate radicals that initiates a polymerization reaction. During propagation 120, an initiator radical attacks the un-saturated bonds of the monomer (e.g., polyethylene Glycol Diacrylate 250) to generate a monomerWSGR Ref. No.55076-707.601 radical. The monomer radical attacks an additional monomer to form another radical. The propagation can be repeated a plurality of times to form a polymer radical. During termination 130, two radicals can form a bond that terminates further addition of a monomer.

[0196] In some embodiments, the present disclosure provides a method of printing a functional 3D microfluidic macrostructure. In some embodiments, prior to printing, the resin mixture may be at a temperature from about 10 °C to about 80 °C. In some embodiments, prior to printing, the resin mixture may be at room temperature. In some embodiments, a magnetic stirring may be performed to dissolve any precipitate in the resin. In some embodiments, the printing process may be performed with a radiation source having a wavelength of 300-800 nm. In some embodiments, the radiation source may have a wavelength of 405 nm.

[0197] FIGS.8A and 8B show example objects printed using Anycubic Photon Mono X N°2 printer. FIG.8A shows example microfluidic bioreactors produced using an example resin. FIG.8B shows scale of example microfluidic macrostructure devices. In some embodiments, a plurality of objects can be printed at the same time. In some embodiments, the resin may be recirculated or stirred to maintain homogeneity during the printing, e.g., for long period. In some embodiments, the resin does not need to be recirculated. In some embodiments, the printing can take minutes to hours, depending on the dimensions and complexity of the objects and printing speed of the printer.

[0198] In some embodiments, to set up the objects for cell culturing, the object may be post processed. In some embodiments, the post processing may comprise using rheologic and biocompatibility enhancer materials that may improve the rheologic characteristics and prevent swelling. In some embodiments, post processing may comprise eliminating any residual cytotoxic substances that may be found in the bioreactor, e.g., when triton has been used.

[0199] In some embodiments, the post processing may comprise prewashing to remove superficial and thick part of the object, and / or excess liquid resin that has not cured.

[0200] In some embodiments, the post processing may comprise light curing. In some embodiments, the object may be radiated superficially in a chamber at 385 and 405 nm collide (Wavelength). The light curing may generate the crust and reduce the possibility of polymerization byproducts.

[0201] In some embodiments, the post processing may comprise thermocuring. In some embodiments, the thermocuring may be performed in water, which enters the object to cure internally to increase the degree of polymerization (curing) of the resin. In some embodiments, thermocuring may be performed at a temperature from 30 °C to 90 °C.

[0202] In some embodiments, the post processing may comprise cyclic washing. In some embodiments, the washing process can be conducted several times, depending on the resin andWSGR Ref. No.55076-707.601 the chemical characteristics of the components.

[0203] In some embodiments, the post processing may comprise drying. In some embodiments, the drying can comprise initial drying at room temperature in a hood and overnight drying. In some embodiments, the drying can be performed for 1 hour to 12 hours. In some embodiments, the drying can be performed at a temperature from 30 °C to 90 °C.

[0204] In some embodiments, the microfluidic macrostructures (MM) or the printed bioreactor may be coated with a coating material.

[0205] FIG.12 shows a set up for a coating procedure. The printed bioreactor 1220 may be placed in a transparent container comprising a coating solution, e.g., PDMS solution 1210. The PDMS solution is sufficient to submerge the printed bioreactor 1220. The printed bioreactor 1220 can be immersed in the PDMS solution from 10 to 120 minutes, e.g., 15 minutes. During the coating process, the container may be covered to reduce solvent evaporation. Alternatively, the bioreactor may be connected to a pump system and the biocompatible coating may be pumped through the channels of the bioreactor.

[0206] After the coating procedure (or immersion), the printed bioreactor can be removed from the container and placed on a vertical stand to drain excess PDMS solution (FIG.13).

[0207] In some embodiments, a connector 1410 can be coupled to the inlets of the printed bioreactor to apply compressed air inside the printed bioreactor’s channel to aid drainage of PDMS solution (FIG.14).

[0208] In some embodiments, air may be introduced using a pressure ramp, starting from 100 mbar until reaching 1 bar in approximately 10 seconds. Once 1 bar of pressure is reached, airflow may be maintained for 40 seconds. In some embodiments, the printed bioreactor may be rotated or flipped and the air drying process in FIG.14 disclosed above can be repeated on the opposite entry of the printed bioreactor. In some embodiments, the airflow may be stopped. After a rest period of 10 to 60 min (e.g., 15 min), any remaining droplets hanging from the connectors may be dried, e.g., with a lint-free cloth. In some embodiments, the printed bioreactor may be placed e.g., vertically in an oven and cured for 15 min to 4 hr (e.g., 2 hr) at a temperature from 30 °C to 90 °C (e.g., 80 °C).

[0209] In some embodiments, a touch-dry test can be performed to confirm that the printed bioreactor is fully cured. The touch-dry test may comprise touching the piece with gloves and ensuring it does not stain the glove, does not stick, or have any material detachment.

[0210] In some embodiments, the printed bioreactor may be removed from the oven and cooled to room temperature.

[0211] In some embodiments, the coating or immersion (FIG.12), drying (FIGS.13 and 14), and oven treatment can be repeated a few times, e.g., 3 to 12 times (e.g., 6 times) to coat aWSGR Ref. No.55076-707.601 plurality of layers of PDMS on the bioreactor. FIG.15 shows a layer-by-layer treatment scheme, comprising (i) a first immersion procedure (or coating procedure as shown in FIG.12); (ii) a first oven treatment; (iii) a second immersion procedure; and (iv) a second oven treatment. In some embodiments, the layer-by-layer treatment can be repeated 3 to 12 times. In some embodiments, the printed object can be flipped in different treatment cycle.

[0212] In some embodiments, the coating solution may be stored in the refrigerator during curing periods. In some embodiments, a solvent may be added to the coating solution to compensate for solvent evaporation at the end of each stage. In some embodiments, 0.5 wt% to 5 wt% solvent (e.g., petroleum ether) of the total amount of coating solution can be added.

[0213] In some embodiments, a PDMS coating solution can comprise 5 wt% to 50 wt% of PDMS. In some embodiments, a PDMS coating solution can comprise 10 wt% to 40 wt% of PDMS. In some embodiments, a PDMS coating solution can comprise 5 wt% to 15 wt% of PDMS. In some embodiments, a PDMS coating solution can comprise 30 wt% to 50 wt% of PDMS. In some embodiments, a PDMS coating solution can comprise 50 wt% to 95 wt% of petroleum ether. In some embodiments, a PDMS coating solution can comprise 60 wt% to 90 wt% of petroleum ether. In some embodiments, a PDMS coating solution can comprise 50 wt% to 70 wt% of petroleum ether. In some embodiments, a PDMS coating solution can comprise 85 wt% to 95 wt% of petroleum ether. Computer systems

[0214] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG.46 shows a computer system 4601 that is programmed or otherwise configured to design, manufacture, or implements methods of running a bioreactor. The computer system 4601 can regulate various aspects of bioreactor manufacturing and implementation of the present disclosure, such as, for example, designing a bioreactor, printing a bioreactor, or operating a bioreactor. The computer system 4601 can be a control system for a 3D printer, control system for a bioreactor, or an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0215] The computer system 4601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 4605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 4601 also includes memory or memory location 4610 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 4615 (e.g., hard disk), communication interface 4620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 4625, suchWSGR Ref. No.55076-707.601 as cache, other memory, data storage and / or electronic display adapters. The memory 4610, storage unit 4615, interface 4620 and peripheral devices 4625 are in communication with the CPU 4605 through a communication bus (solid lines), such as a motherboard. The storage unit 4615 can be a data storage unit (or data repository) for storing data. The computer system 4601 can be operatively coupled to a computer network (“network”) 4630 with the aid of the communication interface 4620. The network 4630 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 4630 in some cases is a telecommunication and / or data network. The network 4630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 4630, in some cases with the aid of the computer system 4601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 4601 to behave as a client or a server.

[0216] The CPU 4605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 4610. The instructions can be directed to the CPU 4605, which can subsequently program or otherwise configure the CPU 4605 to implement methods of the present disclosure. Examples of operations performed by the CPU 4605 can include fetch, decode, execute, and writeback.

[0217] The CPU 4605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 4601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0218] The storage unit 4615 can store files, such as drivers, libraries and saved programs. The storage unit 4615 can store user data, e.g., user preferences and user programs. The computer system 4601 in some cases can include one or more additional data storage units that are external to the computer system 4601, such as located on a remote server that is in communication with the computer system 4601 through an intranet or the Internet.

[0219] The computer system 4601 can communicate with one or more remote computer systems through the network 4630. For instance, the computer system 4601 can communicate with a remote computer system of a user (e.g., e.g., Virtual Private Networks, Computer hosted in services such as Amazon Web Services (AWS), Satellite communication). 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 4601 via the network 4630.

[0220] Methods as described herein can be implemented by way of machine (e.g., computerWSGR Ref. No.55076-707.601 processor) executable code stored on an electronic storage location of the computer system 4601, such as, for example, on the memory 4610 or electronic storage unit 4615. 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 4605. In some cases, the code can be retrieved from the storage unit 4615 and stored on the memory 4610 for ready access by the processor 4605. In some situations, the electronic storage unit 4615 can be precluded, and machine-executable instructions are stored on memory 4610.

[0221] The code can be pre-compiled and configured for use with a machine having a processer 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.

[0222] Aspects of the systems and methods provided herein, such as the computer system 4601, can be embodied in programming. Various aspects of the technology may 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 may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may 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 may 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 may 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.

[0223] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may beWSGR Ref. No.55076-707.601 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 may 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 may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0224] The computer system 4601 can include or be in communication with an electronic display 4635 that comprises a user interface (UI) 4640 for providing, for example, bioreactor design configurations, printing options and status, and operational setting and bioreactor status. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0225] 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 4605. The algorithm can, for example, adjust variables of the control systems of the 3D printer or the bioreactor using feedback loops, detect problems in the bioreactor manufacture or use by image recognition and pattern analysis, fuzzy logic and with hard and soft threshold enforcements, correlate specific and unspecific data through machine learning (e.g., Supervised, Unsupervised and / or Reinforcement) to optimize process conditions within the system, the process outcomes, modelling behavior and simulation. EXAMPLES Example 1: Preparation of a base resin (CF-A0000)

[0226] A base resin is prepared with polyethylene glycol diacrylate 250 (PEGDA), phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) and α,α-azoisobutyronitrile (AIBN). Weight of BAPO is about 2% of weight of PEGDA 250 and weight of AIBN is about 0.002% of weight of PEGDA 250.

