Systems and methods for making microchannel vascular network devices and implanting microchannels

Abstract

A method for manufacturing a microchannel device is provided. The method includes determining a microchannel vascular network design based on multiple design criteria. The microchannel vascular network design includes a first channel network, a second microchannel network based on the first channel network, and a structure for providing fluid communication between the first channel network and the second channel network. The method includes receiving the microchannel vascular network design in electronic form at a manufacturing system. The manufacturing system includes a prepolymer solution. The method includes forming a microchannel vascular network device of a polymer material using the prepolymer solution at the manufacturing system based on the microchannel vascular network design, thereby manufacturing the microchannel vascular network device.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 863,165, filed on June 18, 2019, entitled “Systems and Methods for Retarding or Arresting Flow in a Microchannel,” and U.S. Provisional Application No. 62 / 863,169, filed on June 18, 2019, entitled “Systems and Methods for Directing Flow in a Bioreactor Device,” each of which is incorporated herein by reference in its entirety. Background of the Invention

[0003] Field of the Invention

[0004] The present disclosure relates to vascular networks. More specifically, the present disclosure relates to systems and methods for fabricating microchannel vascular networks and seeding microchannels. Background Art

[0006] Gelatin methacryloyl (GelMA) hydrogels have been widely used in various biomedical applications, including the generation of three-dimensional biofabricated tissue analogs. This widespread adoption is due in part to the combination of the desirable biological properties of gelatin and the variable physical features exhibited by GelMA hydrogels, as well as the cell attachment and matrix metalloproteinase-responsive peptide motifs characteristic of the hydrogels. These attributes closely resemble the various properties of the natural extracellular matrix, which is critical for the various biomedical applications that utilize GelMA hydrogels.

[0007] In some biomedical applications, it is desirable to have a configurable device that includes one or more microchannels and allows a controlled flow of culture medium within the device. For example, in the process of inoculating cells included in such a device, the microchannels of the device need to block or stop the flow through it so as to allow cells to properly attach to the walls of the device without breaking away from the flow itself. Once cells are properly attached to the walls of the device, it may be necessary to accelerate or reopen the flow within the microchannels so as to provide nutrition to the cells and / or remove waste generated by the cells. However, this temporary blocking or stopping of microchannels has not been achieved to date.

[0008] In addition, bioreactors have been developed for culturing various types of cells and subsequently conducting experiments on the cultured cells. For example, conventional bioreactors include wells for culturing cells and fluid flow therethrough. However, these devices lack tools for providing fluid flow in more than one direction and controlling the direction of the flow. In addition, these devices require that the cells be removed from the bioreactor after culturing and can only culture cells in spheroid models.

[0009] In addition, vascular network devices have been developed for culturing various types of cells and performing analysis and / or implantation in subjects. However, these vascular network devices do not yet allow for culturing physiologically relevant substances of different geometries. Furthermore, these vascular network devices cannot use biodegradable materials to cultivate these substances.

[0010] Therefore, prior to the present disclosure, there was a need for a method of preventing or stopping flow in a microchannel. In addition, prior to the present disclosure, there was a need for a system and method for directing flow in a cell culture device capable of culturing physiologically relevant substances of different geometries. In addition, prior to the present disclosure, there was a need for a system and method for manufacturing a microchannel vascular network device capable of culturing physiologically relevant substances of different geometries.

[0011] The information disclosed in this Background of the Invention is only for enhancement of understanding of the general background of the invention and should not be taken as an admission or any form of suggestion that this information forms the prior art already known to a person skilled in the art. Summary of the invention

[0012] In view of the above background, there is a need in the art for systems and methods for fabricating microchannel vascular network devices and seeding a plurality of cells within the corresponding microchannel vascular network devices.

[0013] The present disclosure provides improved systems and methods for fabricating microchannel vascular network devices and seeding a plurality of cells within the corresponding microchannel vascular network devices.

[0014] In more detail, one aspect of the present disclosure provides a method for manufacturing a microchannel vascular network device. The method includes determining a microchannel vascular network design based on multiple design criteria. The microchannel vascular network design includes a first channel network, a second microchannel network based on the first channel network, and a structure for providing fluid communication between the first channel network and the second channel network. The method includes receiving the microchannel vascular network design in electronic form at a manufacturing system. In addition, the manufacturing system includes a prepolymer solution. Furthermore, the method includes forming a microchannel vascular network device of a polymer material using the prepolymer solution at the manufacturing system based on the microchannel vascular network design. In this way, the method is conducive to manufacturing the microchannel vascular network device.

[0015] In some embodiments, the determining further comprises determining one or more design criteria of a plurality of design criteria associated with forming the polymer material from the pre-polymer solution.

[0016] In some embodiments, the polymer material includes polydimethylsiloxane (PDMS), polyglycerol sebacate (PGS), polylactic acid (PLA), poly L-lactic acid (PLLA), poly D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polyglucose ester, polylactic acid-polyethylene oxide copolymer, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxypropionic acid, polyphosphate, poly (α-hydroxy acid), polycaprolactone, polycarbonate, polyamide, polyanhydride, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, degradable urethane, aliphatic polyester, polyacrylate, polymethacrylate, acyl substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonylated polyolefin, polyethylene oxide, polyvinyl alcohol, Nylon silicon and shape memory materials such as poly(styrene-block-butadiene), polynorbornene, hydrogels, metal alloys, and oligo(ε-caprolactone)diol as switching segments / oligo(p-polydioxanone)diol as physical crosslinkers.

[0017] In some embodiments, the polymer is a biodegradable material.

[0018] In some embodiments, the prepolymer solution comprises a photoinitiator. Additionally, the forming comprises exposing the prepolymer solution to ultraviolet light for a predetermined period of time.

[0019] In some embodiments, the polymer material of the microchannel vascular network device is transparent.

[0020] In some embodiments, the structure includes a first end connected to the first channel network, wherein the first end has a first diameter, and a second end connected to the second channel network, wherein the second end has a second diameter. In addition, the first diameter and the second diameter of the structure define an internal transition region of the structure.

[0021] In some embodiments, the first diameter is different from the second diameter.

[0022] In some embodiments, the first diameter is from about 992 micrometers (μm) to about 623 μm, and the second diameter is from about 832 μm to about 553 μm.

[0023] In some embodiments, the interior transition region of the structure comprises an interior surface defined by revolving a continuous smooth curve about an axis of the structure.

[0024] In some embodiments, the continuous smooth curve comprises a cone, an ellipse, or a cylinder.

[0025] In some embodiments, the interior transition region of the structure includes an interior surface defined by a monotonic function.

[0026] In some embodiments, the inner transition region of the structure has a corresponding length of about 399 μm to about 701 μm.

[0027] In some embodiments, the length defined from the first end to the second end of the micro-channel vascular network device is about 15 centimeters (cm) to about 7.5 cm.

[0028] In some embodiments, the plurality of design criteria include one or more length design criteria, one or more mass design criteria, one or more time design criteria, one or more polymer design criteria, one or more time design criteria, one or more illumination design criteria, or a combination thereof.

[0029] In some embodiments, the determining one or more length design criteria of the plurality of design criteria includes a first length associated with the microchannel vascular network design and a second length associated with the microchannel vascular network device. In addition, in some embodiments, the second dimension is smaller than the first dimension.

[0030] In some embodiments, the second length is based on the first length, the polymer, the manufacturing system, or a combination thereof.

[0031] In some embodiments, one or more polymer design criteria in the plurality of design criteria comprises selecting the formed polymer based on a degree of swelling of the polymer.

[0032] In some embodiments, one or more length design criteria of the plurality of design criteria include a length resolution of the microchannel vascular network device.

[0033] In some embodiments, one or more polymer design criteria in the plurality of design criteria include porosity of the microchannel vascular network device.

[0034] In some embodiments, the median size of the pores of the microchannel vascular network device is about 19 μm to about 231 μm.

[0035] In some embodiments, the manufacturing system utilizes an additive manufacturing method for the forming.

[0036] In some embodiments, the forming forms a microchannel vascular network device having a positive mold, thereby forming a void between the first channel network and the second channel network.

[0037] In some embodiments, the forming forms a microchannel vascular network device having a positive mold, thereby forming a void between the first channel network and the second channel network.

[0038] In some embodiments, the additive manufacturing method is selected from the group consisting of binder jetting, material extrusion, material jetting, polyjet, powder bed, sheet lamination, and vat photopolymerization.

[0039] Another aspect of the present disclosure relates to providing a method for preventing or stopping flow in a microchannel device. The microchannel device includes a first channel and a second channel. The first channel and the second channel are interconnected by one or more structures. Therefore, the method includes perfusing a suspension of a plurality of GelMA microgels contained in a solution through the first channel of the device. Therefore, perfusing the solution causes one or more GelMA microgels to prevent or stop flow in at least one of the one or more structures.

[0040] In some embodiments, the plurality of GelMA microgels are fabricated using a photomask technique.

[0041] In some embodiments, the photomask technique includes placing a prepolymer solution of GelMA and a photoinitiator in a buffer solution onto a first glass slide whose outer edge portion is surrounded by a spacer. After completing the placement of the prepolymer solution onto the first glass slide, a second glass slide is disposed above the first glass slide to form a containment structure. The method includes placing a photomask on the top surface of the second glass slide. In addition, the method includes exposing the photomask and the containment structure to ultraviolet light for a predetermined period of time to generate a GelMA microgel.

[0042] In some embodiments, the method further comprises disassembling the containing structure to recover the generated GelMA microgel, thereby producing a plurality of GelMA microgels.

[0043] In some embodiments, the prepolymer solution comprises about 7% to about 20% by weight of GelMA and about 1% of a photoinitiator.

[0044] In some embodiments, a prepolymer solution is formed having a degree of methacryloyl substitution of about 75% to about 90%.

[0045] In some embodiments, the array of transparent members is an array of circles. Each circle in the array of circles has a diameter of about 50 micrometers (μm) to about 650 μm.

[0046] In some embodiments, the spacer has a thickness of about 50 μm to about 550 μm.

[0047] In some embodiments, before disposing the prepolymer solution, for at least one slide and / or spacer, the method further includes disposing a sample of a stock solution of about 0.5 wt% methoxypolyethylene glycol 5000Si (mPEG5000-Si), about 1 wt% acetic acid and ethanol onto at least one slide and / or spacer. In addition, the method includes subjecting at least one slide and / or spacer to incubation at about 70°C for about 30 minutes to form an incubated slide and / or spacer. The immersed slide and / or spacer is formed by immersing the incubated slide and / or spacer in deionized water. In addition, the method includes ultrasonically treating the immersed slide and / or spacer for a predetermined period of time to form an ultrasonically treated slide and / or spacer. In addition, the method includes drying the ultrasonically treated slide and / or spacer to form a prepared slide and / or spacer.

[0048] In some embodiments, the predetermined period of time is less than or equal to about 30 seconds.

[0049] In some embodiments, the ultraviolet light has a wavelength of about 360 nanometers (nm) to about 480 nm and an intensity of about 12.4 milliwatts per cubic centimeter.

[0050] In some embodiments, recovering the GelMA microgel comprises transferring the produced GelMA microgel into a mineral oil bath, and agitating the mineral oil bath.

[0051] In some embodiments, the recovering of the GelMA microgel comprises immersing the generated GelMA microgel in a sodium hydroxide solution. In addition, the recovering comprises immersing the GelMA microgel in an ethanol solution and subjecting the recovered GelMA microgel to ultrasonic treatment for a third predetermined period of time.

[0052] In some embodiments, a plurality of GelMA microgels are fabricated using microfluidics processing.

[0053] In some embodiments, one or more GelMA microgels in the plurality of GelMA microgels are formed as spheres.

[0054] In some embodiments, one or more structures are in the shape of a truncated cone.

[0055] In some embodiments, the method further comprises applying fluid flow in a second channel of the device after perfusing the suspension of the plurality of GelMA microgels and the first solution through the first channel of the device, thereby generating a pressure differential between the first channel and the second channel of the device.

[0056] Another aspect of the present disclosure relates to providing a bioreactor system. The bioreactor control system is configured to control the flow through the microchannel vascular network device. In addition, the bioreactor control system includes one or more processors, a memory coupled to the one or more processors, and a controller coupled to the memory and the one or more processors. In addition, the bioreactor control system includes a microchannel vascular network device. The microchannel vascular network device includes an inlet channel having a first inflow opening and a first outflow opening, a hole configured to accommodate a cell culture insert, and an outlet channel. The hole includes an inlet connected to the first outflow opening, an outlet arranged below the cell culture insert and connected to a second inflow opening of the outlet channel. The outlet channel includes a second inflow opening and a second outflow opening. In addition, the bioreactor system includes a pump connected to the microchannel vascular network device and configured to promote the flow of fluid from the inlet channel through the outlet channel through the microchannel vascular network device based on one or more instructions provided by the controller.

[0057] In some embodiments, the bioreactor system further includes a capsule disposed over the cell culture insert and removably coupled to the microchannel vascular network device.

[0058] In some embodiments, the caplet of the device comprises a second outlet.

[0059] In some embodiments, the caplet is shaped as an inverted funnel.

[0060] In some embodiments, the caplet is coupled to the aperture via an o-ring.

[0061] In some embodiments, each surface of the caplet that contacts at least one wall of the well comprises a hydrophobic material.

[0062] In some embodiments, the O-ring comprises a hydrophobic material.

[0063] In some embodiments, the cell culture insert is a sponge or a mesh.

[0064] In some embodiments, the cell culture insert is coated with gelatin.

[0065] In some embodiments, the bioreactor system includes a plurality of microchannel vascular network devices connected in series and in fluid communication with each other such that a corresponding inlet channel of a second microchannel vascular network device of the plurality of microchannel vascular network devices is coupled to a corresponding outlet channel of a first microchannel vascular network device.

[0066] In some embodiments, the bioreactor system includes a plurality of microchannel vascular network devices, wherein the plurality of microchannel vascular network devices are connected in parallel such that each microchannel vascular network device of the plurality of microchannel vascular network devices is coupled to a pump.

[0067] Another aspect of the present disclosure is to provide a method for culturing multiple cells in a microchannel vascular network device, the method comprising preparing a microchannel vascular network device based on a candidate subject, thereby forming a prepared microchannel vascular network device, wherein the prepared microchannel vascular channel device comprises a first channel network and a second channel network. In addition, the method comprises inoculating multiple cells for culturing in the prepared microchannel vascular device based on the candidate subject, thereby forming multiple seed cells. In addition, the method comprises using a bioreactor system connected to the prepared microchannel vascular network device to allow a culture medium containing multiple seed cells to flow through the prepared microchannel vascular network device, thereby culturing multiple cells in the microchannel vascular network device.

[0068] In some embodiments, the preparing further comprises preparing a plurality of cells for inoculation.

[0069] In some embodiments, preparing the plurality of cells comprises exposing the plurality of cells to a solution. The solution comprises calcium and magnesium.

[0070] In some embodiments, the plurality of cells are exposed to the solution for a period of time ranging from about 15 minutes to about 25 minutes.

[0071] In some embodiments, preparing the microchannel vascular network device includes modifying the surface of the microchannel vascular network device.

[0072] In some embodiments, the surface modification of the microchannel vascular network device is a chemical surface modification.

[0073] In some embodiments, the chemical surface modification comprises forming a peptide conjugate on the surface of the microchannel vascular network device.

[0074] In some embodiments, the peptide conjugation is to an arginylglycylaspartate peptide.

[0075] In some embodiments, the surface modification of the microchannel vascular network device is mechanical surface modification.

[0076] In some embodiments, the mechanical surface modification comprises applying a coating to the surface of the microchannel vascular network device.

[0077] In some embodiments, the coating comprises collagen, fibronectin, gelatin, GelMA, PEGdA, PLL, or a combination thereof.

[0078] In some embodiments, the coating comprises a first weight percent (wt %) of about 8% to about 12% PEGdA and a second wt % of about 0.5% to about 7% GelMa.

[0079] In some embodiments, the first wt % of PEGdA is 10% and the second wt % of GelMA is about 1% to about 5%.

[0080] In some embodiments, the coating includes a first coating comprising PEGdA, PPL, GelMA or a combination thereof. The first coating includes a first upper surface and a first lower surface. The coating also includes a second coating comprising collagen or gelatin. The second coating includes a second upper surface and a second lower surface. In addition, the first lower surface is adjacent to the surface of the microchannel vascular network device, and the second lower surface is adjacent to the first upper surface.

[0081] In some embodiments, seeding comprises encapsulating the plurality of cells in culture medium.

[0082] In some embodiments, the culture medium comprises a photoinitiator, and prior to encapsulating the plurality of cells, seeding further comprises exposing the culture medium and the plurality of cells to ultraviolet light for a predetermined period of time.

[0083] In some embodiments, the culture medium has a thickness of about 5 μm to about 15 μm.

[0084] In some embodiments, the density of the inoculated multiple seed cells is about 1*10 4 cells / cm2 to about 1*10 5 cells / cm2.

[0085] In some embodiments, the flow rate of the flow is about 375 microliters (μL) / minute to about 425 μL / minute.

[0086] In some embodiments, before flowing, a portion of the prepared microchannel vascular network device is in a dry state or a wet state.

[0087] In some embodiments, the portion of the prepared microchannel vascular network device is the lower end portion of the microchannel vascular network device before flowing.

[0088] The present invention of preventing or stopping flow in a microchannel has other features and advantages that will become apparent or more particularly elucidated from the accompanying drawings and the following detailed description incorporated herein, which together serve to explain certain principles of exemplary embodiments of the invention.

[0089] Other features and advantages of the present invention will become apparent or be more fully elucidated from the accompanying drawings and the following detailed description incorporated herein, which together serve to explain certain principles of exemplary embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 A block diagram is shown illustrating an embodiment of a system for fabricating a microchannel vascular network device and / or seeding a plurality of cells in a microchannel device according to an embodiment of the present disclosure;

[0091] Figure 2 A manufacturing system for manufacturing a microchannel vascular network device according to an embodiment of the present disclosure is shown;

[0092] Figure 3 A bioreactor system for controlling flow through a microchannel device according to an embodiment of the present disclosure is shown;

[0093] Figure 4 A client device for designing and / or controlling flow through a microchannel vascular network device according to an embodiment of the present disclosure is shown;

[0094] Figure 5 A first flow chart of a method for manufacturing a microchannel vascular network device according to an embodiment of the present disclosure is shown, wherein optional elements are indicated by dashed boxes and / or lines;

[0095] Figure 6 shows a second flow chart of a method for seeding a plurality of cells in a microchannel vascular network device according to an embodiment of the present disclosure, wherein optional elements are indicated by dashed boxes and / or lines;

[0096] Figure 7 shows a view of an exemplary microchannel device according to an embodiment of the present disclosure;

[0097] Figure 8 shows an exemplary microgel unit according to an embodiment of the present disclosure;

[0098] Fig. 9 An exemplary process for generating a microgel unit according to an embodiment of the present disclosure is shown;

[0099] Fig.10 Various exemplary microgel units according to embodiments of the present disclosure are shown;

[0100] Fig.11 shows a view of an exemplary microchannel device including a microgel unit according to an embodiment of the present disclosure;

[0101] Fig.12 Shows Fig.11Another view of an exemplary microchannel device;

[0102] Fig.13A Shows Fig.11 Still another view of a microchannel device of claim 1, wherein the first image and the second image are merged to highlight one or more microgel units according to an embodiment of the present disclosure; and

[0103] Fig. 13B Shows Fig.11 Yet another view of a microchannel device, wherein the first image and the second image are merged to highlight one or more microgel units according to an embodiment of the present disclosure.

