3D Printing of Organs, Devices and Matrices

Abstract

Provided herein are methods and systems for bioprinting three-dimensional organs and organoids. Also provided herein are uses of bioprinted three-dimensional organs and organoids for generating and / or evaluating immunological products and / or immune responses. Also provided herein are methods and systems for bioprinting three-dimensional matrices.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 907,488, filed on September 27, 2019, which is incorporated herein by reference in its entirety. Background Art

[0003] To date, despite significant advances in cell biology, microfluidics, engineering, and 3D printing, conventional approaches have failed to recreate the functional capillaries that feed and support the thick tissues necessary to build human organs. To date, these approaches in tissue engineering have relied on the ingrowth of blood vessels into tissue-engineered devices to achieve durable vasculature. This strategy is suitable for some very thin tissues (such as bladder wall replacements) or tissues that do not require a working vasculature (such as bone replacements). However, current tissue engineering technologies fall short in generating complex tissues such as large vital organs, including the liver, kidney, thick skin, and heart. Larger tissues can also be viewed as tissues of smaller tissue subunits; for example, the kidney is composed of hundreds of thousands of nephrons, and the functional units of the lungs (i.e., the alveolar cavity) have a total surface area of ​​70 to 80 square meters (m 2 ), but it is only a cell wall with a thickness of 5 to 10 micrometers (μm). Current tissue printing methods are not only unable to recreate the delicate microvasculature necessary to support tissues thicker than 300 micrometers (μm), but also unable to organize cells into the structural orientation and niches necessary for organ function.

[0004] Antibodies are proteins produced and secreted by B cells during an immune response. Antibodies can have high binding specificity and affinity for potential infectious agents and can therefore be used to bind to and isolate, neutralize, or modify the effects of other proteins, viruses, bacteria, chemical-protein combinations, or carbohydrate molecules. This makes antibodies valuable proteins in protecting against pathogens and isolating or neutralizing infectious or other pathological factors or proteins. In addition, antibodies can be used to redirect immune responses by means of modulation of other protein-protein interactions, through the opsonization of phagocytosis, significantly increasing the likelihood of immune recognition and destruction of pathogens or pathological factors.

[0005] In order for B cells to produce high-affinity or high-affinity antigenic antibodies, a multi-step process called affinity maturation is required. During affinity maturation, genetic changes occur in the B cell receptor (BCR). After these genetic changes, a similar evolutionary process of guessing and testing occurs. This is a competitive process in which high binding strength leads to more contact with accessory cells that provide positive survival signals. Accessory cells include, but are not limited to, T cells, B cells, monocytes, macrophages, dendritic cells, natural killer cells, etc. Ineffective BCR rearrangement terminates the affinity maturation process and no longer receives survival signals from accessory cells presenting antigens or self-BCR cross-linking that can provide additional positive feedback. Therefore, higher affinity BCR rearrangements and random mutations give B cells positive feedback, promote B cell division, more receptor rearrangements and more random mutations, while lower affinity BCR rearrangements can lead to cell death or anergy. After several rounds of selection in this guessing and testing sequence, high affinity B cells survive differentially and transform into plasma cells. Plasma cells circulate in the bloodstream and secrete large amounts of antibodies to assist in immune response. Affinity maturation mostly occurs in lymph nodes and lasts for several days.

[0006] Lymph nodes consist of a large number of immune cells within a reticular network, mainly B cells, T cells and follicular dendritic cells (FDC). Lymph nodes achieve the extensive cell-cell interactions required for a comprehensive immune response by increasing the proximity of cells to each other. Secondary survival signals from accessory cells support B cell receptor rearrangement. These proximity-based cell interactions require or are significantly improved by a specific three-dimensional (3D) spatiotemporal arrangement of the immune cells found in the lymph nodes.

[0007] The three-dimensional cell movement and spatiotemporal arrangement of cells are essential for several cell-based processes, including cell differentiation and cellular responses to external or internal stimuli. During affinity maturation of B cells, the immune cells involved are physically compartmentalized in lymph nodes into regions containing dividing B cells (e.g., dark zones), regions containing non-dividing B cells (e.g., light zones), and regions containing supporting accessory cells, such as Fig.16 As shown in Figure 2, the compartmentalization of immune cells and subsequent rearrangement during activation dictates that the proper development of high-affinity antibodies is dependent on this tissue. As B cells undergo affinity maturation, they move between compartments in the lymph node, crawling through other cells and collagen networks, such as Fig.17 The present disclosure describes a process for non-toxic printing of cell-containing collagen networks at millimeter, micrometer or submicrometer resolution, allowing the generation of lymphoid organs or organoids with limited cell compartmentalization containing other cell types for purposes including but not limited to antibody production.

[0008] De novo formation of antigen-specific antibodies in synthetic tissues following antigen challenge or vaccination of organoids indicates functional cell-cell interactions and a functional responsive organization that can support complex cell-cell interactions over the course of days, weeks to months.

[0009] Antibodies have been used for therapeutic purposes due to their high efficacy and versatility in targeting, neutralizing and / or modulating biological factors associated with many disease states including cancer, autoimmune diseases and infectious diseases. However, current methods for discovering and producing antibodies for therapeutic or research purposes are time-consuming and expensive. Standard methods for antibody production require the use of animals, typically mice or other rodents, rabbits, chickens, horses or non-human primates, to which antigens are injected and bled to collect B cells after an immune response is demonstrated. Antibodies intended for use against human targets (e.g., for therapeutic purposes) produced by this method require additional laborious humanization steps that may change the binding affinity of the target while not guaranteeing safety or efficacy in humans. Other methods such as phage display use predefined antibody libraries or sequence sets combined with some methods for selecting proteins of interest. Typically, these libraries do not produce unique or high-affinity sequences. In addition, as predefined protein groups, they may not be able to generate responsiveness to novel infectious agents. Our technology addresses two issues: (a) reliance on animal models for antibody discovery and (b) the inability to use high-throughput human-derived models to generate high-affinity, unique antibodies.

[0010] The methods described involve the use of light sources including, but not limited to, white light, blue light, green light, and single or multi-photon laser sources of any wavelength. The light can be projected in two or three dimensions.

[0011] 2D projection is achieved by two-dimensional (2D) projection of a single axial plane using a digital micromirror device (DMD) or spatial light modulator (SLM) that places light only in specific areas where polymerization of the intended material occurs.

[0012] If three-dimensional projection is used, it can be achieved by holographic projection of light using two light modulation systems in series, as disclosed in the co-invented U.S. Provisional Patent Application No. 62 / 469,948 entitled MULTI-PHOTON TISSUE PRINTING, which is incorporated herein by reference. The polymerization of biomaterials has been described and implemented for bioprinting of cell scaffold materials. The method described herein involves projecting a light source into a bath containing a polymerizable material to encapsulate cells when polymerization occurs. In contrast, alternative in-medium polymerization-based tissue engineering methods use light projection to produce 3D scaffolds, into which cells can then be implanted. Encapsulating cells during the polymerization process rather than implanting them increases the accuracy with which cells can be placed; the resolution that can be achieved within the polymerization space is further increased by using a two-photon light source rather than a standard single-photon light source. The use of a two-photon light source to initiate polymerization eliminates or significantly reduces the toxicity of light to cells, and speeds up printing, thereby improving cell viability and growth. Multiphoton and single-photon laser methods are superior to extrusion printing in terms of the resolution that can be obtained and the speed at which large or complex structures can be printed. Alternatively, longer wavelength photons can be used to reduce damage to the cells, and / or lower intensity light or shorter exposure times can be used. Additionally, simultaneous printing in three dimensions by holographic projection of a light source with a predetermined polymer pattern greatly reduces printing time and also reduces stress on the cells due to exposure or time outside the incubator.

[0013] The most commonly used medical devices for wound closure, wound repair, such as using stents, bone healing or fusion of tissues including bone and skin are made of bioinert materials. Many of these materials dissolve over time, but many retain persistent characteristics for years after surgery and can cause complications or impede the healing process.

[0014] Some of the more advanced materials and medical devices used for surgical wound closure or tissue repair use 3D extrusion printing followed by cell seeding to introduce stem cells or other cell types that may be beneficial to wound healing or closure. However, seeding cells into bioinert materials has low viability and survival characteristics for the cells, so the delivery of beneficial cells is incomplete.

[0015] Tissue implants used to promote tissue healing or improved function are typically in the form of cell suspension injectables or small devices that do not violate the 200-300 micron diffusion limit for oxygen, nutrients, and waste, or are mostly cell-free. In addition, tissue implants do not contain cells printed in place that could remodel and grow in the printed material, which severely hinders the development of functional tissue inserts that can be incorporated into the implant environment.

[0016] The engineering of medical devices containing cells that are able to remodel and grow within implanted devices is limited by printing resolution, the lack of structurally resilient biomaterials available for extrusion printing, the cytotoxicity of high-resolution extrusion printing, and the technology to introduce cells into 3D printed medical devices after printing. In addition, 3D extrusion printing of highly complex devices is slow, often taking hours or days to complete a printing cycle. This makes it difficult to achieve production and scale-up of the desired cell-containing devices.

[0017] The present disclosure describes the development and use of three-dimensional lithography enabled by holographic light projection using a technique known as light wavefront shaping for bioprinting cell-containing structures and materials. The cell-containing structures and materials are specifically designed to maintain structural properties, such as tensile strength, shear and compression resistance, compressibility, or other properties that allow compatibility with surgical techniques, specifications, and native tissue and organ structures while being fully biocompatible. The hardening or polymerization of the biomaterial can be driven by light or laser interaction with the printed material at specific points in three-dimensional space. The printed material includes a monomeric biomaterial and a dopant or driver that is non-cytotoxic but reactive to light or specific wavelengths of light. The printed biocompatible device or structure containing embedded or captured cells allows for remodeling and decomposition or resorption of the implanted device, which is used to deliver cells to a predetermined site for the purpose of, but not limited to, healing, augmentation, or replacement of tissue function. Summary of the invention

[0018] In one aspect, the present disclosure provides a method for printing a three-dimensional (3D) object, comprising (a) directing a first light beam into a medium containing a polymer precursor to generate a holographic projection corresponding to at least a portion of the 3D object in the medium, to solidify a portion of the medium to produce the at least a portion of the 3D object; and (b) directing a second light beam into the medium to solidify at least a portion of the remainder of the medium.

[0019] In some embodiments, the first light beam is a multi-photon light beam. In some embodiments, the multi-photon light beam is a two-photon light beam. In some embodiments, the second light beam is a single-photon light beam. In some embodiments, the first light beam and the second light beam are directed into the medium substantially simultaneously. In some embodiments, the first light beam and the second light beam are directed into the medium through the same optical path. In some embodiments, the first light beam and the second light beam are directed into the medium through the same optical element. In some embodiments, the optical element is a phase and / or amplitude modulator. In some embodiments, the phase and / or amplitude modulator is a digital micromirror device. In some embodiments, the phase and / or amplitude modulator is a spatial light modulator.

[0020] In another aspect, the present disclosure provides a method for printing a three-dimensional (3D) object, comprising (a) generating a first 3D projection corresponding to a first portion of the 3D object in a medium comprising at least one polymer precursor, wherein the first 3D projection comprises a substantially simultaneous holographic array of multiple points; and (b) generating at least one additional projection corresponding to at least one additional portion of the 3D object substantially simultaneously with (a), wherein the first projection and the at least one additional projection form the 3D object in the medium.

[0021] In some embodiments, the first projection and at least one additional projection are projected along the same set of components. In some embodiments, the same set of components includes the same objective lens. In some embodiments, the first projection and at least one additional projection are projected along different sets of components. In some embodiments, different sets of components can generate projections perpendicular to the first 3D projection. In some embodiments, different sets of components can generate projections parallel to the first 3D projection. In some embodiments, different sets of components do not share a common optical axis. In some embodiments, different sets of components include one or more different focal length objective lenses. In some embodiments, the medium is rotating. In some embodiments, at least one additional projection is rotating. In some embodiments, at least one additional projection is rotated by the rotation of one or more optical elements, and the optical elements are configured to generate at least one additional projection. In some embodiments, the first 3D projection and at least one additional projection are projected simultaneously. In some embodiments, the first 3D projection and at least one additional projection are initially simultaneous. In some embodiments, at least one additional projection stops before the first 3D projection. In some embodiments, the first 3D projection and at least one additional projection are continuous. In some embodiments, the first 3D projection is projected after at least one additional projection. In some embodiments, the first 3D projection and at least one additional projection comprise a combined additive and subtractive manufacturing method. In some embodiments, at least one additional projection generates a form of the object in a medium. In some embodiments, the first 3D projection removes material from the form of the object. In some embodiments, at least one additional projection comprises five additional projections. In some embodiments, the first 3D projection is generated without using a digital micromirror device (DMD). In some embodiments, at least one additional projection is generated without using a digital micromirror device (DMD).

[0022] In another aspect, the present disclosure provides a method for producing one or more immune proteins, comprising: (a) providing a medium chamber comprising a medium comprising (i) a plurality of cells and (ii) one or more polymer precursors; (b) directing at least one energy beam to the medium in the medium chamber along at least one energy beam path patterned as a 3D projection according to computer instructions in a computer memory for printing a three-dimensional (3D) lymphoid organ to form at least a portion of the 3D lymphoid organoid, the 3D lymphoid organoid comprising (i) at least a subset of the plurality of cells, and (ii) a polymer formed from the one or more polymer precursors; and (c) subjecting at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate the production of the one or more immune proteins.

[0023] In some embodiments, the conditions include exposing at least a portion of the 3D lymphoid organoid to an antigen so as to stimulate production of the one or more immune proteins. In some embodiments, the method further includes: (d) extracting one or more immune proteins from at least a portion of the 3D lymphoid organoid. In some embodiments, the immune protein is selected from an antibody, a T cell receptor, and a cancer immunotherapeutic agent. In some embodiments, the plurality of cells are from a subject. In some embodiments, the plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or precursors thereof, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermal derived dendritic cells, dendritic cell precursors, monocyte derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D lymphoid organoid is selected from B cell germinal center, thymic development niche, lymph node, Langerhans island, hair follicle, tumor, tumor sphere, nerve bundle or supporting cell, nephron, liver organoid, intestinal crypt, primary lymphoid organ and secondary lymphoid organ. In some embodiments, the shape of the 3D lymphoid organoid is selected from spherical, elliptical, oval, ovoid, square, rectangular, cubic, any polygonal shape, free form and teardrop. In some embodiments, the polymer of at least a portion of the 3D lymphoid organoid forms a network. In some embodiments, the network is reticular, amorphous or a mesh. In some embodiments, the amorphous network is designed to promote cell interactions. In some embodiments, the amorphous network is designed to promote movement between or within cell niches.

[0024] In another aspect, the present disclosure provides a method for producing one or more immune proteins, comprising (i) printing a three-dimensional (3D) lymphoid organoid comprising a matrix comprising a plurality of cells, and (ii) processing the 3D lymphoid organoid to produce the one or more immune proteins.

[0025] In some embodiments, the immune protein is selected from antibodies, T cell receptors and cancer immunotherapeutics. In some embodiments, the multiple cells are from a subject. In some embodiments, the multiple cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subpopulations, effector T cells and their subpopulations, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subpopulations, follicular dendritic cells, Langerhans dendritic cells, dermal derived dendritic cells, dendritic cell precursors, monocyte derived dendritic cells, monocytes and their subpopulations, macrophages and their subpopulations, leukocytes and their subpopulations. In some embodiments, the 3D lymphoid organoid is selected from a B cell germinal center, a thymic developmental niche, a lymph node, an islet of Langerhans, a hair follicle, a tumor, a tumor sphere, a nerve bundle or supporting cell, a nephron, a liver organoid, an intestinal crypt, a primary lymphoid organ, and a secondary lymphoid organ. In another aspect, the present disclosure provides a method for producing one or more immune proteins, comprising: (a) providing a medium chamber comprising a first medium, wherein the first medium comprises a first plurality of cells and a first polymer precursor; (b) directing at least one energy beam along at least one energy beam path to the first medium in the medium chamber according to computer instructions in a computer memory for printing a three-dimensional (3D) lymphoid organ, so that at least a portion of the first medium in the medium chamber forms a first portion of the 3D lymphoid organ; (c) providing a second medium in the medium chamber, wherein the second medium comprises a second plurality of cells and a second polymer precursor, wherein the second plurality of cells are of a different type than the first plurality of cells; (d) directing at least one energy beam along at least one energy beam path to the second medium in the medium chamber according to the computer instructions, so that at least a portion of the second medium in the medium chamber forms a second portion of the 3D lymphoid organ; and (e) subjecting the first portion and the second portion of the 3D lymphoid organ to conditions sufficient to stimulate the production of the one or more immune proteins.

[0026] In some embodiments, the conditions include exposing the first portion and the second portion of the 3D lymphoid organoid to an antigen so as to stimulate the production of one or more immune proteins. In some embodiments, the method further includes: (f) extracting one or more immune proteins from the first portion and the second portion of the 3D lymphoid organoid. In some embodiments, the immune protein is selected from an antibody, a T cell receptor, and a cancer immunotherapeutic agent. In some embodiments, the first plurality of cells and the second plurality of cells are from a subject. In some embodiments, the first plurality of cells and the second plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or precursors thereof, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D lymphoid organoid is selected from B cell germinal center, thymic developmental niche, lymph node, Langerhans island, hair follicle, tumor, tumor sphere, nerve bundle or supporting cell, nephron, liver organoid, intestinal crypt, primary lymphoid organ and secondary lymphoid organ. In some embodiments, the shape of the 3D lymphoid organoid is selected from spherical, elliptical, oval, ovoid, square, rectangular, cubic, any polygonal shape, free form and teardrop. In some embodiments, the polymer of at least a portion of the 3D lymphoid organoid forms a network. In some embodiments, the network is reticular, amorphous or a mesh. In some embodiments, the amorphous network is designed to promote cell interactions. In some embodiments, the amorphous network is designed to promote movement between or within cell niches.

[0027] On the other hand, the present disclosure provides a method for producing one or more immune proteins, comprising (i) printing a three-dimensional (3D) lymphoid organoid comprising a matrix, the matrix comprising a first plurality of cells and a second plurality of cells, and (ii) processing the 3D lymphoid organoid to produce the one or more immune proteins. In some embodiments, the immune protein is selected from an antibody, a T cell receptor, and a cancer immunotherapeutic agent. In some embodiments, the first plurality of cells and the second plurality of cells are from a subject. In some embodiments, the first plurality of cells and the second plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or precursors thereof, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D lymphoid organoid is selected from B cell germinal center, thymic developmental niche, lymph node, Langerhans island, hair follicle, tumor, tumor sphere, nerve bundle or supporting cell, nephron, liver organoid, intestinal crypt, primary lymphoid organ and secondary lymphoid organ.

[0028] In another aspect, the present disclosure provides a method for using a three-dimensional (3D) cell-containing matrix, comprising: (a) providing a medium chamber comprising a medium comprising (i) a plurality of cells and (ii) one or more polymer precursors; (b) directing at least one energy beam to the medium in the medium chamber along at least one energy beam path patterned as a 3D projection according to computer instructions in a computer memory for printing the three-dimensional (3D) cell-containing medical device to form at least a portion of the 3D cell-containing matrix, the 3D cell-containing matrix comprising (i) at least a subpopulation of the plurality of cells, and (ii) a polymer formed from the one or more polymer precursors; and (c) positioning the 3D cell-containing matrix within a subject.

[0029] In some embodiments, the multiple cells are from a subject. In some embodiments, the multiple cells are selected from matrix endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subpopulations, effector T cells and their subpopulations, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subpopulations, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subpopulations, macrophages and their subpopulations, leukocytes and their subpopulations. In some embodiments, the 3D cell-containing matrix forms sutures, stents, nails, clips, strands, patches, grafts, sheets, tubes, pins or screws. In some embodiments, the graft is selected from skin implants, endometrium, neural tissue implants, bladder walls, intestinal tissue, esophageal lining, gastric lining, skin and retinal tissue embedded in hair follicles. In some embodiments, the 3D cell-containing matrix is ​​about 1 μm to about 10 cm. In some embodiments, the 3D cell-containing matrix further comprises an agent that promotes vascular system or nerve growth. In some embodiments, the agent is selected from growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid analgesics, non-opioid analgesics, immunosuppressants, immune inducers, monoclonal antibodies and stem cell proliferation agents.

[0030] In another aspect, the present disclosure provides a method of using a three-dimensional (3D) cell-containing matrix, comprising (i) printing the 3D cell-containing matrix comprising a plurality of cells, and (ii) positioning the 3D cell-containing matrix within a subject.

[0031] In some embodiments, the multiple cells are from a subject. In some embodiments, the multiple cells are selected from matrix endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subpopulations, effector T cells and their subpopulations, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subpopulations, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subpopulations, macrophages and their subpopulations, leukocytes and their subpopulations. In some embodiments, the 3D cell-containing matrix forms sutures, stents, nails, clips, strands, patches, grafts, sheets, tubes, pins or screws. In some embodiments, the graft is selected from skin implants, endometrium, neural tissue implants, bladder walls, intestinal tissue, esophageal lining, gastric lining, skin and retinal tissue embedded in hair follicles. In some embodiments, the 3D cell-containing matrix is ​​about 1 μm to about 10 cm. In some embodiments, the 3D cell-containing matrix further comprises an agent that promotes vascular system or nerve growth. In some embodiments, the agent is selected from growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid analgesics, non-opioid analgesics, immunosuppressants, immune inducers, monoclonal antibodies and stem cell proliferation agents.

[0032] On the other hand, the present disclosure provides a method for using a three-dimensional (3D) cell-containing matrix, comprising: (a) providing a medium chamber containing a first medium, wherein the first medium contains a first plurality of cells and a first polymer precursor; (b) directing at least one energy beam along at least one energy beam path to the first medium in the medium chamber according to computer instructions in a computer memory for printing the 3D cell-containing matrix, so that at least a portion of the first medium in the medium chamber forms a first portion of the 3D cell-containing matrix; (c) providing a second medium in the medium chamber, wherein the second medium contains a second plurality of cells and a second polymer precursor, wherein the second plurality of cells are of a different type from the first plurality of cells; (d) directing at least one energy beam along at least one energy beam path to the second medium in the medium chamber according to the computer instructions, so that at least a portion of the second medium in the medium chamber forms a second portion of the 3D cell-containing matrix; and (e) positioning the first portion and the second portion of the 3D cell-containing matrix within a subject.

[0033] In some embodiments, the first plurality of cells and the second plurality of cells are from a subject. In some embodiments, the first plurality of cells and the second plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermal derived dendritic cells, dendritic cell precursors, monocyte derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D cell-containing matrix forms sutures, scaffolds, nails, clips, strands, patches, grafts, sheets, tubes, pins or screws. In some embodiments, the implant is selected from skin implants, endometrium, neural tissue implants, bladder wall, intestinal tissue, esophageal lining, gastric lining, hair follicle embedded skin and retinal tissue. In some embodiments, the 3D cell-containing matrix is ​​about 1 μm to about 10 cm. In some embodiments, the 3D cell-containing matrix further comprises an agent that promotes vascular system or nerve growth. In some embodiments, the agent is selected from growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid analgesics, non-opioid analgesics, immunosuppressants, immune inducers, monoclonal antibodies and stem cell proliferation agents.

[0034] In another aspect, the present disclosure provides a method of using a three-dimensional (3D) cell-containing matrix, comprising (i) printing the 3D cell-containing matrix comprising a first plurality of cells and a second plurality of cells, wherein the first plurality of cells is different from the second plurality of cells, and (ii) positioning the 3D cell-containing matrix within a subject.

[0035] In some embodiments, the first plurality of cells and the second plurality of cells are from a subject. In some embodiments, the first plurality of cells and the second plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermal derived dendritic cells, dendritic cell precursors, monocyte derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D cell-containing matrix forms sutures, scaffolds, nails, clips, strands, patches, grafts, sheets, tubes, pins or screws. In some embodiments, the implant is selected from skin implants, endometrium, neural tissue implants, bladder wall, intestinal tissue, esophageal lining, gastric lining, hair follicle embedded skin and retinal tissue. In some embodiments, the 3D cell-containing matrix is ​​about 1 μm to about 10 cm. In some embodiments, the 3D cell-containing matrix further comprises an agent that promotes vascular system or nerve growth. In some embodiments, the agent is selected from growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid analgesics, non-opioid analgesics, immunosuppressants, immune inducers, monoclonal antibodies and stem cell proliferation agents.

[0036] In another aspect, the present disclosure provides a system for producing one or more immune proteins, comprising: (a) a medium chamber configured to contain a medium comprising a plurality of cells and one or more polymer precursors; (b) at least one energy source configured to direct at least one energy beam to the medium chamber; and (c) one or more computer processors operably coupled to the at least one energy source, wherein the one or more computer processors are individually or collectively programmed to (i) receive computer instructions for printing a three-dimensional (3D) lymphoid organ from a computer memory; (ii) direct the at least one energy source to direct the at least one energy beam to the medium in the medium chamber along at least one energy beam path according to the computer instructions so that at least a portion of the polymer precursor forms at least a portion of the 3D lymphoid organ, and (iii) subject at least a portion of the 3D lymphoid organ to conditions sufficient to stimulate the production of the one or more immune proteins.

[0037] In some embodiments, the conditions sufficient to stimulate production of the one or more immune proteins comprise exposing at least a portion of the 3D lymphoid organoid to an antigen so as to stimulate production of the one or more immune proteins.

[0038] In another aspect, the present disclosure provides a system for producing one or more immune proteins, comprising: (a) a medium chamber configured to contain a first medium, the first medium comprising a first plurality of cells and a first plurality of polymer precursors; (b) at least one energy source configured to direct at least one energy beam to the medium chamber; and (c) one or more computer processors operably coupled to the at least one energy source, wherein the one or more computer processors are individually or collectively programmed to (i) receive computer instructions for printing a three-dimensional (3D) lymphoid organoid from a computer memory; (ii) direct the at least one energy source to direct the at least one energy beam to the medium chamber along at least one energy beam path according to the computer instructions; (iii) directing the at least one energy source to direct the at least one energy beam along at least one energy beam path to a second medium in the medium chamber in accordance with the computer instructions so that at least a portion of the second medium in the medium chamber forms at least a second portion of the 3D lymphoid organoid, wherein the second medium comprises a second plurality of cells and a second plurality of polymer precursors, wherein the second plurality of cells are of a different type than the first plurality of cells; and (iv) subjecting the first portion and the second portion of the 3D lymphoid organoid to conditions sufficient to stimulate production of the one or more immune proteins.