[0227] 4.5 ± 0.1g of BAPO is weighed on a clean watch glass and added to a 500 ml AmberWSGR Ref. No.55076-707.601 Nalgene Bottle (NB).0.045± 0.001g of AIBN is weighed on a clean and dry watch glass and add to the NB containing BAPO.200 mL PEGDA (225±1 g) is measured using a graduate cylinder and added to the NB containing AIBN and BAPO. The mixture is stirred using a glass rod until the mixture is homogenized. The NB is covered immediately to ensure the mixture is not exposed to light. The NB is placed into an ultrasonic bath using a three-prong clamp and sonicated for 30 minutes. If solids are not completely dissolved, the mixture may be stirred using the glass rod and sonicated for an additional 30 minutes in the ultrasonic bath. The stirring and / or sonication may be repeated until the solids completely dissolve. The mixture can be carried out using an Ultra-turrax (maintaining agitation until complete dissolution is achieved at a speed of 1000 rpm). After the sonication and complete dissolution of the solids, in the event of a rise on the pressure inside the NB, the NB is slowly opened inside the fume hood. Example 2: Preparation of CF-A0028 resin

[0228] CF-A0028 resin is prepared with base resin (CF-A0000, 65% of total weight), modified hydroxyapatite (HApC12, functionalized with carbon, 3% of total weight, structural improvement), 1-decanol (Dc, 4.5% of total weight), and Triton X-100 (Triton, 27.5% of total weight, porogen).

[0229] 27.5 ± 0.1 g Triton is added to a 250 ml Amber Nalgene Bottle (NB).4.5 ± 0.1 g of Dc is added to the NB containing the Triton.3.0 ± 0.1 g of HApC12 is slowly introduced to the NB containing the Dc and Triton. The mixture is sonicated with the Ultra-turrax for 10 minutes at 12000 rpm. In the event of having solids on the NB walls, the solids can be scraped with the glass rod.65.0 ± 0.1 g of the base resin CF-A0000 is added to the mixture and stirred using a glass rod until the resulting mixture is homogenized. The NB is covered immediately to ensure the mixture is not exposed to light.

[0230] In some embodiments, the resin CF-A0028 can comprise CF-A0000 from about 50% to about 100%, from about 70% to about 90%, from about 50% to about 80%, or from about 90% to about 100%. In some embodiments, the resin CF-A0028 can comprise Triton x from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, or from about 20% to about 40%. In some embodiments, the resin CF-A0028 can comprise HApC12 from about 1% to about 15%, from about 1% to about 6%, from about 5% to about 10%, or from about 5% to about 15%.

[0231] Table 1 shows example weight percentage of the components of resin CF-A0028 and coating solution.WSGR Ref. No.55076-707.601 Table 1Example 3: Preparation of CF-A0133 resin

[0232] CF-A0133 is prepared with base resin (CF-A0000) and polyethylene glycol 200 (PEG-200, porogen, 50 wt% of base resin).

[0233] 100.0 ± 0.1 g base resin CF-A0000 is added to a 250 ml Amber Nalgene Bottle (NB). 50.0 ± 0.1 g PEG-200 is slowly introduced to the NB containing the CF-A0000. The mixture is sonicated with the Ultra-turrax for 10 minutes at 12000 rpm. In the event of having solids on the NB walls, the solids can be scraped with the glass rod. The mixture is stirred using a glass rod until homogenized. The NB is covered immediately to ensure the mixture is not exposed to light.

[0234] In some embodiments, the resin CF-A0133 can comprise CF-A0000 from about 40% to about 100%, from about 50% to about 70%, from about 40% to about 60%, or from about 70% to about 100%. In some embodiments, the resin CF-A0133 can comprise PEG200 fromWSGR Ref. No.55076-707.601 about 10% to about 60%, from about 10% to about 40%, from about 30% to about 50%, or from about 50% to about 60%.

[0235] Table 2 shows example weight percentage of the components of resin CF-A0133 and coating solution. Table 2Example 4: Preparation of CF-A0133AVO resin

[0236] CF-A0133AVO resin is prepared with base resin (CF-A0000), polyethylene glycol 200 (PEG-200, porogen, 50 wt% of base resin), and avobenzone (Avo, light blocker, 0.5 wt% of base resin).

[0237] 100.0 ± 0.1 g of CF-A0000 is added to a 250 ml Amber Nalgene Bottle (NB).50.0 ± 0.1 g PEG-200 is slowly introduced into the NB containing the CF-A0000.0.5 ± 0.1 g Avo is slowly introduced into the NB containing the CF-A0000 and PEG-200. The dissolution of Avo is slow. The mixture is sonicated with the Ultra-turrax for 10 minutes at 12000 rpm. In the event of having solids on the NB walls, the solids can be scraped with the glass rod.WSGR Ref. No.55076-707.601

[0238] In some embodiments, the resin CF-A0133AVO can comprise CF-A0000 from about 40% to about 100%, from about 50% to about 70%, from about 40% to about 60%, or from about 70% to about 100%. In some embodiments, the resin CF-A0133AVO can comprise PEG200 from about 10% to about 60%, from about 10% to about 40%, from about 30% to about 50%, or from about 50% to about 60%.

[0239] Table 3 shows example weight percentage of the components of resin CF- A0133AVO and coating solution. Table 3Example 5: Preparation of CF-A0233 resin

[0240] CF-A0233 is prepared with base resin (CF-A0000), polyethylene glycol 400 (PEG- 400), and decanol (Dc).WSGR Ref. No.55076-707.601

[0241] 100.0 ± 0.1 g of CF-A0000 is added to a 250 ml Amber Nalgene Bottle (NB).50.0 ± 0.1 g PEG-400 is slowly introduced into the NB containing the CF-A0000.5.0 ± 0.1 g Dc is slowly introduced into the NB containing the CF-A0000 and PEG-400. The mixture is sonicated for 10 minutes or with the Ultra-turrax for 2 minutes at 10000 rpm. The mixture is then mixed with the glass rod until it is homogenized, followed by degasifying for 5 minutes to eliminate any bubble. The NB is covered to ensure that the mixture is not exposed to light.

[0242] Table 4 shows example weight percentage of the components of resin CF-A0233 and coating solution. Table 4Example 6: Preparation of coating material

[0243] The coating material is prepared with polydimethylsiloxane (PDMS). PDMS is weighed and added to a plastic container. A solvent, e.g., petroleum ether or ethanol, is added toWSGR Ref. No.55076-707.601 the container, and the mixture is stirred until a transparent and homogeneous mixture is obtained. The solution is then stored in the refrigerator until ready to use. Example 7A: Coating well-type printed objects

[0244] The well type printed objects are coated with a coating material to improve the permeability and / or biocompatibility. The coating material can comprise any suitable coating material disclosed in this application. In some embodiments, a coating material comprises PDMS.

[0245] The coating procedure can comprise the following operations:

[0246] (1) Take the PDMS solution prepared previously and ensure its homogeneity and transparency before use.

[0247] (2) Fill a 10mL syringe entirely with the PDMS solution (e.g., 20 or 40% w / w).

[0248] (3) Using the syringe, fill the well until the PDMS solution covers the entire volume of the well, caution is taken not to overflow the well. FIG.2 shows an example 3D printed well filled with the PDMS solution.

[0249] (4) Once filled, let the PDMS-covered well rest for 15 minutes inside a closed container to reduce solvent evaporation.

[0250] (5) After the 15 minutes rest, transfer the excess solution from each well into a plastic waste container and place each piece upside down to drain the remaining PDMS solution for 15 minutes, or until ensuring no further draining occurs (visual verification by reviewing the wetting of the surface / lint-free paper).

[0251] (6) Place the well upright and transfer it to an oven.

[0252] (7) Cure the object inside the oven for 2 hours at 80 °C.

[0253] (8) Confirm that the object is fully cured. Perform a touch-dry test by touching the object with gloves and ensure it does not stain the glove, does not stick, or does not have any material detachment.

[0254] If additional layers are desired, repeat processes 2 to 8 or more times (coating, draining, curing, and curing verification) to coat multiple layers of PDMS on the well. FIG.3 illustrates an example coating process, comprising a first immersion operation 310, a first oven treatment 320, a second immersion operation, and a second oven treatment. In some embodiments, the coating process can be repeated (e.g., 330) 2 to 8, or more times.

[0255] After each coating layer, visually check that the material does not have any fracture or failure during the process. Example 7B: Permeability assay of CF-A0028 resin

[0256] To calculate the permeability of a resin, a properly sealed aluminum chamber whereWSGR Ref. No.55076-707.601 a membrane is placed was used. The permeability to CO2 of a membrane refers to the passage of gas through the membrane. The membrane was printed using the CF-A0028 resin (25 mm diameter x 2 mm thick). At the bottom of the chamber, CO2enters at a known pressure, e.g., 1 atm. The membrane was in the middle of the chamber. At the top of the chamber, a pressure sensor was placed to record the pressure data. Pressure as a function of time (test time 30 min) is Barrer (1 Barrer = 3.35*10-1.m recorded (FIG.4) and permeability data in6m2.s .Pa) is calculated.

[0257] CF-A0028 has a permeability of 1626 Barrer in comparison to a Formlabs commercial resin having a permeability of 5.30 Barrer. Example 8: Biocompatibility assessment

[0258] With the aim of assessing the biocompatibility and cell culture capability of the resins, assays were conducted with each type of resin. All assays were performed with the resin well model (a well that is made from the resin) coated with PDMS. Example 9: Biocompatibility assessment of resin wells made of CF-A0028 and polydopamine (PDA) with N1 Fibroblasts

[0259] With the aim of assessing the biocompatibility and cell culture capability of the resins, assays were conducted with each type of resin. All assays were performed for 72 hours with the resin well model coated with PDMS and polydopamine (PDA).