[0104] Fig.14 An exemplary microchannel vascular network device according to an embodiment of the present disclosure is shown;

[0105] Fig.15 Another exemplary microchannel vascular network device according to an embodiment of the present disclosure is shown;

[0106] Fig.16 shows a portion of a bioreactor system according to an embodiment of the present disclosure, wherein the dashed lines indicate the general flow path of the culture medium;

[0107] Fig.17A A bioreactor system including a first microchannel vascular network device and a second microchannel vascular network device connected in series according to an embodiment of the present disclosure is shown;

[0108] Fig. 17B A bioreactor system including a first microchannel vascular network device and a second microchannel vascular network device connected in parallel is shown according to an embodiment of the present disclosure;

[0109] Fig. 17C A bioreactor system including a first microchannel vascular network device and a second microchannel vascular network device connected in parallel is shown according to an embodiment of the present disclosure;

[0110] Fig.18A , 18B , 18C and 18D show various configurations of bioreactor systems according to embodiments of the present disclosure;

[0111] Fig.19 shows fluorescence lysis induced by controlling various bioreactor system and manufacturing system conditions according to embodiments of the present disclosure;

[0112] Fig. 20 shows additional fluorescent lysis induced by controlling various bioreactor system and manufacturing system conditions according to embodiments of the present disclosure;

[0113] Fig.21 A graph showing fluorescence cleavage induced by controlling bioreactor system and manufacturing system conditions after exposure of Hep G2 cells to fluorescein diacetate according to an embodiment of the present disclosure;

[0114] Fig. 22 Another graph showing fluorescence cleavage induced by controlling bioreactor system and manufacturing system conditions following fluorescein diacetate exposure according to embodiments of the present disclosure;

[0115] Fig.23 shows another fluorescent cleavage induced by controlling bioreactor system and manufacturing system conditions according to embodiments of the present disclosure;

[0116] Fig.24 shows the cell proliferation effect within the PEGdA hydrogel microchannel vascular network device according to an embodiment of the present disclosure;

[0117] Fig.25 , 26 , 27, 28, 29, 30A, 30B, 30C, 30D, 31A, 31B, 31C, 32, 33, 34, 35, 36, 37, 38, 39 and 40 show examples of systems and methods for making a microchannel vascular network device and culturing cells in a microchannel vascular network device according to embodiments of the present disclosure; and

[0118] Fig.41 and 42 A connector for coupling a microchannel vascular network device with a bioreactor system according to an embodiment of the present disclosure is shown.

[0119] It should be understood that the accompanying drawings are not necessarily drawn to scale, presenting a somewhat simplified representation of various features illustrating the basic principles of the invention. The specific design features of the present invention as disclosed herein (for example, including specific dimensions, orientations, locations, and shapes) will be determined in part by the specific intended application and use environment.

[0120] In the drawings, reference numbers refer to the same or equivalent parts of the present invention throughout the several views. DETAILED DESCRIPTION

[0121] Reference will now be made in detail to various embodiments of the present invention, examples of which are shown in the accompanying drawings and described below. Although the present invention will be described in conjunction with exemplary embodiments, it should be understood that this description is not intended to limit the present invention to those exemplary embodiments. On the contrary, the present invention is intended to cover not only the exemplary embodiments, but also various replacements, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present invention as defined by the appended claims.

[0122] Reference will now be made in detail to the embodiments, examples of which are shown in the accompanying drawings. In the following detailed description, many specific details are set forth in order to thoroughly understand the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure can be implemented without these specific details. In other examples, well-known methods, procedures, and methods are not described in detail to avoid unnecessarily obscuring various aspects of the embodiments.

[0123] It should also be understood that although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. For example, without departing from the scope of the present disclosure, a first graphical diagram may be referred to as a second graphical diagram, and similarly, a second graphical diagram may be referred to as a first graphical diagram. Both the first graphical diagram and the second graphical diagram are graphical diagrams, but they are not the same graphical diagram.

[0124] The terms used in this disclosure are only used to describe specific embodiments and are not intended to limit the present invention. As used in the description of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "said" are intended to also include plural forms. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the related enumerated items. It should also be understood that when used in this specification, the term "comprises and / or comprising" specifies the presence of stated features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0125] The foregoing description includes exemplary systems, methods, techniques, instruction sequences, and computing machine program products that embody illustrative embodiments. For purposes of explanation, many specific details are set forth to provide an understanding of the various embodiments of the subject matter of the present invention. However, it will be apparent to those skilled in the art that embodiments of the present invention may be practiced without these specific details. In general, well-known illustrative examples, protocols, structures, and techniques are not shown in detail.

[0126] For the purpose of explanation, the foregoing description has been described with reference to specific embodiments. However, the above illustrative discussion is not intended to be exhaustive or to limit the embodiments to the precise forms disclosed. In view of the above teachings, many modifications and variations are possible. The embodiments are selected and described in order to best explain the principles and their practical application, thereby enabling others skilled in the art to best use the embodiments and various embodiments with various modifications suitable for the intended specific use.

[0127] For the sake of clarity, not all routine features of the embodiments described herein are shown and described. It should be understood that in the development of any such actual embodiment, many specific implementation decisions are made to achieve the designer's specific goals, such as meeting use case and business-related constraints, and these specific goals will vary from one embodiment to another and from one designer to another. In addition, it should be understood that such design work may be complex and time-consuming, but is still a routine engineering task for those skilled in the art who have the benefit of this disclosure.

[0128] As used herein, the term "if" may be interpreted to mean "when" or "based on" or "in response to determining" or "in response to detecting," depending on the context. Similarly, the phrases "if it is determined" or "if [a stated condition or event] is detected" may be interpreted to mean "upon determining" or "in response to determining" or "upon detecting [a stated condition or event]" or "in response to detecting [a stated condition or event]," depending on the context.

[0129] As used herein, the term "about" or "approximately" may mean within an acceptable error range of a particular value determined by a person of ordinary skill in the art, which may depend in part on how the value is measured or determined (e.g., limitations of the measurement system). For example, according to the practice in the art, "about" may mean within or exceeding 1 standard deviation. "About" may mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. Where specific values ​​are described in the application and claims, unless otherwise stated, the term "about" shall mean within an acceptable error range of a particular value. The term "about" may have the meaning commonly understood by a person of ordinary skill in the art. The term "about" may mean ±10%. The term "about" may mean ±5%.

[0130] As used herein, the term "biological sample", "patient sample" or "sample" refers to any sample taken from a subject, which includes nucleic acids reflecting the subject's genotype for the loci described herein. Examples of biological samples include, but are not limited to, blood samples, saliva samples, oral cell samples, etc. The sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample).

[0131] As used herein, unless expressly stated otherwise, the term "equidistant" means that the distance from a first feature to a corresponding second feature is the same for consecutive pairs of features.

[0132] As used herein, "biodegradable" means a material that can be bioresorbed and / or degraded and / or broken down into metabolizable or excretable components by mechanical degradation (e.g., dissolution, resorption, etc.) within a period of minutes to three years, preferably less than one year, after interaction with a physiological environment, while maintaining the necessary structural integrity.

[0133] As used herein, "exchange mechanism" means a material or structure configured to substantially allow or inhibit the flow of material from a first element to a second element, including open walls, permeable membranes, permeable walls, porous walls, porous membranes, perforations, and the like.

[0134] As used herein, "diameter" is meant to include the equivalent characteristic length, including the hydraulic diameter of non-circular structures.

[0135] As used herein, “flush” means that a surface of a first element and a coplanar surface of a second element have a distance or level that separates the first element and the second element within a tolerance of 0 μm, within a tolerance of 5 μm, within a tolerance of 10 μm, within a tolerance of 20 μm, or within a tolerance of 100 μm.

[0136] As used herein, "direct flow" means the transfer or flow of at least one substance or material from a first element to at least a second element.

[0137] As used herein, "indirect flow" means the exchange or flow of at least one substance or material from a first element to at least a second element mediated by an exchange mechanism.

[0138] As used herein, "natural means" means a process or development found in nature.

[0139] As used herein, "polymer" is intended to include polymers and monomers that can be polymerized or adhered to form an integral unit. A polymer may be non-biodegradable or biodegradable, typically via hydrolysis or enzymatic cleavage.

[0140] As used herein, "the next channel" means, for a given channel, the channel from which the material flows out. Thus, as used herein, "the previous channel" means, for a given channel, the channel to which the material flows.

[0141] As used herein, "rigid" means a material that is hard and does not easily deform. As used herein, "elastomeric" means a material or composite material that does not have rigidity as defined herein.

[0142] Additionally, unless expressly stated otherwise, the terms "client," "patient," "subject," and "user" are used interchangeably herein.

[0143] A "disease" is a health state of an animal in which the animal cannot maintain homeostasis, and in which if the disease is not improved, the animal's health continues to deteriorate. In an exemplary embodiment, a device of the invention is implanted in a subject suffering from a disease and being treated by such implantation.

[0144] As used herein, "amino acid" refers to the genus covering hydrophilic amino acids, acidic amino acids, basic amino acids, polar amino acids, hydrophobic amino acids, aromatic amino acids, non-polar amino acids and aliphatic amino acids, including the genus and species therein. "Peptide" is formed by such amino acids connected via peptide bonds. In addition to α-amino acids, amino-carboxylic acid species are also covered by amino acids, such as aminobutyric acid (ABA), aminocaproic acid (AHA), aminomethylbenzoic acid (AMB) and the like. In an exemplary embodiment, the cyanine dye of the present invention is conjugated to a carrier molecule by a linker having one or more than one amino acid. Exemplary amino acids for such linkers include lysine, proline and acidic amino acids.

[0145] "Activated derivatives of carboxyl moieties" and equivalent species refer to moieties on precursor components of the conjugates of the invention (e.g., dyes, adapters, linkers, multivalent moieties) that have a leaving group, such as an active ester, acyl halide, acyl imidazole, and the like.

[0146] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, monounsaturated or polyunsaturated, and may contain divalent and polyvalent radicals having the specified number of carbon atoms (i.e., C1-C 10 Meaning one to ten carbons). Examples of saturated alkyl groups include, but are not limited to, groups such as methyl, methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, such as homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl is an alkyl with one or more double or triple bonds. Examples of unsaturated alkyl include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. Unless otherwise stated, the term "alkyl" is optionally a derivative of those alkyls defined in more detail below, such as "alkenyl", "alkynyl", "alkyldiyl", "bridged alkylene" and "heteroalkyl".

[0147] "Alkenyl" refers to an unsaturated branched, straight chain or cyclic alkyl group having at least one carbon-carbon double bond derived by removing a hydrogen atom from a single carbon atom of a parent alkane. The group may be in the cis or trans configuration about the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl groups, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyl groups, such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1,3-dien-1-yl, etc. In an exemplary embodiment, the alkenyl group is a (C2-C6)alkenyl group.

[0148] "Alkynyl" refers to an unsaturated branched, straight or cyclic alkyl group having at least one carbon-carbon triple bond derived by removing one hydrogen atom from a single carbon atom of a parent olefin. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyl groups, such as but-1-yn-1-yl, but-3-yn-1-yl, etc. In an exemplary embodiment, the alkynyl group is a (C2-C6)alkynyl group.

[0149] "Alkyldiyl" refers to a saturated or unsaturated branched, straight chain or cyclic divalent hydrocarbon radical derived by removing one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene or alkyne, or by removing two hydrogen atoms from a single carbon atom of a parent alkane, alkene or alkyne. Each valency of the two monovalent radical centers or divalent radical centers may form a bond with the same or different atoms. Typical alkyldiyls include, but are not limited to, methanediyl; ethyldiyls, such as ethyl-1,1-diyl, ethyl-1,2-diyl, ethylene-1,1-diyl, ethylene-1,2-diyl; propyldiyls, such as prop-1,1-diyl, prop-1,2-diyl, prop-2,2-diyl, prop-1,3-diyl, cyclopropyl-1,1-diyl, cyclopropyl-1,2-diyl, prop-1-ene-1,1-diyl, prop-1-ene- 1,2-diyl, prop-2-ene-1,2-diyl, prop-1-ene-1,3-diyl, cycloprop-1-ene-1,2-diyl, cycloprop-2-ene-1,2-diyl, cycloprop-2-ene-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyl diyl, such as butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, butane-1,4-diyl, butane-2,2-diyl, 2-methyl -prop-1,1-diyl, 2-methyl-prop-1,2-diyl, cyclobutane-1,1-diyl; cyclobutane-1,2-diyl, cyclobutane-1,3-diyl, but-1-ene-1,1-diyl, but-1-ene-1,2-diyl, but-1-ene-1,3-diyl, but-1-ene-1,4-diyl, 2-methyl-prop-1-ene-1,1-diyl, 2-methylene-prop-1,1-diyl, but-1,3 -diene-1,1-diyl, but-1,3-diene-1,3-diyl, cyclobut-1-ene-1,2-diyl, cyclobut-1-ene-1,3-diyl, cyclobut-2-ene-1,2-diyl, cyclobut-1,3-diene-1,2-diyl, cyclobut-1,3-diene-1,3-diyl, but-1-yne-1,3-diyl, but-1-yne-1,4-diyl, but-1,3-diyne-1,4-diyl, etc. Where a specific level of saturation is desired, the nomenclature alkyldiyl, alkenyldiyl and / or alkynyldiyl is used. In a preferred embodiment, the alkyldiyl is a (C2-C6)alkyldiyl. Also preferred are saturated acyclic alkyldiyl groups in which the radical center is at the terminal carbon, such as methylenediyl (methylene); ethane-1,2-diyl (ethylenediyl); propane-1,3-diyl (propylenediyl); butane-1,4-diyl (butylenediyl), etc. (also referred to as alkylene, as defined below).

[0150] "Alkylene" refers to a linear alkyldiyl radical having two terminal monovalent radical centers derived by removing one hydrogen atom from each of the two terminal carbon atoms of a linear parent alkane, alkene or alkyne. Typical alkylene radicals include, but are not limited to, methylene radicals; ethylene radicals, such as alkylene radicals, vinylene radicals, ethynylene radicals; propylene radicals, such as propylene radicals, propenylene radicals, propenylene radicals [1]enylene radicals, propenylene radicals [1,2]dienylene radicals, propenylene radicals [1]alkynylene radicals, and the like; butenyl radicals, such as butenylene radicals, butenylene radicals [1]enylene radicals, butenylene radicals [2]enylene radicals, butenylene radicals [1,3]dienylene radicals, butenylene radicals [1]alkynylene radicals, butenylene radicals [2]alkynylene radicals, butenylene radicals [1,3]diynylene radicals, and the like. Where a particular level of saturation is desired, the nomenclature alkylene radicals, alkenylene radicals, and / or alkynylene radicals are used. In a preferred embodiment, the alkylene radical is a (C2-C6)alkylene radical.

[0151] Unless otherwise specified, the term "heteroalkyl" alone or in combination with another term means a stable straight or branched chain, or cyclic hydrocarbon group or combination thereof, which consists of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S (and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized). The heteroatoms O, N, S, P and Si may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3 and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl group, as exemplified below but not limited to -CH2-CH2-S-CH2CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, the direction in which the chemical formula of the linking group is written does not imply an orientation of the linking group. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-.

[0152] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, represent cyclic forms of "alkyl" and "heteroalkyl", respectively. Divalent and multivalent species, such as "cycloalkylene", are also included. In addition, for heterocycloalkyl, a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

[0153] Unless otherwise stated, the term "halo" or "halogen" alone or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is meant to include, but is not limited to, species such as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0154] Unless otherwise stated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent that can be a single ring or multiple rings (preferably 1 to 3 rings) fused together or covalently connected. The term "heteroaryl" refers to an aryl (or ring) containing one to four heteroatoms selected from the group consisting of N, O and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atoms can be optionally quaternized. Heteroaryl can be attached to the rest of the molecule by heteroatoms. The limiting examples of aryl and heteroaryl include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4 1-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. Also included are divalent and multivalent linker species such as "arylene". Substituents for each of the above-noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0155] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes aryl and optionally heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those groups in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including those in which a carbon atom (e.g., a methylene group) has been replaced, for example, with an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0156] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Exemplary substituents for each type of radical are provided below.

[0157] Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC( R', -R', -R'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in the group. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or arylalkyl. For example, when a compound of the invention contains more than one R group, each R group is independently selected, as are each of these groups when more than one R', R", R"' and R"" groups are present. When R' and R" are attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Thus, from the above discussion of substituents, those skilled in the art will understand that the terms "substituted alkyl" and "heteroalkyl" are intended to include groups having carbon atoms bonded to groups other than hydrogen atoms, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0158] The substituents listed in the above paragraph are referred to herein as "alkyl substituents".

[0159] Similar to the substituents described for the alkyl groups, the substituents for the aryl and heteroaryl groups are varied and are selected from, for example, halogen, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', SO3R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein R', R", R"' and R"" are preferably independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl. For example, when a compound of the invention contains more than one R group, each R group is independently selected, as are each of these groups when more than one R', R", R"' and R"" groups are present.

[0160] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -TC(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a double bond of the formula -(CRR') s -X-(CR”R’”) d-, wherein s and d are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2- or -S(O)2NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6)alkyl.

[0161] The substituents listed in the above two paragraphs are referred to herein as "aryl substituents".

[0162] A "linking fragment" is a bond, or a group formed by reacting two reactive functional groups with complementary reactivity. Exemplary linking fragments are amides formed by reacting an activated derivative of an amine and a carboxylic acid (e.g., an acyl halide, an acyl imidazole, an active ester, etc.). When the cyanine dyes of the present invention are conjugated to a carrier molecule, they can be conjugated directly via a linking fragment or via a linker comprising one or more linking fragments. For example, a conjugate in which the dye is bound to a carrier molecule via a linker optionally comprises a linking fragment connecting the linker and the dye and / or connecting the linker and the carrier molecule.