[0039] In some embodiments, the conditions sufficient to stimulate production of the one or more immune proteins comprise exposing the first portion and the second portion of the 3D lymphoid organoid to an antigen so as to stimulate production of the one or more immune proteins.

[0040] On the other hand, the present disclosure provides a method for producing a human immune protein population, comprising: bioprinting a three-dimensional lymphoid organoid using a multiphoton laser bioprinting system; exposing the three-dimensional lymphoid organoid to an antigen to stimulate the production of a human immune protein population; and extracting the human immune protein population from the three-dimensional lymphoid organoid.

[0041] In another aspect, the present disclosure provides a method for producing a population of human immune proteins, comprising: (a) providing a medium comprising: (i) a plurality of cells and (ii) one or more polymer precursors; (b) depositing at least one layer of the medium onto a substrate; (c) subjecting the at least one layer of the medium to an energy source to form at least a portion of the 3D lymphoid organoid, the 3D lymphoid organoid comprising (i) at least a subset of the plurality of cells, and (ii) a biogel formed from the one or more polymer precursors; and (d) subjecting at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate production of the one or more immune proteins.

[0042] In some embodiments, the conditions include exposing at least a portion of the 3D lymphoid organoid to an antigen to stimulate the production of the one or more immune proteins. In some embodiments, the method further includes: (d) extracting one or more immune proteins from at least a portion of the 3D lymphoid organoid. In some embodiments, the immune protein is selected from antibodies, T cell receptors, and cancer immunotherapeutics. In some embodiments, the plurality of cells are from a subject. In some embodiments, the plurality of cells are selected from stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermal derived dendritic cells, dendritic cell precursors, monocyte derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets. In some embodiments, the 3D lymphoid organoid is selected from B cell germinal center, thymic developmental niche, lymph node, Langerhans island, hair follicle, tumor, tumor sphere, nerve bundle or supporting cell, nephron, liver organoid, intestinal crypt, primary lymphoid organ and secondary lymphoid organ. In some embodiments, the medium comprises a photoinitiator, a cross-linking agent, collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic acid-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar or any combination thereof. In some embodiments, the energy source is a laser, a heat source, a light source or any combination thereof.

[0043] The present disclosure provides methods and systems for rapidly generating cell-containing structures using spatial light modulation of a multiphoton excitation source. In some embodiments, the cell-containing structure can be a multilayer vascularized tissue, a cell-containing device, or a cell-containing material. Using this method, a method for rapidly generating a cell-containing structure is provided by layering a cell-size-specific mesh with embedded mechanical and / or biological elements (such as a microvascular system). Based on three-dimensional (holographic) projection, two-dimensional projection, and / or on any planar axis (such as x, y, x, z, or y, z), the deposition of cells contained in a collagen mesh or other biocompatible or inert material mesh is a rapid iterative process that can be combined with scanning of multiphoton laser excitation. Three-dimensional scanning, two-dimensional scanning, and raster scanning can be used simultaneously in various combinations to achieve rapid generation of complete structures. The dynamic transition between laser projection modes allows complex structures to be rapidly generated in a large field of view while maintaining fine resolution from micrometers to nanometers. The methods allow for the rapid generation of larger (eg, up to about 5 centimeters (cm)) multilayer vasculature and smaller vasculature (eg, 1-10 micrometers (μm)) monolayer vasculature.

[0044] The present disclosure allows multiple cell types to be layered in two and / or three dimensions, making it possible to construct tissues in a manner that is not limited by multiple cell types, size, or complexity. In some cases, this is achieved by using multiphoton (e.g., two-photon) excitation light, such as can be provided by, for example, a laser.

[0045] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods above or elsewhere herein.

[0046] Another aspect of the present disclosure provides a system including one or more computer processors and a computer memory coupled thereto. The computer memory includes machine executable code that, when executed by the one or more computer processors, implements any method above or elsewhere herein.

[0047] Another aspect of the present disclosure provides for the generation of lymphatic organs and organoids by photolithography, which uses white light, single photon excitation, or multiphoton laser excitation projected into a two-dimensional sequential plane or a three-dimensional hologram. Lymph node organs and organoids can be printed from collagen or other biocompatible materials. Active, responsive synthetic human immune tissues can be produced by the methods disclosed herein. Immune response tissues can be produced by the methods disclosed herein. Synthetic immune response tissues produced by the methods disclosed herein can be used to develop new products and examine the immune response of living individuals. Using this method, a method for rapidly generating new antibodies is provided by exposing lymphatic organs to selected antigens and allowing the physiological process of antibody clone selection and expansion to occur. Synthetic immune response tissues produced by the methods disclosed herein can be used to generate new antibodies from a single blood donation, thereby replacing hundreds of animal or human surrogates for producing antibodies in a set of 96-well plates. Synthetic immune response tissues produced by the methods disclosed herein generate antibody libraries or examine immune system responses in a high-throughput manner. Synthetic organoids produced by the methods disclosed herein can be pharmaceutically tested for biological and / or non-biological drug compounds or drug combinations.

[0048] Another aspect of the present disclosure provides methods for producing a range of cell-containing devices or materials for medical purposes, which are three-dimensionally printed with multiphoton and / or single photon or white light lithography, including but not limited to surgical wound closure and tissue repair. The devices and materials may include sutures, staples, clips, strands, patches, grafts, sheets, tubes, pins, screws, and similar structures intended for use in living subjects. Such devices and materials allow cells, including stem cells, to be delivered into living subjects, potentially accelerating wound healing and / or tissue repair or altering the cellular composition at the site of use in some predetermined manner.

[0049] Another aspect of the present disclosure provides methods for implanting multiphoton 3D printed cell-containing devices or structures having predetermined physiological functions, including duplicating the functions of natural organs and organoids, to supplement the functions naturally existing in a living subject. In this iteration, the device is not intended to deliver cells or regenerate natural structures or functions, but rather to recapitulate the natural functions of the tissue itself, combining natural functions to enhance or assist functions or operating independently in place of natural tissue functions.

[0050] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art through the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative rather than restrictive in nature.

[0051] Incorporation by reference

[0052] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description which sets forth illustrative embodiments utilizing the principles of the invention and the accompanying drawings, in which:

[0054] Figure 1 An embodiment of a system for rapid multiphoton printing of predetermined tissues is illustrated.

[0055] Figure 2A-2D Exemplary stages of generating a predetermined tissue within a media chamber are illustrated. Figure 2A The diagram shows a medium chamber containing medium containing a first population of cells. Figure 2B The diagram shows a media chamber containing media containing a second population of cells. Figure 2C The delivery of pulses of a multiphoton laser beam to a medium is illustrated. Figure 2D An embodiment is illustrated in which a cell-containing scaffold is printed along the bottom of a media chamber containing media.

[0056] Figure 3A-3C Various embodiments of laser systems are illustrated. Figure 3A An embodiment of a laser system having a single multiphoton laser source is illustrated. Figure 3B An embodiment of a laser system having multiple laser lines is illustrated. Figure 3C An embodiment of a laser system including multiple laser lines, a photomultiplier tube (PMT), and an objective lens is illustrated.

[0057] Figure 4A-4C Various embodiments of a printing system are illustrated. Figure 4A An embodiment of such a printing system is illustrated that includes a beam expander, an optical focusing lens, an additional laser focusing lens, and no axicon lens or TAG lens. Figure 4B An embodiment of such a printing system is illustrated, which includes a beam expander, an optical focusing lens, an additional laser focusing lens, and an axicon lens or a TAG lens. Figure 4C A Z-step projection printing setup is illustrated, comprising a single SLM or DMD for 2D x,y sheet or holographic projection, for printing around cells and resulting structures printed at a given Z-step.

[0058] Figure 5A-Figure 5BVarious embodiments of a multiphoton tissue print head are illustrated. Figure 5A An embodiment of a multiphoton tissue print head comprising a single vertical objective lens is illustrated. Figure 5B An embodiment of a multiphoton tissue print head with inverted optics for imaging structures is illustrated.

[0059] Figure 6A-6B An embodiment of a removable and attachable fiber optic cable accessory is illustrated. Fig. 6A Fiber optic cable accessories and fiber optic cables are illustrated. Figure 6B Illustration of a fiber optic cable attachment for printing predetermined complex structures.

[0060] Figure 7 An embodiment is illustrated wherein the print head optics include at least three objective lenses, wherein each objective lens includes a fiber optic cable attachment leading into a single media chamber.

[0061] Figure 8 An embodiment is illustrated in which the print head optics includes at least six objective lenses, each of which includes a fiber optic cable attachment leading into a separate well in a separate media chamber, such as a multi-well plate.

[0062] Fig. 9 An embodiment of a print head optics having an array of objective lenses acting as a print head is illustrated.

[0063] Fig.10 Illustrated is an objective lens programmed to move in the X and Y directions over a multi-well plate to deliver a laser beam projection into each well.

[0064] Fig.11 A computer control system programmed or otherwise configured to implement the methods provided herein is shown.

[0065] Fig.12 The optical components and light paths of an embodiment of a printing system without temporal focusing are illustrated.

[0066] Fig.13 Optical components and light paths of further embodiments of a printing system with temporal focusing are illustrated.

[0067] Fig.14 The optical components and light paths of yet another embodiment of a printing system without temporal focusing are illustrated.

[0068] Fig.15 The light detection system is illustrated.

[0069] Fig.16 The compartmentalization and organization of several types of lymphocytes in a lymph node are illustrated, with additional depictions in cross-section labeling the major structures and cell types.

[0070] Fig.17 Depicted are B cell germinal centers, which include dark areas where B cells proliferate and light areas where B cells interact with antigen presenting cells and accessory cells.

[0071] Figure 18A-18B Illustration of the B cell germinal center and thymic developmental niche. Fig.18A Illustration of a B cell germinal center in response to an antigen, where B cells migrate between compartmentalized areas called dark and light zones as part of the maturation and selection process for the production of high-affinity antibodies. Fig.18B Diagram of the thymic developmental niche, in which T cells undergo selection and maturation in a series of sequential steps as they move from cortical to medullary thymic tissue.

[0072] Fig.19 Illustration of various examples of lymph node organoid structures designed for the purpose of facilitating cellular niche formation and associated cell-cell interactions that occur during immune responses.

[0073] Fig. 20 Illustration of lymph node organoids, or lymphocyte-containing organoids, that can be printed in an asymmetric teardrop-like shape.

[0074] Fig.21 Schematic representation of the sequential process of depositing biogel layers containing lymphocytes for the purpose of constructing lymph node organoids.

[0075] Fig. 22 Shown are enzyme-linked immunosorbent assay (ELISA) results for the study of Zika antibody production in printed lymphoid organoids.

[0076] Fig.23 Microscope images of three-dimensionally printed lymph node organoids produced by the methods disclosed herein are shown. The T cell zone indicates a tissue region containing a mixture of T cells and supporting accessory cells. The B cell zone indicates a tissue region containing a mixture of B cells and supporting accessory cells.

[0077] Fig.24 Examples of staples, sutures, stents, and clips containing cells for dissolving and incorporating into tissue or promoting tissue healing are illustrated.

[0078] Fig.25 Examples of 3D printed bone resorbable screws, pins, and grafts that may contain cells are illustrated.

[0079] Fig.26 Illustrated are examples of functional tissue implants that enhance function by interacting with cells and cellular systems proximal to the implanted cell site.

[0080] Fig. 27 Illustration of cells printed on a lattice structure.

[0081] Figure 28A-Figure 28E Shown are clusters of human pluripotent stem cell-derived insulin-producing cells encapsulated by holographic printing. Fig.28A Images of clusters of encapsulated cells expressing enhanced green fluorescent protein (eGFP) are shown. Fig.28B Shown is a graph corresponding to the amount of human C-peptide produced by encapsulated cells. Fig.28C Shown is the first image of encapsulated cells expressing eGFP five days after encapsulation. Fig.28D A second image showing encapsulated cells expressing eGFP five days after encapsulation. Fig.28E An image of the package structure is shown.

[0082] Figure 29A-29C Shown is a biocompatible microscaffold structure generated by holographic printing. Fig.29A Representative computer-generated renderings of microscaffolds are shown. Fig.29B An image showing a side view of the printed microscaffold. Fig.29C An image showing a cross-sectional view of the printed microscaffold.

[0083] Figure 30A-Figure 30B Images showing compressibility and resilience testing of holographically printed microscaffolds. Fig. 30A Shown is a series of images demonstrating repeated compression of a microscaffold against a solid surface. Fig. 30B A series of images demonstrating the resilience of the microscaffold are shown.

[0084] Figure 31A-Figure 31E A three-dimensional holographically printed micromesh network is shown. Fig.31A A computer-generated image showing a microgrid network. Fig.31B An image of a microgrid network is shown. Fig.31C A close-up image of a microgrid network is shown. Fig.31D A series of images of a micromesh network undergoing lateral compression is shown. Fig.31E A series of images of a micromesh network during handling with tweezers is shown.

[0085] Figure 32A-32H Images of printed lymph node organoids (LNOs) and characterization of their functions are shown. Fig.32A An image of a printed collagen matrix containing B cells, T cells, and antigen presenting cells (APCs) is shown. Fig.32B Shown are images representing a mixed cell population of B cells, T cells, and APCs in a printed collagen matrix. Fig.32CImages showing populations of B cells, T cells, and APCs in tissue culture wells but not in printed collagen matrices. Fig.32D Images of LNO and cell clusters 24 hours after addition of antigen are shown. Fig.32E Shown are images of printed lymph node organoids and cell clusters 72 hours after addition of antigen pulse. Fig.32F Images of printed LNOs and cell clusters 120 hours after antigen addition are shown. Figure 32G Shown is a graph showing the production of interleukin 4 (IL-4) by LNO. Fig.32H Shown is a graph showing the production of IL-2 by LNO.

[0086] Figure 33A-Figure 33C Shown is human immunoglobulin (IgG) purified from printed LNO. Fig.33A Shown is a graph representing the protein concentration in LNO samples. Fig.33B Shown is an image of an SDS-PAGE gel containing purified human IgG isolated from the printed LNO media. Fig.33C Shown is an image of an SDS-PAGE gel containing unpurified human IgG isolated from the printed LNO media.

[0087] Figure 34A-Figure 34B Shown are printed LNO medium aliquots tested for human IgG reactivity with the antigen used in antigen challenge. Fig.34A Shown is a graph showing the absorbance at 450 nanometers (nm) and 570 nm of samples tested using ELISA. Fig.34B Shown are two graphs representing unique hybridomas that were subcloned and further assayed for the presence of specific antigen-reactive human IgG.

[0088] Fig.35 An example of simultaneous projection of a single-photon light source and a multi-photon light source is shown.

[0089] Fig.36 An example of knitted printed structures that do not chemically interact with each other is shown.

[0090] Fig.37 is an example of a first 3D projection and at least one additional projection formed using the same components.

[0091] Fig.38 is an example of two-photon projection as opposed to single-photon projection.

[0092] Fig.39 is an example of a combined one-photon and two-photon printing process involving a rotating media bath.

[0093] Fig.40 is an example of a combined printing process.

[0094] Fig.41 is an example of a light reuse scheme.

[0095] Fig.42 is an example of a 2D projection of a 3D object partitioned in two ways.

[0096] Fig.43 is an example of a 3D clustering operation. DETAILED DESCRIPTION

[0097] Although various embodiments of the present invention have been shown and described herein, it will be readily understood by those skilled in the art that these embodiments are provided by way of example only. A variety of changes, modifications and substitutions may be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0098] definition

[0099] The terminology used herein is for descriptive purposes only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. In addition, where the terms "including," "having," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive, similar to the term "comprising."

[0100] The term "about" or "approximately" refers to an amount that is approximately 10%, 5%, or 1% of the stated amount, including increments therein. For example, "about" or "approximately" may mean a range that includes the specific value and spans from 10% below the specific value to 10% above the specific value.

[0101] As used herein, the term "biomaterial" generally refers to any material that can perform a chemical or biological function. A biomaterial can be a biofunctional tissue or a functional tissue, which can be a biological structure that can perform or exert a biomechanical or biological function. Biofunctional tissues can include cells within a diffusion distance of each other, including at least one cell type, each of which is within a diffusion distance of a capillary or vascular network component, promoting and / or inhibiting the realization of protein function, or any combination thereof. Biofunctional tissues can be at least a portion of a tissue or an organ such as a vital organ. In some examples, biomaterials can facilitate drug development, for example, screening multiple cells or tissues with different therapeutic agents.

[0102] Biomaterials can include matrix such as polymer matrix, biogel, hydrogel or polymer scaffold, which includes one or more other types of materials such as cells. Biomaterials can include lymphoid organs and organoids. Biomaterials can be derived from primary cells, cell lines, stem cells, stem cell lines, differentiated stem cells, transdifferentiated stem cells, autologous cells, allogeneic cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells or any combination thereof from human or animal sources. Biomaterials can have a variety of shapes, sizes or configurations. In some cases, biomaterials such as meat or meat materials can be consumed by an object (e.g., animal).

[0103] As used herein, the term "three-dimensional printing" (also referred to as "3D printing") generally refers to a process or method for generating a 3D part (or object). Such a process can be used to form a 3D part (or object), such as a 3D biomaterial.

[0104] As used herein, the term "energy beam" generally refers to a beam of energy. The energy beam can be a beam of electromagnetic energy or a beam of electromagnetic radiation. The energy beam can be a particle beam. The energy beam can be a light beam (e.g., gamma waves, X-rays, ultraviolet light, visible light, infrared light, microwaves, or radio waves). The light beam can be a coherent light beam, such as can be provided by light amplification by stimulated emission of radiation ("laser"). In some examples, the light beam is generated by a laser diode or a multi-diode laser.

[0105] As used herein, the term "allogeneic" refers to a plurality of cells obtained from a genetically non-identical donor. For example, allogeneic cells are extracted from a donor and returned to a different, genetically non-identical recipient.

[0106] As used herein, the term "autologous" refers to a plurality of cells obtained from a genetically identical donor. For example, autologous cells are extracted from a patient and returned to the same genetically identical individual (e.g., donor).

[0107] As used herein, the term "pluripotent stem cell" (PSC) refers to a cell that is capable of producing different cell types derived from all three germ layers (e.g., endoderm, mesoderm, and ectoderm) under appropriate conditions. The definition of pluripotent stem cells includes various types of embryonic stem cells, including human embryonic stem (hES) cells, human embryonic germ stem (hEG) cells; non-human embryonic stem cells, such as embryonic stem cells from other primates, such as macaque stem cells, marmoset stem cells; mouse stem cells; stem cells produced by nuclear transplantation technology, and induced pluripotent stem cells (iPSC).

[0108] As used herein, the term "embryonic stem cell" (ESC) refers to a pluripotent stem cell derived from a blastocyst before the cells are substantially differentiated into three germ layers (e.g., endoderm, mesoderm, and ectoderm). ESCs include any commercially available or mature ESC cell lines, such as H9, H1, H7, or SA002.

[0109] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a somatic cell that has been reprogrammed to a pluripotent state similar to an embryonic stem cell. The definition of iPSC includes various types of iPSCs, including human iPSCs and non-human iPSCs, such as iPSCs derived from somatic cells such as primate somatic cells or mouse somatic cells.

[0110] As used herein, the term "energy source" refers to a laser, such as a fiber laser, a short pulse laser, or a femtosecond pulse laser; a heat source, such as a hot plate, a lamp, an oven, a hot water bath, a cell culture incubator, a heating chamber, a furnace, or a drying oven; a light source, such as white light, infrared light, ultraviolet (UV) light, near infrared (NIR) light, visible light, or a light emitting diode (LED); an acoustic energy source, such as an ultrasound probe, a sonicator, or an ultrasound bath; an electromagnetic radiation source, such as a microwave source; or any combination thereof.

[0111] As used herein, the term "biogel" refers to a hydrogel, a biocompatible hydrogel, a polymer hydrogel, a hydrogel bead, a hydrogel nanoparticle, a hydrogel droplet, having a viscosity of at least about 10 x 10 when measured at 25 degrees Celsius (°C). -4 Solutions of Pascal·seconds (Pa·s) to 100 Pa·s or more, hydrogels comprising non-hydrogel beads, nanoparticles, microparticles, nanorods, nanoshells, liposomes, nanowires, nanotubes or combinations thereof; gels in which the liquid component is water; degradable hydrogels; non-degradable hydrogels; resorbable hydrogels; hydrogels comprising polymers of natural origin; or any combination thereof.

[0112] The present disclosure provides methods and systems for printing three-dimensional (3D) biomaterials. On the one hand, the method for printing 3D biomaterials includes providing a medium chamber containing a medium, the medium containing (i) multiple cells and (ii) one or more polymer precursors. Subsequently, according to computer instructions for printing 3D biomaterials in a computer memory, at least one energy beam can be directed to the medium in the medium chamber along at least one energy beam path patterned as a 3D projection. This can form at least a portion of the 3D biomaterial, the 3D biomaterial including (i) at least a subset of multiple cells, at least a subset of the multiple cells containing at least two different types of cells, and (ii) a polymer formed by one or more polymer precursors.

[0113] The method and system of the present disclosure can be used to print multiple layers of 3D objects such as 3D biomaterials simultaneously. Such 3D objects can be formed by polymer materials, metals, metal alloys, composite materials or any combination thereof. In some examples, the 3D object is formed by a polymer material, which in some cases includes a biomaterial (e.g., one or more cells or cell components). In some cases, a 3D object can be formed by guiding an energy beam (e.g., a laser) as a 3D projection (e.g., a hologram) to one or more precursors of a polymer material to induce polymerization and / or cross-linking to form at least a portion of a 3D object. This can be used to form multiple layers of a 3D object simultaneously.

[0114] Alternatively, the 3D object can be formed of a metal or metal alloy, such as, for example, gold, silver, platinum, tungsten, titanium or any combination thereof. In such a case, the 3D object can be formed by sintering or melting metal particles, such as can be achieved by, for example, directing an energy beam (e.g., a laser beam) to a powder bed containing particles of a metal or metal alloy. In some cases, the 3D object can be formed by directing such an energy beam as a 3D projection (e.g., a hologram) into a powder bed to promote the sintering or melting of the particles. This can be used to form multiple layers of a 3D object at the same time. The 3D object can be formed of an organic material such as graphene. The 3D object can be formed of an inorganic material such as silicone. In such a case, the 3D object can be formed by sintering or melting organic and / or inorganic particles, such as can be achieved by, for example, directing an energy beam (e.g., a laser beam) to a powder bed containing particles of organic and / or inorganic materials. In some cases, a 3D object can be formed by directing such an energy beam as a 3D projection (eg, a hologram) into a powder bed to promote sintering or melting of organic and / or inorganic particles.

[0115] The depth of energy beam penetration can be determined by the interaction of the beam wavelength with the electron field of a given metal, metal alloy, inorganic material and / or organic material. The organic material can be graphene. The inorganic material can be silicone. The particles can be functionalized or combined to allow greater interaction or less interaction with a given energy beam.

[0116] In some examples, at least one energy beam is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more energy beams. At least one energy beam can be or include coherent light. In some cases, at least one energy beam is a laser beam.

[0117] At least one energy beam may be directed as an image or a collection of images. The image may be fixed over time or change over time. At least one energy beam may be directed as a video.

[0118] The computer instructions may correspond to a computer model or representation of a 3D biological material. The computer instructions may be part of a computer model. The computer instructions may include a collection of images corresponding to the 3D biological material.

[0119] At least one energy beam can be directed as a holographic image or video. This can enable different points in the medium to be simultaneously exposed to at least one energy beam to, for example, induce the formation of a polymer matrix (e.g., by polymerization) in multiple layers simultaneously. In some cases, a 3D image or video can be projected into the medium at different focal points using, for example, a spatial light modulator (SLM).

[0120] The computer instructions may include and / or direct adjustment of one or more parameters of at least one energy beam as a function of time during formation of the 3D biomaterial, such as, for example, applying power to a source of at least one energy beam (e.g., turning a laser on / off). Such adjustments may be made as a function of an image or video (e.g., a holographic image or video) corresponding to the 3D biomaterial. Alternatively or additionally, the computer instructions may include and / or direct adjustment of a position of a stage on which the 3D biomaterial is formed.

[0121] In some cases, during or after the formation of the 3D biomaterial, at least a portion of at least a subset of the plurality of cells can be subjected to differentiation to form at least two different types of cells. For example, this can be employed by exposing the cells to an agent or subjecting the cells to conditions that induce differentiation. Alternatively or additionally, the cells can be subjected to dedifferentiation or induced to be quiescent.

[0122] Another aspect of the present disclosure provides a method for printing 3D biomaterials, the method providing a medium chamber containing a first medium. The first medium may include a first plurality of cells and a first polymer precursor. According to a computer instruction for printing the 3D biomaterial, at least one energy beam may be directed to the first medium in the medium chamber along at least one energy beam path so that at least a portion of the first medium in the medium chamber forms a first portion of the 3D biomaterial. Subsequently, a second medium may be provided in the medium chamber. The second medium may include a second plurality of cells and a second polymer precursor. The second plurality of cells may be of a different type from the first plurality of cells. Subsequently, according to a computer instruction, at least one energy beam may be directed to the second medium in the medium chamber along at least one energy beam path so that at least a portion of the second medium in the medium chamber forms at least a second portion of the 3D biomaterial.

[0123] In another aspect of the present disclosure, a system for printing 3D biomaterials includes: a media chamber configured to contain a media comprising a plurality of cells comprising at least two different types of cells and one or more polymer precursors; at least one energy source configured to direct at least one energy beam to the media chamber; and one or more computer processors operably coupled to the at least one energy source, wherein the one or more computer processors are individually or collectively programmed to (i) receive computer instructions for printing 3D biomaterials from a computer memory; and (ii) direct at least one energy source to direct at least one energy beam to the media in the media chamber along at least one energy beam path in accordance with the computer instructions so that at least a portion of the polymer precursor forms at least a portion of the 3D biomaterial.