[0260] In order to analyze the adhesion of human fibroblasts to resins, fibroblasts were plated in 3 resin wells with 6 layers of PDMS and coated with PDA for 72 hours at room temperature.50,000 cells were seeded per control well (made of polystyrene) and 38,500 cells were seeded per resin well. At 24 hours, staining was performed with calcein, and cells with good morphology were observed. A lot of background was observed in the images of the wells, which may be due to effect of polydopamine. At 72 hours, the cells were lifted (detached form the wall of the well), and the cell values were obtained. FIG.5 shows the control well had 45000 cells (4688 cells / cm2) and the resin well had 16833 cells (2301 cells / cm2). The number of cells were reduced in comparison to the number of cells / well that were seeded. The cell density in the resin well was about half of the cell density in the control well. Example 10: Adherence of induced pluripotent stem cell (iPSC) to CF-A0028 N1 resin

[0261] CF-A0028 resin was functionalized with polydopamine by a 72-hour treatment at room temperature (CF-A0028 N1 resin). Resin wells were made from the CF-A0028 N1 resin. The wells were coated with Matrigel (solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells).150,000 cells were seeded per control well (made of polystyrene) and 110,000 cells were seeded per resin well, resulting in a cell density ofWSGR Ref. No.55076-707.601 approximately 15,000 cells / cm². At 24 hours, staining with Alkaline Phosphatase was performed (a lot of background is observed in the images of the wells - possible effect of polydopamine), and cells with good morphology were observed. At 72 hours, the cells were lifted and counted. FIG.6A shows the control well had 650000 cells (67708 cells / cm2) and the resin well had about 250000 cells (34199 cells / cm2). The cell density in the resin well was about half of that in the control well. Example 11: Adherence of iPSCs to CF-A0028 N2 resin

[0262] CF-A0028 resin was functionalized with a polydopamine by a 4-hour treatment at 60 °C (CF-A0028 N2 resin). Resin wells were made from the CF-A0028 N2 resin. The wells were coated with Matrigel.135,000 cells were seeded per control well and 105,000 cells were seeded per resin well, resulting in a cell density of approximately 14,000 cells / cm². At 24 hours, staining with Alkaline Phosphatase was performed (a lot of background is observed in the images of the wells - possible effect of polydopamine), and cells with good morphology were observed. At 72 hours, the cells were lifted and counted. FIG.6B shows the control well had 195000 cells (20312 cells / cm2) and the resin well had about 60000 cells (8207 cells / cm2). The cell density in the resin well was about half of the cell density in the control well. One resin well (highlighted by the ellipse shape) had 175,000 cells (23,923 cells / cm²) which is similar to those of the control wells. Example 12: Resin wells CF-A0133 + DOPA with N1 fibroblasts

[0263] In order to analyze the adhesion of human fibroblasts to resins disclosed herein, fibroblasts were plated in 3 resin wells made of CF-A0133 with 6 layers of PDMS and coated with polydopamine for 72 hours at room temperature.50,000 cells were seeded per well. At 24 hours, staining was performed with calcein (a lot of background is observed in the images of the wells - possible effect of polydopamine), and cells with good morphology were observed. At 72 hours, the cells were lifted, and counted. FIG.7 shows the numbers of lifted cells of the control well and resin well (control well: 87666 cells / well or 9132 cells / cm2; resin well: 37000 cells / well or 5058 cells / cm2). The cell density in the resin well was about half of that in the control well. Example 13: Resin wells CF-A0133 + DOPA with iPSC N1

[0264] In order to analyze the adherence of human iPSCs to resins disclosed herein, cells were plated in 3 resin wells made of resin CF-A0133 with 6 layers of PDMS and coated with polydopamine for 72 hours at room temperature. The wells were further coated with Matrigel. 150,000 cells were seeded per well. At 24 hours, staining was performed with AlkalineWSGR Ref. No.55076-707.601 Phosphatase (a lot of background is observed in the images of the wells - possible effect of polydopamine), and cells with good morphology were observed. At 72 hours, the cells were lifted and counted, and a portion of these cells were seeded to perform an immunofluorescence. In the immunofluorescence, a similar expression of SOX2, a pluripotency marker was observed. This indicates that the cells do not lose their potential when cultured in this resin coated with polydopamine. Example 14: Using a 3D printed device as a bioreactor in a bioprocessor

[0265] A 3D printed bioreactor (or device) was used in a bioprocessor to (i) characterize the growth and viability of hgCHO cells (cells derived from Chinese hamster ovary from the laboratory of Dr. Hagens Gerrit); (ii) quantify Trastuzumab production in every inoculation; and (iii) determine daily metabolite concentrations (e.g., Glucose, Glutamine, Glutamate, Lactate and Ammonium), osmolarity, and pH of the extracellular medium.

[0266] The bioreactor (e.g., 2030 in FIG.20) was printed with resin CF-A0028 and coated with 6 layers of PDMS (using PDMS solution in 20% ethanol). The bioreactor was sterilized by ethylene oxide (ETO).

[0267] BalanCD CHO growth medium was supplemented with 4 mM L-glutamine and 0.01% Pluronic F-68. SCHOTT bottles (e.g., from SCHOTT AG) were adapted for supplying sterilized (e.g., by autoclave or ETO) medium. Blue caps must include connector, while red caps with septum are sterilized without them, because they are not required to connect the hoses. T75 or T175 “collector” bottles with ETO sterilized venting. Two peristaltic pumps were set to control the flow rate: 1 for medium circulation and 1 for inoculation + corresponding controllers.

[0268] The incubator was connected with a flipper and a holder adapted to the bioreactor and controller.

[0269] FIG.19 shows a continuous inoculation module (CIM). The CIM comprises an inoculator 1910, a connector 1930 to supply inoculum, and a connector 1920 to direct inoculum out of the inoculator. The inoculator 1910 comprises a U shaped cavity to hold the inoculum and cells. the CIM further comprises an Erlenmeyer 125 mL plastic sterilized by ETO and 50 mL glass / plastic sterilized by autoclave, 125 mL Erlenmeyer cap modified for CIM use, CIM microfluidic connections sterilized by ETO, 0.22 mm filters, needles, and peristaltic pumps set to the required flow rate.

[0270] The inoculum had 2.00×106cells / mL hgCHO. The exit flow was 60-65 mL / day. The rocking velocity was 180° / 40 sec. The incubator wherein the CIM was encased in had a temperature 37 °C, a CO2concentration of 5%, and a pressure of 1 atm. The inoculator was set to rock at a rocking velocity of 180° per 40 seconds or 4.5° / sec. The peristaltic pump was setWSGR Ref. No.55076-707.601 with corresponding flow with 1 volume of the device / day using the sequence “Push&Pull”.

[0271] The biosafety cabinet (BC) was cleaned with UV for 15 min. The bioreactor and connections previously sterilized with ETO were assembled in the BC. The inlets were the smooth side, last printed part) and the outlets were the rough side, first printed.

[0272] The bioreactor was filled with BalanCD CHO growth medium and antibiotic- antimycotic (1x) vertically. The average volume of the bioreactor was approximately 60 ml. The total flow rate at the inlet was 75-80 ml / day (variable depending on swelling and evaporation) and 60-65 ml / day at the outlet (fixed established by the peristaltic pump) using a “Push&Push” sequence with the following parameters: (i) “Push” flow rate of 2 ml / min and a total volume of 1.4 ml (duration 42 s) and (ii) “Low” flow rate of 0.022 ml / min and a total volume of 1.3 ml (duration 59 min). The sequence repeats once.

[0273] FIG.20 left panel shows the filling of the bioreactor. The filing can be performed 48-72 hours before inoculating the device with cells. The bioreactor 2040 was placed upright and verified that the outlet hose was not “clamped” by the peristaltic pump 1690 (see FIG.16).

[0274] The filing can comprise the following processes: (1) placing a 3-way medium inlet valve in the open direction for the bioreactor and closed for the medium bottle, (2) infusing medium slowly, verifying absence of losses (the volume of the bioreactor can be determined based on when culture medium begins to come out), (3) at the end of filling, placing the outlet hose on the peristaltic pump, (4) with the setup in a horizontal position, opening the medium feed valve and filling the section of hose that connects the supply container to the T of the feed circuit (priming), and then closing the valve, (5) once filling was complete, filling the feeding bottle 2050 with complete BalanCD CHO Growth medium supplemented with antibiotic- antimycotic up to an equivalent of 2 internal volumes of the bioreactor, (6) before moving the bioreactor to the incubator, verifying that the circulation system was closed by return valves, (7) placing the bioreactor in the incubator, (8) restoring normal circulation flow by opening the media feed valve and turning on the pump, and (9) verifying that the “flipping” is correct and that there are no obstructions in the path.

[0275] In the case of the feeding bottle, the graduation was used and in the case of the “T output collector"” measure with a falcon.10 ml of culture medium that flows into the T collector must be reserved every 24 hours until the day of inoculation (including the day of inoculation). The 1st inoculation has a study up to 72 hours prior to inoculation of the state of the culture medium and its capacity to maintain both the growth and normal viability of the cells while the following inoculations has one study 24 hours prior to inoculation after having washed the device. These media are called “conditioned media”.

[0276] Day 0- procedure for automatic inoculation of the device horizontally with flippingWSGR Ref. No.55076-707.601

[0277] Five 15 mL falcons (1.1, 1.2, 1.3, 1.4, 1.5) and 450 mL falcons (2.1, 2.2, 3.1, 3.2) are labeled in advance.

[0278] The procedure comprises the following operations.

[0279] 1. Close the valves and take the setup to the CBS.

[0280] 2. Enter the flipper 2060 into the CBS and verify that it rotates correctly before beginning to inoculate.

[0281] 3. Set a peristaltic pump 2040 at a flow rate of 1.2-1.3 mL / min and enter it into the CBS.

[0282] 4. Set a 19 mm orbital shaker at 120 rpm to place the inoculum Erlenmeyer flask.

[0283] 5. Take note of the volume found in the "T output collector" and the feeding container. In the case of the feeding bottle, the graduation is used and in the case of the "T output collector" measure with a falcon.

[0284] 6. Reserve culture medium that comes from the bioreactor (10 mL) to carry out a control of the conditioned medium inside after swelling / evaporation or after previous inoculation*.

[0285] 7. Release the pump hose and verify by means of a hydraulic test with culture medium that the system does not have obstructions that could cause an increase in internal pressure before inoculation.

[0286] 8. Add antibiotic-antimycotic to the inoculum (1x).

[0287] 9. Homogenize the inoculum.

[0288] 10. Plate 2 mL of the culture in triplicate in a 12-well plate as an inoculum control.

[0289] 11. Centrifuge 10 mL of the culture (10 min at 200 g at RT) and then resuspend it in 10 mL of “conditioned” medium. The 1st inoculation can have a medium of 24, 48 and 72hours pre-inoculation. The following inoculations only have culture medium 24 hours pre-inoculation, taken before the washes of the previous inoculation. Plate 2 mL of the culture in triplicate.