[0163] As used herein, the term "linker" or "L" refers to a single covalent bond or a series of stable covalent bonds incorporating 1-40, e.g., 10-30, non-hydrogen atoms selected from the group consisting of C, N, O, S, and P, which covalently attach a fluorogenic or fluorescent compound to another moiety, such as a chemically reactive group or a biological or non-biological component, e.g., a carrier molecule. Exemplary linkers include one or more linking fragments, e.g., -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, which connect the dye to the linker and / or connect the linker to a carrier molecule, etc. Linkers are also used to connect the cyanine core component of the compound to a reactive functional group or a component of a reactive functional group.

[0164] As used herein, "fluorophore" refers to a fluorescent substance.

[0165] "Moiety" refers to a group of a molecule that is attached to another moiety.

[0166] Additionally, when a reference numeral is assigned an "i-th" designation, the reference numeral refers to a common component, group, or embodiment. For example, a graphical icon referred to as "graphic icon i" refers to the i-th graphical icon in a plurality of graphical icons (e.g., graphical icon 616-i in plurality of graphical icons 616).

[0167] The compounds, probes, and methods discussed in the following sections are generally representative of compositions of the invention and methods in which such compositions may be used. The following discussion is intended to illustrate selected aspects and embodiments of the invention, and it should not be construed as limiting the scope of the invention.

[0168] In this disclosure, unless otherwise explicitly stated, descriptions of devices and systems will include one or more computer implementations. Figure 1 In the example, a client device (e.g., Figure 4 The client device 400 of the present disclosure is represented as a single device that includes all the functionality of the client device 400. However, the present disclosure is not limited in this regard. For example, the functionality of the client device 400 can be distributed across any number of networked computers, and / or reside on each of several networked computers, and / or hosted on one or more virtual machines and / or containers located at a remote location accessible via a communication network (e.g., communication network 106). Those skilled in the art will appreciate that a variety of different computer topologies are possible for the client device 400 and other devices and systems of the present disclosure, and all such topologies are within the scope of the present disclosure.

[0169] Figure 1 A block diagram of a distributed client-server system (e.g., distributed client-server system 100) according to some embodiments of the present disclosure is depicted. System 100 facilitates manufacturing of microchannel vascular network devices (e.g., Figure 7 Microchannel vascular network device 700-1, Fig.14 In addition, the system 100 facilitates the seeding and culturing of multiple cells within the microchannel vascular network device 700. In this way, the present disclosure provides systems and methods for manufacturing a microchannel vascular network device 700 and seeding cells therein that were not previously feasible in other ways.

[0170] Of course, other topologies of system 100 are possible. For example, in some embodiments, any of the illustrated devices and systems may actually constitute multiple computer systems linked together in a network, or virtual machines or containers in a cloud computing environment. In addition, the illustrated devices and systems may wirelessly transmit information between each other, rather than relying on a physical communication network 106. Thus, Figure 1 The exemplary topologies shown are merely intended to describe features of embodiments of the present disclosure in a manner that is readily understood by those skilled in the art.

[0171] refer to Figure 1 In some embodiments, the distributed client-server system 100 includes a manufacturing system 200 for facilitating the manufacturing of a microchannel vascular network device 700, a bioreactor system 200 for facilitating the cultivation of cells within the microchannel vascular network device 700, and one or more client devices 400 (e.g., a first client device 400-1), hereinafter referred to as "client devices",

[0172] In some embodiments, the communication network 106 optionally includes the Internet, one or more local area networks (LANs), one or more wide area networks (WANs), other types of networks, or combinations of such networks.

[0173] Examples of the communication network 106 include the World Wide Web (WWW), an intranet and / or a wireless network (such as a cellular telephone network), a wireless local area network (LAN) and / or a metropolitan area network (MAN), and other means of wireless communication. Wireless communication optionally uses any of a variety of communication standards, protocols, and technologies, including Global System for Mobile Communications (GSM), Enhanced Data GSM Environment (EDGE), High Speed ​​Downlink Packet Access (HSDPA), High Speed ​​Uplink Packet Access (HSUPA), Evolution-Data Only (EV-DO), HSPA, HSPA+, Dual Cell HSPA (DC-HSPDA), Long Term Evolution (LTE), Near Field Communication (NFC), Wideband Code Division Multiple Access (W-CDMA), Code Division Multiple Access (CDMA), Time Division Multiple Access (TDMA), Bluetooth, Wireless Fidelity (Wi-Fi) (e.g., IEEE 802.11a, IEEE 802.11ac, IEEE 802.11ax, IEEE 802.11b, IEEE 802.11d, IEEE 802.11e, IEEE 802.11f, IEEE 802.11f, IEEE 802.11g, IEEE 802.11h ... 802.11g and / or IEEE 802.11n), Voice over Internet Protocol (VoIP), Wi-MAX, email protocols (e.g., Internet Message Access Protocol (IMAP) and / or Post Office Protocol (POP)), instant messaging (e.g., Extensible Messaging and Presence Protocol (XMPP), Session Initiation Protocol with extensions for Instant Messaging and Presence Support (SIMPLE), Instant Messaging and Presence Service (IMPS)) and / or Short Message Service (SMS), or any other suitable communication protocol, including communication protocols that have not been developed as of the filing date of this document.

[0174] Now that the distributed client-server system 100 has been generally described, reference will be made to Figure 2 An exemplary manufacturing system 200 for manufacturing a microchannel vascular network device 700 is described.

[0175] In various embodiments, manufacturing system 200 includes one or more processing units (CPUs) 202 , a network or other communication interface 204 , and memory 212 .

[0176] The memory 212 includes a high-speed random access memory, such as DRAM, SRAM, DDR RAM or other random access solid-state memory devices, and optionally also includes a non-volatile memory, such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices or other non-volatile solid-state storage devices. The memory 212 may optionally include one or more storage devices located away from the CPU 202. The memory 212 or alternatively the non-volatile memory device in the memory 212 includes a non-temporary computer-readable storage medium. The memory 212 is optionally controlled by a controller by accessing other components (such as CPU 202) of the manufacturing system 200. In some embodiments, the memory 212 may include a large-capacity memory remotely located relative to the CPU 202. In other words, some data stored in the memory 212 can actually be hosted on a device located outside the allele detection system 200, but can be electronically accessed by the manufacturing system 200 through the Internet, an intranet or other form of network 106 or a cable using a communication interface 204.

[0177] In some embodiments, the memory 212 of the manufacturing system 200 for generating the microchannel vascular network device 700 stores:

[0178] Operating system 216 (e.g., ANDROID, iOS, DARWIN, RTXC, LINUX, UNIX, OS X, WINDOWS, or an embedded operating system such as VxWorks), which includes programs for handling various basic system services;

[0179] An electronic address 218 associated with the manufacturing system 200 that identifies the manufacturing system 200 ;

[0180] A design control module 220 for forming a corresponding micro-channel vascular network device based on one or more design criteria 230;

[0181] a design memory 240 for storing a plurality of designs 240, each design being associated with a corresponding micro-channel vascular network device; and

[0182] A fabrication module 260 for facilitating fabrication of a microchannel vascular network device.

[0183] An electronic address 218 is associated with the allele detection system 200 and is used to uniquely identify the allele detection system 200 from at least the other devices and components of the distributed system 100. For example, in some embodiments, the electronic address 218 is used to receive a request from a client device 300 to generate and / or transmit a patient-specific report 600.

[0184] The design control module 220 includes a plurality of design standards 230, which serve as the basis for manufacturing the corresponding microchannel vascular network device. For example, in some embodiments, the corresponding design standard 230 in the plurality of design standards 230 is associated with the parameters of the microchannel vascular network device (e.g., the length of the microchannel vascular network device, the porosity of the material forming the microchannel vascular network device, etc.), the parameters of the manufacturing system 200 (e.g., the resolution of the manufacturing system 200, etc.), or both. In some embodiments, the plurality of design standards 230 include one or more predetermined design standards. In some embodiments, the predetermined design standards include a predetermined range of choices for the subject to choose from when manufacturing the microchannel vascular network device. In this way, the subject is prevented from selecting design standards outside the functional implementation scheme of the microchannel vascular network device 700.

[0185] For example, in some embodiments, a plurality of design criteria are utilized and / or based on empirical parameters for simulating the flow in the corresponding microchannel device. For example, in some embodiments, a plurality of design criteria include a plurality of flow design criteria, including flow rate, diffusion rate, or both (e.g., diffusion rate during flow and / or diffusion rate without flow).

[0186] The design memory 240 retains one or more microchannel vascular network device designs 250. Each corresponding design 250 includes a corresponding selection of one or more design criteria 230. In this way, the subject can select a design 250 from the designs 240 without having to individually select a corresponding design standard 230 to manufacture the microchannel vascular network device 700. For example, in some embodiments, the subject determines a corresponding plurality of design criteria 230 to manufacture a corresponding microchannel vascular network. Therefore, in response to determining a corresponding plurality of design criteria 230, the manufacturing system 200 can retain the corresponding device design 250 associated with the corresponding microchannel vascular network. Therefore, the subject can manufacture multiple corresponding microchannel vascular networks without having to determine the design criteria for each manufacturing instance.

[0187] For example, in some embodiments, one or more device designs 250 include a predetermined microchannel vascular network device design 250 configured for a candidate subject. As a non-limiting example, in some embodiments, the first microchannel vascular network device design is configured for a first mammal candidate subject, and the second microchannel vascular network device design is configured for a second mammal candidate subject. In some embodiments, the first mammal candidate subject and the second mammal candidate subject are mammals of the same species (e.g., both are configured for human mammal candidate subjects, both are configured for rat mammal candidate subjects, etc.). In addition, in some embodiments, the first mammal candidate subject and the second mammal candidate subject are mammals of the same species, but different candidate subjects (e.g., the first candidate subject is a first human, and the second candidate subject is a second human different from the first human). However, the present disclosure is not limited thereto. For example, in some embodiments, the first mammal candidate subject and the second mammal candidate subject belong to mammals of different species and / or different genera.

[0188] As another non-limiting example, in some embodiments, the device design 250 includes one or more device designs associated with in vivo implementation, and one or more device designs associated with in vitro implementation. For example, in some embodiments, the first device design 250-1 is associated with a first microchannel vascular network device, a layer thickness of 50 μm, an exposure time of 2 seconds, a first layer time scaling factor (FLTSF) of about 2x to about 4x, and a manufacturing time of about 48 minutes, the first microchannel vascular network device including a first plurality of design criteria 230 associated with a first channel, a second channel, and a structure between the first channel and the second channel. In some embodiments, the second device design 250-2 is associated with a second microchannel vascular network device, a layer thickness of 100 μm, an exposure time of 4 seconds, a FLTSF of about 2x to about 4x, and a manufacturing time of about 24 minutes, the second microchannel vascular network device including a second plurality of design criteria 230 associated with a first channel, a second channel, and a structure between the first channel and the second channel. In some embodiments, the third device design 250-3 is associated with a third microchannel vascular network device, a layer thickness of 100 μm, an exposure time of 4 seconds, a FLTSF of about 4x to about 8x, and a manufacturing time of about 72 minutes to about 77 minutes, the third microchannel vascular network device including a third plurality of design criteria 230 associated with a first channel, a second channel, and a structure between the first channel and the second channel. In some embodiments, the third device design 250-3 is associated with a rat mammal candidate subject.

[0189] The manufacturing system 200 includes a manufacturing module 260 that facilitates controlling a mechanism for manufacturing a microchannel vascular network device. For example, in some embodiments, the manufacturing module transmits one or more instructions to the mechanism, which passes through an area in response to the one or more instructions and performs the manufacturing of the microchannel vascular network device. As a non-limiting example, consider a manufacturing system 200 that includes a VAT photopolymerization mechanism. Therefore, the manufacturing module 260 transmits one or more instructions to the VAT photopolymerization structure, the instructions being associated with: the light intensity associated with the VAT photopolymerization mechanism, the light exposure time associated with the VAT photopolymerization mechanism, the supply of a prepolymer solution of the VAT photopolymerization mechanism, the supply of a photoinitiator associated with the VAT photopolymerization mechanism, and the like.

[0190] Each of the modules and applications identified above corresponds to a set of executable instructions for performing one or more of the functions described above and the methods described in this disclosure (e.g., the computer-implemented methods and other information processing methods described herein; Figure 5 Method 500; Figure 6 These modules (e.g., instruction sets) need not be implemented as separate software programs, procedures, or modules, and thus various subsets of these modules are optionally combined or otherwise rearranged in various embodiments of the present disclosure. In some embodiments, memory 212 optionally stores a subset of the modules and data structures identified above. In addition, in some embodiments, memory 212 stores other modules and data structures not described above.

[0191] It should be understood that Figure 2 The manufacturing system 200 is merely one example of a manufacturing system 200 , and the manufacturing system 200 optionally has more or fewer components than shown, optionally combines two or more components, or optionally has a different configuration or arrangement of components. Figure 2 The various components shown are implemented in hardware, software, firmware, or a combination thereof, including one or more signal processing and / or application specific integrated circuits.

[0192] refer to Figure 3 In various embodiments, the present disclosure includes a bioreactor system 300. The bioreactor system 300 includes one or more processing units (CPUs) 302, a network or other communication interface 304, and a memory 312.

[0193] The memory 312 includes high-speed random access memory, such as DRAM, SRAM, DDR RAM or other random access solid-state memory devices, and optionally also includes non-volatile memory, such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices or other non-volatile solid-state storage devices. The memory 312 may optionally include one or more storage devices located away from the CPU 202. The memory 312 or alternatively the non-volatile memory device within the memory 312 includes a non-transitory computer-readable storage medium. Access to the memory 312 by other components of the bioreactor system 300 (such as the CPU 302) is optionally controlled by the controller. In some embodiments, the memory 312 may include a large capacity memory located remotely relative to the CPU 302. In other words, some of the data stored in the memory 312 may actually be hosted on a device located outside the bioreactor system 300, but can be accessed electronically by the bioreactor system 300 via the Internet, an intranet or other form of network 106 or a cable using the communication interface 304.

[0194] In some embodiments, the memory 312 of the bioreactor system 300 for culturing a plurality of cells in the microchannel vascular network device 700 stores:

[0195] Operating system 316 (e.g., ANDROID, iOS, DARWIN, RTXC, LINUX, UNIX, OS X, WINDOWS, or an embedded operating system such as VxWorks), which includes programs for handling various basic system services;

[0196] an electronic address 318 associated with the bioreactor system 300 identifying the bioreactor system 300; and

[0197] A control module for controlling one or more system parameters of the bioreactor system 300 .

[0198] The electronic address 318 is associated with the bioreactor system 300 and is used to uniquely identify the allele detection system 300 from at least the other devices and components of the distributed system 100. For example, in some embodiments, the electronic address 318 is used to receive the design of the microchannel vascular network device 700 from the client device 400.

[0199] The control module 320 facilitates communicating one or more instructions to components of the bioreactor system 300. For example, in some embodiments, the bioreactor system 300 includes a pump (e.g., pump 1720 of FIG. 17 ) and / or one or more reservoirs (e.g., reservoir 1710 of FIG. 17 ), the operation of which is controlled by the control module 320. For example, in some embodiments, the control module 320 controls the flow rate of the bioreactor system 300.

[0200] Brief reference Fig.17A , 17B 17C, showing various arrangements of the bioreactor system 300, which include one or more devices of the bioreactor system 300, the microchannel vascular network device 700. For example, Fig.17A A system is shown in which a first device 700-1 and a second device 700-2 are connected in series so that culture medium 130 is pumped from a first reservoir 1720-1 through the first device, through the second device, and collected by the second reservoir 1720-2. Thus, cells housed by the second device 700-2 are subjected to materials (e.g., waste) output from the first device 700-1 in order to determine how the materials output from the first device affect the cells of the second device. This determination can be used as a component of toxicity assays for a variety of compounds, including pharmaceutical compositions, unknown substances, putative toxins, known toxins, warfare agents, etc. Similarly, Fig. 17B An embodiment of a bioreactor system is shown, in which a first device 700-1 and a second device 700-2 are connected in parallel, so that the culture medium 130 is sucked from the first reservoir 1720-1 by a pump and dispersed to flow through the first device and through the second device, which is in turn collected by the second reservoir 1720-2. Fig. 17B This configuration as shown can be used to test how the conditions of the culture medium 130 affect different cells stored in the respective devices 700 (e.g., to determine how a pharmaceutical composition contained in the culture medium 130 affects a first cell in a first device and a second cell in a second device simultaneously). Fig. 17C In some embodiments, one or more devices 700 of the bioreactor system are assigned a corresponding reservoir 1720. For example, in some embodiments, each device 700 or a subset of devices is provided with a dedicated reservoir 1720 that collects culture medium 130 output by the corresponding device (e.g., output via an outlet of a channel of the corresponding device or an outlet of a caplet) so that the collected culture medium can be tested (e.g., for toxicity assay).

[0201] refer to 18A to 18D , showing various embodiments of bioreactor systems and flow paths. 18A to 18DIn various systems of the invention, any useful apparatus configuration (eg, multiple apparatuses in series, multiple apparatuses in parallel, etc.) is represented by a portion of a bioreactor system indicated by “apparatus 100”. Fig.18A One embodiment of a system is shown in which reservoir 1720-1 supplies a medium stream 130, which is collected by reservoir 1720-2 after flowing through the system. Fig.18B Shown with Fig.18A A similar embodiment as depicted in , but utilizing pump 1720 to draw culture medium 130 through the system rather than pushing or driving the culture medium. Fig. 18C A bioreactor system is shown in which a portion of the outlet of the device 700 is open (e.g., exposed to the external environment). In some embodiments, this open configuration is used to form a siphon pressure difference or collect culture medium 130 outside the corresponding reservoir. In addition, Fig.18D A bioreactor system is shown in which a portion of the outlet of the device 700 is in communication with the reservoir 200, thereby forming a closed flow path for the culture medium 130. In some embodiments, this closed configuration is used to determine the effect of the device 700 on cells (e.g., exposure to the culture medium 130) over a period of time (e.g., the culture medium 130 flows continuously through the system over a period of time).

[0202] Although FIG. 17A to FIG. 18D The systems of each illustrate an arrangement that includes at least one reservoir, but those skilled in the art will recognize that these systems are merely exemplary and that the present disclosure includes systems that use no reservoirs or that use multiple reservoirs (e.g., a reservoir for each device of the system).

[0203] Each of the modules and applications identified above corresponds to a set of executable instructions for performing one or more of the functions described above and the methods described in this disclosure (e.g., the computer-implemented methods and other information processing methods described herein; Figure 5 Method 500; Figure 6 These modules (e.g., instruction sets) need not be implemented as separate software programs, procedures, or modules, and thus various subsets of these modules are optionally combined or otherwise rearranged in various embodiments of the present disclosure. In some embodiments, memory 312 optionally stores a subset of the modules and data structures identified above. In addition, in some embodiments, memory 312 stores other modules and data structures not described above.

[0204] It should be understood that Figure 3The bioreactor system 300 is merely one example of a bioreactor system 300 , and the bioreactor system 300 optionally has more or fewer components than shown, optionally combines two or more components, or optionally has a different configuration or arrangement of components. Figure 3 The various components shown are implemented in hardware, software, firmware, or a combination thereof, including one or more signal processing and / or application specific integrated circuits.