[0124] In another aspect, a system for printing 3D biomaterials comprises: a media chamber configured to contain a media comprising a plurality of cells and a plurality of polymer precursors; at least one energy source configured to direct at least one energy beam to the media chamber; and one or more computer processors operably coupled to the at least one energy source, wherein the one or more computer processors are individually or collectively programmed to (i) receive computer instructions for printing 3D biomaterials from a computer memory; (ii) direct at least one energy source to direct at least one energy beam to a media in the media chamber along at least one energy beam path in accordance with the computer instructions so that at least a portion of the polymer precursors form at least a portion of the 3D biomaterial; and (iii) direct at least one energy source to direct at least one energy beam to a second media in the media chamber along at least one energy beam path in accordance with the computer instructions so that at least a portion of the second media in the media chamber forms at least a second portion of the 3D biomaterial, wherein the second media comprises a second plurality of cells and a second polymer precursor, wherein the second plurality of cells are of a different type than the first plurality of cells.

[0125] In another aspect of the present disclosure, a method for printing a three-dimensional (3D) object may include directing at least one energy beam into a medium containing one or more precursors to generate a 3D object containing a material formed from the one or more precursors, wherein the at least one energy beam is directed into the medium as a 3D projection corresponding to the 3D object.

[0126] In another aspect, a method for printing three-dimensional (3D) biomaterials may include directing at least one energy beam to: 1) a first medium comprising a first plurality of cells and a first polymer precursor, and 2) a second medium comprising a second plurality of cells and a second polymer precursor to generate a first portion of the 3D biomaterial and a second portion of the 3D biomaterial.

[0127] In another aspect, the present disclosure provides a method for producing a population of human immune proteins, comprising: (a) providing a medium comprising: (i) a plurality of cells and (ii) one or more polymer precursors; (b) depositing at least one layer of the medium onto a substrate; (c) subjecting at least one layer of the medium to an energy source to form at least a portion of a 3D lymphoid organoid comprising (i) at least a subset of the plurality of cells, and (ii) a biogel formed from one or more polymer precursors; and (d) subjecting at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate production of one or more immune proteins.

[0128] refer to Figure 1 , illustrates an embodiment of a system 100 for rapid multiphoton printing of a predetermined tissue. Here, the system 100 includes a laser printing system 110 driven by a solid model computer-aided design (CAD) modeling system 112. In this embodiment, the CAD modeling system 112 includes a computer 114 that controls the laser printing system 110 based on the CAD model of the predetermined tissue and additional parameters. The laser printing system 110 includes a laser system 116 in communication with a multiphoton tissue printing printhead 118, which projects a waveform of a multiphoton laser beam 120 into a media chamber 122 to match a predetermined structure completely or in a specific portion. The multiphoton tissue printing head 118 includes at least one objective lens 124 that delivers the multiphoton laser beam 120 in the transverse and axial planes of the media chamber 122 to provide a two-dimensional and / or three-dimensional and thus holographic projection of the CAD-modeled tissue within the media chamber 122. The objective lens 124 can be a water immersion objective lens, an air objective lens or an oil immersion objective lens. Two-dimensional and three-dimensional holographic projections can be generated simultaneously, and projected to different regions by lens control. The medium chamber 122 contains a medium composed of cells, polymerizable materials and culture medium. The polymerizable materials can include biocompatible, soluble and in some cases bioinert polymerizable monomer units. The monomer units (or subunits) can be polymerized, cross-linked or reacted in response to the multiphoton laser beam 120, thereby producing a cell-containing structure for the tissue to be generated, such as a cell matrix and a basement membrane structure. The monomer units can be polymerized and / or cross-linked to form a matrix. In some cases, the polymerizable monomer units can include a mixture of collagen and other extracellular matrix components, and the other extracellular matrix components include but are not limited to elastin and hyaluronic acid, and their percentages vary depending on a predetermined tissue matrix.

[0129] The non-limiting example of the extracellular matrix components for producing the cell structure can include proteoglycans such as heparan sulfate, chondroitin sulfate and keratan sulfate, non-proteoglycan polysaccharides such as hyaluronic acid, collagen, and elastin, fibronectin, laminin, nidogen, or any combination thereof. These extracellular matrix components can be functionalized with acrylate, diacrylate, methacrylate, cinnamoyl, coumarin, thymine or other side groups or chemically reactive parts to promote the crosslinking induced by multiphoton excitation direct induction or by multiphoton excitation induction of one or more chemical dopants. In some cases, photopolymerizable macromonomers and / or photopolymerizable monomers can be used in combination with the extracellular matrix components to produce the cell structure. The non-limiting example of photopolymerizable macromonomers can include polyethylene glycol (PEG) acrylate derivatives, PEG methacrylate derivatives and polyvinyl alcohol (PVA) derivatives. In some cases, the collagen used to produce the cell-containing structure can be a fibrillar collagen such as type I, type II, type III, type V, and type XI collagen, a facit collagen such as type IX, type XII, and type XIV collagen, a short chain collagen such as type VIII and type X collagen, a basement membrane collagen such as type IV collagen, type VI collagen, type VII collagen, type XIII collagen, or any combination thereof.

[0130] Specific mixtures of monomer units can be produced to change the final properties of the polymerized biogel. The base printing mixture can contain other polymerizable monomers that are synthetic and not native to mammalian tissue, including mixtures of biological and synthetic materials. An example mixture can include about 0.4% w / v collagen methacrylate plus about 50% w / v polyethylene glycol diacrylate (PEGDA) added. The photoinitiator that directs the polymerization can be reactive in the ultraviolet (UV), infrared (IR), or visible light range. Examples of two such photoinitiators are eosin Y (EY) and triethanolamine (TEA), which when combined can polymerize in response to exposure to visible light (e.g., wavelengths of about 390 to 700 nanometers). Non-limiting examples of photoinitiators may include azobisisobutyronitrile (AIBN), benzoin derivatives, benziketals, hydroxyalkyl phenones, acetophenone derivatives, trimethylolpropane triacrylate (TPT), acryloyl chloride, benzoyl peroxide, camphorquinone, benzophenone, thioxanthone, and 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone. Hydroxyalkyl phenones may include 4-(2-hydroxyethylethoxy)-phenyl-(2-hydroxy-2-methylpropyl)ketone ( 295), 1-hydroxycyclohexyl-1-benzophenone ( 184) and 2,2-dimethoxy-2-phenylacetophenone ( 651). The acetophenone derivative may include 2,2-dimethoxy-2-phenylacetophenone (DMPA). The thioxanthone may include isopropylthioxanthone.

[0131] Specific mixtures of monomeric units of biomaterials can be produced to alter the final properties of the polymerized biogel, an exemplary mixture can include about 1 mg / mL type I collagen-methacrylate, about 0.5 mg / mL type III collagen, about 0.2 mg / mL methacrylated hyaluronic acid, about 0.1% Eosin Y, and about 0.1% triethanolamine.

[0132] In some cases, the polymerized biogel can include at least about 0.01% photoinitiator. In some cases, the polymerized biogel can include about 10% photoinitiator or more. In some cases, the polymerized biogel includes about 0.1% photoinitiator. In some cases, the polymerized biogel can contain about 0.01% to about 0.05%, about 0.01% to about 0.1%, about 0.01% to about 0.2%, about 0.01% to about 0.3%, about 0.01% to about 0.4%, about 0.01% to about 0.5%, about 0.01% to about 0.6%, about 0.7% to about 0.8%, about 0.9% to about 1%, about 0.01% to about 2%, about 0.01% to about 3%, about 0.01% to about 4%, about 0.01% to about 5%, about 0.01% to about 6%, about 0.01% to about 7%, about 0.01% to about 8%, about 0.01% to about 9%, or about 0.01% to about 10% of a photoinitiator.

[0133] The polymerized biogel may contain about 0.05% photoinitiator. The polymerized biogel may contain about 0.1% photoinitiator. The polymerized biogel may contain about 0.2% photoinitiator. The polymerized biogel may contain about 0.3% photoinitiator. The polymerized biogel may contain about 0.4% photoinitiator. The polymerized biogel may contain about 0.5% photoinitiator. The polymerized biogel may contain about 0.6% photoinitiator. The polymerized biogel may contain about 0.7% photoinitiator. The polymerized biogel may contain about 0.8% photoinitiator. The polymerized biogel may contain about 0.9% photoinitiator. The polymerized biogel may contain about 1% photoinitiator. The polymerized biogel may contain about 1.1% photoinitiator. The polymerized biogel may contain about 1.2% photoinitiator. The polymerized biogel may contain about 1.3% photoinitiator. The polymerized biogel may contain about 1.4% photoinitiator. The polymerized biogel may contain about 1.5% photoinitiator. The polymerized biogel may contain about 1.6% photoinitiator. The polymerized biogel may contain about 1.7% photoinitiator. The polymerized biogel may contain about 1.8% photoinitiator. The polymerized biogel may contain about 1.9% photoinitiator. The polymerized biogel may contain about 2% photoinitiator. The polymerized biogel may contain about 2.5% photoinitiator. The polymerized biogel may contain about 3% photoinitiator. The polymerized biogel may contain about 3.5% photoinitiator. The polymerized biogel may contain about 4% photoinitiator. The polymerized biogel may contain about 4.5% photoinitiator. The polymerized biogel may contain about 5% photoinitiator. The polymerized biogel may contain about 5.5% photoinitiator. The polymerized biogel may contain about 6% photoinitiator. The polymerized biogel may contain about 6.5% photoinitiator. The polymerized biogel may contain about 7% photoinitiator. The polymerized biogel may contain about 7.5% photoinitiator. The polymerized biogel may contain about 8% photoinitiator. The polymerized biogel may contain about 8.5% photoinitiator. The polymerized biogel may contain about 9% photoinitiator. The polymerized biogel may contain about 9.5% photoinitiator. The polymerized biogel may contain about 10% photoinitiator.

[0134] In some cases, the polymerized biogel may contain at least about 10% photopolymerizable macromers and / or photopolymerizable monomers. In some cases, the polymerized biogel may contain about 99% or more photopolymerizable macromers and / or photopolymerizable monomers. In some cases, the polymerized biogel may contain about 50% photopolymerizable macromers and / or photopolymerizable monomers. In some cases, the polymerized biogel can contain about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to about 40%, about 10% to about 45%, about 10% to about 50%, about 10% to about 55%, about 10% to about 60%, about 10% to about 65%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 95%, or about 10% to about 99% photopolymerizable macromers and / or photopolymerizable monomers.

[0135] The polymerized biogel may contain about 10% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 15% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 20% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 25% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 30% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 35% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 40% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 45% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 50% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 55% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 60% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 65% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 70% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 75% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 80% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 85% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 90% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may contain about 95% photopolymerizable macromonomers and / or photopolymerizable monomers. The polymerized biogel may comprise about 96% photopolymerizable macromers and / or photopolymerizable monomers. The polymerized biogel may comprise about 97% photopolymerizable macromers and / or photopolymerizable monomers. The polymerized biogel may comprise about 98% photopolymerizable macromers and / or photopolymerizable monomers. The polymerized biogel may comprise about 99% photopolymerizable macromers and / or photopolymerizable monomers.

[0136] Two-photon absorption is nonlinear and cannot be accurately predicted or calculated based on the single-photon absorption properties of a chemical. Photoreactive chemicals may have a peak in two-photon absorption at or near twice the single-photon absorption, or slightly red-shifted in the absorption spectrum. Therefore, by stimulating a polymerization catalyst such as a mixture of EY or TEA, a wavelength of 900 nanometers or about 900 nanometers to about 1400 nanometers may be used to polymerize the monomer material. Single-wavelength polymerization may be sufficient to produce all structural elements, but to further accelerate the printing process, multiple wavelengths can be used for printing simultaneously through the same printing device and into the same printing chamber.

[0137] Premixing or prereacting polymerizable monomer units with catalysts containing different absorption bands can allow printing at different wavelengths, thereby forming different substrate-based structural elements simultaneously within the media chamber 122. Therefore, certain structural elements can be generated by tuning the excitation wavelength of the laser to a specific wavelength, and then other structural elements can be generated around the existing elements by tuning another or the same laser to a different excitation wavelength that can interact with a different photoinitiator that can initiate polymerization of one material substrate with higher efficiency. Similarly, different wavelengths can be used for different structural elements, where increased stiffness is predetermined in some locations, and soft or elastic structures are predetermined in other locations. Due to the different physical properties of the polymerizable materials, this can allow the same cell to produce potentially more rigid, softer, or more elastic structures in the same printing step by simply electronically tuning the excitation wavelength of the laser, by switching between different lasers, or by projecting two different wavelengths simultaneously.

[0138] Figure 2A-2C Exemplary stages of generating a predetermined tissue within the media chamber 122 are illustrated. Figure 2AA medium chamber 122 containing a medium 126 is shown, and the medium 126 is composed of a first cell group, a polymerizable material, and a culture medium. In this embodiment, the pulse of the multiphoton laser beam 120 can be delivered to the medium 126 according to the CAD model of the vascular structure and microvasculature corresponding to the predetermined tissue. In some cases, the first cell group can include vascular and / or microvascular cells, including but not limited to endothelial cells, microvascular endothelial cells, pericytes, smooth muscle cells, fibroblasts, endothelial progenitor cells, stem cells, or any combination thereof. Therefore, a portion of the medium 126 can be polymerized, cross-linked, or reacted to form a cell-containing scaffold 128, which represents the vascular system and microvasculature of the predetermined tissue. In this embodiment, the medium 126 can then be discharged through the first port 130a, the second port 130b, the third port 130c, the fourth port 130d, and the fifth port 130e to remove the first cell group and the associated medium. In some cases, the medium chamber 122 can include at least one port. In some cases, the media chamber 122 may include a plurality of ports ranging from at least one port to up to 100 ports. The media chamber 122 may include at least two ports. The media chamber 122 may include at least three ports. The media chamber 122 may include at least four ports. The media chamber 122 may include at least five ports.

[0139] refer to Figure 2B , the medium chamber 122 can be filled with a medium 126 containing a second cell group, a polymerizable material, and a culture medium through a port 130. The second cell group can be used to generate a tissue structure around an existing cell-containing scaffold 128. In some cases, the cell-containing scaffold 128 can be a vascular scaffold. The printed vascular scaffold can include endothelial cells, vascular endothelial cells, pericytes, smooth muscle cells, fibroblasts, endothelial progenitor cells, stem cells, or any combination thereof.

[0140] The first cell group and / or the second cell group can include endothelial cells, microvascular endothelial cells, pericytes, smooth muscle cells, fibroblasts, endothelial progenitor cells, lymphocytes, T cells such as helper T cells and cytotoxic T cells, B cells, natural killer (NK) cells, reticular cells, hepatocytes or any combination thereof. The first cell group and / or the second cell group can include exocrine secretory epithelial cells, hormone secreting cells, epithelial cells, neural cells, adipocytes, kidney cells, pancreatic cells, lung cells, extracellular matrix cells, muscle cells, blood cells, immune cells, reproductive cells, interstitial cells or any combination thereof.

[0141] The first cell group and / or the second cell group may include exocrine secretory epithelial cells, including but not limited to salivary gland mucous cells, mammary gland cells, sweat gland cells such as eccrine sweat gland cells and apocrine sweat gland cells, sebaceous gland cells, type II pneumocytes, or any combination thereof.

[0142] The first cell group and / or the second cell group may include hormone-secreting cells, including but not limited to anterior pituitary cells, middle pituitary cells, magnocellular neurosecretory cells, intestinal cells, respiratory tract cells, thyroid cells, parathyroid cells, adrenal cells, Leydig cells, theca interna cells, luteal cells, juxtaglomerular cells, macula densa cells, peripolar cells, mesangial cells, pancreatic islet cells such as α cells, β cells, δ cells, PP cells and ε cells or any combination thereof.

[0143] The first cell group and / or the second cell group may include epithelial cells, including but not limited to cornified epithelial cells such as keratinocytes, basal cells and hair stem cells, stratified barrier epithelial cells such as surface epithelial cells of stratified squamous epithelium, basal cells of epithelium and urothelial cells, or any combination thereof.

[0144] The first cell group and / or the second cell group may include neural cells or neurons, including but not limited to sensory transduction cells, autonomic neuronal cells, peripheral neuronal supporting cells, central nervous system neurons such as interneurons, spindle neurons, pyramidal cells, astrocytes, astrocytes, oligodendrocytes, ependymal cells, glial cells, or any combination thereof.

[0145] The first cell group and / or the second cell group may include renal cells, including but not limited to parietal cells, podocytes, mesangial cells, distal tubule cells, proximal tubule cells, Loop of Henle thin segment cells, collecting duct cells, interstitial renal cells, or any combination thereof.

[0146] The first cell group and / or the second cell group may include lung cells, including but not limited to type I pneumocytes, alveolar cells, capillary endothelial cells, alveolar macrophages, bronchial epithelial cells, bronchial smooth muscle cells, tracheal epithelial cells, small airway epithelial cells, or any combination thereof.

[0147] The first cell group and / or the second cell group may include extracellular matrix cells including, but not limited to, epithelial cells, fibroblasts, pericytes, chondrocytes, osteoblasts, osteocytes, osteoprogenitor cells, stellate cells, hepatic stellate cells, or any combination thereof.

[0148] The first cell group and / or the second cell group may include muscle cells, including but not limited to skeletal muscle cells, cardiomyocytes, Purkinje fiber cells, smooth muscle cells, myoepithelial cells, or any combination thereof.

[0149] The first cell group and / or the second cell group may include blood cells and / or immune cells, including but not limited to erythrocytes, megakaryocytes, monocytes, macrophages, osteoclasts, dendritic cells, microglia, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer (NK) cells, reticulocytes or any combination thereof.

[0150] Figure 2C The diagram shows that pulses of a multiphoton laser beam 120 are delivered to the medium 126 according to the CAD model of the remaining tissue. As a result, additional portions of the medium 126 can polymerize, crosslink, or react to form a cell-containing structure 132 around the existing cell-containing scaffold 128 (no longer visible) without damaging or affecting the existing vascular scaffold 128. The steps of draining the medium 126, refilling with new medium 126, and delivering laser energy can be repeated any number of times to produce a predetermined complex tissue.

[0151] Figure 2D An embodiment is illustrated in which a cell-containing scaffold 128 may be printed along the bottom of a media chamber 122 containing media 126. Thus, the scaffold 128 may not be free-standing or free-floating. Multichannel input can reduce shear forces associated with bulk flow from one direction, uneven washing of fine structures (because the bulk flow may not wash unwanted cells from small features), and uneven distribution of new cell-containing media (because it circulates into the tissue printing chamber). Multiple inputs can be from the top, bottom, side, or all three simultaneously. Multiple inputs are particularly desirable for tissue printing because cell-containing structures are relatively fragile and potentially damaged by applied fluid forces associated with media exchange through the chamber. Figure 2D It is shown that tissue can be printed above the floor of the media chamber. In some embodiments, cells and tissue can be printed flush against the bottom of the media chamber. In addition, the design can allow the printed tissue to be easily transported and positioned under the laser print head (focusing objective), and is a closed system that can allow media exchange and printing without exposure to room air. This may be desirable because exposure to room air may introduce infectious agents into the cell culture medium, which may damage or completely destroy the development of useful tissue.

[0152] Laser Printing System

[0153] In one aspect, the present disclosure provides a system for printing three-dimensional (3D) biomaterials. The x, y, and z dimensions can be accessed simultaneously by the system provided herein. The system for printing 3D biomaterials may include a medium chamber configured to contain a medium, the medium containing a plurality of cells containing cells and one or more polymer precursors. The plurality of cells may include at least one type of cells. The plurality of cells may include at least two different types of cells. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber and / or the cell-containing chamber. The system may include one or more computer processors operably coupled to at least one energy source, wherein the one or more computer processors may be individually or collectively programmed to: receive computer instructions for printing 3D biomaterials from a computer memory; and guide at least one energy source to direct at least one energy beam to the medium in the medium chamber along at least one energy beam path according to the computer instructions so that at least a portion of the polymer precursor forms at least a portion of the 3D biomaterial.

[0154] On the other hand, the present disclosure provides another system for printing 3D biomaterials, comprising a medium chamber configured to contain a medium containing a plurality of cells and a plurality of polymer precursors. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber. In addition, the system may include one or more computer processors that can be operably coupled to at least one energy source. The one or more computer processors may be programmed individually or collectively for: (i) receiving computer instructions for printing 3D biomaterials from a computer memory; (ii) directing at least one energy source to direct at least one energy beam to a medium in the medium chamber along at least one energy beam path according to the computer instructions so that at least a portion of the polymer precursor forms at least a portion of the 3D biomaterial; and (iii) directing at least one energy source to direct at least one energy beam to a second medium in the medium chamber along at least one energy beam path according to the computer instructions so that at least a portion of the second medium in the medium chamber forms at least a second portion of the 3D biomaterial, wherein the second medium contains a second plurality of cells and a second polymer precursor, wherein the second plurality of cells are of a different type from the first plurality of cells.

[0155] The one or more computer processors are individually or collectively programmed to generate a point cloud representation or line-based representation of the 3D biomaterial in a computer memory and use the point cloud representation or line-based representation to generate computer instructions in the computer memory for printing the 3D biomaterial. The one or more computer processors may be individually or collectively programmed to direct at least one energy source to direct at least one energy beam along one or more additional energy beam paths to form at least another portion of the 3D biomaterial.

[0156] The system may include one or more computer processors operably coupled to at least one energy source and / or at least one light patterning element. The point cloud representation or line-based representation of the computer model may be a holographic point cloud representation or a holographic line-based representation. The one or more computer processors may be programmed individually or collectively to re-project a holographic image illuminated by at least one energy source using the light patterning element.

[0157] In some cases, one or more computer processors may be individually or collectively programmed to convert a point cloud representation or a line-based representation into an image. One or more computer processors may be individually or collectively programmed to project an image in a holographic manner. One or more computer processors may be individually or collectively programmed to project an image as a hologram. One or more computer processors may be individually or collectively programmed to project an image as a partial hologram. In some cases, one or more computer processors may be individually or collectively programmed to convert a point cloud representation or a line-based representation of a complete image set into a series of holographic images via an algorithmic transformation. Then, such a transformed image set may be sequentially projected through the system by a light patterning element such as a spatial light modulator (SLM) or a digital mirror device (DMD), thereby reproducing a projection image in a printing room using projection light distributed simultaneously in 2D and / or 3D. The expanded or widened laser beam may be projected onto an SLM and / or DMD used as a projection system for a holographic image. In some cases, one or more computer processors may be individually or collectively programmed to project an image in a holographic manner. In some cases, one or more computer processors may be programmed individually or collectively to project the images all at once, or played continuously as a video, to form a larger 3D structure in a holographic manner.

[0158] Holography is the art of projecting multi-dimensional (e.g., 2D and / or 3D) holographic images or holograms. When a laser capable of photopolymerizing a medium is projected as a hologram, the laser can photopolymerize, cure, crosslink, bond, harden, and / or change the physical properties of the medium along the projected laser path; thus, the laser can allow the printing of 3D structures. Holography may require a light source such as a laser or a coherent light source to produce a holographic image. The holographic image can be constant over time or vary over time (e.g., holographic video). In addition, holography may require a shutter to open or move the laser path, a beam splitter to split the laser into separate paths, a mirror to direct the laser path, a diverging lens to expand the beam, and additional patterning or light directing elements.

[0159] A holographic image of an object can be produced by expanding a laser beam with a diverging lens and directing the expanded laser beam to a hologram and / or at least one patterning element (such as, for example, a spatial light modulator or SLM). The patterning element can encode a pattern including a holographic image as a laser beam path. The patterning element can encode a pattern including a portion of a hologram as a laser beam path. Subsequently, the pattern can be directed to and focused in a medium chamber containing a printing material (e.g., a medium containing a plurality of cells and polymer precursors), in which a photoreactive photoinitiator found in the printing material (e.g., in the medium) can be excited. Subsequently, the excitation of the photoreactive photoinitiator can cause photopolymerization of the polymer-based printing material and form a structure in a predetermined pattern (e.g., a holographic image). In some cases, one or more computer processors can be programmed individually or collectively to project a holographic image by directing an energy source along different energy beam paths.

[0160] In some cases, the at least one energy source may be a plurality of energy sources. The plurality of energy sources may direct a plurality of at least one energy beam. The energy source may be a laser. In some examples, the laser may be a fiber laser. For example, the fiber laser may be a laser having an active gain medium comprising an optical fiber doped with a rare earth element such as, for example, erbium, ytterbium, neodymium, dysprosium, praseodymium, thulium, and / or holmium. The energy source may be a short pulse laser. The energy source may be a femtosecond pulse laser. The femtosecond pulse laser can have a pulse width of less than or equal to about 500 femtoseconds (fs), 250 fs, 240 fs, 230 fs, 220 fs, 210 fs, 200 fs, 150 fs, 100 fs, 50 fs, 40 fs, 30 fs, 20 fs, 10 fs, 9 fs, 8 fs, 7 fs, 6 fs, 5 fs, 4 fs, 3 fs, 2 fs, 1 fs or less. The femtosecond pulse laser can be, for example, a titanium: sapphire (Ti: Sa) laser. At least one energy source can be derived from a coherent light source.

[0161] Coherent light sources can provide light with a wavelength of about 300 nanometers (nm) to about 5 millimeters (mm). Coherent light sources can include a wavelength of about 350nm to about 1800nm ​​or about 1800nm ​​to about 5mm. Coherent light sources can provide light with a wavelength of at least about 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 3mm, 4mm, 5mm or more.

[0162] The computer processors may be programmed, individually or collectively, to direct at least one energy source to direct at least one energy beam along one or more additional energy beam paths to form at least another portion of the 3D biomaterial. The one or more additional energy beam paths may be along the x-axis, the x and y planes, or the x, y, and z planes. The one or more additional energy beam paths may be along the x-axis. The one or more additional energy beam paths may be along the y-axis. The one or more additional energy beam paths may be along the z-axis. The energy beam path may converge with one or more other beams on the same axis. The one or more additional energy beam paths may be in the x and y planes. The one or more additional energy beam paths may be in the x and z planes. The one or more additional energy beam paths may be in the y and z planes. The one or more additional energy beam paths may be in the x, y, and z planes.