[0290] 12. Carefully introduce the hose with a magnetic davit into the inoculum Erlenmeyer flask, secure it with an external magnet in the desired position and place it on the orbital shaker.

[0291] 13. Place the Tronqueta output hose in the 15 mL falcon (label 1.1).

[0292] 14. Place the hose on the peristaltic pump and circulate the inoculum until the entire hose system is full.

[0293] 15. Connect the inoculation hose to the inoculation port of the device (3-way needleless valve on the device holder that connects device inlet ports 1-2). Verify that the inoculation valve is open and the culture medium valve is closed.

[0294] 16. Turn on the pump and introduce the inoculum into the device until 3 volumes have passed (50 mL each), collecting each volume separately in a sterile container. These cellsWSGR Ref. No.55076-707.601 must be counted, and the volumes obtained recorded in order to calculate by difference the number of cells inside the device. The first volume will be counted every 10 mL. The second and third volume will be counted every 25 mL. After counting the falcons separately, a new integrated measurement of them will be carried out every 50 mL. To do this, add 5 ml of each 15 mL falcon (1.1-1.5) and 5 ml of falcons 2.1 and 2.2 and 5 ml of falcons 3.1 and 3.2. These are the average samples. The third volume should NOT leave the CBS to be counted if it was not posted before.

[0295] 17. Plate in triplicate 2 mL of the culture from the third volume (combine samples 3.1 and 3.2) at the outlet of the device (this will serve as a control of the state of the cells).

[0296] 18. Also record the volume and concentration of the cells that remained in the Erlenmeyer (remnant).

[0297] 19. Fill the feed container with the equivalent of at least 2 volumes of daily flow before leaving it running.

[0298] 20. Place the output hose in the T collector bottle T, the hose inside the peristaltic pump and return the setup to the incubator.

[0299] 21. Return the setup to the incubator.

[0300] 22. Restore normal circulation flow by opening the medium inlet valve and turning on the pump.

[0301] Device assembly: the device is mounted in a type 2 biosafety cabinet vertically on the microfluidic lid of the roller. When the device is mounted on the microfluidic lid support, the device is filled through the 4 inlet ports with culture medium SCUP002 + Antibiotic- Antimycotic (GIBCO, 15240062) at a flow rate of 3 ml / min. The device is filled once the exit of the culture medium is detected through the outlet ports simultaneously, and the internal volume is then recorded. Before removing it from the cabin, it is verified that the device does not present leaks, that the inoculation valve T is closed, that the valve T of the culture medium bottle is open and that the culture medium bottle has more than 120 ml of medium reservoir (equivalent to 2 device volumes). After this filling, the pharmed hose of the peristaltic pump is blocked so that the device does not go empty and the microfluidic lid with the support and the device is taken to the roller to adjust it with 10 wing nuts. The pharmed hose is adjusted for the peristaltic pump and the continuous perfusion test values are set in the SCADA user interface: (a) roller flipping condition: +360° in 80 sec / -360° in 80 sec (half-turn), without acceleration or pause, (b) total flow rate (push +nominal): 45 mL / day, (c) “Push” flow rate: 1000 µL at flow rate 2880 mL / day, and (d) nominal flow rate: 400 µL at a flow rate of 10 mL / day.

[0302] The environmental values of the test are set considering the following parameters: temperature of 37 °C, CO2 concentration of 5%, and humidity of 95-97%. To obtain the requiredWSGR Ref. No.55076-707.601 humidity, 10 ml of sterile miliQ water is placed through a needle port of the microfluidic roller cap, which serves to maintain the atmosphere. After setting the parameters, the continuous perfusion system is started with the parameters running for 48 hours to allow the device to absorb the culture medium and be ready for inoculation.

[0303] FIG.25 shows example inoculum concentration as a function of outlet volume during an inoculation process for an example culture system. The device is filled with medium, and medium with cells is subsequently introduced into the device. During the inoculation process, the concentration of the cells increases. After 1 volume (of the internal volume of the device, about 50 mL), the cell concentration is about 13% of the inoculum concentration. After 2 volumes, the cell concentration is about 75% of the inoculum concentration. After 3 volumes, the cell concentration is about 90% of the inoculum concentration.

[0304] FIG.26 shows example cell recovery as a function of time for an example culture system. The recovery of hgCHO cells in the device on day 13 was 1117% before washing and 1728% afterwards (FIG.26). The output concentration increased throughout the days, going from approx.1.50E+06 total cell / mL to approx.3.00E+06 total cell / mL. The general viability of the assay was good throughout the days (92.7% average) with a maximum of 99.12% (sample taken in the afternoon, day 1) and a minimum of 78.45% (overnight (ON) sample, day 13).

[0305] FIG.29 shows example cell recovery and percent viability as a function of time for an example culture system, comparing CHO mAb cells (TRQ 19.1, experiments carried out in Buenos Aires Argentina with the same hydraulic and mechanical parameters but using CHOmAbxience cells) and hgCHO cells. Concentration of total cells at nominal flow rate of thesamples was taken throughout the days. The values in the viability graph represent the daily mean ± SD. Measurements were performed in duplicate using the microscope. In comparison with TRQ 19.1, hgCHO cells had a higher percentage of recovery than CHO mAB cells from day 4, which may be due to the difference in the cell doubling time of the hgCHO line (16 h) compared to the CHO mAb line (22 h). No significant difference was observed for both cells (FIG.29 right panel).

[0306] After completing the washings inside the device, 9.92E+08 total cells were found, so the internal concentration of the device would be around 1.6E+07 cell / mL in 62 mL of bioreactor volume.

[0307] It is hypothesized that the presence of 0.01% pluronic acid in the culture medium could positively contribute to both viability (less variation ON samples / late sample) and cell migration within the device.

[0308] Inoculation: the starting point is an inoculum in an Erlenmeyer flask in general of 200 ml to 2 million cells / ml of Trastuzumab-producing CHO, which after being supplementedWSGR Ref. No.55076-707.601 with Anti-Anti, a 60 cm long inoculation hose is placed. Inoculation is carried out through the inoculation port on the microfluidic lid of the rocking system, at a flow rate of 1.25 ml / min after opening the T valve of this port. About 150 ml of the inoculum is inoculated to ensure that the entire device has a homogeneous concentration of cells and to be able to quantify the exact number of cells left in the device. Once inoculated, the continuous perfusion test is maintained, after opening the T valve of the culture medium bottle.

[0309] Test follow-up: the device is sampled twice a day, in the morning taking a sample recovered throughout the night (AM), and a “push” is performed to take another sample and a sample in the afternoon (PM) where the following is determined: cell concentration, pH, % O2, Glucose concentration, and Trastuzumab concentration. In the case of environmental parameters, the following criteria were taken: humidity (supplement with 5 ml if the humidity falls below 95% and with 10 ml if it falls below 92%) and glucose (supplement with SCUP002 culture medium with 13 gr / L of glucose if it falls below 6 gr / L or return to SCUP002 without supplementing in the event that more than 9 gr / L is registered at the exit).

[0310] FIG.27 shows the concentrations of total cells at nominal flow rate and during washes of the samples taken throughout the days and viability of the samples. Plotted values represent daily mean ± SD. Measurements were performed in duplicate using the microscope.

[0311] FIG.28 shows example cell concentration prior to and after culture in an example continuous flow bioreactor. Cell concentration (live cells) and cell viability evaluated in samples of the original inoculum prior to entering the device (control) and the third volume after passing through it (V3) for 4 days. Additionally, fresh cells were grown in culture medium from the bioreactor 24 hours prior to inoculation (from the swelling / evaporation study). Plotted values represent daily mean ± SD. Measurements were performed in duplicate using the microscope.

[0312] FIG.30 shows example nitrogen metabolism as a function of time for an example culture system, including glutamine (Gln), glutamate (Glu), ammonium (NH4+), and pH. Arrow shows the wash / harvest point.

[0313] FIG.31 shows example ion concentration as a function of time for an example culture system, including sodium, potassium, and calcium.

[0314] FIG.32 shows an example specific productivity and titer as a function of time for an example culture system, including daily producing cells, antibody concentration (Tz), and specific productivity (Qph). The cells and antibody increased during the bioproduction process.

[0315] FIG.33 shows example harvest and total cells as a function of time for an example culture system. Both cell number and antibody were accumulated in the harvest.

[0316] FIG.34 shows example carbon metabolism as a function of time for an example culture system, including glucose, lactate, and pH. Arrow shows the wash / harvest point.WSGR Ref. No.55076-707.601

[0317] The samples that come out of the device (am, instantaneous, pm) are centrifuged and the concentration of Trastuzumab (Tz) is quantified from the supernatant by densitometry using SDS-PAGE gels. It is graphed to compare the concentrations of Tz with the viability or concentration of viable cells (depending on the requirement). Subsequently, an integrated analysis of the metabolites that have been measured is carried out, along with the cells, the production of Trastuzumab, cellular metabolism, and productivity of those cells in that device can be obtained. FIG.35 shows example cellular viability and monoclonal antibody production (Tz) as a function of time for an example culture system. Example 15: Designing a 3D bioreactor using equalization layers

[0318] This example introduces the idea of the tool that may be used to control the flow structure. The mathematical formulation may be applicable for any given TPMS. This example uses the Schwarz P crystal and the Gyroid crystal.

[0319] For a given micro-vascular system defined by a TPMS, a connection point to consider is the minimum passage between two pores. Precise control of the size of this connection allows control of the patterns generated in the flow structure.

[0320] To find a surface for any TPMS, the value of bias for which the topology of the surface changes from a connected pore into an unconnected one may be used.

[0321] At this value of bias, there may be points P where the loss of connectivity is reached. At normal values of bias, at the point P, a surface S may be defined that passes through it and has a normal ^^ and is limited by its intersection with the TPMS. Then, the surface S is a function of ^^. The hydraulic resistance to the flow passage is then defined by the minimum surface. Once founded the point P and the normal ^^, there are two strategies to determine the hydraulic diameter of the passage for a given bias value.

[0322] The first strategy comprises determining S and the perimeter to determine thehydraulic diameter (D4^^ h), which is a S and perimeter: ^^ℎ =^^^^^^^^^^^^^^^^^^ .