[0205] refer to Figure 4 , an exemplary client device 400 (e.g., a first client device 400-1) is provided. The client device 400 includes one or more processing units (CPUs) 402, one or more network or other communication interfaces 404, a memory 411 (e.g., random access memory and / or non-volatile memory) optionally accessed by one or more controllers, and one or more controllers, and one or more communication buses 414 interconnecting the above components.

[0206] In some embodiments, the client device 400 includes a mobile device such as a mobile phone, a tablet computer, a laptop computer, a wearable device such as a smart watch, etc. Alternatively, in some embodiments, the client device 400 is a desktop computer or other similar device. In addition, in some embodiments, the client device 400 (e.g., the first user device 300-1, the second user device 400-2, the third user device 400-3) communicates with a centralized client device 400 (e.g., a server client device 400) that facilitates transmitting the design of the microchannel vascular network device 700 to the manufacturing system 200.

[0207] In addition, the client device 400 includes a user interface 406. The user interface 406 typically includes a display device 408 for presenting media, such as a graphical user interface associated with the client application 420 and receiving instructions from an object operating the client device 400. In some embodiments, the display device 408 is optionally integrated into the client device 400 (e.g., installed in the same chassis as the CPU 402 and the memory 412), such as a smart (e.g., smart phone) device. In some embodiments, the client device 400 includes one or more input devices 410, which allow objects to interact with the client device 400. In some embodiments, the input device 410 includes a keyboard, a mouse, and / or other input mechanisms. Alternatively or in addition, in some embodiments, the display device 408 includes a touch-sensitive surface, for example, where the display 408 is a touch-sensitive display, or the client device 400 includes a touchpad.

[0208] In some embodiments, the client device 400 includes an input / output (I / O) subsystem 430 for interfacing with one or more peripheral devices through the client device 400. For example, in some embodiments, audio is presented by an external device (e.g., a speaker, headphones, etc.) that receives audio information from the client device 400 and / or a remote device (e.g., the manufacturing system 200) and presents data based on the audio information. In some embodiments, the input / output (I / O) subsystem 430 also includes an audio output device such as a speaker or an audio output device for connecting to or interfacing with a speaker, headphones, or headset. In some embodiments, the input / output (I / O) subsystem 430 also includes speech recognition capabilities (e.g., to supplement or replace the input device 410).

[0209] In some embodiments, the client device 400 also includes one or more sensors (e.g., accelerometers, magnetometers, proximity sensors, gyroscopes, etc.), image capture devices (e.g., camera devices or image capture modules and related components), positioning modules (e.g., global positioning system (GPS) receivers or other navigation or geographic positioning system modules / devices and related components), or combinations thereof, etc.

[0210] The memory 412 includes a high-speed random access memory, such as DRAM, SRAM, DDR RAM or other random access solid-state memory devices, and optionally also includes a non-volatile memory, such as one or more magnetic disk storage devices, optical disk storage devices, flash memory devices or other non-volatile solid-state storage devices. The memory 412 may optionally include one or more storage devices located away from the CPU 402. The memory 412 or alternatively the non-volatile memory device in the memory 412 includes a non-temporary computer-readable storage medium. Access to the memory 412 by other components of the client device 400 (such as the CPU 402 and the I / O subsystem 330) is optionally controlled by a controller. In some embodiments, the memory 412 may include a large-capacity memory remotely located relative to the CPU 402. In other words, some data stored in the memory 412 can actually be hosted on a device located outside the client device 400, but can be accessed electronically by the client device 400 through the Internet, an intranet or other form of network 106 or a cable using the communication interface 404.

[0211] In some embodiments, the memory 412 of the client device 400 stores:

[0212] Operating system 416, which includes programs for handling various basic system services;

[0213] An electronic address 418 associated with the client device identifying the client device; and

[0214] A client application 420 for generating content for display via a graphical user interface presented on the display 408 of the client device 400 .

[0215] An electronic address 418 is associated with the client device 400 and is used to uniquely identify the client device 400 from at least other devices and components of the distributed system 100. In some embodiments, the electronic address 418 associated with the client device 400 is used to determine the source of communications received from and / or provided to the client device 400.

[0216] In some embodiments, the client application 420 is a set of instructions that, when executed by a processor (e.g., CPU 402), generates content (e.g., a graphical user interface for selecting one or more design criteria of the microchannel vascular network device 700) for presentation to a subject. In some embodiments, the client application 420 generates the content in response to one or more inputs received from the subject via the user interface 406 of the client device 400. For example, in some embodiments, the client application 420 includes a media presentation application for viewing the content of a file or a web application.

[0217] Each of the modules and applications identified above corresponds to a set of executable instructions for performing one or more of the functions described above and the methods described in this disclosure (e.g., the computer-implemented methods and other information processing methods described herein; Figure 5 Method 500; Figure 6 These modules (e.g., instruction sets) need not be implemented as separate software programs, procedures, or modules, and thus various subsets of these modules are optionally combined or otherwise rearranged in various embodiments of the present disclosure. In some embodiments, memory 312 optionally stores a subset of the modules and data structures identified above. In addition, in some embodiments, memory 312 stores other modules and data structures not described above.

[0218] It should be understood that Figure 4 The client device 400 is merely one example of a client device 400 , and the client device 400 optionally has more or fewer components than shown, optionally combines two or more components, or optionally has a different configuration or arrangement of components. Figure 3 The various components shown are implemented in hardware, software, firmware, or a combination thereof, including one or more signal processing and / or application specific integrated circuits.

[0219] Now that the general topology of the distributed system 100 has been described according to various embodiments of the present disclosure, the following description will be given of a method for manufacturing a distributed system according to the present disclosure. Figure 5 Some methodological details of the microchannel vascular network device. Figure 5 A first flow chart of a method (e.g., method 500) for manufacturing a microchannel vascular network device 700 according to an embodiment of the present disclosure is shown. In the flow chart, preferred parts of the method are shown in solid boxes, while optional variations of the method or optional equipment used in the method are shown in dashed boxes.

[0220] Unless otherwise explicitly stated, the various modules in the memory 212 of the manufacturing detection system 200, the memory 412 of the client device 400, or both perform Figure 5 In addition, it will be understood that Figure 5 The processes in can be encoded in a single module or in any combination of modules.

[0221] Box 502. Reference Figure 5 At block 502, method 500 includes providing a method based on a plurality of design criteria (e.g., Figure 2 The design criteria 230) determine the microchannel vascular network device (e.g., Figure 7 The microchannel vascular network design of the microchannel vascular device 700 (e.g., Figure 2 Device design standards 250).

[0222] The microchannel vascular network design includes a first channel network (e.g., Figure 7 The first channel network 702-1) and the first channel network (eg, Figure 7 A second microchannel network of the first channel network 702-1).

[0223] In some embodiments, a structure is provided between the first channel network and the second channel network for providing fluid communication between the first channel network and the second channel network (e.g., Figure 7 In some embodiments, the structure is a material (e.g., a polymer material) of a microchannel vascular network device. In other embodiments, the structure comprises a membrane. In some embodiments, the structure comprises one or more intermediate channels between the first channel network and the second channel network.

[0224] As a non-limiting example, refer to Figure 25 to Figure 27 as well as Fig.37 and Fig.38In some embodiments, the corresponding microchannel vascular design is associated with a candidate subject of a mammal rat. Therefore, the corresponding microchannel vascular design is configured to form a microchannel vascular network device having a first channel network configured as a hepatic vein (HB) and a second channel network configured as a portal vein (PV). A structure between the first channel network and the second channel network is formed, which allows fluid communication between the first channel network and the second channel network. In some embodiments, the first channel network is fluidly connected to the second channel network. In some embodiments, the structure includes one or more intermediate channels, which are configured to exchange the mechanism within the internal structure of one or more intermediate channels. Here, the corresponding microchannel vascular design manufactures a microchannel vascular network device with a length of 31 millimeters (mm) (e.g., y-axis length), a width of 11.4 mm (e.g., x-axis length) and a height of 11 mm (e.g., z-axis depth). Each of the first channel network and the second channel network has a corresponding diameter.

[0225] In some embodiments, the structure includes a first end connected to the first channel network, wherein the first end has a first diameter, and a second end connected to the second channel network, wherein the second end has a second diameter. In addition, the first diameter and the second diameter of the structure define an internal transition region of the structure.

[0226] In some embodiments, the first diameter is different from the second diameter. For example, in some embodiments, the first diameter is about 992 micrometers (μm) to about 623 μm, and the second diameter is about 832 μm to about 553 μm. In some embodiments, the first diameter and / or the second diameter is about 1,000 μm to about 399 μm. In some embodiments, the first diameter and / or the second diameter is about 900 μm to about 199 μm.

[0227] In some embodiments, the internal transition region of the structure comprises an internal surface defined by rotating a continuous smooth curve around the axis of the structure. In some embodiments, the continuous smooth curve comprises a cone, an ellipse, or a cylinder. As a non-limiting example, Fig. 30A A number of structures are shown, each having an inner transition region that is conical in shape. Fig. 30B A plurality of structures are shown, each having an inner transition region that is cylindrical. In addition, Fig. 30C A plurality of structures are shown, each having an inner transition region that is elliptical in shape. Fig.30D , providing a plurality of structures including conical smooth continuous curves, wherein the corresponding diameters range from about 1000 μm to about 573 μm. Briefly referring to Fig.32, a cross-section of a corresponding microchannel vascular network device is provided, wherein the first structure comprises a corresponding diameter of about 982 μm to about 805 μm, and the second structure comprises a corresponding diameter of about 960 μm to about 815 μm.

[0228] In some embodiments, the internal transition region of the structure includes an internal surface defined by a monotonic function. In some embodiments, the monotonic function is a linear function (eg, a frustum of a cone), a square root function, or a logarithmic function.

[0229] In some embodiments, the internal transition region of the structure has a corresponding length of about 399 μm to about 701 μm. Thus, this length allows for the exchange of culture medium between the first channel network and the second channel network without deteriorating one aspect of the culture medium.

[0230] In some embodiments, the length defined from the first end to the second end of the microchannel vascular network device is about 15 centimeters (cm) to about 7.5 cm.

[0231] In some embodiments, the plurality of design criteria include one or more length design criteria, one or more mass design criteria, one or more time design criteria, one or more polymer design criteria, one or more time design criteria, one or more illumination design criteria, or a combination thereof.

[0232] For example, in some embodiments, one or more design criteria include the molecular weight of the prepolymer solution, the molecular weight of the polymer material, the concentration of the prepolymer solution, the concentration of the polymer material, exposure time, power (e.g., the intensity of light emitted from the manufacturing system), thickness, a time scale factor (e.g., a first layer time scale factor), or a combination thereof. As a non-limiting example, in some embodiments, the molecular weight of the prepolymer solution and / or the molecular weight of the polymer material is about 2,000 molecular weight PEGdA-500. In some embodiments, the concentration of the prepolymer solution and / or the concentration of the polymer material is about 20 wt %. In some embodiments, the exposure time is in the range of about 1.5 seconds to about 10 seconds. In some embodiments, the power is about 20 milliwatts per square centimeter (mW / cm 2 In some embodiments, the thickness (eg, layer thickness or slice thickness) is about 50 μm to about 100 μm. In some embodiments, the first layer time scale factor (eg, first layer time scale factor) is about 2x seconds to about 8x seconds.

[0233] In some embodiments, one or more length design criteria of the determined plurality of design criteria include a first length associated with a microchannel vascular network design and a second length associated with a microchannel vascular network device. In addition, in some embodiments, the second dimension is smaller than the first dimension.

[0234] In some embodiments, the second length is based on the first length, polymer, manufacturing system, or a combination thereof. In some embodiments, the second length forms a microchannel vascular network device with a volume between 1.49 milliliters (mL) and 4.51 mL.

[0235] In some embodiments, one or more polymer design criteria in a plurality of design criteria include selecting the formed polymer based on the swelling degree of the polymer. In some embodiments, the swelling degree is based on one or more parameters, including one or more dimensional parameters (e.g., planar swelling, volume swelling), one or more mass parameters (e.g., mass swelling), time parameters (e.g., per unit time) or a combination thereof. In some embodiments, the swelling degree is the ratio of the difference between wet weight and dry weight compared to the wet weight. For example, the swelling ratio of a hydrogel is an important factor for tissue engineering applications. In order to demonstrate the swelling properties of a PEGDA hydrogel microchannel device, the device was applied to a PBS solution and the swelling weight of the device was determined. The hydrogel was then freeze-dried to obtain a dry weight. The swelling degree was calculated as follows: Swelling ratio = W wet – W dry W wet. After being immersed in PBS at 4'C for one week, the dimensions w, h, d1, and d2 of the printed device increased by 18%, 15%, 16%, and 13%, respectively. After three weeks, the swelling rate remained stable, indicating that the swelling rate and degradation rate reached a state of equilibrium.

[0236] In some embodiments, one or more length design criteria in the plurality of design criteria include a length resolution of the microchannel vascular network device. In some embodiments, the resolution is between 0.005 inches and 0.010 inches. In some embodiments, one or more length design criteria in the plurality of design criteria include a first resolution of a first length of the microchannel vascular network device and a second resolution of a second length of the microchannel vascular network (e.g., a 0.005 inch resolution of the xy plane of the microchannel vascular network device 700).

[0237] In some embodiments, one or more polymer design criteria in a plurality of design criteria include the porosity of the microchannel vascular network device. In some embodiments, the porosity of the microchannel vascular network device is determined based on a candidate subject associated with the microchannel vascular network device. For example, in some embodiments, the porosity of the microchannel vascular network device is determined to allow candidate subject cells to attach to the pores of the microchannel vascular network device. In some embodiments, the median size of the pores of the microchannel vascular network device is about 19 μm to about 231 μm. For example, in some embodiments, a plurality of design criteria include the permeability of the porous material of the microchannel device.

[0238] In some embodiments, the determining further comprises determining one or more design criteria of the plurality of design criteria associated with forming a polymer material from a prepolymer solution. For example, in some embodiments, the design criteria associated with forming a polymer material from a prepolymer solution comprises a material design property of the prepolymer solution.

[0239] Block 504. Referring to block 504, method 500 includes receiving the microchannel vascular network design in electronic form at the manufacturing system. In some embodiments, the receiving includes transmitting one or more instructions based on the microchannel vascular network design to a manufacturing facility associated with a manufacturing control facility of the manufacturing system.

[0240] In some embodiments, the manufacturing system 200 includes a prepolymer solution. For example, in some embodiments, the manufacturing system 200 includes a plurality of prepolymer solutions, thereby allowing the manufacturing system to provide a prepolymer solution for manufacturing the microchannel vascular network device 700. In some embodiments, the prepolymer solution provided to the manufacturing system 200 is preformed. However, the present disclosure is not limited thereto. For example, in some embodiments, the manufacturing system forms a prepolymer solution for forming the microchannel vascular network device 700.

[0241] In some embodiments, the prepolymer solution includes a photoinitiator, such that exposing the prepolymer solution with the photoinitiator to light causes a cross-linking effect, which in turn forms a polymer material.

[0242] Block 506. Referring to block 506, the method includes forming a microchannel vascular network device of a polymer material using a prepolymer solution at a manufacturing system based on the microchannel vascular network design. In this manner, the method facilitates manufacturing of a microchannel vascular network device.

[0243] In some embodiments, the polymer material includes polydimethylsiloxane (PDMS), polyglycerol sebacate (PGS), polylactic acid (PLA), poly L-lactic acid (PLLA), poly D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polyglucose ester, polylactic acid-polyethylene oxide copolymer, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxypropionic acid, polyphosphate, poly (α-hydroxy acid), polycaprolactone, polycarbonate, polyamide, polyanhydride, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, degradable urethane, aliphatic polyester, polyacrylate, polymethacrylate, acyl substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonylated polyolefin, polyethylene oxide, polyvinyl alcohol, Nylon silicon and shape memory materials such as poly (styrene-block-butadiene), polynorbornene, hydrogel, metal alloy, and oligo (ε-caprolactone) diol as switching segment / oligo (p-polydioxanone) diol as physical cross-linker. For example, in some embodiments, the PLGA polymer is a PLA / PGA polymer = 50 / 50 with a molecular weight of 30,000 Da.

[0244] For example, by utilizing PLGA polymer, the present disclosure

[0245] In some embodiments, the polymer material of the microchannel vascular network device is transparent. In some embodiments, the polymer material of the microchannel vascular network device is transparent, translucent, opaque, or a combination thereof. By having a transparent and / or translucent microchannel vascular device, a subject can optically inspect the interior of the microchannel vascular network device without disturbing the contents therein. For example, referring to Fig. 27 , Figure 29-34 and Figure 36-40 , providing an embodiment of transparent and or translucent manufacturing including corresponding microchannel vascular networks. Specifically, Figure 31 shows multiple microscopic images of multiple PLGA microspheres (e.g., beads) in the following: (A) twenty conical structures between the corresponding first channel network and the corresponding second channel network; (B) twenty spherical structures between the corresponding first channel network and the corresponding second channel network; and twenty cylindrical structures between the corresponding first channel network and the corresponding second channel network. For example, Figure 31 (A) depicts that, in contrast to the corresponding devices of Figures 31 (B) and (C), a portion of the microspheres are trapped inside the structure without size differences from top to bottom.

[0246] In some embodiments, the polymer is a biodegradable material. In some embodiments, the polymer is non-resorbable, resorbable, absorbable, biodegradable or a combination thereof. By utilizing a biodegradable material such as PEGdA, the corresponding microchannel vascular network device can be implanted in a subject without the need to remove the microchannel vascular network device later (e.g., by an invasive procedure).

[0247] In some embodiments, the forming comprises exposing the prepolymer solution to ultraviolet light for a predetermined period of time. For example, in some embodiments, the manufacturing system comprises a VAT photopolymerization mechanism that facilitates exposing the prepolymer solution to ultraviolet light. In addition, in some embodiments, the manufacturing system utilizes volumetric printing.

[0248] In some embodiments, the manufacturing system is formed using an additive manufacturing method. In some embodiments, the additive manufacturing method is selected from the group consisting of: binder jetting, material extrusion, material jetting, polyjet, powder bed, sheet lamination, and VAT photopolymerization. In some embodiments, the additive manufacturing method of VAT photopolymerization includes stereolithography (e.g., projection stereolithography).

[0249] In some embodiments, the forming forms a microchannel vascular network device having a positive mold, thereby forming a gap between the first channel network and the second channel network. In some embodiments, the forming forms a microchannel vascular network device having a positive mold, thereby forming a gap between the first channel network and the second channel network. For example, Fig.38 The corresponding microchannel vascular network device formed as a positive mold is shown, and Fig.39 A corresponding microchannel vascular network device formed as a negative mold is shown.