[0163] The system may also include at least one objective lens for directing at least one energy beam to the medium in the medium chamber. In some cases, at least one objective lens may include a water immersion objective lens. In some cases, at least one objective lens may include a water immersion objective lens. In some cases, at least one objective lens may include a water immersion objective lens. In some cases, at least one objective lens may include an oil immersion objective lens. In some cases, at least one objective lens may include an achromatic objective lens, a semi-apochromatic objective lens, a plan objective lens, an immersion objective lens, a Huygens objective lens, a Ramsden objective lens, a periplan objective lens, a compensating objective lens, a widefield objective lens, a superfield objective lens, a condenser objective lens, or any combination thereof. Non-limiting examples of condenser objective lenses may include an Abbe condenser, an achromatic condenser, and a universal condenser.

[0164] One or more computer processors may be individually or collectively programmed to receive an image of an edge of a 3D biomaterial. One or more computer processors may be individually or collectively programmed to receive an image of an outer surface of a 3D biomaterial. One or more computer processors may be individually or collectively programmed to receive an image of an inner surface of a 3D biomaterial. One or more computer processors may be individually or collectively programmed to receive an image of an interior of a 3D biomaterial.

[0165] One or more computer processors may be individually or collectively programmed to direct the connection of the 3D biomaterial to other tissues, the connection may be in accordance with computer instructions. One or more computer processors may be individually or collectively programmed to directly connect, merge, bond or weld the 3D printed material to the printed structure, the connection in accordance with computer instructions. In some cases, connecting the 3D biomaterial to other tissues may involve chemical cross-linking, mechanical connection and / or adhesive coupling.

[0166] On the other hand, the system may include a medium chamber configured to contain a medium containing a plurality of cells and a plurality of polymer precursors. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber. The system may include one or more computer processors operably coupled to the at least one energy source, wherein the one or more computer processors are individually or collectively programmed to: receive a computer model of a 3D biomaterial in a computer memory; generate a point cloud representation or a line-based representation of the computer model of the 3D biomaterial in the computer memory; guide at least one energy source to guide at least one energy beam to the medium in the medium chamber along at least one energy beam path according to the computer model of the 3D biomaterial so that at least a portion of the polymer precursor forms at least a portion of the 3D biomaterial; and guide at least one energy source to guide at least one energy beam to a second medium in the medium chamber along at least one energy beam path according to the computer model of the 3D biomaterial so that at least a portion of the second medium in the medium chamber forms at least a second portion of the 3D biomaterial, wherein the second medium contains a second plurality of cells and a second polymer precursor, wherein the second plurality of cells are of a different type than the first plurality of cells.

[0167] In laser printing of cellular structures, rapid generation of three-dimensional structures using minimally toxic laser excitation is critical to maintaining cell viability and, in the case of functional tissue printing, is essential for large-format, high-resolution multicellular tissue generation. Other approaches to two-photon printing can rely on raster scanning of two-photon excitation in a two-dimensional plane (x,y) (e.g., selective laser sintering) while moving a microscope or stage in the z direction to generate three-dimensional structures. This technique may be too slow for large-format multicellular tissue printing, making it unlikely to maintain cell viability during the printing of complex structures. Certain hydrogels with high polymerization rates can also be used for two-dimensional projection of tissue slices that are timed so that each step projects a slice of the structure in the x, y, or z plane. Additionally, mixed plane angles representing slices or including orthogonal slices can also be utilized. In the case of rapidly polymerizing hydrogels, these projections can function on timescales compatible with tissue printing, while laser sintering or raster scanning (e.g., layer-by-layer deposition) may be too slow to build complex structures.

[0168] The laser printing system 110 of the present disclosure can be equipped with an objective lens 124 that can allow focusing of a three-dimensional or two-dimensional holographic projection in the lateral and axial planes to quickly generate cell-containing structures. The objective lens 124 can be a water immersion objective lens, an air objective lens, or an oil immersion objective lens. In some cases, the laser printing system 110 can include a laser system 116 with multiple laser lines and can be capable of performing three-dimensional holographic projection of an image via holographic projection into a cell-containing medium for lithography.

[0169] Figure 3A An embodiment of a laser system 116 having a first multiphoton laser source 140a is illustrated. Here, a multiphoton laser beam laser line 1 can be reflected by a spatial light modulator (SLM) at video rates or faster refresh rates for image projection, allowing rapid changes in the projected three-dimensional structure.

[0170] In some cases, a spatial light modulator (SLM) can be used to print 3D biomaterials. In some cases, the method proposed herein may include receiving a computer model of a 3D biomaterial in a computer memory, and further processing the computer model so as to "slice" the computer model into layers, thereby generating a two-dimensional (2D) image of each layer. The computer model may be a computer-aided design (CAD) model. The system disclosed herein may include at least one computer processor that may be programmed individually or collectively to calculate a laser scanning path based on a "sliced" computer model that determines the boundary contour and / or filling order of the 3D biomaterial to be printed. Holographic 3D printing can be used with one or more polymer precursors described herein. SLM can be used with two or more polymer precursors described herein.

[0171] A spatial light modulator (SLM) is an electrically programmable device that can modulate the amplitude, phase, polarization, propagation direction, intensity, or any combination thereof of a light wave in space and time according to a fixed spatial (e.g., pixel) pattern. The SLM can be based on translucency, such as a liquid crystal display (LCD) microdisplay. The SLM can be based on reflection, such as a liquid crystal on silicon (LCOS) microdisplay. The SLM can be a microchannel spatial light modulator (MSLM), a parallel aligned nematic liquid crystal spatial light modulator (PAL-SLM), a programmable phase modulator (PPM), a phase spatial light modulator (LCOS-SLM), or any combination thereof. The LCOS-SLM can include a chip including a liquid crystal layer arranged on top of a silicon substrate. Circuits can be built on the silicon substrate of the chip using semiconductor technology. The top layer of the LCOS-SLM chip can contain aluminum electrodes that can independently control their electric potential. A glass substrate can be placed on a silicon substrate while maintaining a constant gap filled by a liquid crystal material. The liquid crystal molecules can be aligned in parallel by alignment control techniques provided in the silicon and glass substrates. The electric field across the liquid crystal layer can be controlled pixel by pixel. The phase of light can be modulated by controlling the electric field; changes in the electric field may cause the liquid crystal molecules to tilt accordingly. When the liquid crystal molecules tilt, the liquid crystal refractive index may change, further changing the optical path length and, in turn, causing a phase difference.

[0172] 3D biomaterials can be printed using an SLM. 3D biomaterials can be printed using a liquid crystal on silicon (LCOS)-SLM. 3D biomaterials can be printed using a liquid crystal SLM. A point cloud representation or a line-based representation of a computer model of a 3D biomaterial can be projected using an SLM. The methods disclosed herein can include converting a point cloud representation or a line-based representation into a holographic image. A holographic image of a computer model of a 3D biomaterial can be projected using an SLM. A phase of light of a point cloud representation or a line-based representation of a computer model of a 3D biomaterial can be modulated using an SLM. A phase of light of a holographic image of a computer model of a 3D biomaterial can be modulated using an SLM.

[0173] The projection of three-dimensional multi-photon excitation can also be achieved by using a dual digital micromirror device (DMD) system alone or in combination with a spatial light modulator (SLM). A pair of DMDs can be used together with a pair of SLMs using the methods described herein to print 3D materials. 3D materials can be printed using at least one SLM and at least one DMD using the methods described herein. 3D materials can be printed using a pair of SLMs using the methods described herein. 3D materials can be printed using a pair of DMDs using the methods described herein. 3D materials can be printed using at least one SLM using the methods described herein. 3D materials can be printed using at least one DMD using the methods described herein. DMD is an electrical input, optical output micro-electromechanical system (MEMS) that allows high-speed, efficient and reliable spatial light modulation. DMDs can include a plurality of micro-mirrors (usually in the order of hundreds of thousands or millions) arranged in a rectangular array. Each micro-mirror in a DMD can correspond to a pixel of an image to be displayed, and can be rotated by about, for example, 10-12° to an "on" or "off" state. In the "on" state, light from a projector bulb can be reflected into the micro-mirrors, so that its corresponding pixels appear bright on the screen. In the "off" state, light can be directed elsewhere (usually onto a heat sink), causing the corresponding pixel of the micromirror to appear dark. The micromirrors in a DMD can be made of highly reflective aluminum and have a lateral length of about 16 micrometers (μm). Each micromirror can be built on top of an associated semiconductor memory cell and mounted on a yoke, which in turn is connected to a pair of pillars via a torsion hinge. The angle of motion of each micromirror can be controlled by loading a "1" or "0" to each underlying semiconductor memory cell. A voltage is then applied, which can cause each micromirror to be electrostatically deflected around the torsion hinge to an associated + / - angle state via electrostatic attraction.

[0174] refer to Figure 3A-3C, an optional beam expander can be added, followed by a Bessel beam generating lens, which is a fixed axicon lens or an adjustable acoustic gradient (TAG) lens, to change the properties of the laser to achieve higher resolution and greater tissue printing depth, especially in turbid solutions. The laser line, which may include an optional beam expander and / or a Bessel beam generating lens, is guided to different projection systems using fast switching mirrors, which have material advantages in forming specific structures associated with tissue printing. In some cases, a high-resolution DMD mirror combined with an SLM system can achieve higher axial resolution than the resolution that can be achieved by two SLM systems. Finally, the laser line can be used with a single DMD or SLM system in combination with a mirror to allow scanless projection of a two-dimensional image in any axial plane. The 3D projection pattern can also be raster scanned over a larger field of view by a scanning mirror, where the wavelength and / or power is controlled in the laser emission pattern to match the raster scanning speed, so that a bonded and complex structure can be deposited. In systems containing more than one laser line, the configuration can be any combination of dual SLM, dual DMD, single SLM, single DMD, or simple planar scanning.

[0175] In some cases, such as Figure 3A-3C One or more of the optical paths shown may be used independently or in conjunction. Lenses, gratings and mirrors that focus and distribute the light or energy beam within the optical path may be placed between the main wavefront shaping elements to distribute the light passing through the key element or to modulate the incident light (in the case of gratings), such as Figure 3A For the purpose of focusing, distributing or clipping the input laser light, at least one grating or reflector may be placed between the wavefront shaping elements "F" (e.g., between the SLM, DMD and / or TAG lenses). The optical wavefront shaping device F may include an SLM, an LCOS-SLM, a DMD, a TAG lens or any combination thereof.

[0176] In some cases, a DMD can be used to print 3D biomaterials. A DMD can be used to project a point cloud representation or a line-based representation of a computer model of a 3D biomaterial. The methods disclosed herein may include converting the point cloud representation or the line-based representation into a holographic image. A DMD can be used to project a holographic image of a computer model of a 3D biomaterial. A DMD can be used to print 3D biomaterials.

[0177] In some cases, a combination of at least one SLM and at least one DMD can be used to print 3D biomaterials in the methods disclosed herein. The combination of at least one SLM and at least one DMD can be arranged in series. The combination of at least one SLM and at least one DMD can be arranged in parallel. When used to print 3D biomaterials, any number of SLMs and any number of DMDs can be arranged in series. When used to print 3D biomaterials, any number of SLMs and any number of DMDs can be arranged in parallel.

[0178] A 3D biomaterial can be printed using a combination of at least two SLMs and at least one DMD. A 3D biomaterial can be printed using a combination of at least three SLMs and at least one DMD. A 3D biomaterial can be printed using a combination of at least four SLMs and at least one DMD. A 3D biomaterial can be printed using a combination of at least five SLMs and at least one DMD. A 3D biomaterial can be printed using a combination of at least ten SLMs and at least one DMD. A 3D biomaterial can be printed using a combination of at least twenty SLMs and at least one DMD.

[0179] A 3D biomaterial can be printed using a combination of at least one SLM and at least two DMDs. A 3D biomaterial can be printed using a combination of at least one SLM and at least three DMDs. A 3D biomaterial can be printed using a combination of at least one SLM and at least four DMDs. A 3D biomaterial can be printed using a combination of at least one SLM and at least five DMDs. A 3D biomaterial can be printed using a combination of at least one SLM and at least ten DMDs. A 3D biomaterial can be printed using a combination of at least one SLM and at least twenty DMDs.

[0180] A 3D biomaterial can be printed using a combination of at least two SLMs and at least two DMDs. A 3D biomaterial can be printed using a combination of at least three SLMs and at least three DMDs. A 3D biomaterial can be printed using a combination of at least four SLMs and at least four DMDs. A 3D biomaterial can be printed using a combination of at least five SLMs and at least five DMDs. A 3D biomaterial can be printed using a combination of at least ten SLMs and at least ten DMDs. A 3D biomaterial can be printed using a combination of at least twenty SLMs and at least twenty DMDs.

[0181] A liquid crystal SLM may be used to print 3D biomaterials. A plurality of SLMs may be used to print 3D biomaterials. A plurality of SLMs may be arranged in series. A plurality of SLMs may be arranged in parallel. A 3D biomaterial may be printed using at least one or more SLMs. A 3D biomaterial may be printed using at least two or more SLMs. A 3D biomaterial may be printed using at least three or more SLMs. A 3D biomaterial may be printed using at least four or more SLMs. A 3D biomaterial may be printed using at least five or more SLMs. A 3D biomaterial may be printed using at least ten or more SLMs. A 3D biomaterial may be printed using at least twenty or more SLMs. A 3D biomaterial may be printed using at least one to about fifty or more SLMs. A 3D biomaterial may be printed using at least one to about twenty or more SLMs. A 3D biomaterial may be printed using at least one to about fifteen or more SLMs. A 3D biomaterial may be printed using at least one to about ten or more SLMs. A 3D biomaterial may be printed using at least one to about five or more SLMs.

[0182] A plurality of DMDs may be used to print 3D biomaterials. A plurality of DMDs may be arranged in series. A plurality of DMDs may be arranged in parallel. A 3D biomaterial may be printed using at least one or more DMDs. A 3D biomaterial may be printed using at least two or more DMDs. A 3D biomaterial may be printed using at least three or more DMDs. A 3D biomaterial may be printed using at least four or more DMDs. A 3D biomaterial may be printed using at least five or more DMDs. A 3D biomaterial may be printed using at least ten or more DMDs. A 3D biomaterial may be printed using at least twenty or more DMDs. A 3D biomaterial may be printed using at least one to about fifty or more DMDs. A 3D biomaterial may be printed using at least one to about twenty or more DMDs. A 3D biomaterial may be printed using at least one to about fifteen or more DMDs. A 3D biomaterial may be printed using at least one to about ten or more DMDs. A 3D biomaterial may be printed using at least one to about five or more DMDs.

[0183] In this design, the SLM may refer to a liquid crystal SLM, and the DMD may function similarly to an SLM. The lasers may be controlled by one or more computer inputs to handle the position and printing timing of the multiple laser lines. Figure 3AAn exemplary overall design of an optical path including an optional tandem excitation path is illustrated in FIG, and further descriptions of the elements are provided in Table 1. Due to the large pulse width between packets of two-photon excitation light, any combination of these laser lines (which may be non-interfering) can be used for printing simultaneously and printing with simultaneous imaging. This can allow very low interference between the beams, so that the beams do not intersect. Thus, as Figure 3B-Figure 3C As shown, it is possible to use multiple laser lines with minimal to no interference, and further description of the elements is also provided in Table 1. The group delay dispersion optical element in this configuration can be used to disperse the two-photon packet so that the peak power output will not damage the fiber optic cable if it is to be used in certain configurations. In addition, the group delay dispersion can concentrate the photons into a shorter pulse width, giving more energy at the focus or in the projected image, allowing faster printing.

[0184] The two-photon excitation pulses can be controlled temporally so that excitation at a single point occurs with pulses with lengths in the femtosecond to nanosecond range (depending on laser tuning), with the timing between these photon packets being three to six orders of magnitude longer than the pulse width. This can allow for minimal cross-path interference of laser excitation when using multiple laser lines in series, making it possible to print simultaneously using multiple lasers. An example of multi-laser projection at three different theoretical wavelengths for the purpose of structure deposition is shown in Figure 3B Multiphoton lasers are tunable; therefore, they can allow a range of wavelengths to be selected. This is advantageous in tissue printing, where different photoinitiators for polymerization that respond to different wavelengths can be used in combination or in series to prevent unwanted polymerization of residual material. Therefore, each of these laser lines can be tuned to a different multiphoton output wavelength, can have a different peak power output, and can project different elements of a CAD image including a tissue structure.

[0185] Table 1. Figure 3A-3C Component Description

[0186]

[0187]

[0188]

[0189]

[0190] Figure 4A-4BAn optional beam expander placement before an axicon or adjustable acoustic gradient (TAG) lens is shown. This can allow for the generation of a Bessel beam with the goal of increasing deep penetration in tissue and turbid media during printing without loss of focus fidelity. This feature can increase the printing depth through turbid media or established tissue without loss of power.

[0191] After the dual SLM or DMD combination, a lens can be used to broaden or pre-focus the laser. In addition, a computer-controlled laser attenuation device or filter wheel can be added before the focusing optics to control the laser power output at the printing site.

[0192] Figure 4C A laser source A is illustrated that projects a laser beam onto a beam collector B. After leaving the beam collector B, the laser beam can be directed to an optical TAG or an axicon C, and further directed to a movable single SLM or DMD D to perform 2D x, y sheet projection for printing collagen mesh around cells and resulting structures printed with a given Z step length. The laser beam can be directed from the SLM or DMD D to a reflector G, and then reflected onto the print head optical device H. In this example, a single SLM can be used to produce a two-dimensional (2D) projection with a z motor stepping movement that matches the frame rate of the projection. The two-dimensional video projection of the Z stack slice can be achieved by a single DMD or a single SLM (with a timed z movement) so that each step projects a different image of the printed 2D image from top to bottom. In another embodiment, a multi-photon or alternative laser excitation source can be used to project complex structures from the side, from bottom to top, or at different joints and slice by slice, 2D projection, and printing. The source of the CAD image F can be guided to the system from a computer E. The system can include a motorized stage I that can match the step rate (milliseconds to seconds) and the step size of the Z projection. The step size can be in the order of micrometers to nanometers. Figure 4C 1, 2 and 3 illustrate examples of planar projection construction steps.

[0193] Fig.12 The optical components and light paths of an embodiment of a three-dimensional printing system are illustrated. Fig.12The optical components and optical paths shown in can provide a three-dimensional printing system that can use no time focusing. The three-dimensional printing system may include an energy source 1000. The energy source 1000 may be a coherent light source. The energy source 1000 may be a laser. The energy source 1000 may be a femtosecond pulse laser light source. The energy source 1000 may be a first laser source 140a, a second laser source 140b, or a third laser source 140c. The energy source 1000 may be a multi-photon laser beam 120. The energy source 1000 may be a two-photon laser beam. The energy source 1000 may be controlled by a computer system 1101. The energy source 1000 may be tuned by the computer system 1101. The computer system 1101 may control and / or set the energy wavelength of the energy source 1000 before or during the printing process. The computer system 1101 may generate different excitation wavelengths by setting the wavelength of the energy source 1000.

[0194] The energy source 1000 can be pulsed. The energy source 1000 can be pulsed at a rate of about 500 kilohertz (kHz). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100,000 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 10 microjoules (μJ) to 100 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 20 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 40 microjoules (μJ) to 80 μJ or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 120 microjoules (μJ) to 160 μJ or more.

[0195] The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 10 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 20 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 30 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 40 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 50 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 60 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 70 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 80 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 90 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 100 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 110 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 120 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 130 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 140 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 150 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 160 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 170 μJ (per packet).The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 180 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 190 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 200 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 20,000 μJ (per packet). The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 100,000 μJ (per packet). The energy source 1000 (eg, a laser) may provide energy (eg, a laser beam) having energy packets having a pulse energy (per packet) of about 1,000,000 μJ.

[0196] The energy source 1000 (e.g., a laser) can provide an energy beam (e.g., a light beam) having a wavelength of, for example, about at least 300 nm to about 5 mm or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 600 nm to about 1500 nm or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 350 nm to about 1800 nm or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 1800 nm to about 5 mm or more. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 300 nm. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 400 nm. The energy source 1000 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 600 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 700 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 800 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 900 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1000 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1100 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1200 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1300 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1400 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1500 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1600 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1700 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1800 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1900 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 2000 nm. The energy source 1000 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 3000 nm.The energy source 1000 (eg, a laser) may provide energy (eg, a laser beam) having a wavelength of about 4000 nm. The energy source 1000 (eg, a laser) may provide energy (eg, a laser beam) having a wavelength of about 5000 nm.

[0197] like Fig.12 As shown, the energy source 1000 can project a laser beam 1002 through a shutter 1004. Once the laser beam 1002 leaves the shutter 1004, the laser beam 1002 can be directed through a rotating half-wave plate 1006. The rotating half-wave plate can be a transparent plate with a specific amount of birefringence, which can be mainly used to manipulate the polarization state of the beam. The rotating half-wave plate can have a slow axis and a fast axis (e.g., two polarization directions), which can be both perpendicular to the direction of the laser beam 1002. The rotating half-wave plate 1006 can change the polarization state of the laser beam 1002 so that the difference in phase delay between the two linear polarization directions is π. The difference in phase delay can correspond to a propagation phase shift over a distance of λ / 2. Other types of wave plates can be used with the system disclosed herein; for example, a rotating quarter-wave plate can be used. The rotating half-wave plate 1006 can be a true zero-order wave plate, a low-order wave plate, or a multi-order wave plate. The rotated half-wave plate 1006 may be composed of crystalline quartz (SiO2), calcite (CaCO3), magnesium fluoride (MgF2), sapphire (Al2O3), mica, or a birefringent polymer.

[0198] Laser beam 1002 may exit rotating half wave plate 1006 and may be directed through polarizing beam splitter 1008. Polarizing beam splitter 1008 may split laser beam 1002 into a first laser beam 1002a and a second laser beam 1002b. First laser beam 1002a may be directed to beam dump 1010. Beam dump 1010 is an optical element that may be used to absorb stray portions of a laser beam. Beam dump 1010 may absorb first laser beam 1002a. First laser beam 1002a may be a stray laser beam. Beam dump 1010 may absorb second laser beam 1002b. Second laser beam 1002b may be a stray laser beam. Laser beam 1002 may be directed as a whole into beam dump 1010 and thus may be used as a default "off" state for a printing system. Second laser beam 1002b may be directed to beam expander 1012. Beam expander 1012 may expand the size of laser beam 1002b. The beam expander 1012 may increase the diameter of the input second laser beam 1002b to the larger diameter of the output expanded laser beam 1054. The beam expander 1012 may be a prismatic beam expander. The beam expander 1012 may be a telescopic beam expander. The beam expander 1012 may be a multi-prism beam expander. The beam expander 1012 may be a Galilean beam expander. The beam expander 1012 may provide a beam expander capability of about 2X, 3X, 5X, 10X, 20X, or 40X. The beam expander 1012 may provide a beam expander capability of about 2X to about 5X. The beam expander 1012 may provide a continuous beam expansion of about 2X to about 5X. The beam expander 1012 may provide a beam expander capability of about 5X to about 10X. The beam expander 1012 may provide a continuous beam expansion of about 5X to about 10X. The expanded laser beam 1054 may be collimated upon exiting the beam expander 1012.

[0199] After exiting the beam expander 1012, the expanded laser beam 1054 may be directed to a first mirror 1014a, which may redirect the expanded laser beam 1054 to a spatial light modulator (SLM) 1016. The SLM 1016 may be controlled by the computer system 1101. The SLM 1016 may be directed to project a specific image or a specific portion of an image of a material to be printed using the methods and systems disclosed herein. The material to be printed may be a biological material. The biological material may be a three-dimensional biological material. The specific image or specific portion of an image may be one-dimensional, two-dimensional, and / or three-dimensional. The SLM 1016 may be directed to simultaneously project at least one image in light of different wavelengths. The SLM 1016 may be directed to project different aspects of the material to be printed by using a mirror rather than using the computer system 1101. In some cases, at least one mirror may be used to redirect or "turn off" or "turn on" a specific optical path or laser beam in order to print different aspects or portions of the material to be printed.

[0200] After leaving SLM 1016, the expanded laser beam 1054 can be directed to f1 lens 1018. f1 lens 1018 can be a focusing lens. After leaving f1 lens 1018, the expanded laser beam 1054 can be directed to blocking element 1020. Blocking element 1020 can be immovable. Blocking element 1020 can suppress illumination from the zero order spot. The zero order can be a portion of the energy from the expanded laser beam 1054 that is not diffracted and behaves according to the laws of reflection and refraction. After leaving blocking element 1020, the expanded laser beam 1054 can be directed through f2 lens 1022. f2 lens can be a focusing lens.

[0201] After leaving the f2 lens 1022, the expanded laser beam 1054 can be directed onto the second reflector 1014b and can be subsequently directed onto the third reflector 1014c. The third reflector 1014c can redirect the expanded laser beam 1054 through the long pass dichroic mirror 1024. The first reflector 1014a, the second reflector 1014b, and / or the third reflector 1014c can include an infrared (IR) coating to improve reflectivity. The first reflector 1014a, the second reflector 1014b, and / or the third reflector 1014c may not include an infrared (IR) coating. Non-limiting examples of IR coatings include protective gold-based coatings and protective silver-based coatings. The first reflector 1014a, the second reflector 1014b, and / or the third reflector 1014c can be controlled by the computer system 1101. The computer system 1101 may turn the first mirror 1014a, the second mirror 1014b, and / or the third mirror 1014c “on” or “off” to redirect the expanded laser beam 1054 according to a predetermined direction.

[0202] The dichroic mirror can be a short pass dichroic mirror. The long pass dichroic mirror 1024 can reflect the expanded laser beam 1054 into the focusing objective 1032. In some cases, a beam combiner can be used to redirect the expanded laser beam 1054 into the focusing objective 1032 instead of using the long pass dichroic mirror 1024. The long pass dichroic mirror 1024 can be controlled by the computer system 1101 to redirect the expanded laser beam 1054 into the focusing objective 1032. The focusing objective 1032 can focus the expanded laser beam 1054 when it is projected into the printing chamber 1034. The printing chamber 1034 can be a medium chamber 122. The printing chamber 1034 can include a medium containing cells, a plurality of cells, a cell component (e.g., an organelle), and / or at least one polymer precursor.