[0323] The second strategy comprises finding the family of directions ^^ tangential to S. ^^ canbe founded as ^̂^ ∙ ^̂^ = 0. Departing from P in any direction ^^ will take a distance d to reach theTPMS. FIG.38A schematically illustrates an example of a Schwarz P crystal when the diameter is 0. FIG.38B schematically illustrates an example of a Schwarz P crystal when the diameter is 1. When the diameter changes, the bias changes. Both figures are of the same crystal but with different diameters, and therefore different biases. This is a way to graphically show how the structure changes by modifying the diameter, which in turn modifies the bias.

[0324] The distance can vary in the family ^̂^, so it’s needed to average it for all ^^. ThisWSGR Ref. No.55076-707.601operation yields the value of ^^ =^^ℎ2. This yields a direct correlation between the bias value and the hydraulic diameter and is the reason why the value of bias (x,y,z) can be controlled to have different values of ^^ℎ (^^,^^,^^) in the micro-vascular system.

[0325] For the Schwarz P crystal defined as cos(ρ^^) + cos(ρ^^) + cos ρ^^ = ^^^^^^^^ (^^, ^^, ^^), one ^^ of the points P is in ( ^^, 0,0). The normal ^^ for this point is (1,0,0). Along the family of vectors^̂^ =bias for a distance R is ^^^^^^^^ = ^^^^^^(^^^^). Then, to have a certain structure with ^^ ^^(^^, ^^, ^^), the function of bias results in ^^^^^^^^ ^^, ^^, ^^ = cos(^^2 ). For the gyroid, the calculation is more complex, but follows the same logical construction. The bias function in this case results in.

[0326] The number 0.94415 is due to the fact that for the gyroid the surface S is an ellipsoid, and the direction used in the formula presented is the one that calculates the shorter diameter. Since the exectricity (a measure of how much a conic section, e.g., an ellipse, a parabola, or a hyperbola, deviates from being a circle) of this figure is known, the relation between the shorter diameter and a given hydraulic diameter can be defined (FIG.38B).

[0327] In these simulations, the problem of achieving uniform residence time was solved by using equalization layers. The device was made of gyroids with a scanning radius R of 1.8 mm, a channel diameter D of 2 mm, following the ratio R / D = 0.9. The device comprises a macrostructure made of 20 * 16 * 20 single gyroids, with a ratio L / W = 0.8. If the channel diameters were even, the whole device would have a capacity of 503 mL.

[0328] This change in the design was conducted due to the low uniformity of the flow within the device. These changes allow to improve the flow uniformity by at least 35% by making the residence time even within the device. In a further improvement, the equalization layers help the macrostructure to be printed evenly because these changes allow the resin to drain from the structure during the printing process, the washing process, and the posttreatment process. The change may achieve as uniform as possible flow through the microvascular structure to assure homogeneous properties for the fluid as well as a similar residence time inside the structure. In any channel, the flow profile may affect the velocity of fluid distribution and one factor that influences the flow profile in a closed channel is its diameter. Considering laminar flow with a parabolic velocity profile, through the microvascular structure in each channel, the fluid close to the walls has a velocity that is close to zero and the fluid close to the central area has theWSGR Ref. No.55076-707.601 maximum velocity. To gain uniformity in the structure, the structure can be equalized to obtain a flat velocity profile.

[0329] Uniformity metric is a metric that compares the quantity of channels in the system that have its flow rate inside a given threshold compared to the quantity of channels that have its flow rate inside the threshold for a Darcy flow condition. For any given TPMS that defines a microvascular / pore structure, a layer is a zone composed by an array of crystals of width L. The layers can be stacked together to amplify its effect on the flow. Equalization is a strategy that has the purpose to distribute the flow uniformly to reach a higher uniformity metric. Another possible definition can be that equalization is the change of diameter between two channels with the main purpose of obtaining uniform flow.

[0330] Equalization layer is a layer that is designed to meet a better uniformity metric. It is composed by the change in diameter in two of the three directions x,y,z considering a reference diameter and reducing or increasing it in any direction in the plane. This allows the regions with less flow rate to increase its flow rate and equalize its values. One example contains a lower diameter in the center which increases radially from the center to the perimeter. The structure can be composed with more than one equalization layer based on a threshold of uniformity. These layers tend to be closer to the inlets and outlets of the structure but can be contemplated in any position of the structure.

[0331] FIG.39 shows mesh equalization layers. A mesh was built using Cäster direct simulation module, version 1.0.0. The diameter of the core channel was about 2 mm, with a scanning radius of 1.8 mm. The pyramid, in every case, had an optimization to reach a final diameter of 3 mm on the top.

[0332] For equalization layers, the diameter of the corners was set equal to the core diameter 2 mm. The central diameter was customized from 1.0 mm up to 2.0 mm with 0.05 mm steps. The module was set to scan the equalization potencies.1.0, 1.5 and 2.0.

[0333] The uniformity curves as a function of the central diameter for the quantities of layers tested are presented below. The threshold to consider that the flow is considerably uniform is that the core channels have ±5% of the nominal flow of the device. This univocality is evaluated in the entire device (device core plus the equalization layer), not layer by layer, for this first analysis. FIG.40 shows a total uniformity of flow as a function of central diameter for different amounts of equalization layers. A device having the same channel size throughout the structure results in non-uniform flow. If equalization layers are added, the flow uniformity improves but decreases as the diameters become larger. A device having a single equalization layer (1 EL) has the highest uniformity (95%) when a central diameter is 1.05 mm. A device having 2 equalization layers (2 EL) has the highest uniformity of 95% when a central diameter isWSGR Ref. No.55076-707.601 1.3 mm. A device having 3 equalization layers (3 EL) has the highest uniformity of 88% when a central diameter is 1.45 mm. If the diameter sizes are to be increased, more equalization layers need to be added to maintain flow uniformity. In designing the device, to achieve uniform flow distribution, designing parameters may comprise the number of equalization layers, the diameter of the channels, the variation of the diameter across a layer, and the variation of the diameter across the flow direction.

[0334] To give more perspective, the following graph is presented on how the flow evolves for various proposed devices:

[0335] FIG.41 shows the flow rate distribution for each layer in the Z direction, plotted as a percentage of the nominal flow rate (no equalization layer). In the first layer of the central zone of the device (Z layer 0), the flow rate in the central zone reaches 1.65 times the desired (nominal) flow rate, while in the corners it drops to 0.35 of the nominal flow rate. The flow rate has low uniformity, which affects the residence time in this layer, making it uneven. In subsequent layers, the uniformity increases. However, it does not reach a uniform distribution, e.g., the maximum flow rate is above 1.05 of the nominal flow rate and the minimum flow rate is below 1.05 of the nominal flow rate.

[0336] The flow was not even. Moreover, the flow was not even in the core of the device (layers = 7). This explains the non-uniformity of this device. The graph is not symmetrical, since the face symmetrical to face “0” in the opposite corner is face “1”.

[0337] FIG.42 shows flow rate sampling of a device with three layers of equalization. The central diameter was 1.45 mm and the diameter was varied quadratically. At layer 0, the maximum flow rate is about 1.21 of the nominal flow rate and the minimum flow rate is about 0.59 of the nominal flow rate. Starting from layer 3, the flow rate becomes uniform, which are close to the nominal flow rate.

[0338] FIG.43 shows example diameter variation in an example equalization layer. The center of the equalization layer has a smaller diameter, and the diameter increases radially from the center to the perimeter.

[0339] FIG.44 shows example flow uniformity as a function of gyroid width and number of layers. The flow is not distributed uniformly considering the ratio gyroid width and quantity of layers in Z. There are unutilized dead zones. The ratio R / D supposed to be used to gain volume, impacts negatively the gas exchange.

[0340] FIG.45 schematically illustrates an example biologic production workflow. The production process can comprise (1) decide the production run plan, comprising fluidic diagram, experimental protocol, and setup status check; (2) request of the inoculum; (3) preparation of fluidic connections; (4) sterilization (e.g., by ETO); and (5) visual check of the bioprocessorWSGR Ref. No.55076-707.601 setup. The production process further comprises (6) assembly of the fluidic circuit. After the assembly in (6), the production process can comprise (7) filling the fluidic circuit with media. After the filling in (7), the production process can comprise (8) mounting the component into the bioprocessor. The production process can further comprise (9) calibrating pumps, which can be automated.

[0341] After (10) startup of the system, the production process can further comprise (11) swelling and control of parameters and (12) collection of a conditioned media. The production process can further comprise (13) inoculation of the system and (14) inoculation of the controls.

[0342] When the bioprocessor is running, the production process can further comprise (15) daily harvest and instant sampling.

[0343] The daily harvest and instant sampling can further comprise (16) daily analytic run molecules (e.g., for Flex, CC, ATP, and viability). The daily harvest and instant sampling can further comprise (17) downstream analysis of molecules which may comprise analysis by SDS page, capture ELISA, and indirect ELISA. The daily harvest and instant sampling can further comprise purification of mAb. The daily harvest and instant sampling can further comprise protein analysis.

[0344] The production process can further comprise (18) final cell harvest. The final cell harvest can further comprise (19) daily analytic run (e.g., for Flex, CC, ATP, and viability). The final cells harvest can further comprise (20) downstream analysis which may comprise analysis by SDS page, capture ELISA, and indirect ELISA. The final cell harvest can further comprise purification of mAb. The final cell harvest can further comprise protein analysis.

[0345] After the final cell harvest, the production process can further comprise (21) inactivation and discard of the medium and (22) production run report.

[0346] The proposed device for printing is the following: Channel diameter = 2 mm, Scanning radius = 1.58 mm, Optimization diameter of thunderbolts = 3 mm, Central diameter of equalization layer = 1.45 mm, Number of equalization layers = 3, Variation exponent = 2, Core height = 16 layers of gyroids, Core width = 20 layers of gyroid, L / W ratio = 0.8, and R / D ratio = 0.9.

[0347] Finally, taking into account the sensitivity of the result to the real values of the channel diameters, it would be advisable to try to ensure that the final dimensions are those mentioned, and not necessarily those set in the design software, as long as there is some predictability of how the channel diameter varies between the setting and the actual printed one. Example 16: Method for Harvesting Induced Pluripotent Stem Cells Using Ultrasonic- Assisted Detachment in a Functionalized Microfluidic BioreactorWSGR Ref. No.55076-707.601

[0348] Harvesting efficiency of adherent cells may be improved by using ultrasonic-assisted cell detachment methods. In an example, human induced pluripotent stem cells (iPSCs) are harvested from a microfluidic bioreactor using two distinct detachment strategies: (i) ultrasonic detachment and (ii) enzymatic detachment. Human induced pluripotent stem cells (iPSCs) were cultivated in a perfusable microfluidic bioreactor. The bioreactor comprised an internal surface functionalized with a polydopamine (PDA) layer to enhance cellular adherence. The PDA coating was applied at a concentration of 2 mg / mL in Tris-HCl buffer (10 mM, pH 8.5) at ambient temperature for a period of four hours. Bioreactors were inverted hourly to ensure uniform deposition of PDA and subsequently washed with 30 mL of sterile distilled water. The coated chambers were subjected to ethylene oxide (ETO) sterilization and aerated for a minimum of thirty days prior to use.