[0250] Now that a general method 500 for making a microchannel vascular network device 700 according to various embodiments of the present disclosure has been described, a method for seeding a microchannel vascular network device according to the present disclosure will be described. Figure 6 Some methodological details of the microchannel vascular network device. Figure 6 A second flow chart of a method (e.g., method 600) for seeding a microchannel vascular network device 700 according to an embodiment of the present disclosure is shown. In the flow chart, preferred portions of the method are shown in solid boxes, while optional variations of the method or optional equipment used in the method are shown in dashed boxes.

[0251] Box 602. Reference Figure 6 In block 602, method 600 includes preparing a microchannel vascular network device based on a candidate subject, thereby forming a prepared microchannel vascular network device. The prepared microchannel vascular channel device includes a first channel network and a second channel network.

[0252] In some embodiments, the preparing further comprises preparing a plurality of cells for inoculation.

[0253] In some embodiments, preparing the plurality of cells comprises exposing the plurality of cells to a solution. In some embodiments, the solution comprises calcium and magnesium.

[0254] In some embodiments, the plurality of cells are exposed to the solution for a period of about 15 minutes to about 25 minutes. In some embodiments, the plurality of cells are exposed to the solution for a period of about 20 minutes.

[0255] In some embodiments, the preparing the microchannel vascular network device includes modifying the surface of the microchannel vascular network device.

[0256] In some embodiments, the surface modification of the microchannel vascular network device is chemical surface modification, mechanical surface modification or both. In some embodiments, chemical surface modification is performed before mechanical surface modification. In other embodiments, mechanical surface modification is performed before or simultaneously with chemical surface modification.

[0257] In some embodiments, chemical surface modification includes forming a conjugate on the surface of the microchannel vascular network device. It is within the ability of those skilled in the art to conjugate the compounds of the present disclosure to donors or acceptors through reactive functional groups on the conjugation partner and appropriate linkers, adapters, carrier molecules or combinations thereof.

[0258] For example, in some embodiments, the surfactants of the present disclosure are conjugated to a carrier molecule via a linker having one or more than one amino acid. Exemplary amino acids for such linkers include lysine, proline, and acidic amino acids.

[0259] In some embodiments, the conjugation is a peptide conjugation. For example, in some embodiments, the surfactant of the microchannel vascular network device 700 comprises both polar groups and non-polar groups. Therefore, by forming a peptide conjugation, the non-polar group of the surfactant is conjugated to the surface of the microchannel vascular network device 700. On the other hand, the polar group of the surfactant captures one or more proteins and / or one or more cells within the microchannel vascular network device 700. Therefore, by forming a peptide conjugation, the wettability of the surface of the microchannel vascular device 700 is increased, which improves the ability to culture cells within the microchannel vascular network device 700.

[0260] In exemplary embodiments, the peptide is a tripeptide. In various embodiments, the peptide contains one or more of arginyl, glycyl or aspartic acid residues. In some embodiments, the peptide comprises a member selected from: arginyl and glycyl, arginyl and aspartic acid, and glycyl and aspartic acid. In exemplary embodiments, the peptide comprises a dipeptide residue selected from arginylglycyl, glycyl aspartic acid and combinations thereof. For example, in some embodiments, the peptide conjugation is an arginylglycyl aspartic acid peptide. The peptide is conjugated to the surface directly or through a joint. Many approaches to conjugating peptides to the surface are known. In one example, the carboxylic acid group on the peptide is activated and reacts with a surface or joint with an amine or hydroxyl moiety. In another example, the carboxylic acid moiety on the surface is activated and subsequently reacts with an amine on the peptide. Obviously, this is also true for those embodiments using a joint. The peptide can be conjugated to a joint that is bonded to the surface, or alternatively, the peptide-joint box can be bonded to the surface or two second joints that are already present on the surface.

[0261] Additional non-limiting information regarding conjugation strategies in various embodiments is set forth below.

[0262] Compounds of the present invention and conjugates are assembled by covalent bonding reactions between precursors with reactive functional groups, and the reactive functional groups are sites for forming covalent bonds between precursors. The precursors of compounds of the present invention carry reactive functional groups, which can be located at any position on the compound. The dye conjugates completed can include other reactive functional groups at any point of the molecule. In various embodiments, the reactive functional groups on the peptide react with the reactive functional groups on the surface (or the joint attached to the surface) to covalently couple two components together by connecting fragments.

[0263] Exemplary substances include reactive functional groups that are directly attached to the peptide core or attached to the joint attached to the surface component. Exemplary reactive functional groups are attached to alkyl or heteroalkyl moieties. When reactive groups are attached to substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl joint moieties, reactive groups are preferably located at the terminal position of alkyl or heteroalkyl chains. Reactive groups and reaction categories that can be used for implementing the present invention are generally those known in the field of bioconjugation chemistry. Currently available preferred reaction categories with the active dye-based compounds of the present invention are those reactions carried out under relatively mild conditions. These include but are not limited to nucleophilic substitutions (for example, amine and alcohol and the reaction of acyl halides, active esters), electrophilic substitutions (for example, enamine reaction) and addition to carbon-carbon and carbon-heteroatom multiple bonds (for example, Michael reaction (Michael reaction), Diels-Alder addition). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0264] Useful reactive functional groups include, for example:

[0265] (a) carboxyl groups and their derivatives, including but not limited to activated esters, such as N-hydroxysuccinimide esters, N-hydroxyphthalimide, N-hydroxybenzotriazole esters, p-nitrophenyl esters; acyl halides; acyl imidazoles; thioesters; alkyl, alkenyl, alkynyl and aromatic esters; and activated groups for peptide synthesis;

[0266] (b) Hydroxyl and hydroxylamine, which can be converted into esters, sulfonates, phosphoramidates, ethers, aldehydes, etc.

[0267] (c) haloalkyl, where the halide can be replaced by a nucleophilic group such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion or an alkoxide ion, resulting in a new group covalently attached in place of the halogen atom;

[0268] (d) a dienophilic group capable of participating in a Diels-Alder reaction, such as, for example, a maleimide group;

[0269] (e) aldehyde or ketone groups, allowing derivatization via formation of carbonyl derivatives (e.g., imines, hydrazones, semicarbazones, or oximes) or via mechanisms such as Grignard addition or alkyllithium addition;

[0270] (f) sulfonyl halide groups for reaction with amines, e.g. to form sulfonamides;

[0271] (g) thiol groups, which can, for example, be converted into disulfides or reacted with acyl halides;

[0272] (h) amines, hydrazines or thiols, which may, for example, be acylated, alkylated or oxidized;

[0273] (i) olefins, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.;

[0274] (j) epoxides, which can react with, for example, amines and hydroxy compounds; and

[0275] (k) Phosphoramidites and other standard functional groups used in nucleic acid synthesis.

[0276] In various embodiments, the reactive functional group is a member selected from:

[0277]

[0278] wherein each r is independently selected from an integer from 1 to 10; G is halogen; and R 30 and R 31 are independently selected from H members and halogen, and R 30 and R 31 At least one of them is a halogen.

[0279] Reactive functional groups can be selected so that they do not participate in or interfere with the reaction required for assembling or utilizing functionalized surfaces or peptides. Alternatively, reactive functional groups can be prevented from participating in the reaction by the presence of blocking groups. Those skilled in the art understand how to protect specific functional groups so that they do not interfere with the reaction conditions of the selected group. For examples of useful blocking groups, see, for example, Greene et al., Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0280] In various embodiments, the peptide is attached to the surface via a linker or a linker comprising at least one linker. Exemplary linkers include bonds and moieties comprising at least one heteroatom, which are formed by the reaction of two reactive functional groups having complementary reactivities. Exemplary linkers for use in the conjugates of the invention include, but are not limited to:

[0281] -(CH2) t S(CH2) z -、-(CH2) x SC(O)NR(CH2) z -、-(CH2) x SC(O)O(CH2) z -、-(CH2) x NR(CH2) z -、-(CH2) x NRC(O)(CH2) z -、-(CH2) x NRC(O)O(CH2) z -、-(CH2) x O(CH2) z -、-(CH2) o T-PEG-, -(CH2) x C(O)CH2S-, -S-maleimide-N-, -RNC(O)NR-, -RNS(O)NR-, -S(O)2NR-,

[0282] wherein T is a member selected from S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH and O. The index o is an integer from 1 to 50; and the indices t and z are independently selected from integers from 0 to 10.

[0283] Linking fragments can also be formed via "click chemistry between one component having an azide moiety and another component having an alkyne moiety." Either the donor or the acceptor can be derivatized with any reactive functional group, as can the carrier molecule.

[0284] As a non-limiting example, consider that the microchannel vascular network device includes a PEG-based hydrogel polymer material, which is well known for its biologically inert protein and cell repelling properties. Therefore, in order to improve the cell attachment properties of the PEGdA material, the present disclosure chemically activates the surface of the microchannel vascular network device by performing a chemical surface modification, which forms a biologically functional surface by conjugation of an RGD peptide. For example, by RGD conjugation chemical surface modification, the ability of multiple cells in the microchannel vascular network device 700 to proliferate (e.g., culture) is greatly improved.

[0285] In some embodiments, the chemical surface modification includes surface grafting modification. For example, in some embodiments, the surface of the microchannel vascular network device 700 includes an acrylic surface, so that the chemical surface modification includes surface grafting via opening epoxy groups and / or kenotic groups on the surface, thereby increasing the number of hydroxyl groups on the surface of the microchannel vascular network device 700.

[0286] In some embodiments, the surface modification of the microchannel vascular network device is mechanical surface modification. In some embodiments, mechanical surface modification is applied to the microchannel vascular network device 700 alone or in combination with chemical surface treatment. For example, in some embodiments, chemical surface treatment forms one or more contaminant particles, which need to be processed by mechanical surface modification. In this way, mechanical surface modification can generate a structure between the surface of the microchannel vascular network device 700 and the multiple cells in the microchannel vascular network device 700 to prevent the interaction between the corresponding cells and the corresponding contaminant particles.

[0287] In some embodiments, the mechanical surface modification comprises applying a coating to the surface of the microchannel vascular network device. In some embodiments, the coating comprises collagen, fibronectin, gelatin, GelMA, PEGdA, PLL or a combination thereof.

[0288] In some embodiments, the coating comprises a first weight percent (wt%) of PEGdA of about 8% to about 12% and a second wt% of GelMA of about 0.5% to about 7%. In some embodiments, the first wt% of PEGdA is 10%, and the second wt% of GelMA is about 1% to about 5%. In some embodiments, the second wt% of GelMA is between 4% and 5%.

[0289] In some embodiments, the coating includes a first coating comprising PEGdA, PPL, GelMA or a combination thereof. The first coating includes a first upper surface and a first lower surface. The coating also includes a second coating comprising collagen or gelatin. The second coating includes a second upper surface and a second lower surface. In addition, the first lower surface is adjacent to the surface of the microchannel vascular network device, and the second lower surface is adjacent to the first upper surface.

[0290] For example, in some embodiments, the coating comprises 10% PEGdA, 5% GelMA, and 0.25% photoinitiator. In some embodiments, the coating comprises 10% PEGdA coated with GelMA, and the coating is then exposed to UV light. In some embodiments, the coating comprises 10% PEGdA further coated with 10% gelatin. In some embodiments, the coating comprises 10% gelatin. In some embodiments, the coating comprises aortic endothelial cells.

[0291] In some embodiments, the surface modification includes applying a NaOH-EtOH solution treatment for about 10 minutes. Thus, an effective surface morphology change is provided by generating new cracks in the surface of the microchannel device.

[0292] For example, briefly refer to Fig.35 As a non-limiting example, the perfusion of cells within the corresponding microchannel devices of PLGA polymer, the internal porous structure of the PLGA device, and the cell attachment of the device were characterized by SEM images. For sample preparation of SEM samples, the HepG2-perfused PLGA device was frozen and manually broken into small fragments for microscopic observation. PLGA printing filaments were observed in almost all the broken PLGA fragments. The BioX printer piston size is 100um, and the size of the real printing filament is almost the same. Fig.35 As shown, irregularly shaped pores (holes) were observed between the filaments; the size varied from 20 um to 200 um. Based on this, HepG2 cells were observed on the surface of the microchannel device.

[0293] Block 604. Referring to block 604, method 600 includes seeding a plurality of cells for culture in the prepared microchannel vascular device based on the candidate subject, thereby forming a plurality of seed cells.

[0294] In some embodiments, seeding comprises encapsulating the plurality of cells in culture medium.

[0295] In some embodiments, the culture medium comprises a photoinitiator, and prior to encapsulating the plurality of cells, seeding further comprises exposing the culture medium and the plurality of cells to ultraviolet light for a predetermined period of time.

[0296] Brief reference Fig.24, provides the effect of exposure time on cell proliferation in a PEGDA hydrogel microchannel vascular network device. Specifically, Fig.24 Shown are multiple fluorescent images of encapsulated cells in a PEGDA microchannel vascular network device, with different exposure times (e.g., 30 seconds, 60 seconds, and 120 seconds). A mixture of 1.6M / mL RAEC, 0.8M / mL H4-II-EC3, and 0.4M / mL rMSC is encapsulated in a 20wt% PEGDA hydrogel and cross-linked for about 30sec to about 120sec. Fluorescent images are captured and compared on the 1st and 7th days, as shown. RAEC cells are marked in red, H4-II-EC3 cells are marked in green, and the scale bar indicates 200um. In order to demonstrate cell proliferation in a hydrogel microchannel vascular network device 700, cells are encapsulated in a 150um thick hydrogel and cultured for 1 to 7 days (e.g., Figure 6 Method 600). Light irradiation was varied from about 30 seconds to about 2 minutes to evaluate the effect of light exposure time on cell proliferation. On day 1, all samples showed high cell viability regardless of light exposure time. However, on day 7, cells in hydrogels exposed for 2 minutes showed less than half of cell viability compared to day 1. This difference is due to the phototoxicity of long-term irradiation and the large amount of free radicals generated by the photoinitiator molecules. In contrast, cells in hydrogels exposed for 30 sec and 60 sec still maintained their cell viability after 7 days of culture. This indicates that exposure times up to 60 min have minimal cytotoxicity to encapsulated cells.

[0297] In some embodiments, the culture medium has a thickness of about 5 μm to about 15 μm.

[0298] In some embodiments, the density of the inoculated multiple seed cells is about 1*10 4 cells / cm2 to about 1*10 5 cells / cm2.

[0299] In some embodiments, the culture medium comprises a plurality of cells encapsulated in collagen. Such encapsulation provides sufficient support structure within the microchannel device to support cell culture.

[0300] Cells are evenly distributed throughout the cross section

[0301] Block 606. Referring to block 606, method 600 includes using a bioreactor system coupled to the prepared microchannel vascular network device to flow a culture medium containing a plurality of seed cells through the prepared microchannel vascular network device, thereby culturing a plurality of cells in the microchannel vascular network device.

[0302] For example, briefly refer to Fig.33, an image of a first channel of a microchannel device is provided. As shown, movement of cells within the device is provided by a bioreactor system. Here, the diameter of the channel is about 920 μm. Thus, the present disclosure allows culturing cells within a microchannel vascular network device by allowing control of flow within the device as facilitated by a bioreactor system.

[0303] In some embodiments, the flow rate of the flow is about 375 microliters (μL) / minute to about 425 μL / minute.For example, in some embodiments, the bioreactor system 200 transmits one or more instructions to the pump 1720, which facilitates controlling the flow rate.

[0304] In some embodiments, before flowing, a portion of the prepared microchannel vascular network device is in a dry state or a wet state.

[0305] In some embodiments, the portion of the prepared microchannel vascular network device is the lower end portion of the microchannel vascular network device before flowing.

[0306] Various aspects of the present disclosure relate to providing systems and methods for preventing or stopping the flow of culture medium in a device. The device of the present disclosure includes an artificial organ device (e.g., an implant), a bioreactor device, a chip lab device (e.g., a chip organ device), etc. Therefore, an exemplary device includes at least a first channel and a second channel through which the culture medium flows. In addition, in some embodiments, each of the first channel and the second channel includes a first opening and a second opening arranged at the first end of the device. The corresponding second openings of the first channel and the second channel can be arranged on the same end of the device (e.g., the second end of the device) or on different ends of the device (e.g., respectively at the second end of the device and the third end of the device). Therefore, each opening of the corresponding channel is used as an inlet or outlet for the flow of culture medium. However, the present disclosure is not limited thereto. In some embodiments, the device of the present disclosure includes a main inlet and a main inlet communicated with the first channel and the second channel.

[0307] As previously described, in some embodiments, exemplary devices are components of systems, such as bioreactor systems. Therefore, in some embodiments, each opening of corresponding passage includes a part for being conducive to being connected to another component of the system, such as a pipeline communicated with a reservoir or pump of a culture medium. In some embodiments, the connection portion of each corresponding passage has a connection diameter different from the channel diameter of the corresponding passage. In some embodiments, the first opening and the second opening of the first channel and the second channel each have about 500 microns (μm) to about 10,000 μm, about 500 μm to about 5,000 μm, about 500 μm to about 2,500 μm, about 500 μm to about 1,500 μm, about 500 μm to about 1,000 μm, about 650 μm to about 2,500 μm, about 650 μm to about 1,500 μm, about 650 μm to about 1,000 μm or about 650 μm to about 750 μm in the range of channel diameter. Additionally, in some embodiments, the first opening and the second opening of the first channel have different diameters than the first opening and the second opening of the second channel.

[0308] One or more intermediate channels are interconnected with the first channel and the second channel. One or more intermediate channels are configured to diffuse the material in the culture medium or culture medium such as nutrients and / or waste materials to the first channel and the second channel of the device or diffuse therefrom. In some embodiments, the diffusion rate of the culture medium is proportional to the number of the intermediate channels included in the device (for example, the more intermediate channels, the higher the diffusion rate of the culture medium). In some embodiments, the device includes one intermediate channel, two intermediate channels, three or less intermediate channels, five or less intermediate channels, ten or less intermediate channels, fifteen or less intermediate channels, twenty or less intermediate channels, fifty or less intermediate channels, one hundred or less intermediate channels, two hundred or less intermediate channels, five hundred or less intermediate channels, one thousand or less intermediate channels, or ten thousand or less intermediate channels. In addition, one or more intermediate channels can be formed into various shapes, including cylinders, cuboids, truncated cones, three-dimensional grids, etc. In some embodiments, one or more intermediate channels are formed with rounded or chamfered edges to provide more uniform flow in the intermediate channel. However, each intermediate channel is formed with an opening wide enough to allow the medium to flow through the intermediate channel and to receive at least one microgel unit. In some embodiments, each of the one or more intermediate channels is formed with an opening of about 50 μm to about 1,500 μm, about 50 μm to about 1,250 μm, about 50 μm to about 1,000 μm, about 100 μm to about 1,250 μm, about 100 μm to about 1,000 μm, about 100 μm to about 800 μm, about 400 μm to about 1,000 μm, about 300 μm to about 800 μm, about 300 μm to about 600 μm, or about 400 μm to about 400 μm. In addition, in some embodiments, each of the one or more intermediate channels is formed with a variable diameter so that the diameter of each corresponding intermediate channel decreases from the first diameter of the first opening to the second diameter of the second opening. In some embodiments, the second opening is about 50% to about 90% of the size of the first opening. For example, in some embodiments, the first opening of the corresponding intermediate channel is 600 μm, and the second opening of the corresponding intermediate channel is 500 μm (e.g., the second opening is approximately 83% of the size of the first opening). In addition, in some embodiments, the diameter of the first opening of each corresponding intermediate channel is greater than or equal to the diameter of the microgel unit in the plurality of microgel units, and the diameter of the second opening is less than the diameter of the microgel unit in the plurality of microgel units. For example, in some embodiments, a plurality of microgel units in the range of 300 μm to 500 μm are formed. Therefore, the first opening of each intermediate channel is greater than or equal to 500 μm, and the second opening of each intermediate channel is less than or equal to 300 μm.The above configuration of the variable diameter of the one or more intermediate channels allows one or more microgel units among the plurality of microgel units suspended in the first solution to enter the intermediate channel through the first opening and then remain in a portion of the intermediate channel having a smaller diameter than the microgel.