[0203] A light emitting diode (LED) collimator 1040 may be used as a source of collimated LED light 1056. The LED collimator 1040 may include a collimating lens and an LED emitter. The LED may be an inorganic LED, a high brightness LED, a quantum dot LED, or an organic LED. The LED may be a single color LED, a dual color LED, or a tri-color LED. The LED may be a blue LED, an ultraviolet LED, a white LED, an infrared LED, a red LED, an orange LED, a yellow LED, a green LED, a blue-violet LED, a pink LED, or a violet LED. The LED collimator 1040 may project a beam of collimated LED light 1056 through an f4 lens 1038. The f4 lens 1038 may be a focusing lens. Once the collimated LED light 1056 is transmitted through the f4 lens 1038, the collimated LED light 1056 may be directed into a focusing objective lens 1036. The focusing objective lens 1036 may focus the collimated LED light 1056 into the printing chamber 1034. The focusing objective 1036 can focus the collimated LED light 1056 into the sample medium. The focusing objective 1036 can focus the collimated LED light 1056 into the medium containing cells. The collimated LED light 1056 can be transmitted through the print chamber 1034 and enter the focusing objective 1032. Once the collimated LED light 1056 leaves the focusing objective 1032, the collimated LED light 1056 can be directed to the long pass dichroic mirror 1024. The collimated LED light 1056 reflected off the long pass dichroic mirror 1024 can be the sample emission 1026. The long pass dichroic mirror 1024 can redirect the sample emission 1026 into the f3 lens 1028. The f3 lens 1028 can be a focusing lens. Once the sample emission 1026 is transmitted through the f3 lens 1028, the detection system 1030 detects and / or collects the sample emission 1026 for imaging. The detection system 1030 may include at least one photomultiplier tube (PMT). The detection system 1030 may include at least one camera. The camera may be a complementary metal oxide semiconductor (CMOS) camera, a scientific CMOS camera, a charge coupled device (CCD) camera, or an electron multiplying charge coupled device (EM-CCD). The detection system 1030 may include at least one array-based detector.

[0204] Fig.13 The optical components and optical paths of yet another embodiment of a three-dimensional printing system are illustrated. Fig.13The optical components and optical paths shown in provide a three-dimensional printing system that can use time focusing. The three-dimensional printing system may include an energy source 1100. The energy source 1100 may be a coherent light source. The energy source 1100 may be a laser. The energy source 1100 may be a femtosecond pulse laser light source. The energy source 1100 may be a first laser source 140a, a second laser source 140b, or a third laser source 140c. The energy source 1100 may be a multi-photon laser beam 120. The energy source 1100 may be a two-photon laser beam. The energy source 1100 may be controlled by a computer system 1101. The energy source 1100 may be tuned by the computer system 1101. The computer system 1101 may control and / or set the energy wavelength of the energy source 1100 before or during the printing process. The computer system 1101 may generate different excitation wavelengths by setting the wavelength of the energy source 1100.

[0205] The energy source 1100 can be pulsed. The energy source 1100 can be pulsed at a rate of about 500 kilohertz (kHz). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100,000 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet having a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 10 microjoules (μJ) to 100 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 20 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 40 microjoules (μJ) to 80 μJ or more. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 120 microjoules (μJ) to 160 μJ or more.

[0206] The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 10 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 20 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 30 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 40 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 50 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 60 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 70 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 80 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 90 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 100 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 110 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 120 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 130 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 140 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 150 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 160 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 170 μJ (per packet).The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 180 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 190 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 200 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 20,000 μJ (per packet). The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 100,000 μJ (per packet). The energy source 1100 (eg, a laser) may provide energy (eg, a laser beam) having energy packets having a pulse energy (per packet).

[0207] The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 300nm to 5mm, 600nm to 1500nm, 350nm to 1800nm, or 1800nm ​​to 5mm. The energy source 1100 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of at least about 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 3mm, 4mm, 5mm, or more.

[0208] like Fig.13 As shown, the energy source 1100 can project a laser beam 1102 through a shutter 1104. Once the laser beam 1102 leaves the shutter 1104, the laser beam 1102 can be directed through a rotating half-wave plate 1106. The rotating half-wave plate 1106 can change the polarization state of the laser beam 1102 so that the difference in phase delay between the two linear polarization directions is π. The difference in phase delay can correspond to a propagation phase shift over a distance of λ / 2. Other types of wave plates can be used with the system disclosed herein; for example, a rotating quarter-wave plate can be used. The rotating half-wave plate 1106 can be a true zero-order wave plate, a low-order wave plate, or a multi-order wave plate. The rotating half-wave plate 1106 can be composed of crystalline quartz (SiO2), calcite (CaCO3), magnesium fluoride (MgF2), sapphire (Al2O3), mica, or a birefringent polymer.

[0209] Laser beam 1102 may exit rotating half wave plate 1106 and may be directed through polarizing beam splitter 1108. Polarizing beam splitter 1108 may split laser beam 1102 into a first laser beam 1102a and a second laser beam 1102b. First laser beam 1102a may be directed to beam dump 1110. Beam dump 1110 is an optical element that may be used to absorb stray portions of a laser beam. Beam dump 1110 may absorb first laser beam 1102a. First laser beam 1102a may be a stray laser beam. Beam dump 1110 may absorb second laser beam 1102b. Second laser beam 1102b may be a stray laser beam. Laser beam 1102 may be directed as a whole into beam dump 1110 and may therefore be used as a default "off" state for a printing system. Second laser beam 1102b may be directed to beam expander 1112. Beam expander 1112 may expand the size of second laser beam 1102b. The beam expander 1112 may increase the diameter of the input second laser beam 1102b to the larger diameter of the output expanded laser beam 1154. The beam expander 1112 may be a prismatic beam expander. The beam expander 1112 may be a telescopic beam expander. The beam expander 1112 may be a multi-prism beam expander. The beam expander 1112 may be a Galilean beam expander. The beam expander 1112 may provide a beam expander capability of about 2X, 3X, 5X, 10X, 20X, or 40X. The beam expander 1112 may provide a beam expander capability of about 2X to about 5X. The beam expander 1112 may provide a continuous beam expansion of about 2X to about 5X. The beam expander 1112 may provide a beam expander capability of about 5X to about 10X. The beam expander 1112 may provide a continuous beam expansion of about 5X to about 10X. The expanded laser beam 1154 may be collimated upon exiting the beam expander 1112.

[0210] After leaving the beam expander 1112, the expanded laser beam 1154 can be directed to a first mirror 1114a, which can redirect the expanded laser beam 1154 to a first spatial light modulator (SLM) 1116a. After leaving the first SLM 1116, the expanded laser beam 1154 can be directed to an f1 lens 1118. The f1 lens 1118 can be a focusing lens. After leaving the f1 lens, the expanded laser beam 1154 can be directed to a grating 1142. The grating 1142 can be a diffractive laser beam splitter. The grating 1142 can be a holographic grating. The grating 1142 can be a ruled grating. The grating 1142 can split and / or diffract the expanded laser beam 1154 into multiple expanded laser beams (at Fig.131101). The grating 1142 may act as a dispersive element. Once the expanded laser beam 1154 is split, diffracted, and / or dispersed by the grating 1142, the expanded laser beam 1154 may be transmitted through the f2 lens 1122. The f2 lens 1122 may be a focusing lens. After leaving the f2 lens 1122, the expanded laser beam 1154 may be directed to the second SLM 1116b. The SLMs (e.g., the first SLM 1116a and the second SLM 1116b) may be controlled by the computer system 1101. The SLMs described above may perform Fig.12 All functions of the SLM1016 are shown in .

[0211] After leaving the second SLM 1116b, the expanded laser beam 1154 can be directed to the f3 lens 1128. The f3 lens 1128 can be a focusing lens. After leaving the f3 lens, the expanded laser beam 1154 can be directed to the blocking element 1120. The blocking element 1120 can be immovable. The blocking element 1120 can be used to suppress illumination from the zero-order spot. After leaving the blocking element 1120, the expanded laser beam 1154 can be directed through the f4 lens 1138. The f4 lens 1138 can be a focusing lens. After leaving the f4 lens 1138, the expanded laser beam 1154 can be directed onto the second reflector 1114b and can then be directed onto the third reflector 1114c. The third reflector 1114c can redirect the expanded laser beam 1154 through the long pass dichroic mirror 1124. The first reflector 1114a, the second reflector 1114b, and / or the third reflector 1114c can be controlled by the computer system 1101. The computer system 1101 can turn the first reflector 1114a, the second reflector 1114b, and / or the third reflector 1114c "on" or "off" to redirect the expanded laser beam 1154 according to a predetermined direction. The dichroic mirror can be a short-pass dichroic mirror. The long-pass dichroic mirror 1124 can reflect the expanded laser beam 1154 into the focusing objective 1132. In some cases, a beam combiner can be used to redirect the expanded laser beam 1154 into the focusing objective 1132 instead of using the long-pass dichroic mirror 1124. The long-pass dichroic mirror 1124 can be controlled by the computer system 1101 to redirect the expanded laser beam 1154 into the focusing objective 1132. The focusing objective 1132 can focus the expanded laser beam 1154 as it is projected into the printing chamber 1134. The printing chamber 1134 can be the medium chamber 122. The printing chamber 1134 can include a cell-containing medium, a plurality of cells, cell components (e.g., organelles), and / or at least one polymer precursor.

[0212] The print chamber 1134 can be mounted on a movable stage 1146. The movable stage 1146 can be an xy stage, a z stage, and / or an xyz stage. The movable stage 1146 can be manually positioned. The movable stage 1146 can be automatically positioned. The movable stage 1146 can be a motorized stage. The movable stage 1146 can be controlled by the computer system 1101. The computer system 1101 can control the movement of the movable stage 1146 in the x, y, and / or z directions. The computer system 1101 can automatically position the movable stage 1146 at a predetermined x, y, and / or z position. The computer system 1101 can position the movable stage 1146 at a predetermined x, y, and / or z position with a positional accuracy of at most about 3 μm. The computer system 1101 can position the movable stage 1146 at a predetermined x, y, and / or z position with a positional accuracy of at most about 2 μm. The computer system 1101 can position the movable stage 1146 at a predetermined x, y, and / or z position with a positional accuracy of up to about 1 μm. The computer system 1101 can automatically adjust the position of the movable stage 1146 before or during three-dimensional printing. The computer system 1101 may include a piezoelectric (piezoelectric) controller to provide computer-controlled z-axis (e.g., vertical direction) positioning and active position feedback. The computer system 1101 may include a joystick console to enable a user to control the position of the movable stage 1146. The joystick console may be a z-axis console and / or an x-axis and y-axis console. The movable stage 1146 may include a print chamber holder. The print chamber holder may be a bracket, a clamp, and / or a recessed sample holder. The movable stage 1146 may include a multi-slide holder, a slide holder, and / or a Petri dish holder. The movable stage 1146 may include a sensor to provide position feedback. The sensor may be a capacitive sensor. The sensor may be a piezoresistive sensor. The movable stage 1146 may include at least one actuator (eg, a piezoelectric actuator) that moves (or positions) the movable stage 1146 .

[0213] A light emitting diode (LED) collimator 1140 may be used as a source of collimated LED light 1156. The LED collimator 1140 may include a collimating lens and an LED emitter. The LED may be an inorganic LED, a high brightness LED, a quantum dot LED, or an organic LED. The LED may be a single color LED, a dual color LED, or a tri-color LED. The LED may be a blue LED, an ultraviolet LED, a white LED, an infrared LED, a red LED, an orange LED, a yellow LED, a green LED, a blue-violet LED, a pink LED, or a violet LED. The LED collimator 1140 may project a beam of collimated LED light 1156 through an f6 lens 1148. The f6 lens 1148 may be a focusing lens. Once the collimated LED light 1156 is transmitted through the f6 lens 1148, the collimated LED light 1156 may be directed into the focusing objective 1136. The focusing objective 1136 may focus the collimated LED light 1156 into the printing chamber 1134. The focusing objective 1136 can focus the collimated LED light 1156 into the sample medium. The focusing objective 1136 can focus the collimated LED light 1156 into the medium containing cells. The collimated LED light 1156 can be transmitted through the print chamber 1134 and enter the focusing objective 1132. Once the collimated LED light 1156 leaves the focusing objective 1132, the collimated LED light 1156 can be directed to the long pass dichroic mirror 1124. The collimated LED light 1156 reflected off the long pass dichroic mirror 1124 can be the sample emission 1126. The long pass dichroic mirror 1124 can redirect the sample emission 1126 into the f5 lens 1144. The f5 lens 1144 can be a focusing lens. Once the sample emission 1126 is transmitted through the f5 lens 1144, the detection system 1130 detects and / or collects the sample emission 1126 for imaging. The detection system 1130 may include at least one photomultiplier tube (PMT). The detection system 1130 may include at least one camera. The camera may be a complementary metal oxide semiconductor (CMOS) camera, a scientific CMOS camera, a charge coupled device (CCD) camera, or an electron multiplying charge coupled device (EM-CCD). The detection system 1130 may include at least one array-based detector.

[0214] Fig.14 Optical components and light paths of additional embodiments of a three-dimensional printing system are illustrated. Fig.14The optical components and optical paths shown in provide a three-dimensional printing system that can be used without time focusing. The three-dimensional printing system may include an energy source 1200. The energy source 1200 may be a coherent light source. The energy source 1200 may be a laser. The energy source 1200 may be a femtosecond pulse laser light source. The energy source 1200 may be a first laser source 140a, a second laser source 140b, or a third laser source 140c. The energy source 1200 may be a multiphoton laser beam 120. The energy source 1200 may be controlled by a computer system 1101. The energy source 1200 may be tuned by the computer system 1101. The computer system 1101 may control and / or set the energy wavelength of the energy source 1200 before or during the printing process. The computer system 1101 may generate different excitation wavelengths by setting the wavelength of the energy source 1200.

[0215] The energy source 1200 can be pulsed. The energy source 1200 can be pulsed at a rate of about 500 kilohertz (kHz). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100,000 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 1,000 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 100 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 10 microjoules (μJ) to 100 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 20 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 1 microjoule (μJ) to 50 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 40 microjoules (μJ) to 80 μJ or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy (per packet) of about at least 120 microjoules (μJ) to 160 μJ or more.

[0216] The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 10 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 20 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 30 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 40 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 50 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 60 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 70 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 80 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 90 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 100 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 110 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with an energy packet having a pulse energy of about 120 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with energy packets having a pulse energy of about 130 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with energy packets having a pulse energy of about 140 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with energy packets having a pulse energy of about 150 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with energy packets having a pulse energy of about 160 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) with energy packets having a pulse energy of about 170 μJ (per packet).The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 180 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 190 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 200 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 20,000 μJ (per packet). The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having an energy packet with a pulse energy of about 100,000 μJ (per packet). The energy source 1200 (eg, a laser) may provide energy (eg, a laser beam) having energy packets having a pulse energy (per packet).

[0217] The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of, for example, about at least 300 nm to about 5 mm or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 600 nm to about 1500 nm or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 350 nm to about 1800 nm or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about at least 1800 nm to about 5 mm or more. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 300 nm. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 400 nm. The energy source 1200 (e.g., a laser) can provide energy (e.g., a laser beam) having a wavelength of about 600 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 700 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 800 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 900 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1200 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1200 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1200 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1300 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1400 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1500 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1600 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1700 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1800 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 1900 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 2000 nm. The energy source 1200 (e.g., a laser) may provide energy (e.g., a laser beam) having a wavelength of about 3000 nm.The energy source 1200 (eg, a laser) may provide energy (eg, a laser beam) having a wavelength of about 4000 nm. The energy source 1200 (eg, a laser) may provide energy (eg, a laser beam) having a wavelength of about 5000 nm.

[0218] like Fig.14 As shown, the energy source 1200 can project a laser beam 1202 through a shutter 1104. Once the laser beam 1202 leaves the shutter 1204, the laser beam 1202 can be directed through a rotating half-wave plate 1206. The rotating half-wave plate 1206 can change the polarization state of the laser beam 1202 so that the difference in phase delay between the two linear polarization directions is π. The difference in phase delay can correspond to a propagation phase shift over a distance of λ / 2. Other types of wave plates can be used with the system disclosed herein; for example, a rotating quarter-wave plate can be used. The rotating half-wave plate 1206 can be a true zero-order wave plate, a low-order wave plate, or a multi-order wave plate. The rotating half-wave plate 1206 can be composed of crystalline quartz (SiO2), calcite (CaCO3), magnesium fluoride (MgF2), sapphire (Al2O3), mica, or a birefringent polymer.

[0219] Laser beam 1202 may exit rotating half wave plate 1206 and may be directed through polarizing beam splitter 1208. Polarizing beam splitter 1208 may split laser beam 1202 into a first laser beam 1202a and a second laser beam 1202b. First laser beam 1202a may be directed to beam dump 1210. Beam dump 1210 is an optical element that may be used to absorb stray portions of a laser beam. Beam dump 1210 may absorb first laser beam 1202a. First laser beam 1202a may be a stray laser beam. Beam dump 1210 may absorb second laser beam 1202b. Second laser beam 1202b may be a stray laser beam. Laser beam 1202 may be directed as a whole into beam dump 1210 and thus may be used as a default "off" state for a printing system. Second laser beam 1202b may be directed to beam expander 1212. Beam expander 1212 may expand the size of second laser beam 1202b. The beam expander 1212 may increase the diameter of the input second laser beam 1202b to the larger diameter of the output expanded laser beam 1254. The beam expander 1212 may be a prismatic beam expander. The beam expander 1212 may be a telescopic beam expander. The beam expander 1212 may be a multi-prism beam expander. The beam expander 1212 may be a Galilean beam expander. The beam expander 1212 may provide a beam expander capability of about 2X, 3X, 5X, 10X, 20X, or 40X. The beam expander 1212 may provide a beam expander capability of about 2X to about 5X. The beam expander 1212 may provide a continuous beam expansion of about 2X to about 5X. The beam expander 1212 may provide a beam expander capability of about 5X to about 10X. The beam expander 1212 may provide a continuous beam expansion of about 5X to about 10X. The expanded laser beam 1254 may be collimated upon exiting the beam expander 1212.

[0220] After exiting the beam expander 1212, the expanded laser beam 1254 can be directed to a first mirror 1214a, which can redirect the expanded laser beam 1254 to a first spatial light modulator (SLM) 1216a. After exiting the first SLM 1216, the expanded laser beam 1254 can be directed to an f1 lens 1218. The f1 lens 1218 can be a focusing lens. After exiting the f1 lens, the expanded laser beam 1254 can be directed to a mirror with a blocking element 1250. The mirror with a blocking element 1250 can be used to suppress illumination from a zero-order spot.

[0221] Once the expanded laser beam 1254 is reflected by the reflector 1250 having a blocking element, the expanded laser beam 1254 can be transmitted through the f2 lens 1222. The f2 lens 1222 can be a focusing lens. After leaving the f2 lens 1222, the expanded laser beam 1254 can be directed to the second SLM 1216b. The SLM (e.g., the first SLM 1216a and the second SLM 1216b) can be controlled by the computer system 1101. The SLM as described above can perform all the functions of the SLM 1016 and SLM1116 shown in Figures 44 and 45, respectively.

[0222] After leaving the second SLM 1216b, the expanded laser beam 1254 can be directed to the f3 lens 1228. After leaving the f3 lens, the expanded laser beam 1254 can be directed to the blocking element 1220. The blocking element 1220 can be immovable. The blocking element 1220 can be used to suppress illumination from the zero-order spot. After leaving the blocking element 1220, the expanded laser beam 1254 can be directed through the f4 lens 1238. The f4 lens 1238 can be a focusing lens. After leaving the f4 lens 1238, the expanded laser beam 1254 can be directed onto the second reflector 1214b and can then be directed onto the third reflector 1214c. The third reflector 1214c can redirect the expanded laser beam 1254 through the long pass dichroic mirror 1224. The first reflector 1214a, the second reflector 1214b, and / or the third reflector 1214c can be controlled by the computer system 1101. The computer system 1101 can turn the first reflector 1214a, the second reflector 1214b, and / or the third reflector 1214c "on" or "off" to redirect the expanded laser beam 1254 according to a predetermined setting. The dichroic mirror can be a short-pass dichroic mirror. The long-pass dichroic mirror 1224 can reflect the expanded laser beam 1254 into the focusing objective lens 1232. In some cases, a beam combiner can be used to redirect the expanded laser beam 1254 into the focusing objective lens 1232 instead of using the long-pass dichroic mirror 1224. The long-pass dichroic mirror 1224 can be controlled by the computer system 1101 to redirect the expanded laser beam 1254 into the focusing objective lens 1232. The focusing objective lens 1232 can focus the expanded laser beam 1254 when it is projected into the printing chamber 1234. The printing chamber 1234 can be the medium chamber 122. The printing chamber 1234 can include a cell-containing medium, a plurality of cells, cellular components (eg, organelles), and / or at least one polymer precursor.

[0223] The print chamber 1234 can be mounted on a movable stage 1246. The movable stage 1246 can be an xy stage, a z stage, and / or an xyz stage. The movable stage 1246 can be manually positioned. The movable stage 1246 can be automatically positioned. The movable stage 1246 can be a motorized stage. The movable stage 1246 can be controlled by the computer system 1101. The computer system 1101 can control the movement of the movable stage 1246 in the x, y, and / or z directions. The computer system 1101 can automatically position the movable stage 1246 at a predetermined x, y, and / or z position. The computer system 1101 can position the movable stage 1246 at a predetermined x, y, and / or z position with a positional accuracy of at most about 3 μm. The computer system 1101 can position the movable stage 1246 at a predetermined x, y, and / or z position with a positional accuracy of at most about 2 μm. The computer system 1101 can position the movable stage 1246 at a predetermined x, y, and / or z position with a positional accuracy of up to about 1 μm. The computer system 1101 can automatically adjust the position of the movable stage 1246 before or during three-dimensional printing. The computer system 1101 may include a piezoelectric controller to provide computer-controlled z-axis (e.g., vertical direction) positioning and active position feedback. The computer system 1101 may include a joystick console to enable a user to control the position of the movable stage 1246. The joystick console may be a z-axis console and / or an x-axis and y-axis console. The movable stage 1246 may include a print chamber holder. The print chamber holder may be a bracket, a clamp, and / or a recessed sample holder. The movable stage 1246 may include a multi-slide holder, a slide holder, and / or a Petri dish holder. The movable stage 1246 may include a sensor to provide position feedback. The sensor may be a capacitive sensor. The sensor may be a piezoresistive sensor. The movable stage 1246 may include at least one actuator (eg, a piezoelectric actuator) that moves (or positions) the movable stage 1246 .

[0224] A light emitting diode (LED) collimator 1240 may be used as a source of collimated LED light 1256. The LED collimator 1240 may include a collimating lens and an LED emitter. The LED may be an inorganic LED, a high brightness LED, a quantum dot LED, or an organic LED. The LED may be a single color LED, a dual color LED, or a tri-color LED. The LED may be a blue LED, an ultraviolet LED, a white LED, an infrared LED, a red LED, an orange LED, a yellow LED, a green LED, a blue-violet LED, a pink LED, or a violet LED. The LED collimator 1240 may project a beam of collimated LED light 1256 through an f6 lens 1248. The f6 lens 1248 may be a focusing lens. Once the collimated LED light 1256 is transmitted through the f6 lens 1248, the collimated LED light 1156 may be directed into the focusing objective 1236. The focusing objective 1236 may focus the collimated LED light 1256 into the printing chamber 1234. The focusing objective 1236 can focus the collimated LED light 1256 into the sample medium. The focusing objective 1236 can focus the collimated LED light 1256 into the medium containing cells. The collimated LED light 1256 can be transmitted through the print chamber 1234 and enter the focusing objective 1232. Once the collimated LED light 1256 leaves the focusing objective 1232, the collimated LED light 1256 can be directed to the long pass dichroic mirror 1224. The collimated LED light 1256 reflected off the long pass dichroic mirror 1224 can be the sample emission 1226. The long pass dichroic mirror 1224 can redirect the sample emission 1226 into the f5 lens 1244. The f5 lens can be a focusing lens. Once the sample emission 1226 is transmitted through the f5 lens 1244, the detection system 1230 detects and / or collects the sample emission 1226 for imaging. The detection system 1230 may include at least one photomultiplier tube (PMT). The detection system 1230 may include at least one camera. The camera may be a complementary metal oxide semiconductor (CMOS) camera, a scientific CMOS camera, a charge coupled device (CCD) camera, or an electron multiplying charge coupled device (EM-CCD). The detection system 1230 may include at least one array-based detector.

[0225] Fig.15The light detection system 1330 is illustrated. The light detection system 1330 may include a plurality of long pass dichroic mirrors arranged in series. The light detection system 1330 may include a plurality of long pass dichroic mirrors arranged in parallel. The light detection system 1330 may include a plurality of long pass dichroic mirrors arranged in series and in parallel. As shown in FIGS. 44-46 , the optical path may include an LED collimator that projects a beam of collimated LED light 1356 onto a focusing objective. Once the collimated LED light 1356 is reflected from the first long pass dichroic mirror 1324a, the collimated LED light 1356 may be converted into sample emission 1326. The sample emission 1326 may be directed through an f5 lens 1344. The f5 lens 1344 may be a focusing lens. After sample emission 1326 exits f5 lens 1344, sample emission 1326 may be directed to a series of long pass dichroic mirrors, including a second long pass dichroic mirror 1324b, a third long pass dichroic mirror 1324c, a fourth long pass dichroic mirror 1324d, and a fifth long pass dichroic mirror 1324e, as shown in FIG. Fig.15 As shown. Sample emission 1326 can be reflected off the second long pass dichroic mirror 1324b and reflected onto the first light detector 1352a. Sample emission 1326 can be reflected off the third long pass dichroic mirror 1324c and reflected onto the second light detector 1352b. Sample emission 1326 can be reflected off the fourth long pass dichroic mirror 1324d and reflected onto the third light detector 1352c. Sample emission 1326 can be reflected off the fifth long pass dichroic mirror 1324e and reflected onto the fourth light detector 1352d. Sample emission 1326 can be reflected off the fifth long pass dichroic mirror 1324e and reflected onto the fifth light detector 1352e. The light detector can be a photomultiplier tube (PMT). The light detector can be a camera. The light detector can be a complementary metal oxide semiconductor (CMOS) camera, a scientific CMOS camera, a charge coupled device (CCD) camera, or an electron multiplying charge coupled device (EM-CCD). The light detector can be an array-based detector. The light detection system 1330 can include a plurality of long-pass dichroic mirrors with gradually red-shifted cut-off wavelengths. In some cases, the second long-pass dichroic mirror 1324b can have a cut-off wavelength of about 460 nm, the third long-pass dichroic mirror 1324c can have a cut-off wavelength of about 500 nm, the fourth long-pass dichroic mirror 1324d can have a cut-off wavelength of about 540 nm, and the fifth long-pass dichroic mirror 1324e can have a cut-off wavelength of about 570 nm.