[0349] The bioreactors were preconditioned by continuous perfusion with phosphate- buffered saline (PBS) at a rate of 12 mL / day over four days, followed by perfusion with Dulbecco’s Modified Eagle Medium (DMEM) / F12 medium for 48 hours to acclimate the bioreactor to the cellular microenvironment. One hour prior to cell seeding, a Geltrex® coating was applied at 37°C, followed by perfusion with culture medium (StemFlex™) containing 10 µM ROCK inhibitor (Y-27632) at a flow rate of 1.3 mL / min to prime the bioreactor.

[0350] Cell suspensions containing approximately 3.5 × 10⁶ iPSCs in 8 mL of StemFlex™ medium were prepared and perfused into the bioreactor at a flow rate of 1.2 mL / min. The bioreactors were maintained under standard culture conditions (37°C, 5% CO₂). To improve homogenous adhesion across the three-dimensional internal architecture, the devices were rotated 90° every five minutes for one hour immediately following inoculation.

[0351] For harvesting, an ultrasonic-assisted detachment protocol was applied to Bioreactor. The method included: replacing the fluidic tubing with a conduit of 1.6 mm internal diameter; perfusing 20 mL of pre-warmed (37°C) PBS at a rate of 2.4 mL / min; introducing 20 mL of EDTA solution at 2.4 mL / min, followed by a 5-minute incubation at 37°C; washing with an additional 20 mL of PBS; applying a first ultrasound cycle of 5 minutes at 40 kHz using an external ultrasonic source; performing a “push-pull” wash with 20 mL of PBS, cycled 24 times with alternating 1.6 mL forward and 0.8 mL reverse volumes; perfusing 20 mL of TrypLE™ enzymatic dissociation solution at 2.4 mL / min and incubating for 8 minutes at 37°C; applying a second ultrasound cycle of 5 minutes at 40 kHz; conducting a second push-pull wash with 20 mL PBS under identical or near identical conditions as the first push-pull wash; flushing the device with 50 mL PBS at a rate of 20 mL / min to collect residual cells; and introducing calcein- AM viability dye according to standard staining protocols.

[0352] The detachment process produced a sequence of discrete cell fractions, eachWSGR Ref. No.55076-707.601 corresponding to a defined phase of the protocol. These fractions included cells removed during EDTA exposure, ultrasonic dislodgement, enzymatic digestion, and final PBS washes. Each fraction was collected independently and quantified.

[0353] To confirm the functional viability of the recovered iPSCs, cells from selected fractions (e.g., EDTA only, TrypLE™ plus ultrasound, etc.) were reseeded at a density of 47,500 cells / well in 24-well plates. After 72 hours of culture, cells were fixed and stained for expression of pluripotency markers using anti-SOX2 (1:100), anti-OCT4 (1:100), and appropriate Alexa Fluor-conjugated secondary antibodies. Nuclei were counterstained with Hoechst 33342.

[0354] Microscopic examinations at 100× and 200× magnification revealed preserved morphology and strong expression of pluripotency-associated markers in the harvested cells, consistent with retention of their stem cell identity post-retrieval.

[0355] This example demonstrates that ultrasonic-assisted harvesting, when applied to iPSCs grown within polydopamine-functionalized microfluidic devices, permits effective and non-destructive recovery of viable pluripotent cells, thereby offering an efficient alternative to purely enzymatic detachment methods.

[0356] While preferred embodiments of the present invention 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. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Ref. No.55076-707.601 CLAIMS WHAT IS CLAIMED IS:

1. A bioreactor comprising: a macrostructure comprising a plurality of fluidically connected minimodules, wherein the plurality of fluidically connected minimodules form a plurality of channels, wherein a channel of the plurality of channels comprises a channel wall disposed between an inner surface and an outer surface, and wherein the channel wall is configured to be: (i) permeable to gas diffusion to permit gas to flow between the inner surface and the outer surface and (ii) liquid impermeable to prevent or substantially prevent the flow of liquid between the inner surface and the outer surface.

2. The bioreactor of claim 1, further comprising an inlet fluidically connected to at least one channel of the plurality if channels of the macrostructure.

3. The bioreactor of claim 1 or 2, further comprising an outlet fluidically connected to at least one channel of the plurality of channels of the macrostructure.

4. The bioreactor of any one of claims 1-3, wherein the channel wall has a gas permeability that is at least about 1000 barrers.

5. The bioreactor of any one of claims 1-4, wherein a composition making up the inner surface or the outer surface of the channel exhibits no more than about 30% swelling when exposed to water for about 150 hours.

6. The bioreactor of any one of claims 1-5, wherein the channel is configured to provide laminar flow of a fluid through the channel.

7. The bioreactor of any one of claims 1-5, wherein the channel is configured to provide transitional flow of a fluid through the channel.

8. The bioreactor of any one of claims 1-7, wherein the inner surface of the channel comprises a three-dimensional (3D) structure configured to modify a fluid flow profile of a fluid flowing through the channel as compared to a channel without the 3D structure.

9. The bioreactor of claim 8, wherein the 3D structure comprises protrusions, grooves, or a combination thereof.

10. The bioreactor of claim 8, wherein the 3D structure is configured to generate chaotic advection in a fluid flowing through the channel.

11. The bioreactor of any one of claims 1-10, wherein the channel comprises a biocompatible material.

12. The bioreactor of claim 11, wherein the biocompatible material is a sterilizable or autoclavable material.

13. The bioreactor of any one of claims 1-12, wherein a diameter of the channel is fromWSGR Ref. No.55076-707.601 about 1 micrometers (µm) to about 10 millimeters (mm).

14. The bioreactor of any one of claims 1-13, wherein a diameter of the channel is at least about 100 µm.

15. The bioreactor of any one of claims 1-14, wherein the channel wall is from about 20 µm to about 500 µm thick.

16. The bioreactor of any one of claims 1-15, wherein a thickness of the channel wall varies along a length of the channel.

17. The bioreactor of claim 16, wherein the channel comprises a first end, a center portion, and a second end, and wherein the channel wall is thicker near the first end or the second end than the center portion.

18. The bioreactor of claim 16, wherein the channel comprises a first end, a center portion, and a second end and wherein the channel wall is thicker near the center portion than the first end or the second end.

19. The bioreactor of any one of claims 1-18, wherein the channel wall is at least about 50 µm thick.

20. The bioreactor of any one of claims 1-19, wherein the plurality of fluidically connected minimodules are disposed in at least one fluidically connected layer.

21. The bioreactor of claim 20, wherein a layer of the at least one fluidically connected layer comprises at least one minimodule.

22. The bioreactor of any one of claims 20-21, wherein the at least one fluidically connected layer forms a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, a toroid with a central core, or any combination thereof.

23. The bioreactor of claim 22, wherein the macrostructure comprises a first layer comprising a first shape and a second layer comprising a second shape, and wherein the first shape and the second shape are different shapes.

24. The bioreactor of any one of claims 1–23, wherein the plurality of fluidically connected minimodules are connected at connection points.

25. The bioreactor of claim 24, wherein a diameter of a connection point of the connection points is from about 1 µm to about 10 mm.

26. The bioreactor of claim 24, wherein a diameter of a connection point of the connection points is at least about 50 µm.WSGR Ref. No.55076-707.601 27. The bioreactor of any one of claims 24-26, wherein a connection point of the connection points has a modified internal diameter as compared to another connection point of the connection points.

28. The bioreactor of any one of claims 24-27, wherein an internal diameter of a connection point of the connection points in a first layer of fluidically connected minimodules is less than another internal diameter of another connection point of the connection points in a second layer of fluidically connected minimodules.

29. The bioreactor of claim 27, wherein the modified internal diameter of the connection point is reduced compared to other connection points in the same layer.

30. The bioreactor of claim 27, wherein the modified internal diameter of the channel is reduced compared to channels near to one or more edges of the macrostructure.

31. The bioreactor of claim 27, wherein the modified internal diameter of the channel is reduced compared to channels adjacent to a center of the macrostructure.

32. The bioreactor device of any one of claims 1-31, wherein an internal volume of the macrostructure is from about 10 milliliters (mL) to about 50 mL.

33. The bioreactor device of any one of claims 1-32, wherein an internal volume of the macrostructure is from about 5 L to about 10 L.

34. The bioreactor device of any one of claims 1-32, wherein an internal volume of the macrostructure is at least about 10 mL.

35. The bioreactor of any one of claims 1-34, wherein a minimodule of the plurality of fluidically connected minimodules comprises a triply periodic minimal surface.

36. The bioreactor of claim 35, wherein the triply periodic minimal surface comprises a gyroid structure, and wherein the gyroid structure comprises a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, of any combination thereof.

37. The bioreactor of claim 35, wherein the minimodule comprises a single gyroid structure.

38. The bioreactor of any one of claims 1-37, wherein the macrostructure comprises layers of the plurality of fluidically connected minimodules, and wherein a layer of the layers comprises from 1 to 105triply periodic minimal surfaces in a first axis and from 1 to 105triply periodic minimal surfaces in a second axis perpendicular or substantially perpendicular to the first axis.

39. The bioreactor of any one of claims 1-38, wherein a core region of the macrostructure comprises at least one layer.

40. The bioreactor of any one of claims 1-39, wherein the macrostructure is generated using three-dimensional (3D) printing.

41. The bioreactor of any one of claims 1-40, wherein the channel wall comprises aWSGR Ref. No.55076-707.601 biocompatible material, and wherein the biocompatible material is generated from a three dimensional (3D) printable bioink.

42. The bioreactor of claim 41, wherein the 3D printable bioink comprises a polymerizable component, a photoinitiatior, and a thermocuring agent.

43. The bioreactor of any one of claims 1-42, further comprising a biocompatible enhancer disposed as a layer on the inner surface of the channel.