[0309] For example, refer to Figure 7 , an exemplary device 700 is shown. The device includes a first channel 702-1 and a second channel 702-2. Each channel 702 (e.g., the first channel 702-1 and the second channel 702-2) includes a first opening and a second opening. Therefore, each corresponding opening of each channel 702 includes a coupling portion 710-1 having a coupling diameter, which is configured to be coupled to another component of the system (e.g., a portion of a receiving tube), and a channel portion 710-2 having a channel diameter. In addition, the first channel 702-1 is interconnected with the second channel 702-2 by an intermediate channel 704 (e.g., a first intermediate channel 704-1, a second intermediate channel 704-2, a third intermediate channel 704-3, ..., an nth intermediate channel 704-n). Figure 7 The device 700 shows an embodiment including sixteen intermediate channels (eg, a first intermediate channel 704-1, a second intermediate channel 704-2, ..., and a sixteenth intermediate channel 704-16). In the exemplary embodiment, each intermediate channel is formed in a rectangular parallelepiped shape with an opening of about 400 μm.

[0310] Now that the general structure of various embodiments of the disclosed device 700 has been described, various fabrication techniques and methods for the microgel cells will now be described in detail.

[0311] In some embodiments, the flow of culture medium in the device must be prevented or stopped. For example, in order to inoculate cells in the device and attach the cells to the channels of the device, the flow of the culture medium in which the cells are suspended must be prevented or stopped; otherwise, the cells may be washed through the device without being attached to the device. In order to facilitate such inoculation, a solution (e.g., culture medium) containing a suspension of microgel units flows through at least one channel of the device. This flow arranges one or more microgel units in at least one intermediate channel of the device, which prevents or stops the flow of culture medium between the channels of the device.

[0312] A plurality of gelatin methacryloyl (GelMA) microgel units can be manufactured using a variety of methods. For example, in some embodiments, a plurality of GelMA microgels are manufactured via micromolding technology, photomask technology, bioprinting technology, self-assembly technology, additive manufacturing technology (e.g., three-dimensional printing), microfluidic technology, or a combination thereof. Depending on the goals of the designer of the present disclosure and the manufacturing technology used, at least the degree of methacryloyl substitution, the concentration of the GelMA microgel, the concentration of the photoinitiator, and the time period for which the sample solution is exposed to UV light can be controlled individually or in combination to configure one or more physical properties of the resulting GelMA hydrogel. In some embodiments, the physical properties include the size of the resulting GelMA microgel, so that the first manufacturing technology produces one or more microgels of a first size (e.g., 50 μm), and the second manufacturing technology produces one or more microgels of a second size (e.g., 500 μm). In some embodiments, the physical properties include the degradation rate of the resulting GelMA microgel. Controlling the degradation rate allows the flow of the culture medium through the intermediate channel of the corresponding device to be blocked or stopped within a predetermined time period proportional to the degradation rate. In some embodiments, the physical properties include the shape of the resulting GelMA microgel. In some embodiments, the physical property includes the viscosity of the resulting GelMA microgel. Other controllable physical properties will be known to those skilled in the art of the present disclosure.

[0313] In some embodiments, GelMA is synthesized by direct reaction of gelatin with methacrylic anhydride (MA) in the presence of a buffer. This reaction introduces methacryloyl substituents (MA) on reactive amines and hydroxyl groups of amino acid residues. Figure 2 ). By adjusting the amount of MA added to the reaction mixture, different degrees of methacryloyl substitution can be achieved in GelMA, which produces GelMA with different physical properties. Photocrosslinking of the synthesized GelMA can be performed using a water-soluble initiator under exposure to ultraviolet (UV) light. The degree of substitution, GelMA concentration, photoinitiator (PI) concentration, and exposure time to UV light are some of the parameters that allow tuning the physical properties of the resulting GelMA hydrogel microunits.

[0314] Therefore, reference Figures 8 to 10, a method for manufacturing multiple GelMA microgels via a photomask technique will be described according to an embodiment of the present disclosure. In some embodiments, the photomask technique includes forming a prepolymer solution. Forming the prepolymer solution includes dissolving GelMA in a buffer solution and adding a photoinitiator. In some embodiments, the buffer solution is phosphate buffered saline (PBS). In some embodiments, the dissolved GelMA is in the range of 5% wt / wt to 35% wt / wt, 7% wt / wt to 20% wt / wt, 10% wt / wt to 30% wt / wt, 15% wt / wt to 25% wt / wt, or 18% wt / wt to 22% wt / wt (e.g., 20% wt / wt). Additionally, in some embodiments, the photoinitiator is added in a range of 0.5% wt / wt to 2% wt / wt of the prepolymer solution, 0.5% wt / wt to 1.5% wt / wt of the prepolymer, 1% wt / wt to 1.5% wt / wt of the prepolymer solution, or 0.8% wt / wt to 1.2% wt / wt of the prepolymer solution (e.g., 1% wt / wt). In some embodiments, the photoinitiator includes 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone. In some embodiments, the use of lower weight portions of GelMA (e.g., 7% weight portions compared to 20% weight portions of GelMA) generally produces microgels that are easier to separate from their manufacturing environment than higher weight portions of GelMA microgels. However, these lower weight portions of GelMA microgels generally deform and / or degrade from their manufactured shape at a faster rate than higher weight portions of GelMA microgels. Likewise, higher weight portions of GelMA (e.g., 20% by weight) produce microgels that are generally hard and / or non-sticky to surrounding environments and structures, but may be difficult to separate from their manufacturing environment. In some embodiments, a prepolymer solution is formed having a methacryloyl substitution degree of about 75% to about 90%. As used herein, "GelMA_low" refers to a methacryloyl substitution degree of less than or equal to about 80%, "GelMA_medium" refers to a methacryloyl substitution degree of greater than about 80%, and "GelMA_high" refers to a methacryloyl substitution degree of greater than about 90%.

[0315] In some embodiments, the photomask technique includes manufacturing and / or utilizing a photomask comprising an array of transparent sheets. Each transparent sheet in the transparent sheet array of the photomask forms at least one corresponding GelMA microgel. In some embodiments, the transparent sheet array is formed as a circular array (e.g., a thin cylinder) that produces a cylindrical or disc-shaped microgel. In addition, in some embodiments, each transparent sheet is formed to have a corresponding diameter in the range of 50 μm to 750 μm, 50 μm to 650 μm, 100 μm to 650 μm, 100 μm to 500 μm, 150 μm to 650 μm, 150 μm to 550 μm, 200 μm to 550 μm, or 300 μm to 500 μm. Additional shapes and sizes of each transparent sheet in the transparent sheet array can be used in the present disclosure. In some embodiments, each transparent sheet in the transparent sheet array is formed to have approximately equal diameters (e.g., within an acceptable tolerance range, such as ±10 μm), which allows the photomask to produce multiple GelMA microgels of approximately equal size. Similarly, in some embodiments, the transparent sheet array includes a range of diameters, thereby allowing the resulting GelMA microgels to be manufactured in a variety of sizes (e.g., a range of 300 μm to 500 μm).

[0316] In some embodiments, to make GelMA microgels, a predetermined amount (e.g., volume or mass) of prepolymer solution and photoinitiator is arranged (e.g., sucked) onto a first glass slide. In some embodiments, the predetermined amount is in the range of 5 microliters (μL) to 1,000 μL, 5 μL to 750 μL, 10 μL to 500 μL, 10 μL to 250 μL, 10 μL to 150 μL, 10 μL to 100 μL, 5 μL to 100 μL, 10 μL to 80 μL, 20 μL to 75 μL, 25 μL to 75 μL, or 35 μL to 65 μL (e.g., 50 μL). In some embodiments, the predetermined amount is a volume defined by the internal capacity (e.g., volume) of the structure formed by the glass slide (e.g., I, the internal volume formed by the inner surface of the glass slide). In some embodiments, the predetermined amount is a volume that is less than the volume of the containing structure (eg, about 95% of the volume of the containing structure).

[0317] In some embodiments, the outer edge portion of the first glass slide surrounds one or more spacers. The one or more spacers provide a vertical separation between the first glass slide and the second glass slide (e.g., the height of the volume containing the structure) and help prevent solution leakage. In addition, in some embodiments, after the arrangement of the prepolymer solution and the photoinitiator is completed, the second glass slide is arranged above the first glass slide to form a film containing the structure while also uniformly distributing the prepolymer solution and the photoinitiator therein. By changing the size of the first glass slide and / or the second glass slide and the thickness of the one or more spacers (e.g., the vertical separation formed), microgels with different aspect ratios can be formed. For example, in some embodiments, the thickness of the resulting microgel is in the range of 50 μm to 1,000 μm, 50 μm to 750 μm, 150 μm to 750 μm, 100 μm to 650 μm, 150 μm to 650 μm, 100 μm to 550 μm, 150 μm to 550 μm, 150 μm to 500 μm, 100 μm to 450 μm, 150 μm to 450 μm, 200 μm to 500 μm, or 300 μm to 500 μm. In addition, in some embodiments, after the arrangement of the prepolymer solution and the photoinitiator is completed, a second glass slide is disposed above the first glass slide, which forms a film containing the structure and in which the prepolymer solution and the photoinitiator are uniformly distributed. By varying at least the size of the first glass slide and / or the second glass slide and the thickness of one or more spacers, the microgel can be configured to have different aspect ratios.

[0318] In some embodiments, the photomask is arranged to the top surface of the second slide.Therefore, the photomask and the containing structure are exposed to UV light, and the predetermined time period is continued, thereby allowing the transparent sheet of the photomask to allow UV light to pass through, while blocking the remaining UV light.In some embodiments, the predetermined time period is less than or equal to about 30 seconds.In some embodiments, the predetermined time period is in the range of 1 second to 180 seconds, 1 second to 150 seconds, 1 second to 120 seconds, 10 seconds to 120 seconds, 1 second to 90 seconds, 1 second to 60 seconds, 5 seconds to 60 seconds, 5 seconds to 50 seconds, 5 seconds to 40 seconds, 10 seconds to 40 seconds, 5 seconds to 30 seconds or 10 seconds to 30 seconds. In some embodiments, the light has a wavelength in the range of 10 nanometers (nm) to 480 nm, 100 nm to 480 nm, 100 nm to 400 nm, 200 nm to 480 nm, 200 nm to 400 nm, 300 nm to 480 nm, 300 nm to 400 nm, or 350 nm to 400 nm (e.g., a wavelength of 365 nm). In addition, in some embodiments, the ultraviolet light has a wavelength in the range of 10 milliwatts per cubic centimeter (mW cm -3 ) to 15 mW cm -3 、10.5mW cm -3 Up to 14.5 mW cm-3 , 11mW cm -3 Up to 14mW cm -3 、11.5mW cm -3 Up to 13.5 mW cm -3 , 12mW cm -3 Up to 13mW cm -3 , or 12.2 mW cm -3 Up to 12.8 mW cm -3 Intensity in the range (e.g., 12.4 mW cm -3 ). Exposure to UV light enables cross-linking of the prepolymer solution, which in turn produces GelMA microgels.

[0319] In some embodiments, the containing structure is at least partially disassembled (e.g., decomposed) to retrieve the GelMA microgel, which can mark the completion of the manufacture of multiple GelMA microgels. In some embodiments, the manufactured GelMA microgel is formed into a connected structure (e.g., a network or matrix of microgel) and requires physical separation to ensure that the GelMA microgel flows properly in the device and blocks the intermediate channel. For example, in some embodiments, the manufacture of GelMA microgel produces a wing beam that interconnects the microgel. Therefore, in some embodiments, the recovery of the GelMA microgel includes transferring the generated GelMA microgel to a mineral oil bath, subjecting it to mechanical agitation (e.g., agitation to remove the wing beam or remove the microgel). In some embodiments, the agitation of the mineral oil bath includes agitation with a pipette (e.g., stirring the pipette with a back-and-forth and / or circular motion). In some embodiments, the agitation of the mineral oil bath includes ultrasonic treatment. In addition, in some embodiments, the agitation of the mineral oil bath includes the use of a magnetic stirrer. The above-mentioned agitation includes manual agitation and / or automatic agitation (e.g., agitation using an automated system).

[0320] In some embodiments, the GelMA microgel manufactured by retrieving the glass slide and / or the gasket containing the structure needs to be processed (for example, pre-treated) before the prepolymer solution is arranged. For example, in some embodiments, the microgel manufactured by failing to process the glass slide and / or the gasket containing the structure cannot be refined from the corresponding glass slide (for example, the microgel manufactured is sticky). Therefore, in some embodiments, the recovery of the GelMA microgel includes immersing the glass slide and / or the gasket in sodium hydroxide (NaOH) solution. In some embodiments, the glass slide and / or the gasket are immersed for a predetermined time period. In some embodiments, this predetermined time period for immersion is in the range of 5 minutes to 60 minutes, 5 minutes to 50 minutes, 10 minutes to 45 minutes, 20 minutes to 40 minutes, or 25 minutes to 35 minutes (for example, 30 minutes). After the glass slide and / or the gasket are immersed in the sodium hydroxide solution, the glass slide and / or the gasket are immersed in an ethanol solution. Subsequently, the submerged slide and / or pad is subjected to ultrasonic treatment for a predetermined period of time, which forms an ultrasonically treated slide and / or pad. In some embodiments, this predetermined period of time is in the range of 5 minutes to 60 minutes, 5 minutes to 50 minutes, 10 minutes to 45 minutes, 5 minutes to 30 minutes, or 5 minutes to 20 minutes (e.g., 15 minutes).

[0321] In addition, in some embodiments, in order to facilitate the recovery (e.g., separation) of the manufactured microgel, before arranging the prepolymer solution, for at least one glass slide and / or spacer, the method also includes preparing a stock solution of about 0.5wt% methoxypolyethylene glycol 5000Si (mPEG5000-Si), about 1wt% acetic acid and ethanol. The stock solution sample is arranged on at least one glass slide and / or spacer. In some embodiments, the sample has a volume in the range of 5 μL to 500 μL, 5 μL to 250 μL, 10 μL to 250 μL, 5 μL to 100 μL, 10 μL to 100 μL, 5 μL to 750 μL, 25 μL to 70 μL, or 40 μL to 60 μL (e.g., 50 μL). In some embodiments, the size of the sample will depend on the size of the corresponding glass slide and / or spacer. However, at least one slide and / or pad is subjected to a heating environment within a predetermined time period, which forms a slide and / or pad of incubation. In some embodiments, the heating environment has a temperature of 50°C to 110°C, 50°C to 100°C, 50°C to 90°C, 60°C to 100°C, 50°C to 80°C, or 65°C to 75°C (e.g., 70°C). In addition, in some embodiments, the predetermined heating time period is within the range of 5 minutes to 60 minutes, 5 minutes to 50 minutes, 10 minutes to 45 minutes, 20 minutes to 40 minutes, or 25 minutes to 35 minutes (e.g., 30 minutes). However, the slide and / or pad of incubation is immersed in deionized water, which forms an immersed slide and / or pad. This immersed slide and / or pad carries out ultrasonic treatment to each corresponding immersed slide and / or pad within a predetermined time period, which forms an ultrasonically treated slide and / or pad. In some embodiments, the predetermined time period is in the range of 5 minutes to 60 minutes, 5 minutes to 50 minutes, 10 minutes to 45 minutes, 5 minutes to 30 minutes, or 5 minutes to 20 minutes (e.g., 15 minutes). In addition, the ultrasonically treated glass slide and / or pad is dried to form a prepared glass slide and / or pad for future use in making GelMA microgels. In some embodiments, the drying of the ultrasonically treated glass slide and / or pad is performed via a dryer.

[0322] In some embodiments, multiple GelMA microgels are manufactured using microfluidics. Microfluidics includes suspending a first solution (e.g., GelMA) in a second solution (e.g., a non-reactive fluid, such as mineral oil or glycerol) to form a suspension of droplets of the first solution. In some embodiments, a combination of channels that aggregate the flow of the first solution and the second solution is used to form droplets. For example, the first channel produces a GelMA flow, and the second channel and the third channel are each orthogonal to the first channel, thereby producing a second solution flow. The second solution flow helps to form droplets of the first solution. The droplets are further exposed to UV light to form the manufactured GelMA microgel. In some embodiments, one or more GelMA microgels in a plurality of GelMA microgels are formed as spheres or ellipsoids. In some embodiments, a plurality of GelMA microgels include a combination of cuboid microgels, cylindrical microgels and / or spheroid (e.g., ellipsoid) microgels. In addition, in some embodiments, a plurality of GelMA microgels include one or more microgels in the shape of a pyramid.

[0323] In addition, in some embodiments of the present disclosure, the microgel includes one or more pluronic hydrogel microgels. In some embodiments, the pluronic microgel is manufactured via a method comprising dissolving pluronic F127 (pl F127, which is a triblock copolymer of poly(ethylene oxide) and poly(propylene oxide) with a nominal formula EO100-PO65-EO100) in pure water (e.g., deionized water) (similar to the above-mentioned dissolution of GelMA). Therefore, in some embodiments, making pluronic F127 GelMA includes dissolving pluronic F127 in a 10% by weight phosphate buffered saline (PBS) solution containing about 0.25% photoinitiator. In some embodiments, the dissolution includes about 20wt% pluronic F127 in deionized water. In some embodiments, the dissolution includes using an organic solvent (e.g., hexane, toluene, chloroform (CHCl3), dichloromethane, acetone, dimethyl sulfoxide (DMSO)) as a solution bath. In some embodiments, the solution bath includes a non-organic solution and / or a non-aqueous solution (e.g., an oil phase solution, such as mineral oil, olive oil, glycerol, etc.). In addition, in some embodiments, the size of one or more microgels in a plurality of microgels is determined according to a stirring mechanism during the preparation of the microgel. For example, in some embodiments, a slower stirring rate (RPM) (e.g., 4 RPM) produces a larger microgel. In addition, in some embodiments, a surface tension reducing agent surfactant (e.g., polysorbate 20 (TWEEN 20)) and / or an emulsifier (e.g., sorbitan monostearate (SPAN 60)) is included in the manufacture of the microgel to increase the stability of the microgel.