[0226] The light detection system 1330 can be controlled by the computer system 1101. The computer system 1101 can collect and / or process the signals obtained by the first light detector 1352a, the second light detector 1352b, the third light detector 1352c, and the fourth light detector 1352d. The computer system 1101 can provide control feedback to the three-dimensional printing system based on the light detector signals of the light detection system 1330, which can be collected and / or processed by the computer system 1101. The computer system 1101 can have control feedback for any optical components and / or hardware of the light paths described in Figures 44-46. The computer system 1101 can have control feedback for any optical components and / or hardware of the light paths described in Figures 44-46. Fig.15 The computer system 1101 may control, for example, an SLM, a shutter, a movable stage, a mirror, a lens, a focusing objective, a beam expander, an LED collimator, a grating, and / or a blocking element in response to signals from the light detection system 1330.

[0227] Figure 5A An embodiment of a multiphoton tissue print head 118 is illustrated. The multiphoton print head 118 can receive a multiphoton laser beam 120 (comprising one or more wavelengths) from the laser system 116 and can focus the beam 120 through a final optical path consisting of final optics consisting of an optional scan head, a long pass mirror for collecting and recording backscattered light, and a focusing objective 200, thereby projecting the beam 120 into a media chamber 122. The light can be collected by the same objective as used for printing and then split via a long pass mirror to a single or array of PMT or CCD cameras.

[0228] In some designs, the optics can send laser light through a fiber optic cable to more easily control where the light is deposited in the tissue printing vessel.

[0229] The systems disclosed herein can utilize a range of focusing objectives, for example, with gradually decreasing magnifications; the field of view may gradually increase. In some cases, the field of view may be the print area that the microscope can use in a single projection area. In some cases, 5x, 10x, or 20x objectives may be used. In some cases, objectives with a high numerical aperture of at least about 0.6 to about 1.2 or greater may be used. The systems disclosed herein can use objectives with magnifications ranging from, for example, about 1x to about 100x. The systems disclosed herein can use objectives with magnifications of about 1x. The systems disclosed herein can use objectives with magnifications of about 2x. The systems disclosed herein can use objectives with magnifications of about 3x. The systems disclosed herein can use objectives with magnifications of about 4x. The systems disclosed herein can use objectives with magnifications of about 10x. The systems disclosed herein can use objectives with magnifications of about 20x. The systems disclosed herein can use objectives with magnifications of about 40x. The systems disclosed herein can use objectives with magnifications of about 60x. The systems disclosed herein may use an objective lens with a magnification of approximately 100x.

[0230] To maintain structural fidelity of the printed tissue, a water immersion objective may be desirable to substantially match the angle of incidence within the cell-containing liquid biogel medium 126. A water immersion objective that is corrected for refractive index variations may be used for printing in liquid media that have a refractive index that is significantly different from that of air.

[0231] Figure 5B A print head 118 including a first objective lens 200a and a second objective lens 200b is illustrated. Figure 5B An inverted optical device for an imaging structure is shown. In this embodiment, light can be collected by the inverted optical device and transmitted to a Figure 5B A CCD camera, a single PMT, or an array of PMTs as shown can be used to create a multicolor image. In some embodiments, the second objective lens can be inverted and can collect images from the bottom side of the tissue, and the incident light is read by the PMTs with a series of long pass or band pass mirrors.

[0232] To switch a multiphoton based printer from printing mode to imaging mode, an x,y raster scan can be performed and the DMD or SLM paths can be bypassed, either by placing the devices in an off or inactive position, or by removing them from the optical path so that a single laser line hits the x,y scanning optics. In some cases, the DMD or SLM paths can also be used for imaging.

[0233] During the printing process, switching to imaging mode may have multiple uses: 1) imaging can be used to monitor the rate of collagen production since collagen naturally produces emission via second harmonic generation, a process when two-photon excitation is scanned across the structure, 2) using imaging mode the edges of the printed tissue can be found, thereby facilitating proper connection of blood vessels and other tissue structures along the edges of the projection space, 3) the structural integrity of the printed tissue structure and the fidelity of the projected image can be verified in real time, and 4) if temporarily labeled cells are used, they can be located within the printed tissue for process verification or monitoring. It will be appreciated that the laser system 116 of the above embodiment may have various software controlled points, including but not limited to: may be programmed to project CAD images by hard-wired changes to the SLM and / or DMD devices; if a TAG lens is used to generate a Bessel beam, the current generated to induce a tunable acoustic gradient (TAG) in the TAG lens may be controlled by computer software; a mirror that directs laser excitation in a single beam entity and may act as an off / on switch for a multi-laser design may be controlled by computer software; laser intensity and tuning of different frequencies via an attenuation wheel may be controlled by software input; movement of a microscope stage may be software controlled; movement of a microscope objective or associated fiber optics may be software controlled; edge finding, illumination and control of an inverted objective by movement or on / off state may be software controlled; any imaging or optical path control components (mirrors, shutters, scanning optics, SLM, DMD, etc.) may be software controlled.

[0234] To accommodate fast printing, objective 200 may be equipped with a fiber optic cable. Fig. 6A An embodiment of a removable and attachable fiber optic cable accessory 250 is illustrated. In this embodiment, the accessory 250 may include a fiber optic cable 252 and an accessory ( Figure 6A-6B ), which can be attached to a multiphoton tissue printing printhead ( Figure 6A-6B ). Figure 6B As shown, the fiber optic cable 252 can then be positioned within the medium 126 of the medium chamber 122. Thus, the multiphoton laser beam 120 can pass through the objective lens 200 and the fiber optic cable 252 to deliver laser energy to the medium 126, thereby generating a predetermined complex tissue structure 260. To avoid moving the microscope objective lens or the printing vessel containing the delicate tissue structure during the printing process, the fiber optic cable itself can be moved if a larger tissue area needs to be printed. In some cases, the attachment 250 can be sterilized or replaced so that direct insertion into the medium 126 does not compromise sterility or cross-contaminate the printed cells.

[0235] Depending on the power input to the fiber optic cable, a multiphoton laser may be able to cause irreversible damage to the inner core of the fiber optic cable. Therefore, in some cases, a wavelength chirp induced by group delay dispersion (GDD) can be provided by effectively dispersing the photons to extend the laser pulse to minimize this potential damage. This can be used to minimize damage to cells in the print media or to extend the life of the fiber optic cable. In such a case, a GDD device can be provided in the laser system 116 after the SLM or DMD and before entering the print head optical device 118.

[0236] In some cases, a single objective lens 200 or an objective lens 200 with an attached fiber optic attachment 250 can be used to perform three-dimensional printing of a predetermined tissue, with one to three different configurations, each of which is associated with a different laser line and represents a different shape or portion of the tissue that can be pulsed by the same objective lens 200. In such a case, a timed shutter system can be installed so that there is no interference or minimal interference between the projected images. Therefore, laser multiplexing can be used to allow portions of the tissue structure to be generated simultaneously at multiple points while utilizing the same CAD model of the tissue structure. Similarly, laser multiplexing can utilize different but adjacent CAD-based tissue models, thereby minimizing the movement required for printing larger structures while further reducing the overall printing time. For example, a vascular bed can have internal structures, such as valves in larger blood vessels that prevent venous reflux in normal circulation. These valve structures can be printed simultaneously with the vessel wall. In such a case, stents associated with the valve structure and / or the vessel wall may be difficult to print separately.

[0237] During a printing run, the instantaneously formed three-dimensional structure can be repeated throughout the printing volume. In biological systems, small units can often be repeated throughout the structure. Therefore, repeatedly generating the same structure in a printing run may be useful for generating functional tissues. Additional non-repetitive fine feature structures and subsequent structures from the same cell-printed material can be generated, which are aligned or connected to the first structure printed.

[0238] In some embodiments, the multiphoton tissue printing printhead 118 may include multiple printing "heads" or multiphoton excitation sources via a first laser objective 200a, a second laser objective 200b, and a third laser objective 200c, such as Figure 7-Figure 8 shown. Figure 7An embodiment is illustrated in which a multiphoton tissue printing print head 118 may include a first laser objective 200a, a second laser objective 200b, and a third laser objective 200c, wherein the first laser objective 200a may include a first fiber optic cable attachment 250a, the second laser objective 200b may include a second fiber optic cable attachment 250b, and the third laser objective 200c may include a third fiber optic cable attachment 250c. The first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, and the third fiber optic cable attachment 250c may be directed into a single media chamber 122. The media chamber 122 may have an open top or a sealed top with port access by each attachment fiber optic cable attachment (e.g., via the first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, and the third fiber optic cable attachment 250c). This arrangement may increase the speed of large, rapid tissue printing while maintaining control over the final tissue structure. In some cases, the first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, and the third fiber optic cable attachment 250c can deliver projections of the same tissue structure. In other cases, each of the first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, and the third fiber optic cable attachment 250c can deliver a first laser beam projection 120a, a second laser beam projection 120b, and a third laser beam projection 120c of a different tissue structure, respectively. Given the flexible arrangement of multiple laser objective lenses and the ability to direct fiber optic cables to the same area within the media chamber 122, tissue structures can be printed simultaneously. The resulting tissue structures can be connected together or not. With some considerations of motion constraints and considerations for each additional excitation source to be considered, the printing time of a given tissue structure may have an inverse relationship with the number of laser transmission elements.

[0239] Figure 8An embodiment is illustrated in which a multiphoton tissue printing print head 118 may include a first objective lens 200a, a second objective lens 200b, a third objective lens 200c, a fourth objective lens 200d, a fifth objective lens 200e, and a sixth objective lens 200f, wherein each objective lens may include a first fiber optic cable attachment 250a, a second fiber optic cable attachment 250b, a third fiber optic cable attachment 250c, a fourth fiber optic cable attachment 250d, a fifth fiber optic cable attachment 250e, and a sixth fiber optic cable attachment 250f directed to a separate first medium chamber 122a, a second medium chamber 122b, a third medium chamber 122c, a fourth medium chamber 122d, a fifth medium chamber 122e, and a sixth medium chamber 122f, respectively. The plurality of medium chambers may be a multi-well plate in which each well of the multi-well plate is a separate single medium chamber. In some cases, the first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, the third fiber optic cable attachment 250c, the fourth fiber optic cable attachment 250d, the fifth fiber optic cable attachment 250e, and the sixth fiber optic cable attachment 250f can deliver at least one projection of the same tissue structure. This provides multiple copies of the tissue structure simultaneously. In other cases, the first fiber optic cable attachment 250a, the second fiber optic cable attachment 250b, the third fiber optic cable attachment 250c, the fourth fiber optic cable attachment 250d, the fifth fiber optic cable attachment 250e, and the sixth fiber optic cable attachment 250f can deliver the first multi-photon laser beam projection 120a, the second multi-photon laser beam projection 120b, and the third multi-photon laser beam projection 120c of different tissue structures. In some cases, due to the ability to produce multiple copies simultaneously, printing time can be greatly reduced.

[0240] In some embodiments, the multiphoton tissue printing print head 118 may include a continuous array of objective lenses, including a first objective lens 200a, a second objective lens 200b, and a third objective lens 200c, such as Fig. 9 As shown. In this embodiment, each objective lens can be aligned with a separate medium chamber. For example, the first objective lens 200a can be aligned with the first medium chamber 122a, the second objective lens 200b can be aligned with the second medium chamber 122b, and the third objective lens 200c can be aligned with the third medium chamber 122c. In some cases, the multiple medium chambers can be holes in the multi-well plate 300. In some embodiments, the first objective lens 200a, the second objective lens 200b, and the third objective lens 200c can deliver projections of the same tissue structure. In other cases, the laser beam projection of each hole may be different. The first objective lens 200a, the second objective lens 200b ( Fig.10 ) and the third objective lens 200c ( Fig.10 ) can be programmed to move over the multiwell plate 300 in the x and y directions, such as Fig.10As shown, to deliver the laser beam projection to each hole. Alternatively, it is understood that the object lens can remain stationary while the porous plate 300 moves in the x and y directions. Therefore, for example, a continuous array with three object lenses can print tissue in a six-well plate in the following two steps: print three tissue structures at the same time, and then print another three tissue structures at the same time. It is understood that a plate with any number of holes can be used, which includes but is not limited to at least about 96 holes to about 394 holes, or more. The porous plate 300 may include at least the first medium chamber 122a. The porous plate 300 may include at least 1 hole. The porous plate 300 may include at least 4 holes. The porous plate 300 may include at least 6 holes. The porous plate 300 may include at least 8 holes. The porous plate 300 may include at least 12 holes. The porous plate 300 may include at least 16 holes. The porous plate 300 may include at least 24 holes. The porous plate 300 may include at least 48 holes. The multiwell plate 300 may include at least 96 holes. The multiwell plate 300 may include at least 384 holes. The multiwell plate 300 may include at least 1536 holes. It will be appreciated that in the embodiments described herein, the microscope stage may be able to move, the microscope lens may be able to move, and / or the associated fiber optic cable attached to the printing objective may be able to move in order to print a larger space.

[0241] Methods for printing organs and organoids

[0242] The present disclosure provides methods and systems for producing one or more immune proteins. On the one hand, the method for producing one or more immune proteins includes providing a medium chamber containing a medium, wherein the medium includes: (i) multiple cells and (ii) one or more polymer precursors. Subsequently, according to computer instructions for printing three-dimensional (3D) lymphoid organoids in a computer memory, at least one energy beam can be directed to the medium in the medium chamber along at least one energy beam path patterned as a 3D projection. This can form at least a portion of a 3D lymphoid organoid, which includes: (i) at least a subset of multiple cells, and (ii) a polymer formed from one or more polymer precursors. Subsequently, the method for producing one or more immune proteins may include subjecting at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate the production of one or more immune proteins.

[0243] In another aspect, a method for producing one or more immune proteins comprises: (i) printing a three-dimensional (3D) lymphoid organoid comprising a matrix comprising a plurality of cells, and (ii) processing the 3D lymphoid organoid to produce the one or more immune proteins.

[0244] On the other hand, a method for producing one or more immune proteins includes providing a medium chamber comprising a first medium. The first medium may include a first plurality of cells and a first polymer precursor. Subsequently, according to computer instructions for printing a three-dimensional (3D) lymphoid organ in a computer memory, at least one energy beam may be directed to the first medium in the medium chamber along at least one energy beam path so that at least a portion of the first medium in the medium chamber forms a first portion of the 3D lymphoid organ. Subsequently, the method may provide a second medium in the medium chamber. The second medium may include a second plurality of cells and a second polymer precursor. The second plurality of cells may be of a different type than the first plurality of cells. Subsequently, the method may include directing at least one energy beam to the second medium in the medium chamber along at least one energy beam path according to computer instructions so that at least a portion of the second medium in the medium chamber forms a second portion of the 3D lymphoid organ. Subsequently, the method may include subjecting the first portion and the second portion of the 3D lymphoid organ to conditions sufficient to stimulate the production of one or more immune proteins.

[0245] In another aspect, a method of producing one or more immune proteins comprises: (i) printing a three-dimensional (3D) lymphoid organoid comprising a matrix comprising a first plurality of cells and a second plurality of cells, and (ii) processing the 3D lymphoid organoid to produce the one or more immune proteins.

[0246] Another aspect of the present disclosure provides a system for producing one or more immune proteins, including a medium chamber configured to contain a medium, the medium comprising a plurality of cells and one or more polymer precursors. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber. The system may include one or more computer processors operably coupled to at least one energy source. The one or more computer processors may be individually or collectively programmed to receive computer instructions for printing three-dimensional (3D) lymphoid organoids from a computer memory. The one or more computer processors may be individually or collectively programmed to guide at least one energy source to direct at least one energy beam to the medium in the medium chamber along at least one energy beam path according to the computer instructions, so that at least a portion of the polymer precursor forms at least a portion of the 3D lymphoid organoid. The one or more computer processors may be individually or collectively programmed to subject at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate the production of one or more immune proteins. The one or more computer processors may be further programmed individually or collectively to extract one or more immune proteins from at least a portion of the 3D lymphoid organoid.

[0247] Another aspect of the present disclosure provides a method for producing a human immune protein population, comprising: using a multiphoton laser bioprinting system to bioprint a three-dimensional lymphoid organoid. Subsequently, the method may include exposing the three-dimensional lymphoid organoid to an antigen to stimulate the production of the human immune protein population. Subsequently, the method may include extracting the human immune protein population from the three-dimensional lymphoid organoid.

[0248] Conditions sufficient to stimulate the production of one or more immune proteins may include exposing at least a portion of the 3D lymphoid organoid to an antigen to stimulate the production of one or more immune proteins. The antigen may be selected from a whole peptide, a partial peptide, a glycopeptide, a whole protein or a protein subunit, a carbohydrate, a nucleic acid, a live virus, a heat-inactivated virus, a viral particle, a membrane-bound or stabilized protein, a phage-displayed antigen, and a whole cell. The antigen may be an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, or a combination thereof. A neoantigen is defined herein as an antigen that is not present in the normal human genome. A neoantigen may be a tumor antigen, a viral antigen, an engineered antigen, or a synthetic antigen.

[0249] The method of the present disclosure may further include extracting one or more immune proteins from at least a portion of the 3D lymphoid organoid. The one or more immune proteins may be human immune proteins. The immune protein may be selected from an antibody, a T cell receptor, and a cancer immunotherapeutic agent. The antibody may be an immunoglobulin G (IgG) antibody. The IgG antibody may be a human IgG antibody. The immune protein may be an IgM, IgA, IgE, IgD antibody, or a combination thereof. The immune protein may be an antibody fragment, an antibody domain, an immunoglobulin heavy chain, an immunoglobulin light chain, or a combination thereof. The antibody fragment may be an antigen binding fragment (Fab), a single chain variable fragment (scFv), or a combination thereof. The immune protein may be a multivalent recombinant antibody. The multivalent recombinant antibody may be a diabody (e.g., a small recombinant bispecific antibody), a microbody (e.g., an engineered antibody fragment), a triabody, a tetrabody, or a combination thereof. The immune protein may be an engineered immune protein, a synthetic immune protein, or a combination thereof. The synthetic immune protein may be a nucleic acid aptamer, a non-immunoglobulin protein scaffold, a non-immunoglobulin peptide aptamer, an affimer protein, or a combination thereof.

[0250] Multiple cells can be from a subject. Multiple cells can be autologous. Multiple cells can be allogeneic. Multiple cells can be selected from stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, immature B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subgroups, effector T cells and their subgroups, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, immature T cells or other immature T cells, dendritic cells and their subgroups, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subgroups, macrophages and their subgroups, leukocytes and their subgroups. B cells can be selected from immature B cells, mature B cells, plasma B cells, B1 B cells and B2 B cells. T cells can be selected from CD8+ and CD4+.

[0251] 3D lymphoid organoids can be selected from B cell germinal centers, thymic-like development niches, lymph nodes, Langerhans islands, hair follicles, tumors, tumor spheres, nerve bundles or supporting cells, nephrons, liver organoids, intestinal crypts, primary lymphoid organs and secondary lymphoid organs. The shape of the 3D lymphoid organoids can be selected from spheres, ellipses, ovoids, ovoids, squares, rectangles, cubes, any polygonal shape, free forms and teardrops. The shape of the 3D lymphoid organoids can be teardrops.

[0252] The polymer of at least a portion of the 3D lymphoid organoid can form a network. The polymer can be collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic acid-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar or a combination thereof. The polymer may include an extracellular matrix component. Non-limiting examples of extracellular matrix components for generating 3D lymphoid organoids may include proteoglycans such as heparan sulfate, chondroitin sulfate and keratan sulfate, non-proteoglycan polysaccharides such as hyaluronic acid, collagen, and elastin, fibronectin, laminin, nidogen, or any combination thereof. These extracellular matrix components can be functionalized with acrylates, diacrylates, methacrylates, cinnamoyl, coumarin, thymine or other side groups or chemically reactive moieties to promote crosslinking induced by multiphoton excitation directly or by multiphoton excitation of one or more chemical dopants. In some cases, photopolymerizable macromonomers and / or photopolymerizable monomers can be used in combination with extracellular matrix components to produce cell-containing structures. Non-limiting examples of photopolymerizable macromonomers can include polyethylene glycol (PEG) acrylate derivatives, PEG methacrylate derivatives, and polyvinyl alcohol (PVA) derivatives. In some cases, the collagen used to produce cell-containing structures can be fibrous collagen such as type I, type II, type III, type V, and type XI collagen, facit collagen such as type IX, type XII, and type XIV collagen, short-chain collagen such as type VIII and type X collagen, basement membrane collagen such as type IV collagen, type VI collagen, type VII collagen, type XIII collagen, or any combination thereof.

[0253] The polymer of at least a portion of the 3D lymphoid organoid can include other polymerizable monomers that are synthetic and not native to mammalian tissue, including mixtures of biological and synthetic materials. An example mixture can include about 0.4% w / v collagen methacrylate plus about 50% w / v polyethylene glycol diacrylate (PEGDA) added. The photoinitiator that directs the polymerization can be reactive in the ultraviolet (UV), infrared (IR), or visible light range. Examples of two such photoinitiators are eosin Y (EY) and triethanolamine (TEA), which when combined can polymerize in response to exposure to visible light (e.g., wavelengths of about 390 to 700 nanometers). Non-limiting examples of photoinitiators may include azobisisobutyronitrile (AIBN), benzoin derivatives, benzoketal, hydroxyalkyl phenone, acetophenone derivatives, trimethylolpropane triacrylate (TPT), acryloyl chloride, benzoyl peroxide, camphorquinone, benzophenone, thioxanthone, and 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone. Hydroxyalkyl phenone may include 4-(2-hydroxyethylethoxy)-phenyl-(2-hydroxy-2-methylpropyl) ketone ( 295), 1-hydroxycyclohexyl-1-benzophenone ( 184) and 2,2-dimethoxy-2-phenylacetophenone ( 651). The acetophenone derivative may include 2,2-dimethoxy-2-phenylacetophenone (DMPA). The thioxanthone may include isopropylthioxanthone.

[0254] The network formed by the polymer can be a mesh, amorphous or a net. The net can be an organized net. The organized net can include a repeated pattern. The network can be a structured network. The network can be an unstructured network. The network can be a mixed grid, wherein it contains a mixture of structured and unstructured parts. The network can be a two-dimensional network. The network can be a three-dimensional network. The three-dimensional network can be a tetrahedral network, a pyramidal network, a hexahedral network, a polyhedral network or a combination thereof. The network formed by the polymer can be a grid. The grid can be a triangular grid, an octagonal grid, a hexagonal grid, a rectangular grid, a square grid, a diamond grid, a circular grid or a combination thereof. The grid can have a variable size per cell per unit area. Amorphous networks can be designed to promote cell interactions. Cell interactions can be B cell and T cell conjugate formation, B cell and B cell interaction, B cell and macrophage, T cell and dendritic cell interaction, matrix cell and T cell interaction, matrix cell and B cell interaction or matrix cell and dendritic cell interaction. Amorphous networks can be designed to promote movement between or within cell niches.

[0255] In one aspect, the present disclosure provides a method for printing an organ and / or organoid. The method may include polymerization of a photopolymerizable material by a laser light source. The organ and / or organoid may be two-dimensional or three-dimensional. The organ and / or organoid may be a lymph node. The organoid may be an islet of Langerhans. The organoid may be a hair follicle. The organ and / or organoid may be a tumor and / or a tumor spheroid. The organoid may be a neural bundle and supporting cells, such as but not limited to Schwann cells and glial cells, including satellite cells, olfactory ensheathing cells, enteric glia, oligodendrocytes, astrocytes and / or microglia. The organoid may be a nephron. The organoid may be a liver organoid. The organoid may be an intestinal crypt. The organ and / or organoid can be a primary lymphoid organ, a secondary lymphoid organ such as spleen, liver, pancreas, gall bladder, appendix, brain, small intestine, large intestine, heart, lung, bladder, kidney, bone, cochlea, ovary, thymus, trachea, cornea, heart valve, skin, ligament, tendon, muscle, thyroid, nerve and / or blood vessel.

[0256] The organization of an organ or organoid by a printing process disclosed herein may require or be achieved by sequentially depositing at least about 1, 10, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000 or more layers of cells. The organization of a lymphoid organ by a printing process may require or be achieved by sequentially depositing 1 to 100 layers of cells. The size of the cell layer may depend on the tissue. The size of the cell layer may include a larger three-dimensional structure, which may be a single layer of cells or may include multiple layers of cells. The cell layer may include about at least 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more cells. If precise placement of each cell type relative to another is required, the cells can be printed in sequential steps with a wash step in between to remove the previously used media. Alternatively, two or more cell types of different sizes can be printed simultaneously using two photopolymerizable materials with different polymerization wavelengths and pore sizes, so that larger cell types can be encapsulated in larger pore sizes and smaller cell types can be encapsulated in smaller pore sizes. Cells are encapsulated in pores based on the size of their nuclei because the cytoskeleton is able to reshape based on the available space.

[0257] Laser light sources can use high energy green, blue, white or lower frequency ultraviolet light to induce polymerization of photopolymerizable materials, or high resolution multiphoton light sources of any wavelength can be used. High resolution, non-toxic multiphoton projection technology is particularly suitable for printing detailed germinal centers, allowing the generation of light and dark areas that recapitulate natural B cell affinity maturation. The method can be used in conjunction with microfluidic manipulation of the vasculature (whether lymphatic or circulatory) to produce functional collagen-based organs and / or organoids, such as lymph node organoids. Alternatively, non-toxic wavelengths of visible and ultraviolet light can be used to print biological gels containing cell structures or cells to be seeded.