44. The bioreactor of claim 43, wherein the biocompatible enhancer comprises between 2 and 12 layers.

45. The bioreactor of any one of claims 43-44, wherein the biocompatible enhancer comprises at least 6 layers.

46. The bioreactor of any one of claims 43-45, wherein the biocompatible enhancer comprises polydimethylsiloxane (PDMS).

47. The bioreactor of any one of claims 1-46, wherein the channel wall comprises a metal.

48. The bioreactor of claim 47, wherein the metal comprises steel, a nickel-based superalloy, aluminum, low-carbon austenitic stainless steel, or any combination thereof.

49. The bioreactor of any one of claims 1-48, wherein the bioreactor is generated using one or more 3D printing techniques selected from the group consisting of metal powder bed fusion (PBF), direct metal laser sintering (DMLS), selective laser melting (SLM), electron beam melting (EBM), directed energy deposition (DED), binder jetting of metallic or ceramic powders, cold spray additive manufacturing, ultraviolet vat photopolymerization, stereolithography (SLA), digital light processing (DLP), fused filament fabrication (FFF), fused deposition modeling (FDM), multi-jet printing (MJP), PolyJet printing, two-photon polymerization (2PP), electrohydrodynamic printing (EHD), inkjet bioprinting, laser-assisted bioprinting, robocasting, and material extrusion of ceramic pastes or slurries.

50. The bioreactor of any one of claims 1-49, wherein the bioreactor comprises at least one surface compatible with establishment, maintenance, growth, differentiation, adhesion, migration, proliferation, metabolic activity, extracellular matrix deposition, signal transduction, morphogenesis, polarization, secretion of biomolecules, or viability of living cells.

51. A bioprocessor comprising the bioreactor of any one of claims 1-50.

52. The bioprocessor of claim 51, further comprising a bioproduction chamber, wherein the bioreactor is enclosed in the bioproduction chamber.

53. The bioprocessor of claim 51 or 52, further comprising a bioproduction cartridge, wherein the bioproduction cartridge comprises the bioreactor.

54. The bioprocessor of claim 53, wherein the bioproduction cartridge comprises an environmental control system.WSGR Ref. No.55076-707.601 55. The bioprocessor of claim 54, wherein the environmental control system is configured to control at least one sensor.

56. The bioprocessor of any one of claims 51-55, further comprising at least one consumables reservoir.

57. The bioprocessor of any one of claims 51-56, further comprising a formulator.

58. The bioprocessor of claim 57, wherein the formulator is configured to generate tailored culture media formulations.

59. The bioprocessor of any one of claims 51-58, further comprising an inoculation module.

60. The bioprocessor of any one of claims 51-59, further comprising a production module comprising the bioreactor.

61. The bioprocessor of any one of claims 51-60, further comprising a harvesting module.

62. The bioprocessor of claim 61, wherein the harvesting module comprises a reservoir configured to retain a product produced by the bioreactor.

63. The bioprocessor of any one of claims 61-62, further comprising a counter suspension module configured to rotate, rock, vibrate, or otherwise disrupt sedimentation in the bioreactor.

64. The bioprocessor of any one of claims 51-63, further comprising at least one sensor, and wherein the at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter.

65. The bioprocessor of claim 64, wherein the biological parameter is selected from the group consisting of cell division rate, cell growth rate, a cell stress response, cell protein content, cell carbohydrate content, cell lipid content, cell viability, cell count, and cell nucleic acid content.

66. The bioprocessor of claim 64, wherein the physical parameter is selected from the group consisting of cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity, temperature, and pressure.

67. The bioprocessor of claim 64, wherein the chemical parameter is selected from the group consisting of pH, liquid media composition, concentration of individual liquid media component, gas composition, gas concentration, and dissolved gas concentration.

68. The bioprocessor of any one of claims 64-67, further comprising at least one temperature sensor, one pH sensor, one glucose sensor, one cell production sensor, one metabolite production sensor, one ultrasound sensor, one pressure sensor, one infrared sensor, one current sensor, one gas sensor, one light sensor, one position sensor, one sound sensor, one color sensor, one hyper spectral sensor, one level sensor, one moisture sensor; and / or any combination thereof.

69. The bioprocessor of any one of claims 51-68, further comprising at least one system toWSGR Ref. No.55076-707.601 control, nutrient concentration, temperature, pH, ultrasound, pressure, current, gas flow, gas concentration, light, position, moisture, or any combination thereof.

70. A 3D printable bioink comprising: a polymerizable component; a photoinitiatior; and a thermocuring agent, wherein the 3D printable bioink is configured to (i) polymerize and (ii) be biocompatible upon polymerization.

71. The 3D printable bioink of claim 70, wherein the polymerizable component comprises Polyethylene Glycol Diacrylate 250 (PEGDA MW 250).

72. The 3D printable bioink of claim 71, wherein the PEGDA MW 250 is from about 70% to about 98% of the bioink (wt / wt).

73. The 3D printable bioink of any one of claims 70-72, wherein the photoinitiator component comprises 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO).

74. The 3D printable bioink of claim 73, wherein the 2,2phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) is from about 2% to about 30 % wt / wt.

75. The 3D printable bioink of any one of claims 70-74, wherein the thermocuring component comprises 2,2′-Azobis(2-methylpropionitrile) (AIBN).

76. The 3D printable bioink of claim 75, wherein the 2,2′-Azobis(2-methylpropionitrile) (AIBN) is from about 0.01% to about 10 % wt / wt.

77. The 3D printable bioink of any one of claims 70-76 further comprising a porogen.

78. The 3D printable bioink of claim 77, wherein the porogen component comprises polyethylene glycol 200 (PEG-200).

79. The 3D printable bioink of claim 78, wherein the polyethylene glycol 200 (PEG-200) is from about 30% to about 70 % wt / wt.

80. The 3D printable bioink of claim 77, wherein the porogen component comprises polyethylene glycol 400 (PEG-400).

81. The 3D printable bioink of claim 80, wherein polyethylene glycol 400 (PEG-400) is from about 30% to about 70 % wt / wt.

82. The 3D printable bioink of any one of the claims 70-81, further comprising a light blocker.

83. The 3D printable bioink of claim 82, wherein the light blocker comprises Avobenzone (Avo).

84. The 3D printable bioink of claim 83, wherein the Avobenzone (Avo) is from about 0.001% to about 1% wt / wt.WSGR Ref. No.55076-707.601 85. The 3D printable bioink of any one of the claims 70-84, further comprising a plasticizer.

86. The 3D printable bioink of claim 85, wherein the plasticizer comprises Decanol (n- decanol, decan-1-ol, capric alcohol).

87. The 3D printable bioink of claim 86, wherein the Decanol (n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt.

88. The 3D printable bioink of claim 77, wherein the porogen is about 5% wt / wt Triton X at a concentration of from about 10% to about 50% wt / wt.

89. The 3D printable bioink of any one of the claims 70-88, further comprising a filler.

90. The 3D printable bioink of claim 89, wherein the filler comprises hydroxyapatite.

91. The 3D printable bioink of claim 90, wherein the hydroxyapatite is from about 1% to about 10% wt / wt.

92. The 3D printable bioink of claim 91, wherein the filler is about 5% wt / wt hydroxyapatite.

93. The 3D printable bioink of any one of claims 90-92, wherein the hydroxyapatite comprises modified hydroxyapatite.

94. The 3D printable bioink of claim 93, wherein the modification of the hydroxyapatite comprises functionalization with carbon (HApC12)-dodecanol-.

95. The 3D printable bioink of any one of the claims 70-94, further comprising a rheological enhancer.

96. The 3D printable bioink of claim 95, wherein the rheological enhancer comprises Decanol (n-decanol, decan-1-ol, capric alcohol).

97. The 3D printable bioink of claim 96 wherein the Decanol(n-decanol, decan-1-ol, capric alcohol) is from about 15% to about 30% wt / wt.

98. The 3D printable bioink of claim 95, wherein the rheological enhancer comprises SILICA.

99. The 3D printable bioink of claim 98, wherein the SILICA is from about 15% to about 30%.

100. The 3D printable bioink of claim 70-98, wherein rheological enhancer comprises modified SILICA.

101. The 3D printable bioink of claim 100, wherein the modified SILICA comprises KH550 or (3-aminopropil) trietoxisilano).

102. The 3D printable bioink of any one of claims 70-101, further comprising a biocompatibility enhancer.

103. The 3D printable bioink of claim 102, wherein the biocompatibility enhancer is applied to a surface of a polymerized composition generated from the 3D printable bioink in layers.WSGR Ref. No.55076-707.601 104. The 3D printable bioink of claim 103, wherein the biocompatibility enhancer comprises from 2 to 12 layers.

105. The 3D printable bioink of claim 103, wherein the biocompatibility enhancer comprises at least 6 layers.

106. The 3D printable bioink of any one of claims 102-105, wherein the biocompatibility enhancer comprises polydimethylsiloxane (PDMS).

107. The 3D printable bioink of any one of claims 70-106, further comprising a metal.

108. The bioreactor of claim 107, wherein the metal comprises steel, a nickel-based superalloy, aluminum, low-carbon austenitic stainless steel, or any combination thereof.

109. The 3D printable bioink of any one of claims 70-108, wherein the 3D printable bioink is configured to be printed using one or more techniques selected from the group consisting of metal powder bed fusion (PBF), direct metal laser sintering (DMLS), selective laser melting (SLM), electron beam melting (EBM), directed energy deposition (DED), binder jetting of metallic or ceramic powders, cold spray additive manufacturing, ultraviolet vat photopolymerization, stereolithography (SLA), digital light processing (DLP), fused filament fabrication (FFF), fused deposition modeling (FDM), multi-jet printing (MJP), PolyJet printing, two-photon polymerization (2PP), electrohydrodynamic printing (EHD), inkjet bioprinting, laser-assisted bioprinting, robocasting, and material extrusion of ceramic pastes or slurries.

110. The 3D printable bioink of any one of claims 70-109, wherein a polymerized component generated from the 3D printable bioink comprises at least one surface compatible with establishment, maintenance, growth, differentiation, adhesion, migration, proliferation, metabolic activity, extracellular matrix deposition, signal transduction, morphogenesis, polarization, secretion of biomolecules, and viability of living cells.