[0324] In some embodiments, one or more microgels in the plurality of microgels are manufactured using additive manufacturing. In some embodiments, one or more microgels include poly(lactic-co-glycolic acid), PGLA, polycaprolactone (PCL), or similar polymers. In some embodiments, one or more microgels include GelMA, fibrin, and collagen.

[0325] In some embodiments, a plurality of GelMA microgels are suspended in a first solution in order to block and / or stop flow in a middle channel of the device. In some embodiments, the first solution contains between 10 microgels / mL working solution and 10,000 microgels / mL working solution, between 10 microgels / mL working solution and 5,000 microgels / mL working solution, between 100 microgels / mL working solution and 5,000 microgels / mL working solution, between 500 microgels / mL working solution and 5,000 microgels / mL working solution, between 100 microgels / mL working solution and 2,500 microgels / mL working solution. working solution, 500 microgels / mL working solution to 2,500 microgels / mL working solution, 500 microgels / mL working solution to 2,000 microgels / mL working solution, 500 microgels / mL working solution to 1,500 microgels / mL working solution, or 750 microgels / mL working solution to 1,250 microgels / mL working solution (e.g., approximately 1,000 microgels / mL working solution). In some embodiments, once a suspension of a plurality of GelMA microgels in a first solution is formed, the suspension of a plurality of GelMA microgels in the first solution is perfused through a first channel of the device. Perfusion of the plurality of GelMA microgel suspensions in the first solution causes one or more of the plurality of GelMA microgels to block and / or stop flow in at least one of the one or more intermediate channels. In some embodiments, a single GelMA microgel is sufficient to block and / or stop flow in the corresponding intermediate channel. In some embodiments, a plurality of GelMA microgels are sufficient to prevent and / or stop flow in a corresponding intermediate channel. For example, in some embodiments, a corresponding GelMA microgel among a plurality of GelMA microgels has a size (e.g., diameter) approximately equal to that of the intermediate channel. Thus, the corresponding GelMA microgel is deformed at the opening of the intermediate channel and / or is retained within the opening of the intermediate channel to prevent and / or stop flow within the intermediate channel. In some embodiments, a corresponding GelMA microgel among a plurality of GelMA microgels has a size smaller than that of the opening of the intermediate channel. Thus, one or more GelMA microgels flow into the opening of the intermediate channel and are retained within the intermediate channel, thereby preventing and / or stopping flow in the intermediate channel. For example, briefly referring to Figure 7B, the structure of one or more intermediate channels 704 includes at least three GelMA microgels 802 (e.g., a first GelMA microgel 202-1, a second GelMA microgel 802-2, and a third GelMA microgel 802-3) that block and / or stop flow within the intermediate channel. Once it is no longer necessary to block or stop the flow of culture medium through the intermediate channel of the device, the microgel that blocks or stops the flow can be removed. In some embodiments, removing the microgel includes applying a fluid flow through the device so that a pressure differential is formed that is conducive to the removal of the microgel. Similarly, in some embodiments, removing the microgel includes allowing a period of time to pass so that the microgel degrades within the device (e.g., based on the degradation rate of the microgel).

[0326] In some embodiments, after a suspension of a plurality of GelMA microgels in a first solution is perfused through a first channel of the device, a fluid (e.g., culture medium) flow is applied to a second channel of the device. This fluid flow creates a pressure differential between the first channel and the second channel of the device, which facilitates the flow of the GelMA microgels into one or more intermediate channels and blocks and / or stops the flow therein. In some embodiments, this pressure differential is required to arrange the GelMA microgels in the intermediate channels because gravity is not sufficient to facilitate blocking or stopping the flow in the intermediate channels via the GelMA microgels.

[0327] refer to Figure 11 to Figure 1 3 and Fig.31A , 31B and 31C, depicting various views of a microchannel vascular network device filled with GelMA microgels. Figure 11 to Figure 1 3. Fig.31A , 31B 31C , a first channel is filled with a plurality of GelMA microgels 802 , which subsequently block and / or stop flow in one or more intermediate channels 704 (eg, structures).

[0328] Thus, the GelMA microgels of the present disclosure allow for temporary blocking and / or stopping of flow within microchannels of various devices. GelMA microgels can be fabricated by a variety of techniques and in a variety of shapes and sizes, allowing for blocking and / or stopping of flow according to the goals of the designer of the present disclosure.

[0329] Furthermore, because GelMA microgels are degradable, blocking and / or stopping flow within the device can be temporary, thereby expanding the use of such devices.

[0330] PLGA stops flowing. As a non-limiting example, PLGA degrades by hydrolysis of its ester bonds in aqueous solution, thereby producing PLA and PGA monomers. In some embodiments, the initial average size of the PLGA microspheres is about 533.7 ± 58.62 μm at day 0, and decreases to about 520.6 ± 117.04 μm after 24 hours of cultivation, and decreases to 509.9 ± 63.24 μm after 48 hours of cultivation. These microspheres continue to decrease after 8 days, indicating that the PLGA microspheres are hydrolyzed in the PBS solution ( Figure 1 B). However, the average diameter increased and maintained its increase at day 11, indicating that the microspheres swelled and degradation continued throughout the culture period. As previously described, swelling is an important process in the degradation process of PLGA microspheres, which is caused by the diffusion of water from the outside and inside (e.g., water absorption) due to the increase in osmotic pressure. At day 16, the microspheres were completely split into small broken pieces due to the degradation process. At day 22, the PLGA microspheres no longer maintained their spherical shape, and the polymer itself completely diffused and attached to the surface of the 96-well plate and remained unchanged. Therefore, the microspheres disclosed herein allow for the prevention and / or cessation of flow within a microchannel device.

[0331] Various aspects of the present disclosure are directed to providing a system for directing the flow of culture medium through one or more devices. The system includes one or more devices configured to cause culture medium to flow therethrough. The arrangement and flow of culture medium through the one or more devices is configurable so that the system provides a supply of nutrients and / or removes waste from various cells cultured in the one or more devices according to the arrangement of the system.

[0332] The device of the present disclosure includes a channel with an inlet and an outlet. Between the inlet and outlet of the device is a hole, which is configured to accommodate a cannula including a structure for culturing cells. In some embodiments, the shape of the inlet and outlet of the device is generally tubular and has the same inner diameter. In addition, in some embodiments, the inlet and outlet of the device include a planar flow nozzle arranged in the corresponding channel. The planar flow nozzle allows the culture medium flowing in the system to have a planar or linear flow path. In an exemplary embodiment, the planar flow path provides a substantially consistent culture medium flow rate in the system. Typically, the speed of the culture medium in the corresponding device determines the concentration of nutrients received by the cells of the corresponding device. This consistent speed provides a consistent flow rate in one or more devices of the system, and therefore provides a consistent nutrient and / or waste concentration. Therefore, in some embodiments, the device of the present disclosure is symmetrical about a vertical axis. In some embodiments, the flow of the culture medium is laminar flow in the device of the system.

[0333] refer to Fig.14, shows an exemplary microchannel vascular network device 700-2 of the present disclosure. Device 700-1 includes a channel 110 that accommodates a culture medium 130 flowing through the device. Channel 100 includes an inlet 112 at a first end of the channel and an outlet 114 at least at a second end of the channel. In some embodiments, one or more of the inlet and outlet of the device is configured to be connected to a silicon tube through which the culture medium flows. For example, in some embodiments, the end of the inlet or outlet is received by a silicon tube (e.g., the end of the inlet or outlet is a convex portion received by a silicon tube), which forms a seal between the pipeline and the inlet or outlet of the channel. In some embodiments, the silicon tube is received by the end of the inlet or outlet (e.g., the end of the inlet or outlet is a concave portion). In some embodiments, the channels of the corresponding devices are connected to each other by a plurality of connecting pins (e.g., corresponding female connecting pins and male connecting pins).

[0334] In some embodiments, the device includes a hole configured to accommodate a cannula for culturing cells. The hole is inserted between the inlet and outlet of the channel so that the culture medium of the system flows from the inlet, through at least a portion of the hole, and flows to the outlet of the device. In some embodiments, the channel of the device is connected to the hole using multiple metal connecting pins, which minimizes the risk of accidental disconnection between the components of the device. In some embodiments, the channel of the device is connected to the hole via a reversible fastener or joint, such as a Luer lock. In some embodiments, the channel of the device is connected to the hole via a standardized fitting. For example, in some embodiments, the standardized fittings are specified by the International Organization for Standardization (ISO) 80369, (2017), "Small bore connectors for liquids and gases in healthcare applications," ISO / TC 210 Quality management of medical devices and corresponding general aspects, ICS 11.040.25, Print.

[0335] refer to Fig.41 and 42 In some embodiments, the bioreactor system includes a plurality of connectors that facilitate coupling the corresponding microchannel vascular network device to the bioreactor system. As shown, a plurality of ratchet-free zones allow an integer number of ratchets in utilizing a coupling. In some embodiments, the port length (e.g., the larger diameter portion) of the coupling is about 0.3 mm to about 1.8 mm. In some embodiments, the port length is 0.5 mm, 0.8 mm, or 1.8 mm (e.g., corresponding to a predetermined size of tubing, such as size #3 or #5 tubing).

[0336] In addition, in some embodiments, the bioreactor system includes a shell or is contained in a shell. In this way, the bioreactor system is isolated from the environment, and the internal features of this protection bioreactor system are protected from pollution. For example, in some embodiments, a PMMA shell surrounds the bioreactor system so that a pump has a suitable opening for being connected to one or more components, such as a microchannel device and / or a filter (for example, a HEPA filter) provided for gas exchange, and one or more ports (for example, a coupler) to allow culture medium to flow into and / or flow out of the inlet and outlet channels of the microchannel device.

[0337] In some embodiments, the hole is configured to accommodate a cell culture cannula in the cavity of the hole. The cannula includes a structure for inoculating multiple cells and other biomaterials. In some embodiments, the cannula includes multiple legs protruding outward from the lower end of the cannula. The multiple legs of the cannula lean against the edge portion of the hole, which in turn provides space between the hole and the cannula. The space allows the culture medium to flow between the channel, hole and cannula of the device accordingly. Alternatively, the cell culture cannula can be a hanging cannula (for example, the cannula leans against the upper end of the hole), or other types of cannula that do not require legs to provide enough space for fluid flow and discharge. In addition, the edge portion of the hole can include multiple gaskets, and the cannula is located on the gasket to provide enough space for the flow and discharge of the culture medium in the device. However, in some embodiments, a part of the cannula includes a membrane, such as a semipermeable membrane. When the cannula includes living cells, there will be a nutrient gradient between the cell and the culture medium, so the membrane provides the diffusion of the culture medium through the membrane driven by the nutrient gradient. In some embodiments, the lower end of the cannula includes a membrane. In some embodiments, one or more side walls of the cannula include a membrane. The well is in communication with the channel, and a capsule is disposed over the cell culture insert of the well to control the flow path of the culture medium therein.

[0338] In some embodiments, the cannula is a polystyrene cannula. In some embodiments, the cannula of the hole has a height of about 10.5 millimeters (mm). In some embodiments, the cannula of the hole has a height in the range of 9mm to 11mm. In some embodiments, the diameter of the cannula is about 12mm. In some embodiments, the diameter of the cannula is in the range of 10mm to 14mm, 11mm to 13mm or 11.5mm to 12.5mm. In addition, in some embodiments, the cannula of the hole includes a plurality of holes. The holes of the cannula are configured to allow the cells maintained in the cannula to exchange materials with the culture medium of the system (e.g., provide nutrients and / or remove waste). In some embodiments, the cannula is configured to accommodate a medium having approximately 1,000 cubic millimeters (mm 3 ) of a volume (e.g., a plurality of cells, a sponge, a hydrogel, etc.). In some embodiments, the cannula is configured to accommodate a volume of 1*10-12 mm 3 Up to 1,500mm 3 , 1*10 -5 mm 3 Up to 1,500mm 3 , or 1mm 3 Up to 1,500mm 3 In some embodiments, the cannula is configured to accommodate objects with a volume within 1 mm. -3 Up to 200mm 2 , 1mm 2 Up to 150mm 2 or 1mm -2 To 00mm 2 Objects with a cross-sectional area within the range.

[0339] The systems and methods of the present disclosure are configured to culture microorganisms that occupy, in some embodiments, a large spatial area (e.g., at least 100 mm 2 In some embodiments, the cannula of the hole is a cannula and / or membrane provided by MilliporeSigma of400Summit Drive,Burlington,MA 01803. In some embodiments, the cannula includes a membrane. For example, in some embodiments, the cannula is configured to accommodate a membrane that also includes a plurality of units. Similarly, in some embodiments, the cannula itself includes a membrane portion that allows the exchange of materials between the culture medium and a plurality of cells. However, in some embodiments, the cannula includes a mesh portion to allow the culture medium to flow through the cannula while preventing the cells from escaping. In some embodiments, the upper portion of the cannula (for example, the portion of the cannula near the capsular sheet of the device), the lower portion of the cannula (for example, the portion of the cannula near the channel of the device), or the upper and lower portions of the cannula include a mesh. For example, in some embodiments, the cannula includes a mesh with a mesh opening of about 30 nanometers (nm), about 40nm, about 50nm, about 60nm, about 70nm, about 80nm or about 90nm. In some embodiments, the cannula comprises a mesh having mesh openings in the range of about 50 nm to 1,000 nm, about 50 nm to 200 nm, or about 60 nm to 150 nm. However, the systems and methods of the present disclosure are capable of culturing a relatively large mass (e.g., a cannula having about 500 mm 3 Up to 1,000mm 3 This relatively large mass allows the system to culture cells with physiologically relevant materials and to culture multiple cell types within one system, whereas previous systems could only culture spheroid materials or models.

[0340] like Figure 1As shown in FIG. 1A , device 100 - 1 includes a well 120 configured to receive a cannula 122. Culture medium 130 is received through an inlet 112 of the device, flows through the well 120, flows at least partially through the cannula 122, and exits via an outlet 114. Figure 1 As shown in Figure A, the device 100-1 is symmetrical about a vertical axis, so that the flow conditions within the device are uniform about the planar axis. This planar flow of the culture medium allows the cells in the tubes to receive uniform and consistent nutrient concentrations.

[0341] In some embodiments, the device of the present disclosure includes a caplet disposed above the hole of the device. The caplet is configured to seal at least a portion of the device (e.g., the hole of the device) relative to the external environment (e.g., to prevent contamination of culture medium and / or cells), and because in some embodiments, the caplet is removably connected to the device, it is convenient to operate and observe the cannula in the device (e.g., insert the cannula, check the cells cultured in the cannula, remove the cannula, etc.). In some embodiments, the caplet is accommodated by the hole (e.g., a portion of the caplet is connected to the inner edge portion of the hole). For example, in some embodiments, the caplet includes a concave portion configured to receive the corresponding convex portion of the hole. In some embodiments, the caplet and the hole of the device include corresponding threaded portions, so that the caplet and the hole are connected via the rotational movement of the corresponding threaded portions. However, the connection of the caplet and the hole is preferably carried out using a mechanism (e.g., press fit, thread, etc.) that prevents contamination of culture medium and cells while also preventing culture medium from leaking from the outside of the device or preventing liquid from entering the device.

[0342] In some embodiments, the caplet is configured to provide an additional flow path for the culture medium to pass through the device. For example, in some embodiments, the caplet includes a second outlet, which is configured to provide an additional flow path for the culture medium in the device to pass through. For example, in some embodiments, the culture medium flow through the first outlet (e.g., the outlet of the channel) is blocked or prevented so that the culture medium through the device flows from the entrance, through the hole and toward the second outlet of the caplet. In some embodiments, the culture medium flows from the entrance, through the hole and toward the first outlet of the channel and the second outlet of the hole. In some embodiments, the culture medium flows from the entrance, through the hole and selectively toward one or both of the first outlet of the channel and the second outlet of the hole. This selective flow depends on the pressure gradient provided by one or more pumps of the system (e.g., the suction pressure of the pump connected to the corresponding outlet). In some embodiments, the first inflow opening and the second outflow opening each include a fastening mechanism, which is configured to removably connect the microchannel vascular network device to the pump.

[0343] In some embodiments, the second outlet of the caplet comprises a decrease in inner diameter from a first diameter to a second diameter. Thus, the second outlet of the caplet decreases from a first diameter, which in some embodiments is approximately equal to the diameter of the aperture, to a second diameter, which is approximately equal to the inner diameter of the device inlet. In some embodiments, the decrease in the inner diameter of the caplet from the first diameter to the second diameter is a linear decrease (e.g., the decrease occurs with a linear slope). In some embodiments, the decrease is a discrete (e.g., abrupt or stepped) decrease in diameter. However, the decrease in the inner diameter of the caplet outlet focuses (e.g., converges) the flow path of the culture medium through the device while maintaining a planar flow feature therein (e.g., maintaining a laminar planar flow path). In some embodiments, the second outlet of the caplet comprises the same coupling mechanism as described above with respect to the inlet and outlet of the channel (e.g., a female coupling mechanism with a corresponding male coupling mechanism).

[0344] In some embodiments, the system includes one or more pumps configured to create a pressure differential within the system and provide a flow of culture medium through the system. For example, in some embodiments, a pump is disposed upstream of the system to push (e.g., discharge) or drive culture medium through the system (e.g., Figure 4 In some embodiments, a pump is provided downstream of the system to pull (e.g., suck) or aspirate the culture medium through the system (e.g., Figure 4 B). For example, in some embodiments, a pump is disposed downstream of a channel outlet of a corresponding device, which allows culture medium to be drawn through the outlet (e.g., Figure 2 In some embodiments, a pump is disposed downstream of the second outlet of the caplet of the corresponding device, which allows the culture medium to be drawn through the second outlet of the caplet (e.g., as shown in FIG. Figure 2 In some embodiments, a first pump is disposed downstream of an outlet of a device channel, and a second pump is disposed downstream of a second outlet of a caplet of the device. This first pump and second pump configuration allows medium to be pumped through the outlet of the channel and the second outlet of the caplet (e.g., medium flows from an inlet of the device and flows out of both the outlet of the channel and the outlet of the caplet, as shown in FIG. 1 ). Figure 21 and 132-2). Depending on the configuration of each pump, the flow of culture medium pumped through each outlet of the device can be uniform (e.g., the flow rate through both outlets is the same) or non-uniform (e.g., a first flow rate of culture medium through a first outlet of the device and a second flow rate of culture medium are different from the flow rate of culture medium through a second outlet of the device). In some embodiments, the pump has a maximum discharge pressure of about 200 kilopascals (kPa) or less, about 100 kPa or less, about 90 kPa or less, about 80 kPa or less, about 70 kPa or less, or about 50 kPa or less. In some embodiments, the pump has a maximum suction pressure of about 200 kilopascals (kPa) or less, about 100 kPa or less, about 90 kPa or less, about 80 kPa or less, about 70 kPa or less, or about 50 kPa or less. In some embodiments, the pump is a Welco WPM2 pump (e.g., a Welco WPM2-P1EA-CP pump supplied by Welco of 3-3-1 Sumiyoshi-cho, Fuchu-shi, Tokyo 183-0034, Japan.