[0258] The present disclosure includes printing lymphoid organs or organoids by two-dimensional or three-dimensional projection of a laser beam 1002 from an energy source 1000 (e.g., a laser, particularly a high-resolution multiphoton laser beam, but also including other possible light sources). The laser beam 1002 is intended to induce polymerization of a cell-containing medium 126 in a predetermined pattern to produce a final product that is structurally or functionally similar to a natural one, particularly a human lymphoid organ or organoid. A lymphoid organ is defined herein as a small, fully functional structure containing immune cells that is capable of initiating and executing a functional and complete immune response, which is defined as the production of antibodies, chemicals (e.g., cytokines), or a cellular response to an antigen. A lymphoid organoid is defined herein as a partially intact lymphoid organ that is capable of exhibiting any type of immune activity at the cellular level. Immune activity includes, but is not limited to: (a) cellular activity, defined as up- or down-regulation of cell surface proteins; (b) mitotic cell division; (c) changes in cell motility; (d) functional cell motility within the printed constructs; (e) the development of an immune response as measured by changes in protein production (such as antibodies, cytokines, or chemokines); and (f) the generation of new proteins through activation-associated mutations such as somatic hypermutation typical of B cells.

[0259] Lymph node organoids, or structures containing lymphocytes, are designed to recapitulate essential lymph node functions and provide cellular niches and microarchitecture to support lymphocyte interactions and the onset of functional immune responses; such as B cell germinal centers (GCs; Fig.18A ) and thymic developmental niche ( Fig.18B ) formation. Lymphoid organoids can be any semi-functional aggregate of immune cells, including Fig.18A and Fig.18B Partial structures of those depicted in .

[0260] refer to Fig.18A, the B cell germinal center 105 can be functionally separated into a B cell-dense dark zone 106, where B cells 107 proliferate and undergo somatic hypermutation, and a light zone 108, where B cells interact with cells carrying full antigens and / or supporting cells 109 and / or T cells 111, including but not limited to dendritic cells, monocytes, other B cells 103, to receive positive signals, including but not limited to soluble factors after functional receptor mutation or rearrangement and ligand-based cell surface interactions 112. Once the positive signal 112 is received, the B cell 107 returns to the dark zone 106 and continues the process of proliferation and receptor mutation. This process is repeated until one or more dominant B cell clones are selected and become plasma B cells 113, i.e., secret mature, class-switched highly specific antibodies 114. Movement between the light zone and the dark zone occurs through the movement of individual cells guided by the endogenous chemokine gradient set by the supporting cells and / or materials included in the bioprinting matrix 115.

[0261] Fig.18B Depicted is the thymic developmental niche. The printed structures mimic the sequential development of T cells in the thymus, where as they proliferate and mature, they migrate from the cortical tissue to the medullary tissue in the thymic organoid. The direction of this migration is determined by Fig.18B denoted by arrows 230 in the figure. These movements are guided by cells sensing local chemokine gradients established by local cell populations and introducing reagents into the cell printing matrix to assist in establishing a cellular niche. The distribution of a mixture of accessory cells including but not limited to cortical epithelial cells 216, medullary epithelial cells 117, dendritic cells 218, and macrophages 119 ensures that T cells are in close proximity to the most important accessory cells at this stage of T cell development. The structure consists of the thymic capsule 220, the cortical region 121, and the medullary region 123. Immature thymocytes, double negative T cells, and macrophages ( Fig.18B In the thymus, the medullary epithelial cells, the more abundant macrophages and the dendritic cells derived from the bone marrow are closely connected with the mature thymocytes and promote further development. During the development process, the double negative immature thymocytes 125 move toward the medulla 123 through the cortical structure 131 and the accessory cells 216, differentiate into single positive thymocytes 135, enter the medullary region and become CD4 or CD8 positive mature thymocytes 137. In this process, some cells undergo cell death and may become apoptotic cells 133.

[0262] like Fig.19As shown, the shapes of these printed structures can be spherical, elliptical, oval or ovoid, which can have a flat or torus-shaped bottom and can contain a hollow or recessed center to allow for varying surface area configurations134; square, rectangular, cubic or any polygon135; free-form, particularly where the free-form design is intended to promote the formation of asymmetric spheres136 of multicellular niches; or teardrop-shaped137 with long tails from any direction.

[0263] Can be as Fig. 20 The lymphoid organoids and organoids are shown printed in teardrop-like shapes 137, such that B cells 138 are clustered in a spherical or hemispherical structure at the larger end 139 of the structure, while accessory cells 143 taper off to one or both sides. During affinity maturation, B cells can move independently through the organized cell niche in response to local chemokine gradients. The figures are illustrated as cross-sections of 3D structures in top views of asymmetric teardrop-like shapes with a single tail 141 and a double tail 142.

[0264] Can be as Fig.23 Printing of lymphoid organs and organoids as shown. Fig.23 Microscope images of three-dimensionally printed lymph node organoids produced by the methods disclosed herein are shown. T cells and B cells are shown to be physically separated into separate regions of the lymph node organoid. The T cell region indicates a tissue region containing a mixture of T cells and supporting cells. The B cell region indicates a tissue region containing a mixture of B cells and supporting cells.

[0265] The chemokine gradient can be established by cells as part of an encapsulated cell network, or the chemokine gradient can be deposited as part of the printing process.

[0266] B cells, T cells, follicular dendritic cells, and other cell types can be printed in suspension, adhered to the bottom or sides of a dish / plate, or printed in a network of collagen or another biological, biocompatible, or bioinert material.

[0267] When cells are printed within a mesh, the mesh may be arranged as a mesh, amorphous, or organized mesh. An organized mesh is any mesh with a repeating geometric or other pattern, including hexagons, squares / rectangles, diamonds, circles, semicircles, spheres, hemispheres, or any combination of shapes therein. The mesh or amorphous mesh is produced without much regard for geometric pattern, the primary purpose being rapid production and the ability to encapsulate and contain cells. Additionally, some meshes may appear amorphous to an untrained observer, but actually have a specific shape or design designed to facilitate cell interaction or movement between or within niches.

[0268] The native architecture can be obtained from the imaging data and rendered as a two-dimensional or three-dimensional image with defined edges and / or grayscale areas, which are edges that are not precisely defined but fall somewhere within a specified range so as to be projected onto a polymerizable hydrogel. Such imaging data can provide sufficient details to enable accurate reproduction of multicellular niches that support cell-cell interactions during immune responses. In the immune system, multicellular niches are developed for single B cells or T cells based on receptor recognition of foreign pathogens or substances. High-reactivity of the receptor or high-affinity recognition during the immune response leads to the selection of the B or T cell and further cell division and expansion of the number of cells expressing highly reactive receptors. In these multicellular niches, competition for survival signals transmitted by highly reactive receptors leads to positive selection of the most reactive B or T cells. The native lymph node architecture can support the occurrence of this selection process, which depends on a series of specific cell-cell interactions that support the selection and proliferation of highly reactive cells. Thus, a three-dimensional native architecture that allows cell-cell interactions and independent cell movement is a key component of the B-cell and T-cell clonal selection process. This architecture is therefore an important component of the printed lymph nodes and is provided in particular by the use of multiphoton lasers in the printing process, although it is also possible to achieve functionality without printing at this resolution level through projection of wavefront-shaped multiphoton lasers.

[0269] Cell-cell interactions that may occur within a multicellular niche include, but are not limited to, B cell-T cell conjugate formation, B cell-B cell interactions, B cell-macrophage, T cell-dendritic cell interactions, and interactions of stromal cells with T cells, B cells, and dendritic cells. The interactions are not clearly paired interactions, and various types of cell clusters or clumps are often formed during an immune response, particularly in established cell niches or tissue-like structures.

[0270] As used herein, T cells may refer to any form of T cells, including but not limited to CD8+ or CD4+ T cells. B cells may refer to B cells at any stage of development, including but not limited to naive B cells, mature B cells, plasma B cells, B1 B cells or B2 B cells.

[0271] Fig.17 General details of lymphoid organs are depicted as their structure is currently understood and do not necessarily include every structural detail obtainable from imaging data, nor will the final product necessarily include every structural detail depicted; lymphoid organs and organoids are ultimately defined herein by their function.

[0272] Multiple organoid units can be printed within a single structure to produce larger organs, up to and including full-sized organs. Multiple lymphoid units can be printed within a single structure to produce larger immune organs, up to and including full-sized lymph nodes or thymus. The limiting factor for size is vascularization, which is essential for tissues with a width greater than 200 microns due to diffusion restrictions for most gases and nutrients. The thickness of a complete lymphoid organ or organoid without vascularization can be between 50 microns and 200 microns. If vascularized, the tissue can be 50 microns to 10 centimeters thick, can be of any shape or size, and can contain circulatory vasculature and lymphatic vasculature. The vasculature can include valves and / or sphincters. In some embodiments, the vasculature can be obtained by printing endothelial cells or their precursors in a mesh 500 that is intended to be very similar to the natural microvasculature, the structure of which is obtained from high-resolution imaging data. The capillary bed can branch from larger arterioles and arteries and branch into venules and veins according to the relevant anatomy.

[0273] In one aspect, the present disclosure provides a method of producing a human immune protein population. The method may include providing a medium. The medium may include a plurality of cells and one or more polymer precursors. The polymer precursor may be a biogel precursor. The method may include depositing at least one layer of the medium onto a substrate. The substrate may be a medium chamber. The substrate may be a tissue culture plate or well. The substrate may be a microfluidic chamber. The substrate may be a microfluidic chip. The substrate may be a polymer scaffold.

[0274] The method may include subjecting at least one layer of medium to an energy source to form a 3D lymphoid organoid comprising at least a subpopulation of multiple cells and a biogel formed by one or more polymer precursors. The method may include layer-by-layer deposition of a medium patterned according to a three-dimensional (3D) projection. The 3D projection may be based on computer instructions for printing a 3D lymphoid organoid in a computer memory. The layer-by-layer deposition of a medium patterned according to a three-dimensional (3D) projection and the formation of a biogel may be accomplished by subjecting the medium to an energy source (e.g., a laser). For example, a laser may be projected along an optical path according to a 3D projection so that a polymer precursor is polymerized in the medium and at least a portion of a 3D lymphoid organoid comprising multiple cells and a biogel is formed. On the other hand, the method may include manually depositing the medium layer by layer using a pipette or capillary to deposit at least one droplet of the medium on a substrate. In this example, a 3D projection including a pattern to be printed may not be necessary, instead, once deposited, the droplets of the medium may be subjected to an energy source (e.g., a heat or light source) to form at least a portion of a 3D lymphoid organoid comprising a biogel and multiple cells. On the other hand, the method may include layer-by-layer deposition of a medium using a microfluidic device. The microfluidic device can control the total volume of droplets of the medium deposited on the substrate in a layer-by-layer manner. The microfluidic device can control the total number of cells in each droplet of the medium deposited on the substrate in a layer-by-layer manner. On the other hand, the method may include layer-by-layer deposition of the medium using a printer. The printer can be a laser printer, a layer-by-layer inkjet printer (e.g., a thermal inkjet printer or a piezoelectric inkjet printer), a layer-by-layer extrusion 3D printer (e.g., a pneumatic extrusion bioprinter or a mechanical extrusion bioprinter) or any combination thereof. Droplets of the medium can be combined with other droplets so that cells can be organized into functional multicellular tissue niches.

[0275] The layered droplets can be dried, melted, solidified, gelled, cross-linked, polymerized or photopolymerized sequentially or at once using an energy source or by a chemical substance (e.g., a cross-linking agent or a photoinitiator). The energy source can be an energy beam, a heat source or a light source. The energy source can be a laser, such as a fiber laser, a short pulse laser or a femtosecond pulse laser. The energy source can be a heat source, such as a hot plate, a lamp, an oven, a hot water bath, a cell culture incubator, a heating chamber, a furnace, a drying oven or any combination thereof. The energy source can be a light source, such as white light, infrared light, ultraviolet (UV) light, near infrared (NIR) light, visible light, a light emitting diode (LED) or any combination thereof. The energy source can be an acoustic energy source, such as an ultrasonic probe, an ultrasound instrument, an ultrasonic bath or any combination thereof. The energy source can be an electromagnetic radiation source, such as a microwave source or any combination thereof.

[0276] The medium can be physically polymerized to form a biogel. The medium can be polymerized by a heat source to form a biogel. The medium can be chemically polymerized to form a biogel; for example, by using a crosslinking agent. Non-limiting examples of crosslinking agents include 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), glutaraldehyde, and 1-ethyl-3-3-dimethylaminopropylcarbodiimide (EDAC). The medium can include a photoinitiator, a crosslinking agent, collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic acid-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar, or any combination thereof. The biogel can include a photoinitiator, a crosslinking agent, collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic acid-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar, or any combination thereof. The polymer precursor can be collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar, or any combination thereof.

[0277] The biogel may be a hydrogel. The biogel may be a biocompatible hydrogel. The biogel may be a polymer hydrogel. The biogel may be a hydrogel bead. The biogel may be a hydrogel nanoparticle. The biogel may be a hydrogel droplet. The biogel may be a hydrogel microdroplet.

[0278] The droplets may have a diameter measuring at least about 10 micrometers (μm) to about 1000 μm. The droplets may have a diameter measuring at least about 10 μm. The droplets may have a diameter measuring up to about 1,000 μm.The droplets may have a size measuring from about 10 μm to about 50 μm, from about 10 μm to about 100 μm, from about 10 μm to about 200 μm, from about 10 μm to about 300 μm, from about 10 μm to about 400 μm, from about 10 μm to about 500 μm, from about 10 μm to about 600 μm, from about 10 μm to about 700 μm, from about 10 μm to about 800 μm, from about 10 μm to about 900 μm, from about 10 μm to about 1,000 μm, from about 50 μm to about 100 μm, from about 50 μm to about 200 μm, from about 50 μm to about 300 μm, from about 50 μm to about 400 μm, from about 50 μm to about 500 μm, from about 50 μm to about 600 μm, from about 10 μm to about 700 μm, from about 10 μm to about 800 μm, from about 10 μm to about 900 μm, from about 10 μm to about 1,000 μm. μm to about 700μm, about 50μm to about 800μm, about 50μm to about 900μm, about 50μm to about 1,000μm, about 100μm to about 200μm, about 100μm to about 300μm, about 100μm to about 400μm, about 100μm to about 500μm, about 100μm to about 600μm, about 100μm to about 700μm, about 100μm to about 800μm, about 100μm to about 900μm, about 100μm to about 1,000μm, about 200μm to about 300μm, about 200μm to about 400μm, about 200μm to about 500μm, about 200μm to about 600μm , about 200 μm to about 700 μm, about 200 μm to about 800 μm, about 200 μm to about 900 μm, about 200 μm to about 1,000 μm, about 300 μm to about 400 μm, about 300 μm to about 500 μm, about 300 μm to about 600 μm, about 300 μm to about 700 μm, about 300 μm to about 800 μm, about 300 μm to about 900 μm, about 300 μm to about 1,000 μm, about 400 μm to about 500 μm, about 400 μm to about 600 μm, about 400 μm to about 700 μm, about 400 μm to about 800 μm, about 400 μm to about 900 μm, about 400 μm to about In some embodiments, the present invention relates to a diameter of about 1,000 μm to about 1,000 μm, about 500 μm to about 600 μm, about 500 μm to about 700 μm, about 500 μm to about 800 μm, about 500 μm to about 900 μm, about 500 μm to about 1,000 μm, about 600 μm to about 700 μm, about 600 μm to about 800 μm, about 600 μm to about 900 μm, about 600 μm to about 1,000 μm, about 700 μm to about 800 μm, about 700 μm to about 900 μm, about 700 μm to about 1,000 μm, about 800 μm to about 900 μm, about 800 μm to about 1,000 μm, or about 900 μm to about 1,000 μm.The droplets may have a diameter measuring about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1,000 μm.

[0279] The droplet may have a volume of about 1 microliter (μl) to about 500 μl. The droplet may have a volume of at least about 1 μl. The droplet may have a volume of up to about 500 μl. The droplet may have a volume of about 1 μl to about 2 μl, about 1 μl to about 3 μl, about 1 μl to about 4 μl, about 1 μl to about 5 μl, about 1 μl to about 10 μl, about 1 μl to about 20 μl, about 1 μl to about 25 μl, about 1 μl to about 50 μl, about 1 μl to about 75 μl, about 1 μl to about 100 μl, about 1 μl to about 500 μl, about 2 μl to about 3 μl, about 2 μl to about 4 μl, about 2 μl to about 5 μl, about 2 μl to about 10 μl, about 2 μl to about 20 μl, about 2 μl to about 25 μl, about 2 μl From about 2 μl to about 50 μl, from about 2 μl to about 75 μl, from about 2 μl to about 100 μl, from about 2 μl to about 500 μl, from about 3 μl to about 4 μl, from about 3 μl to about 5 μl, from about 3 μl to about 10 μl, from about 3 μl to about 20 μl, from about 3 μl to about 25 μl, from about 3 μl to about 50 μl, from about 3 μl to about 75 μl, from about 3 μl to about 100 μl, from about 3 μl to about 500 μl, from about 4 μl to about 5 μl, from about 4 μl to about 10 μl, from about 4 μl to about 20 μl, from about 4 μl to about 25 μl, from about 4 μl to about 50 μl, about 4 μl to about 75 μl, about 4 μl to about 100 μl, about 4 μl to about 500 μl, about 5 μl to about 10 μl, about 5 μl to about 20 μl, about 5 μl to about 25 μl, about 5 μl to about 50 μl, about 5 μl to about 75 μl, about 5 μl to about 100 μl, about 5 μl to about 500 μl, about 10 μl to about 20 μl, about 10 μl to about 25 μl, about 10 μl to about 50 μl, about 10 μl to about 75 μl, about 10 μl to about 100 μl, about 10 μl to about 500 μl, about A volume of 20 μl to about 25 μl, about 20 μl to about 50 μl, about 20 μl to about 75 μl, about 20 μl to about 100 μl, about 20 μl to about 500 μl, about 25 μl to about 50 μl, about 25 μl to about 75 μl, about 25 μl to about 100 μl, about 25 μl to about 500 μl, about 50 μl to about 75 μl, about 50 μl to about 100 μl, about 50 μl to about 500 μl, about 75 μl to about 100 μl, about 75 μl to about 500 μl, or about 100 μl to about 500 μl. The droplet may have a volume of about 1 μl, about 2 μl, about 3 μl, about 4 μl, about 5 μl, about 10 μl, about 20 μl, about 25 μl, about 50 μl, about 75 μl, about 100 μl, or about 500 μl.

[0280] The biogel may have a carbon content of at least about 1 x 10 -3The biogel may have a viscosity of about 0.001 Pa·s to about 100,000 Pa·s or more when measured at about 25 degrees Celsius (°C). The biogel may have a viscosity of at least about 0.001 Pa·s when measured at about 25 degrees Celsius (°C). The biogel may have a viscosity of up to about 100,000 Pa·s when measured at about 25 degrees Celsius (°C).When measured at about 25 degrees Celsius (° C.), the biogel can have a relative humidity of about 0.001 Pa·s to about 0.01 Pa·s, about 0.001 Pa·s to about 0.1 Pa·s, about 0.001 Pa·s to about 1 Pa·s, about 0.001 Pa·s to about 10 Pa·s, about 0.001 Pa·s to about 100 Pa·s, about 0.001 Pa·s to about 1,000 Pa·s, about 0.001 Pa·s to about 10,000 Pa·s, about 0.001 Pa·s to about 50,000 Pa·s, about 0.001 Pa·s to about 100,000 Pa·s, about 0.01 Pa·s to about 0.1 Pa·s, about 0.001 Pa·s to about 100,000 Pa·s. 1Pa·s to about 1Pa·s, about 0.01Pa·s to about 10Pa·s, about 0.01Pa·s to about 100Pa·s, about 0.01Pa·s to about 1,000Pa·s, about 0.01Pa·s to about 10,000Pa·s, about 0.01Pa·s to about 50,000Pa·s, about 0.01Pa·s to about 100,000Pa·s, about 0.1Pa·s to about 1Pa·s, about 0.1Pa·s to about 10Pa·s, about 0.1Pa·s to about 100Pa·s, about 0.1Pa·s to about 1,000Pa·s, about 0.1Pa·s to about 10,000Pa·s, about 0.1Pa·s ·s to about 50,000 Pa·s, about 0.1 Pa·s to about 100,000 Pa·s, about 1 Pa·s to about 10 Pa·s, about 1 Pa·s to about 100 Pa·s, about 1 Pa·s to about 1,000 Pa·s, about 1 Pa·s to about 10,000 Pa·s, about 1 Pa·s to about 50,000 Pa·s, about 1 Pa·s to about 100,000 Pa·s, about 10 Pa·s to about 100 Pa·s, about 10 Pa·s to about 1,000 Pa·s, about 10 Pa·s to about 10,000 Pa·s, about 10 Pa·s to about 50,000 Pa·s, about 10 Pa·s to about 100,000 Pa·s, about 10 Pa·s to about 1,000 Pa·s, about 10 Pa·s to about 10,000 Pa·s In some embodiments, the viscosity of the present invention is from about 1 Pa·s to about 1,000 Pa·s, from about 100 Pa·s to about 10,000 Pa·s, from about 100 Pa·s to about 50,000 Pa·s, from about 100 Pa·s to about 100,000 Pa·s, from about 1,000 Pa·s to about 10,000 Pa·s, from about 1,000 Pa·s to about 50,000 Pa·s, from about 1,000 Pa·s to about 100,000 Pa·s, from about 10,000 Pa·s to about 50,000 Pa·s, from about 1,000 Pa·s to about 100,000 Pa·s, from about 10,000 Pa·s to about 50,000 Pa·s, from about 10,000 Pa·s to about 100,000 Pa·s, or from about 50,000 Pa·s to about 100,000 Pa·s.When measured at about 25 degrees Celsius (° C.), the biogel may have a viscosity of about 0.001 Pa·s, about 0.01 Pa·s, about 0.1 Pa·s, about 1 Pa·s, about 10 Pa·s, about 100 Pa·s, about 1,000 Pa·s, about 10,000 Pa·s, about 50,000 Pa·s, or about 100,000 Pa·s.

[0281] The biogel may be a hydrogel comprising a plurality of cells. The biogel may be a hydrogel comprising a plurality of non-hydrogel beads. The biogel may be a hydrogel comprising a plurality of non-hydrogel nanoparticles. The biogel may be a hydrogel comprising a plurality of non-hydrogel microparticles. The biogel may be a hydrogel comprising a plurality of non-hydrogel nanorods. The biogel may be a hydrogel comprising a plurality of non-hydrogel nanoshells. The biogel may be a hydrogel comprising a plurality of liposomes. The biogel may be a hydrogel comprising a plurality of non-hydrogel nanowires. The biogel may be a hydrogel comprising a plurality of non-hydrogel nanotubes. The biogel may be a gel in which the liquid component is water. The biogel may be a network of polymer chains in which water is the dispersion medium. The network of polymer chains may be a network of hydrophilic polymer chains. The network of polymer chains may be a network of hydrophobic polymer chains. The biogel may be a degradable hydrogel. The biogel may be a non-degradable hydrogel. The biogel may be a resorbable hydrogel. The biogel may be a hydrogel comprising a polymer of natural origin, such as collagen.

[0282] The method may include subjecting at least a portion of the 3D lymphoid organoid to conditions sufficient to stimulate the production of one or more immune proteins. Conditions sufficient to stimulate the production of one or more immune proteins may include exposing at least a portion of the 3D lymphoid organoid to an antigen so as to stimulate the production of one or more immune proteins. The method may further include extracting one or more immune proteins from at least a portion of the 3D lymphoid organoid. The immune protein may be selected from antibodies, T cell receptors, and cancer immunotherapeutics. The multiple cells in the medium may be from a subject. The multiple cells may be autologous cells. The multiple cells may be allogeneic cells. The multiple cells may be stem cells. The multiple cells may be induced pluripotent stem cells, pluripotent stem cells, embryonic stem cells, or a combination thereof. The multiple cells may be stem cells that can differentiate into B cells, T cells, or a combination thereof. The plurality of cells may be selected from stromal endothelial cells, endothelial cells, follicular reticular cells or precursors thereof, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD+8 T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subsets, macrophages and their subsets, leukocytes and their subsets.

[0283] 3D lymphoid organoids can be selected from B cell germinal centers, thymic developmental niches, lymph nodes, islets of Langerhans, hair follicles, tumors, tumor spheroids, neural bundles or supporting cells, nephrons, liver organoids, intestinal crypts, primary lymphoid organs, and secondary lymphoid organs.

[0284] Fig.21 A four-step process is illustrated in which a biogel is polymerized by an energy source, including but not limited to white light, single-photon or multiphoton laser, to print lymph node organoids or lymphocyte-containing organs in sequential layers 146 .

[0285] like Fig.21 As shown, a base layer 147, two cell layers, a basal cell layer 148, and a secondary cell layer 149 are printed in a sequential order. The layer-by-layer deposition of the biogel layers may use an energy source such as white light or heat to fuse each biogel layer to each other. The secondary cell layer may include one or more sequential layers. The secondary cell layer 149 may include a mixture of accessory cells, T cells, and antigens. The secondary cell layer 149 may include a protective capsule 150. Fig.21, step 1 shows a base layer 147. Base layer 147 may include collagen or other biological, bio-inert or biocompatible materials. Base layer 147 may include cells. Base layer 147 may not include cells. Base layer 147 may be polymerized by light source 1000. In some embodiments, the biogel may not include base layer 147. Base layer 147 may serve as an anchor or link for subsequent layers such as basal cell layer 148 and secondary cell layer 149.

[0286] exist Fig.21 In, step 2 shows one or more new photopolymerizable biopolymers in their monomeric form (e.g., photopolymerizable polymer precursors). The photopolymerizable polymer precursors may contain one or more cell types. In some embodiments, the one or more cell types are lymphocytes or other eukaryotic sources. The photopolymerizable biopolymer may contain any combination of growth factors or cell-responsive proteins. The photopolymerizable biopolymer may include any of the aforementioned energy sources that can be projected by the device for printing. At this stage, the antigen may be contained in the print medium. At this stage, the antigen may not be contained in the print medium.