111. A bioreactor comprising: an input configured to receive a fluid; an output configured to output the fluid; and a macrostructure fluidically connected to the input and the output, wherein the macrostructure comprises a plurality of minimodules fluidically connected to form a plurality of channels, wherein the macrostructure comprises (i) a first equalization region in fluid communication with the input, iii) a second equalization region in fluid communication with the output, and (iii) a core region disposed between the first equalization region and the second equalization region, wherein the first equalization region comprises diverging channels of the plurality of channels and is configured to distribute fluid from the input to the core region, wherein the core region is configured to maintain uniform fluid flow through the core region, and wherein the second equalization region comprises converging channels of the plurality ofWSGR Ref. No.55076-707.601 channels and is configured to provide the fluid from the core region to the output.

112. The bioreactor of claim 111, wherein the macrostructure comprises a plurality of layers of fluidically connected minimodules of the plurality of fluidically connected minimodules.

113. The bioreactor of claim 112, wherein a layer of the plurality of layers comprises at least one minimodule.

114. The bioreactor of any one of claims 111-113. wherein the plurality of layers forms a shape comprising a stack of a pyramid, a lamella, core, inverted pyramid, hollow pyramid, lamella pyramid, chessboard arrangement, log, , cylindrical vessel, conical bottom, a spherical chamber, a toroidal (doughnut-shaped) configuration, a flat-panel structure, a cube or rectangular prism, a tubular body, a helical coil, a honeycomb, a lattice framework, a modified lattice framework, a capsule or elliptical form, a toroid with a central core, or any combination thereof.

115. The bioreactor of any one of claims 111–114, wherein the plurality of fluidically connected minimodules are connected at connection points.

116. The bioreactor of claim 115, wherein a diameter of a connection point of the connection points is from about 1 µm to about 10 mm.

117. The bioreactor of claim 115, wherein a diameter of a connection point of the connection points is at least about 50 µm.

118. The bioreactor of any one of claims 115-117, wherein a connection point of the connection points has a modified internal diameter as compared to another connection point of the connection points.

119. The bioreactor of claim 115-118, wherein an internal diameter of a connection point of the connection points in a first layer of fluidically connected minimodules is less than another internal diameter of another connection point of the connection points in a second layer of fluidically connected minimodules.

120. The bioreactor of any one of claims 111-119, wherein the macrostructure comprises a plurality of layers, and wherein internal diameters of the plurality of channels varies across a layer of the plurality of layers.

121. The bioreactor of claim 120, wherein internal diameters of channels adjacent to a center of the macrostructure are on average less than internal diameters of channels adjacent to an edge of the macrostructure.

122. The bioreactor of claim 120, wherein internal diameters of channels adjacent to a center of the macrostructure are on average greater than internal diameters of channels adjacent to an edge of the macrostructure.

123. The bioreactor of any one of claims 111-122, wherein the core region comprises at leastWSGR Ref. No.55076-707.601 one layer of minimodules of the plurality of fluidically connected minimodules.

124. The bioreactor device of any one of claims 111-123, wherein an internal volume of the macrostructure is from about 100 mL to about 500 mL.

125. The bioreactor device of any one of claims 111-123, wherein an internal volume of the macrostructure is at least about 10 mL.

126. The bioreactor of any one of claims 111-125, wherein a minimodule of the plurality of fluidically connected minimodules comprises a triply periodic minimal surface.

127. The bioreactor of claim 126, wherein a triply periodic minimal surface comprises a gyroid structure, wherein a gyroid structure comprises a single gyroid, a modified single gyroid, a double gyroid, a modified double gyroid, or any combination thereof.

128. The bioreactor of claim 127, wherein minimodule comprises a single gyroid.

129. The bioreactor of any one of claims 126-128, wherein the layers of the core region comprise between 1 and 105triply periodic minimal surfaces in a first axis and between 1 and 105triply periodic minimal surfaces in a second axis perpendicular to or substantially perpendicular to the first axis.

130. A system for biologic production comprising: a first module comprising a reservoir configured to contain a consumable material; a second module in fluid communication with the first module, wherein the second module comprises an inoculation device configure to (i) contain and generate a plurality of cells from at least one cell, (ii) direct cells from the plurality of cells to different segments of the inoculation device, wherein cell growth conditions in the different segments are individually configurable, and (iii) iteratively generate a set of growth conditions for the plurality of cells; a third module in fluid communication with the first module and the second module, wherein the third module comprises a bioproduction chamber enclosing a bioreactor, wherein the bioproduction chamber is configured to: (i) receive the consumable material from the reservoir, (ii) receive cell from the inoculation module, (iii) control biologic production conditions within the bioreactor, (iv) rotate, rock, vibrate, or otherwise disrupt sedimentation with the bioreactor, and (v) direct a product generated in the bioreactor to a harvesting module; and a fourth module in fluid communication with the third module, wherein the fourth module comprises the harvesting module.

131. The system of claim 130, wherein the third module comprises a plurality of bioreactors comprising the bioreactor.

132. The system of claim 131, wherein biologic production conditions are individually configurable for each bioreactor.WSGR Ref. No.55076-707.601 133. The system of any one of claims 130-132, further comprising a plurality of third modules comprising the third module.

134. The system of any one of claims 130-133, wherein the first module, second module, third module, and fourth module are fluidically interconnected.

135. The system of any one of claims 130-134, wherein the reservoir comprises a cartridge system comprising the consumable material.

136. The system of any one of claims 130-135, wherein the inoculation module comprises a continuous microbioreactor.

137. The system of any one of claims 130-136, wherein the bioproduction chamber is a sealed chamber.

138. The system of any one of claims 130-137, wherein the harvesting module comprises a cartridge system.

139. The system of any one of claims 130-138, wherein the harvesting module comprises an ultrasonic harvesting system.

140. The system of any one of claims 130-139, further comprising a counter suspension system, wherein the counter suspension system is configured to rotate, rock, vibrate, or otherwise disrupt sedimentation in the bioreactor.

141. The system of claim 140, wherein the counter suspension system comprises a rocking system.

142. The system of claim 140, wherein the counter suspension system comprises an ultrasonic system.

143. The system of claim 140, wherein the counter suspension system comprises a vibratory system.

144. The system of claim 140, wherein the counter suspension system comprises a density modified system.

145. The system of claim 140, wherein the counter suspension system comprises a push and pull system.

146. The system of any one of claims 130-145, further comprising at least one sensor, and wherein the at least one sensor measures a biological parameter, a physical parameter, or a chemical parameter.

147. The system of claim 146, wherein the biological parameter is selected from the group consisting of cell division rate, cell growth rate, a cell stress response, cell protein content, cell carbohydrate content, cell lipid content, cell viability, cell count, and cell nucleic acid content.

148. The system of claim 146, wherein the physical parameter is selected from the group consisting of cell size, cell density, cell flow rate, liquid media flow rate, mixing rate, turbidity,WSGR Ref. No.55076-707.601 temperature, and pressure.

149. The system of claim 148, wherein the chemical parameter is selected from the group consisting of pH, liquid media composition, concentration of individual liquid media component, gas composition, gas concentration, and dissolved gas concentration.

150. The system of any one of claims 130-149, further comprising at least one temperature sensor, one pH sensor, one glucose sensor, one cell production sensor, one metabolite production sensor, one ultrasound sensor, one pressure sensor, one infrared sensor, one current sensor, one gas sensor, one light sensor, one position sensor, one sound sensor, one color sensor, one hyper spectral sensor, one level sensor, one moisture sensor, or any combination thereof.

151. The system of any one of claims 130-150, further comprising at least one system to control, nutrient concentration, temperature, pH, ultrasound, pressure, current, gas flow, gas concentration, light, position, moisture, or any combination thereof.

152. The system of any one of claims 130–151, further comprising at least one electronic circuit to control configured to control one or more of the first module, the second module, the third module, and the fourth module.

153. The system of any one of claims 130–152, wherein the bioreactor comprises a macrostructure.

154. The system of any one of claims 153, wherein the macrostructure comprises a plurality of fluidically connected minimodules, and wherein the plurality of fluidically connected minimodules form a plurality of channels.

155. The system of claim 154, wherein a channel of the plurality of channels comprises a channel wall disposed between an inner surface and an outer surface, and wherein the channel wall is permeable to gas diffusion to permit gas to flow between the inner surface and the outer surface.

156. The system of claim 155, wherein the channel wall is liquid impermeable to prevent or substantially prevent the flow of liquid between the inner surface and the outer surface.

157. The system of any one of claims 155-156, wherein the channel wall has a gas permeability that is at least 1000 barrers.

158. The bioreactor device of any one of claims 155-157, wherein the channel is configured to provide laminar flow of a liquid through the channel.

159. The system of any one of claims 155-158, wherein the inner surface of the channel comprises a three-dimensional (3D) structure configured to modify a fluid flow profile of a fluid flowing through the channel as compared to a channel without the 3D structure.

160. The system of claim 159, wherein the 3D structure comprises protrusions, grooves, or aWSGR Ref. No.55076-707.601 combination thereof.

161. The system of claim 159, wherein the 3D structure is configured to generate chaotic advection in a fluid flowing through the channel.

162. The system of any one of claims 155-161, wherein the channel comprises a biocompatible material.

163. The system of any one of claims 130-162, wherein an internal volume of the bioreactor is from about 10 milliliters (mL) to about 5 L.

164. The system of any one of claims 154-163, wherein a minimodule of the plurality of fluidically connected minimodules comprises a triply periodic minimal surface.

165. The system of any one of claims 130-164, wherein the bioreactor comprises a biocompatible material generated from a 3D printable bioink.

166. The system of claim 165, wherein the 3D printable bioink comprises at least one polymerizable component, one photoinitiatior and one thermocuring agent.

167. The system of any one of claims 130-166, wherein the bioreactor comprises a biocompatibility enhancer.

168. The system of claim 167, wherein the biocompatible enhancer is applied in layers to an inner surface of the bioreactor.

169. The system of claim 167 or 168, wherein the biocompatible enhancer is applies using dip coating the bioreactor or pumping the biocompatible enhancer through the bioreactor.

170. The system of claim 168 or 169, wherein the biocompatible enhancer comprises between 2 and 12 layers.

171. The system of claim 168 or 169, wherein the biocompatible enhancer comprises at least 6 layers.

172. The system of any one of claims 167-171, wherein the biocompatible enhancer comprises polydimethylsiloxane (PDMS).

173. The system of any one of claims 130-172, wherein the bioreactor comprises at least one surface compatible with establishment, maintenance, growth, differentiation, adhesion, migration, proliferation, metabolic activity, extracellular matrix deposition, signal transduction, morphogenesis, polarization, secretion of biomolecules, and viability of living cells.

Images