[0345] In some embodiments, the system includes a controller configured to operate at least one or more pumps of the system. The operation of the pump includes controlling the power state (e.g., open state and closed state) of each corresponding pump and the flow rate provided by each corresponding pump (e.g., controlling the speed of the motor of each corresponding pump). This operation of the pump can be performed by various physical mechanisms (such as a dial configuring the flow rate of each corresponding pump) and by instructions provided to the controller.

[0346] As previously described, in some embodiments of the present disclosure, the system includes more than one device to allow more than one cell to be cultured in the system. For example, in some embodiments, the system of the present disclosure includes multiple devices that can be arranged in various configurations. In some embodiments, one or more devices in a plurality of devices are connected and communicated with one or more adjacent devices in a plurality of devices (for example, the device is connected and fluidly connected so that the culture medium of the system flows from the first device to the second device). In some embodiments, a plurality of devices of the system are connected in parallel so that the outlet of the corresponding channel of the first device is communicated with the inlet of the corresponding channel of the second device. Similarly, in some embodiments, a plurality of devices of the system are connected in series so that the corresponding cells cultured in each device are separated from the cells of another device in the plurality of devices (for example, the culture medium flowing through the first device is separated from the culture medium flowing through the second device). Similarly, in some embodiments, a plurality of devices of the system include at least a first subset of devices connected in parallel and a second subset of devices connected in series. The embodiment using more than one device in the corresponding system allows more than one type of cell to be cultured in the system and / or is used to determine the efficacy of the material on the cultured cells. For example, a network of supply and outlet lines (e.g., silicon tubing) can be configured to connect multiple wells in series, enabling the system to mimic in vivo tissue conditions, where nutrient and waste concentrations vary in decreasing or increasing gradients as they do across the spatial dimensions of an organ.

[0347] In addition, in some embodiments, the system includes one or more culture medium reservoirs. Each of the one or more reservoirs of the system is configured to hold a supply of culture medium and / or various byproducts of the system (e.g., waste materials, such as bile effluent, produced by various cells maintained within the pores of the device). For example, in some embodiments, the one or more reservoirs of the system include a first reservoir (e.g., Figure 4 In some embodiments, the one or more reservoirs of the system include a second reservoir (e.g., Figure 2 In some embodiments, the system forms a closed loop such that the reservoir is configured to hold excess culture medium flowing through the system (e.g., Figure 4 D's storage 200).

[0348] refer to Fig.16 , shows an embodiment of a system in which a first device 700-1 and a second device 700-2 are connected in parallel. Fig.1617 , the dashed lines and arrows represent the flow path of the culture medium 130 through the devices. As shown in FIG. 17 , the culture medium 130 is received via the inlet 112-1 of the first device 700-1 and flows through the hole 120-1 of the first device. The culture medium 130 is received by the outlet 114-1 of the first device 700-1, which in turn is received via the inlet 112-2 of the second device 700-2. Once the culture medium 130 is received by the hole 120-2 of the second device 700-2, the flow of the culture medium can continue to flow to the outlet 114-2 of the second device or to the outlet 114-3 of the caplet of the second device, depending on the pressure gradient within the second device. For example, in some embodiments, the culture medium 130 continues to flow through the outlet 114-2 of the second device 700-2, while the waste generated by the cells contained in the hole 120-2 is removed from the system via the outlet 114-3 of the caplet flowing through the second device.

[0349] refer to Fig.16 In some embodiments, the caplet of the device is connected to the hole via an O-ring. In some embodiments, the caplet, the hole, or both the caplet and the hole include a groove configured to accommodate an O-ring. This O-ring is configured to create a seal between the caplet and the hole of the corresponding device, thereby ensuring that air or similar contaminants do not enter the system and / or culture medium does not leak out of the system, thereby isolating the system from the external environment (e.g., atmospheric conditions) and maintaining the integrity of the internal environment of the device. To further ensure that the system is isolated from the external environment, in some embodiments, each surface of the removable caplet that contacts at least one wall of the hole includes a hydrophobic material. In some embodiments, this inclusion of a hydrophobic material includes coating each surface of the caplet that contacts at least one surface (e.g., wall) of the hole with a hydrophobic material (e.g., applying two coatings of a hydrophobic material). For example, in some embodiments, the hydrophobic material is made by Vernon Hills, Illinois. Provided In some embodiments, the O-ring comprises a hydrophobic material (e.g., a hydrophobic material is coated on the O-ring). In some embodiments, the O-ring has an inner diameter in the range of 0.3 inches (in) to 0.7 in, 0.4 in to 0.6 in, or 0.4 in to 0.5 in. In some embodiments, the O-ring has an inner diameter of 0.489 in. In some embodiments, the O-ring has an outer diameter in the range of 0.4 in to 0.8 in, 0.5 in to 0.7 in, or 0.6 in to 0.7 in. In some embodiments, the O-ring has an outer diameter of 0.629 in.

[0350] As previously described, in some embodiments, the system of the present disclosure is used to culture cells and / or to carry out biological experiments on cells cultured in the system. Therefore, in some embodiments, the cell culture intubation tube includes a sponge or a net that is conducive to cell culture. For example, in some embodiments, the sponge of the intubation tube is a type 1 collagen sponge, such as a collagen sponge prepared from the dermis of a pig. In some embodiments, the cell culture intubation tube is a gel foam, such as a cylindrical gel foam. In some embodiments, the cell culture intubation tube is a gel sponge provided by Pfizer in New York City, New York. However, in some embodiments, the cell culture intubation tube is coated with gelatin. For example, in some embodiments, the cell culture intubation tube is coated with about 2% gelatin, about 1.5% gelatin, about 1% gelatin, about 0.5% gelatin, about 0.2% gelatin, about 0.1% gelatin or about 0.05% gelatin.

[0351] Another aspect of the present disclosure relates to providing a method for culturing cells. In some embodiments, the method utilizes an embodiment of the system as described above. However, the method includes inoculating a plurality of cells to be cultured in one or more devices of the system. In some embodiments, inoculation includes arranging a cannula in a hole of a corresponding device, wherein the cannula accommodates the cells. In some embodiments, inoculation includes suspending the cells in a culture medium of the system, and then allowing the culture medium to flow through the system.

[0352] Therefore, the method also includes allowing the culture medium to flow through the system. In some embodiments, multiple cells are cultured in a two-dimensional manner in a corresponding device (e.g., cultured on a film of an intubation). In some embodiments, multiple cells are cultured in a three-dimensional manner in a corresponding device (e.g., multiple cells are embedded in a cell scaffold device and / or include, for example, suspension in a hydrogel).

[0353] In some embodiments, before the cells are seeded into the wells of the device, a plurality of cells are generally preferably used to culture hepatocytes because calcium helps to separate bile and other similar products into the tubules formed by the hepatocytes. In some embodiments, a plurality of cells are exposed to the solution for a predetermined time of about 20 minutes. The present disclosure encompasses the use of any beneficial exposure time. For example, in some embodiments, the predetermined time is about 10 minutes. In some embodiments, the predetermined time is about 15 minutes. In some embodiments, the predetermined time is about 25 minutes. In some embodiments, the predetermined time is about 30 minutes.

[0354] In some embodiments, the plurality of cells is present in an amount of about 1*10 2 cells / cm2, about 5*10 2 cells / cm2, about 1*10 3 cells / cm2, about 5*103 cells / cm2, about 1*10 4 cells / cm2, about 5*10 4 cells / cm2, about 1*10 5 cells / cm2, about 5*10 5 cells / cm2, about 1*10 6 cells / cm2, or about 5*10 6 The cells were seeded in the wells at a density of 10 cells / cm2.

[0355] In some embodiments, the flow of the culture medium in the system is carried out at a flow rate of about 150 microliters / minute (μL / min), about 200 μL / min, about 250 μL / min, about 300 μL / min, about 350 μL / min, about 400 μL / min, about 450 μL / min, about 500 μL / min, about 550 μL / min, about 600 μL / min, about 650 μL / min, or about 700 μL / min. Typically, the flow of the culture medium in the system is carried out at a flow rate that simulates the flow of fluid in an organ, so that enough nutrients and waste removal are provided to the cells of the system without damaging the cells.

[0356] Having now described the general structures of various embodiments of the systems, apparatus, and methods of the present disclosure, various examples of the present disclosure will now be described in detail.

[0357] Example I - Cultivation of Hepatocytes

[0358] The device of the system includes a plurality of hepatocytes housed in respective cannulas. After being introduced into the culture medium, the hepatocytes will produce blood effluent and bile effluent. Therefore, a pump is coupled to the system so that the outlet of the channel of the device is configured to receive the blood effluent, and the outlet of the channel is configured to receive the bile effluent. This configuration allows various experiments and determinations to be performed on each respective effluent (e.g., to determine whether the respective effluent will kill or not kill subsequent cells).

[0359] Example II - Seeding of cells and internal coating of the device

[0360] Brief reference Fig.19 , shows the results of various experiments in coating the sidewalls of holes. Fig.19 In the experiment, Hep G2 cells were cultured at about 3*10 5The cells were seeded at a density of 10 cells / cubic centimeter and cultured in static or dynamic conditions (e.g., subjected to medium flow). The cells were exposed to fluorescein diacetate (FDA) for one hour to detect polarization within the cells. Samples of the medium were captured from the wells of the device, which included the seeded cells, and samples of the medium were captured from a portion of the device below the wells (e.g., a channel of the device). The results indicated that the trend favored an increase in fluorescence in the upper portion of the device (e.g., the upper portion of the well) compared to the bottom of the device lacking Hep G2 cells. In order to increase cell seeding in the bottom of the device, 0.1% gelatin was coated on the corresponding surface of the device before seeding the cells. Thus, as Fig.19 The lysis measured in the 4 cells / cm3 to about 1*10 5 The cells were seeded at a density of 10 cells / cm3 to achieve the optimal seeding density.

[0361] Example III - Inoculation Parameters

[0362] In some embodiments, prior to seeding cells, the caplets of the device (e.g., the top portion of the device) are dried or pre-wetted, wherein the caplets are subjected to culture medium and / or gelatin prior to seeding. Similarly, in some embodiments, prior to seeding, the bottom portion of the wells of the device are dried or pre-wetted. Fig. 20 , Fig.21 and Fig. 22 , depicting results from various experiments related to the cell seeding protocol. These figures depict the results of experiments in which a cylindrical gel foam sponge was loaded with approximately 1*10 6 Hep G2 cells. These cells were cultured for three or six days under a medium flow at a flow rate of about 400 μL / min. In addition, the cells were exposed to about 3 micrograms / milliliter (μg / mL) of fluorescein diacetate while being subjected to the flow described above. Over an eight-hour period, aliquots of the medium were captured and measured from below the cell cannula (e.g., below the sponge at the bottom portion of the well) and after passing through the cell cannula (e.g., after passing through the sponge at the top portion of the device), and the results are depicted in Fig. 20 and Fig.21 In. Fig. 20 , Fig.21 and Fig. 22 As shown, at the beginning of the assay, the fluorescence measured below the cannula and inside the cells is approximately equal. After about 20 minutes, the cells begin to convert FDA to fluorescein. Therefore, a statistical difference between the top of the device and the bottom of the well was measured after the addition of FDA, as shown in FIG. Fig. 22 shown.

[0363] Brief reference Fig.23, tested on gelatin coated polyethylene terephthalate (PET) holes of about 5*10 4 After about 3 days, the seeding density of Hep G2 cells was about 1*10 5 Compared with 90% confluence of each well inoculated with a cell density of 10 cells / cubic centimeter, a lower inoculation density is about 80% confluence. During each washing step after being exposed to FDA or rhodamine, each well inoculated with a lower cell density is separated, while the wells inoculated with a higher cell density remain attached. In this experiment, cells are placed in a Hank's balanced salt solution (HBSS) containing calcium and magnesium for about 20 minutes, and then exposed to 3 μg / mL fluorescein diacetate or 0.1mM rhodamine 20min in HBSS. After about 20 minutes, the washing holes are imaged in HBSS for about 1 hour. In both cases, bright rings can be observed at the edge of most cells, which indicates cracking. The change of this exposure causes the fluorescein of intracellular cracking to be positioned at the outer boundary of the cell. In addition, this change allows better visualization of the cells in culture.

[0364] Substances diffuse in microchannel devices. Two molecules with different molecular weights were used as substances: FITC-BSA (66500Da) and Rhodamine B (479g / mol). Therefore, 40μL of 1.0wt% FTIC-BSA / Rho B solution was applied to the upper surface of the hydrogel microchannel device and washed 3 times with PBS after about 0 to 30 minutes to remove the fluorescent solution on the surface. A fluorescence microscope was used to observe the cross-section of the hydrogel. The diffusion intensity of nutrient transport in simulated hydrogel microchannel devices is an important factor for tissue engineering materials. Compared with Rho B, FITC-BSA showed faster diffusion within the hydrogel device, indicating that larger molecules of 66.5kDa can be easily transported into and diffused through the hydrogel device.

[0365] Thus, the systems and methods of the present disclosure provide a means for directing flow within one or more cell culture devices. These devices are used to simulate in vivo cell culture and are capable of culturing approximately 1,000 mm 3 Volume of cells.

[0366] For ease of interpretation and accurate definition in the appended claims, the terms "above", "below", "up", "down", "inside", "outside", "inward", "outward", "interior", "exterior", "front", "rear", "rear", "forward" and "rearward" are used to describe the features of the exemplary embodiments with reference to the locations of such features as shown in the accompanying drawings.

[0367] The foregoing description of specific exemplary embodiments of the present invention has been presented for purposes of illustration and description. The foregoing description is not intended to be exhaustive or to limit the invention to the precise form disclosed, and it is apparent that many modifications and variations are possible in view of the above teachings. The exemplary embodiments are selected and described in order to explain certain principles of the present invention and their practical application, thereby enabling others skilled in the art to make and utilize the various exemplary embodiments of the present invention and various alternatives and modifications thereof. It is intended that the scope of the present invention be defined by the claims appended hereto and their equivalents.

Claims

1. A method of preventing or stopping flow in a microchannel device, wherein: The microchannel device comprises a first channel and a second channel, the first channel and the second channel are interconnected by one or more structures, wherein the one or more structures include one or more intermediate channels, and wherein the method comprises: A suspension of a plurality of GelMA microgel units in a solution is perfused through a first channel of the device, wherein the perfusion of the solution causes one or more of the plurality of GelMA microgel units to block or stop the flow of liquid in at least one of the one or more intermediate channels. 2 . The method according to claim 1 , wherein the plurality of GelMA microgel units are fabricated using a photomask technique.

3. The method according to claim 2, wherein the photomask technique comprises: Arranging a prepolymer solution comprising GelMA and a photoinitiator in a buffer solution onto a first glass slide whose outer edge portion is surrounded by a spacer, and wherein after completing the arrangement of the prepolymer solution onto the first glass slide, a second glass slide is disposed above the first glass slide, thereby forming a containing structure; disposing a photomask on a top surface of the second slide; as well as The photomask and the containing structure are exposed to ultraviolet light for a predetermined period of time, thereby generating the plurality of GelMA microgel units. 4 . The method according to claim 3 , wherein the method further comprises disassembling the containing structure to recover the generated GelMA microgel units, thereby manufacturing the plurality of GelMA microgel units.

5. The method according to any one of claims 3 or 4, wherein the prepolymer solution comprises: 7% to 20% by weight of GelMA, and 1% photoinitiator.

6. A method according to any one of claims 3 or 4, wherein a prepolymer solution is formed having a degree of methacryloyl substitution of 75% to 90%.

7. A method according to any one of claims 3 or 4, wherein the photomask technology includes manufacturing and / or utilizing a photomask comprising an array of transparent sheets, wherein the array of transparent pieces is an array of circles, and wherein each circle in the array of circles has a diameter of 50 μm to 650 μm.

8. The method according to any one of claims 3 or 4, wherein the spacer has a thickness of 50 μm to 550 μm.

9. The method according to any one of claims 3 or 4, wherein before disposing the prepolymer solution, for at least one slide and / or spacer, the method further comprises: placing a sample of a stock solution of 0.5 wt % methoxypolyethylene glycol 5000Si (mPEG5000-Si), 1 wt % acetic acid, and ethanol onto the at least one slide and / or spacer; subjecting the at least one slide and / or spacer to incubation at 70° C. for 30 minutes, thereby forming an incubated slide and / or spacer; immersing the incubated slide and / or spacer in deionized water to form an immersed slide and / or spacer; subjecting the immersed glass slide and / or spacer to ultrasonic treatment for a predetermined period of time, thereby forming an ultrasonically treated glass slide and / or spacer; as well as The sonicated glass slide and / or spacer is dried to form a prepared glass slide and / or spacer.

10. The method according to any one of claims 3 or 4, wherein the predetermined time period is less than or equal to 30 seconds.

11. The method according to any one of claims 3 or 4, wherein the ultraviolet light has: wavelengths from 360nm to 480nm, and Intensity of 12.4 mW / cm3.

12. The method according to claim 4, wherein said recovering said GelMA microgel unit comprises: The resulting GelMA microgel unit was transferred into a mineral oil bath, and The mineral oil bath was agitated.

13. The method according to claim 4, wherein said recovering said GelMA microgel unit comprises: (a) immersing the generated GelMA microgel unit in a sodium hydroxide solution; (b) immersing the GelMA microgel unit of (a) in an ethanol solution; and (c) subjecting the GelMA microgel unit of (b) to ultrasonic treatment for a third predetermined period of time.

14. The method according to any one of claims 1-4, wherein the plurality of GelMA microgel units are fabricated using a microfluidic process. 15 . The method according to claim 1 , wherein one or more GelMA microgel units in the plurality of GelMA microgels are formed as spheres.

16. The method of any one of claims 1-4, wherein the one or more structures are in the shape of a truncated cone.

17. The method according to any one of claims 1 to 4, wherein the method further comprises: After perfusing the suspension of the plurality of GelMA microgel units and the solution through the first channel of the device, fluid flow is applied in the second channel of the device to generate a pressure differential between the first channel and the second channel of the device.

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