[0287] Step 3 may include repeating step 2 until the desired number of cell layers / cell types is achieved. Different cell types, media conditions, and / or photopolymerizable biopolymers may be used. Fig.21 , step 3 shows one iteration of producing the secondary cell layer 149. Fig.21 , step 4 shows that the lymph node organoid or lymphocyte-containing organ 146 can be encapsulated in a grid 150 containing few or no cells. This structure can be intended to maintain the integrity of the lymph node organoid or lymphocyte-containing organ during culture and development. Any biological material can be used to generate the grid 150. The cells can be encapsulated in the grid 150. The encapsulated cells can be of any source, including but not limited to antigen presenting cells, stromal cells, or cells previously exposed to antigens and activated for antigen presentation. This final printing step can be used to deposit a structure adjacent to the first layer of anchoring collagen or biomaterial to make a sealed and anchored lymphoid organ.

[0288] Washing steps using a medium that does not contain cells and is not a printable material may occur between any given steps to ensure complete rinsing and removal of previous cells and polymerizable material. The final operation of all processes may require one or more washing steps to remove unwanted biogel and prepare the tissue construct for culture.

[0289] The printing of lymph node organoids or organoids may include 1 to 100 different steps. The printing of lymph node organoids or organoids may include at least 1, 10, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000 or more operations. The printing of organs and / or organoids may include at least 1, 10, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000 or more operations.

[0290] Lymphoid organs and organoids can be printed in a format suitable for high-throughput antibody / receptor production and screening, to include 1, 6, 12, 48, 96 and 384 well plates. So far, there is no method for de novo development of lymphoid organs from human tissue in vitro in a high-throughput form by means of two-dimensional or three-dimensional projection printing of a multicellular environment for the development and receptor screening of B cells or T cells. The introduction of such a high-throughput process has the potential to greatly accelerate antibody production and screening processes. However, larger structures may be needed for specific purposes, or high-throughput methods may not always be needed. Therefore, the scope and utility of the present disclosure are not limited to high-throughput formats.

[0291] At least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10000 or more testable lymphoid organs and / or organoids can be printed by the methods disclosed herein. About 200 testable lymphoid organs and / or organoids can be printed by the methods disclosed herein. About 300 to about 500 testable lymphoid organs and / or organoids can be printed by the methods disclosed herein. About 500 to about 1000 testable lymphoid organs and / or organoids can be printed by the methods disclosed herein. One or more cell types of lymphocytes or other eukaryotic sources separated from a donor can be used to print lymphoid organs and / or organoids. A single donor can be selected based on its genetic traits, disease history, gender and / or race. At least 200, 300, 400, 500, 600, 700, 800, 900, 10000 or more testable lymphoid organs and / or organoids can be generated from a single donor. About 200 testable lymphoid organs and / or organoids can be generated from a single donor. About 300 to about 500 testable lymphoid organs and / or organoids can be generated from a single donor. About 500 to about 1000 testable lymphoid organs and / or organoids can be generated from a single donor.

[0292] Within the printed structure, cells may be encapsulated in or scaffolded to a biological, biocompatible or bioinert gel-forming material or biogel. The materials may be synthetic, partially synthetic or natural. These materials may be independently photopolymerizable, may require a photoinitiator to polymerize, or may be modified to polymerize in the presence of a laser source with or without a photoinitiator. Materials that may be incorporated into the biogel as the sole, primary, secondary or other supplemental component include, but are not limited to: collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin and agar.

[0293] Variable tensile strengths and structures of the cell mesh to be printed can be used in different areas to promote cell motility, cell-cell interactions and recombination. Cells that can participate in peristaltic and cell-cell interaction behaviors can take up, process and deliver antigens to responsive immune cells. Therefore, structures that match the natural lymph node architecture or adopt a mesh framework can be printed to promote cell-cell interactions and motility. Structures with net pore diameters or distances between strands between 2 microns and 50 microns can be printed to promote cell-cell interactions and motility.

[0294] Cell types that may be included in the printed structure or surrounding medium include, but are not limited to, stromal endothelial cells, endothelial cells, follicular reticular cells or their precursors, naive B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subsets, effector T cells and their subsets, CD8+ T cells, CD4+ T cells, regulatory T cells, natural killer T cells, naive T cells or other immature T cells, dendritic cells and their subsets, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subsets, macrophages and their subsets, and leukocytes and their subsets.

[0295] Each of these cell types can be separated from a single donor by blood or tissue donation, and expanded to the number that is enough to print lymphoid tissue by in vitro scheme. Some of these cell types that can be immortalized from a given donor can be immortalized to promote expansion and be used for protein production. In the case of animal-based experiments, cells can also be obtained from cell lines or animal sources. Cells can be derived from circulating cells in the blood, from biopsies of lymph nodes, spleen, bone marrow or other tissues. Cells can be derived from any vertebrate, including companion animals, rodents, large mammals and the human race. Cells for single high-throughput determination can be derived from different individual donors, to increase the possibility of having highly reactive antibodies. Cells can also be derived from donors with desired immunoreactivity, which is defined by medical or disease history, genotype, antibody response or titration or ongoing or induced immune response, to increase the possibility of desired immune response, whether positive or negative. The cell can be derived from a donor based on its age, genetic traits, disease history, sex and / or race.

[0296] Multiple cell layers can be printed with or without chemokines, free-floating or tethered cell signaling molecules, growth factors, cytokines, proteins, biologics, or non-biologics (such as adjuvants or small molecules) added to the print medium. These factors can be stimulatory or inhibitory. Factors can be tethered to collagen or other biogel monomers by cross-linking prior to introduction into the print medium. Adding cell-responsive proteins to specific layers of organoids or the surrounding medium can be used for the purpose of promoting cell organization, cell development, cell motility, and other desired events.

[0297] Growth factors, proinflammatory cytokines, anti-inflammatory chemokines, cell-responsive proteins, soluble receptors and other signaling factors can be incorporated into the printing medium, printing support and / or culture / growth medium. Such factors include, but are not limited to, IFN-γ, TNF-α, TGF-β, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-10, IL-11, IL-13, IL-21, IL-23, soluble TNF receptor p55, soluble TNF receptor p75, soluble IL-1 receptor type 2, IL-18 binding protein, CCL2, CCL1, CCL22, CCL17, CXCR3, CXCL9, CXCL10, CXCL11, etc. The medium conditions can be changed at different time points throughout the printing and / or culture process to promote specific immune events.

[0298] Immune events can be induced by changes in the cell culture medium or components added to the cell culture medium. Examples of induced immune events include cell proliferation; release of specific cytokines and / or chemokines; secretion of receptors and cell secretory proteins (including antibodies); evolution of receptors and cell secretory proteins (including antibodies); or cell changes, including but not limited to cell health, cell morphology, expressed proteins, and cell developmental state.

[0299] Use of printed lymph nodes for generating or evaluating antibodies, T cell receptors, immunological products or immune responses

[0300] Lymphoid organs and organoids can be used to produce new cell-secreted and / or membrane-bound immune proteins, including antibodies and T cell receptors. They can also be used to generate cells or cell lines expressing those proteins and / or genetic sequences, including hybridomas. The lymphoid organs and / or organoids produced by the methods described herein can be used to produce cancer immunotherapeutics. The lymphoid organs and / or organoids produced by the methods described herein can be used to produce T cells. The lymphoid organs and / or organoids produced by the methods described herein can be used to predict the release of cytokines. The lymphoid organs and / or organoids produced by the methods described herein can provide the in vivo cell tissues required for antibody affinity maturation.

[0301] Antibodies and other receptors, cells or cell lines that generate them, and / or genetic sequences that encode them can be generated by antigenic attack. Antibodies and other receptors can be generated from a single blood donation sample. Antibodies and other receptors can be generated from multiple blood donation samples. Antibodies or receptors produced by the methods disclosed herein can be generated in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. Antibodies or receptors produced by the methods disclosed herein can be generated in about six weeks. Antibodies or receptors produced by the methods disclosed herein can be generated in about ten weeks. Antibodies or receptors produced by the methods disclosed herein can be generated in about four weeks to about twelve weeks. Antibodies or receptors produced by the methods disclosed herein can be generated without animal and / or human substitutes. Antibodies produced by the methods disclosed herein can be fully human IgG antibodies. Antibodies produced by the methods disclosed herein can be reactive to protein-based target antigens. The antibodies produced by the methods disclosed herein may have an affinity of at least 0.01 pM, 0.1 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM or more for the target antigen. The antibodies produced by the methods disclosed herein may have an affinity of about 500 nM for the target antigen. The antigens may be introduced into the cell medium and / or the printed scaffold at any stage during the printing process; may be pre-incubated with one or more cell types to be printed (including antigen presenting cells, such as follicular dendritic cells); or may be introduced into the printed lymphoid structure at any point during the development of the lymphoid structure.

[0302] The same antigen can be introduced at multiple time points and / or stages during the printing and development of the lymphoid structure. In addition, multiple different antigens can be introduced into the same lymphoid structure at the same or different time points and / or stages during the printing and development of the lymphoid structure.

[0303] Antigen can be introduced by a variety of methods including, but not limited to, injection, delivery of cells cultured with the antigen, deposition of the antigen on the surface of lymph node organoids in a patch, introduction of the antigen via the lymphatic or circulating microvasculature (if present), or introduction of soluble antigen in the medium surrounding the lymph node.

[0304] The antigens that can be introduced include, but are not limited to, natural or engineered: full peptides, partial peptides, glycopeptides, full proteins or protein subunits, carbohydrates, nucleic acids, live viruses, heat-inactivated viruses, viral particles, membrane-bound or stable proteins, phage-displayed antigens, and whole cells. Antigens can be theoretically harmless stimulants (e.g., in the case of allergic reactions), or can be autoantigens (e.g., in the case of autoimmune diseases or cancer). The generation of antibody libraries can be compatible with phage display, ribosome display, yeast display, bacterial display, mRNA display, and other antibody directed evolution technologies.

[0305] Biological and / or non-biological immune adjuvants intended to promote, prolong, accelerate or elicit an immune response can be introduced simultaneously, before or after the introduction of the antigen. Possible adjuvants include, but are not limited to, alum and Toll-like receptor (TLR) stimulating compounds. The adjuvant can be introduced by the same or different introduction method as the antigen.

[0306] Biological and / or non-biological agents intended to attenuate, delay, slow or stop the immune response may be introduced simultaneously with, before or after the introduction of the antigen. Adjuvants may be introduced simultaneously with, before or after such inhibitors, or may not be introduced in conjunction with the inhibitors. Such agents may include pro-apoptotic agents and / or receptor inhibitors, including small molecules, blocking antibodies and other inhibitors.

[0307] Antibodies, secreted receptors, partial cells or whole cells can be collected after the immune response is complete or partially completed. The extraction method may result in the destruction of the printed lymphoid structures or leave the lymphoid structures intact for future use or analysis. Methods for extracting the desired product may include, but are not limited to: flow cytometry-based cell sorting (FACS), single cell dilution from culture, selection based on surface antibody expression, and selection based on microfluidic methods and channels. Secreted products can be collected by media collection.

[0308] Printed lymphoid organs and / or organoids may alternatively or additionally be used to assess immune events, cellular interactions, cellular changes, and other measurable or perceptible events for purposes including, but not limited to: vaccine development; vaccine evaluation, including the potential to cause damage to the immune system (e.g., safety testing) and evaluating immune responses with or without the introduction of the antigen against which the vaccine is intended to act (e.g., efficacy evaluation); drug testing of biological and / or non-biological drug compounds or drug combinations, including the potential to cause damage to the immune system (e.g., safety testing) and evaluating the effects of drugs on the immune system, whether intentional (e.g., , efficacy evaluation) or unintentional (e.g., side effect testing); screening of biological and non-biological agents with potential immunological effects intended for therapeutic or other uses, especially when using high-throughput printing designs; basic research on immune responses and cellular interactions under various laboratory-induced or naturally occurring conditions; and diagnostic evaluations, including but not limited to mixed donor lymphocyte reactions; for example, for evaluating transplant compatibility and responses to exogenous or self-derived agents or antigens; cancer immunotherapy predictive screening assays; T cell thymic selection assays; T cell clone selection assays from natural repertoires; and / or cytokine storm prediction assays. Antigens used for transplant evaluation and patient immune system responsiveness may include antigens associated with allergies, autoimmune diseases, cancers, beneficial microorganisms and viruses, and / or harmful or potentially harmful microorganisms and viruses.

[0309] Immune events that may be measured or observed for this purpose include, but are not limited to, lymphocyte behavior, activation state, phenotype, proliferation rate, cellular interactions, changes in cellular interactions, and / or expression of internal and external markers of cellular activation.

[0310] Measurements that may be used to assess immune events, depending on existing or non-existing protocols for assessing such immune events, include, but are not limited to: antibody class switching; cytokine-based responses; cellular differentiation; response to adjuvants used in vaccination; response to vaccines; cell proliferation; cell killing; cell phenotype; changes in cell phenotype, whether induced or as part of the natural developmental process; memory cell development; memory cell recall; assessment of donor endogenous memory cell populations; any predictive measurement, including evaluation of immune responses or allergic reactions for drug testing; etc.

[0311] The printed lymphoid organs and organoids produced by the methods described herein can be used to predict cytokine release. Cytokine storm prediction assays can predict cytokine release induced by therapeutic agents. Storm prediction assays can predict cytokine release induced by antibody-based therapeutic agents, small molecule therapeutic agents, cell-based therapeutic agents, peptides, nucleic acids, or any combination thereof. Cytokine storm prediction assays can predict cytokine release in humans, primates, and / or rodents. Cytokine storm prediction assays can measure the levels of inflammatory mediators such as cytokines, oxygen free radicals, and coagulation factors. Cytokine storm prediction assays can measure proinflammatory cytokines, anti-inflammatory cytokines, colony stimulating factors, interferons, interleukins, and / or tumor necrosis factors released due to cytokine storms. The cytokines measured by the cytokine storm prediction assay can be tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-8, IL-10, IL-α, IL-1β, IL-1 receptor antagonist, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte colony stimulating factor (G-CSF), CXCR3, CXCL9, CXCL10 and / or CXCL11.

[0312] Measurement of immune responses can be performed by methods including, but not limited to, using whole cells; sorting or isolating cells based on their cellular phenotype, which can include morphology and / or receptor expression profile; and collecting serum or media for purposes including evaluating secretion rates or specific secreted molecules.

[0313] Methods that can be used to assess cell state or changes in cell state include genetic and phenotypic evaluations, including but not limited to: enzyme-linked immunosorbent assay (ELISA), partial gene sequencing or sequencing of specific genomic sites, whole genome sequencing, gel electrophoresis of proteins or nucleic acids, polymerase chain reaction (PCR), quantitative PCR, reverse transcription PCR, Western blotting, flow cytometry for analyzing cell phenotype or activation state, antibody selection, mass spectrometry, and the like.

[0314] Methods for printing cell-containing structures

[0315] The present disclosure provides methods and systems for printing and using three-dimensional cell-containing matrix. On the one hand, the method of using three-dimensional (3D) cell-containing matrix includes: providing a medium chamber containing a medium, the medium including (i) multiple cells and (ii) one or more polymer precursors. Subsequently, the method may include, according to computer instructions for printing three-dimensional (3D) cell-containing medical devices in a computer memory, guiding at least one energy beam to the medium in the medium chamber along at least one energy beam path patterned as a 3D projection, to form at least a portion of a 3D cell-containing matrix, the 3D cell-containing matrix including (i) at least a subpopulation of multiple cells, and (ii) a polymer formed by one or more polymer precursors. Subsequently, the method may include positioning the 3D cell-containing matrix in the subject.

[0316] In another aspect, a method of using a three-dimensional (3D) cell-containing matrix comprises (i) printing a 3D cell-containing matrix comprising a plurality of cells, and (ii) positioning the 3D cell-containing matrix within a subject.

[0317] On the other hand, a method of using a three-dimensional (3D) cell-containing matrix includes providing a medium chamber containing a first medium. The first medium may include a first plurality of cells and a first polymer precursor. Subsequently, the method may include directing at least one energy beam to the first medium in the medium chamber along at least one energy beam path according to computer instructions for printing a 3D cell-containing matrix in a computer memory, so that at least a portion of the first medium in the medium chamber forms a first portion of the 3D cell-containing matrix. Subsequently, the method may include providing a second medium in the medium chamber. The second medium may include a second plurality of cells and a second polymer precursor. The second plurality of cells may be of a different type from the first plurality of cells. Subsequently, the method may include directing at least one energy beam to the second medium in the medium chamber along at least one energy beam path according to computer instructions, so that at least a portion of the second medium in the medium chamber forms a second portion of the 3D cell-containing matrix. Subsequently, the method may include positioning the first portion and the second portion of the 3D cell-containing matrix in the subject.

[0318] In another aspect, a method of using a three-dimensional (3D) cell-containing matrix includes (i) printing a 3D cell-containing matrix comprising a first plurality of cells and a second plurality of cells. The first plurality of cells may be different from the second plurality of cells. Subsequently, the method may include (ii) positioning the 3D cell-containing matrix in a subject.

[0319] Multiple cells can be from the subject. Multiple cells can be selected from matrix endothelial cells, endothelial cells, follicular reticular cells or their precursors, immature B cells or other immature B cells, memory B cells, plasma B cells, helper T cells and their subgroups, effector T cells and their subgroups, CD+8T cells, CD4+T cells, regulatory T cells, natural killer T cells, immature T cells or other immature T cells, dendritic cells and their subgroups, follicular dendritic cells, Langerhans dendritic cells, dermis-derived dendritic cells, dendritic cell precursors, monocyte-derived dendritic cells, monocytes and their subgroups, macrophages and their subgroups, leukocytes and their subgroups. B cells can be selected from immature B cells, mature B cells, plasma B cells, B1 B cells and B2 B cells. T cells can be selected from CD8+ and CD4+. 3D cell-containing matrix can form sutures, stents, nails, clips, strands, patches, grafts, sheets, tubes, pins or screws. The graft may be selected from skin implants, endometrium, neural tissue implants, bladder wall, intestinal tissue, esophageal lining, stomach lining, skin embedded with hair follicles, and retinal tissue.

[0320] The 3D cell-containing matrix may be about 1 micron (μm) to about 10 centimeters (cm). The 3D cell-containing matrix may be at least about 5 μm to about 10 cm or more. The 3D cell-containing matrix may be at least about 10 μm to about 10 cm or more. The 3D cell-containing matrix may be at least about 100 μm to about 10 cm or more. The 3D cell-containing matrix may be at least about 500 μm to about 10 cm or more. The 3D cell-containing matrix may be at least about 1000 μm to about 10 cm or more. The 3D cell-containing matrix may be at least about 1 cm to about 10 cm or more. The 3D cell-containing matrix may be about at least 5 to about 10 cm or more.

[0321] The 3D cell-containing matrix may be about 1 μm to about 1,000 μm. The 3D cell-containing matrix may be at least about 1 μm. The 3D cell-containing matrix may be at most about 1,000 μm. The 3D cell-containing matrix may be about 1 μm to about 5 μm, about 1 μm to about 10 μm, about 1 μm to about 100 μm, about 1 μm to about 1,000 μm, about 5 μm to about 10 μm, about 5 μm to about 100 μm, about 5 μm to about 1,000 μm, about 10 μm to about 100 μm, about 10 μm to about 1,000 μm, or about 100 μm to about 1,000 μm. The 3D cell-containing matrix may be about 1 μm, about 5 μm, about 10 μm, about 100 μm, or about 1,000 μm.

[0322] The 3D cell-containing matrix may be about 0.5 cm to about 10 cm. The 3D cell-containing matrix may be at least about 0.5 cm. The 3D cell-containing matrix may be at most about 10 cm. The 3D cell-containing matrix can be about 0.5 cm to about 1 cm, about 0.5 cm to about 2 cm, about 0.5 cm to about 3 cm, about 0.5 cm to about 4 cm, about 0.5 cm to about 5 cm, about 0.5 cm to about 6 cm, about 0.5 cm to about 7 cm, about 0.5 cm to about 8 cm, about 0.5 cm to about 9 cm, about 0.5 cm to about 10 cm, about 1 cm to about 2 cm, about 1 cm to about 3 cm, about 1 cm to about 4 cm, about 1 cm to about 5 cm, about 1 cm to about 6 cm, about 1 cm to about 7 cm, about 1 cm to about 8 cm, about 1 cm to about 9 cm, about 1 cm to about 10 cm, about 1 cm to about 2 cm, about 1 cm to about 3 cm, about 1 cm to about 4 cm, about 1 cm to about 5 cm, about 1 cm to about 6 cm, about 1 cm to about 7 cm, about 1 cm to about 8 cm, about 1 cm to about 9 cm, m to about 10cm, about 3cm to about 4cm, about 3cm to about 5cm, about 3cm to about 6cm, about 3cm to about 7cm, about 3cm to about 8cm, about 3cm to about 9cm, about 3cm to about 10cm, about 4cm to about 5cm, about 4cm to about 6cm, about 4cm to about 7cm, about 4cm to about 8cm, about 4cm to about 9cm, about 4cm to about 10cm, about 5cm to about 6cm, about 5cm to about 7cm, about 5cm to about 8cm, about 5cm to about 9cm, about 5cm to about 10cm, about 6cm to about 7cm, about 6cm to about 8cm, about 6cm to about 9cm, about 6cm to about 10cm, about 7cm to about 8cm, about 7cm to about 9cm, about 7cm to about 10cm, about 8cm to about 9cm, about 8cm to about 10cm, or about 9cm to 10cm. The 3D cell-containing matrix can be about 0.5 cm, about 1 cm, about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, or about 10 cm.

[0323] The 3D cell-containing matrix may be at least about 1 μm or larger. The 3D cell-containing matrix may be at least about 5 μm or larger. The 3D cell-containing matrix may be at least about 10 μm or larger. The 3D cell-containing matrix may be at least about 50 μm or larger. The 3D cell-containing matrix may be at least about 100 μm or larger. The 3D cell-containing matrix may be at least about 1000 μm or larger. The 3D cell-containing matrix may be at least about 0.5 cm or larger. The 3D cell-containing matrix may be at least about 1 cm or larger. The 3D cell-containing matrix may be at least about 5 cm or larger. The 3D cell-containing matrix may be at least about 10 cm or larger.

[0324] The 3D cell-containing matrix may contain an agent that promotes vasculature or nerve growth. The agent may be selected from growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid analgesics, non-opioid analgesics, immunosuppressants, immune-inducing agents, monoclonal antibodies, and stem cell proliferation agents.

[0325] Another aspect of the present disclosure provides a system for producing one or more immune proteins, including a medium chamber configured to contain a first medium, the first medium containing a first plurality of cells and a first plurality of polymer precursors. The system may include at least one energy source configured to direct at least one energy beam to the medium chamber. The system may include one or more computer processors operably coupled to at least one energy source. One or more computer processors may be individually or collectively programmed to receive computer instructions for printing three-dimensional (3D) lymphoid organoids from a computer memory. One or more computer processors may be individually or collectively programmed to direct at least one energy source to direct at least one energy beam to the first medium in the medium chamber along at least one energy beam path according to the computer instructions, so that at least a portion of the first polymer precursor forms at least a portion of the 3D lymphoid organoid. One or more computer processors may be individually or collectively programmed to direct at least one energy source to direct at least one energy beam to the second medium in the medium chamber along at least one energy beam path according to the computer instructions, so that at least a portion of the second medium in the medium chamber forms at least a second portion of the 3D lymphoid organoid. The second medium may contain a second plurality of cells and a second plurality of polymer precursors. The second plurality of cells may be of a different type from the first plurality of cells. The one or more computer processors may be individually or collectively programmed to subject the first portion and the second portion of the 3D lymphoid organoid to conditions sufficient to stimulate production of the one or more immune proteins. The one or more computer processors may be individually or collectively further programmed to extract the one or more immune proteins from the first portion and the second portion of the 3D lymphoid organoid.

[0326] Fig.24Devices and materials are illustrated, which may include cells 400 embedded in the materials constituting the device. Materials that can be used to print 3D cell-containing matrices or devices include degradable polymers, non-degradable polymers, biocompatible polymers, extracellular matrix components, bioabsorbable polymers, hydrogels, or any combination thereof. Non-limiting examples of bioabsorbable polymers include polyesters, polyamino acids, polyanhydrides, polyorthoesters, polyurethanes, and polycarbonates. Non-limiting examples of biocompatible polymers include collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic acid-co-glycolic acid (PLGA), poly-1-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar, or a combination thereof. Biocompatible polymers may contain extracellular matrix components. Non-limiting examples of extracellular matrix components may include proteoglycans such as heparan sulfate, chondroitin sulfate, and keratan sulfate, non-proteoglycan polysaccharides such as hyaluronic acid, collagen, and elastin, fibronectin, laminin, nestin, or any combination thereof. These extracellular matrix components can be functionalized with acrylate, diacrylate, methacrylate, cinnamoyl, coumarin, thymine or other side groups or chemically reactive parts to promote crosslinking induced by multiphoton excitation directly or by multiphoton excitation induced by one or more chemical dopants. In some cases, photopolymerizable macromonomers and / or photopolymerizable monomers can be used in combination with extracellular matrix components to produce cell-containing structures. Non-limiting examples of photopolymerizable macromonomers can include polyethylene glycol (PEG) acrylate derivatives, PEG methacrylate derivatives and polyvinyl alcohol (PVA) derivatives. In some cases, the collagen used to produce cell-containing structures can be fibrous collagen such as type I, type II, type III, type V and type XI collagen, facit collagen such as type IX, type XII and type XIV collagen, short-chain collagen such as type VIII and type X collagen, basement membrane collagen such as type IV collagen, type VI collagen, type VII collagen, type XIII collagen or any combination thereof.

[0327] Biocompatible polymers may include other polymerizable monomers that are synthetic and not natural to mammalian tissues, including mixtures of biological materials and synthetic materials. Biocompatible polymers may include photoinitiators. Non-limiting examples of photoinitiators may include azobisisobutyronitrile (AIBN), benzoin derivatives, benzoketal, hydroxyalkyl phenones, acetophenone derivatives, trimethylolpropane triacrylate (TPT), acryloyl chloride, benzoyl peroxide, camphorquinone, benzophenone, thioxanthone and 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone. Hydroxyalkyl phenones may include 4-(2-hydroxyethylethoxy)-phenyl-(2-hydroxy-2-methylpropyl)ketone ( 295), 1-hydroxycyclohexyl-1-benzophenone ( 184) and 2,2-dimethoxy-2-phenylacetophenone ( 651). The acetophenone derivative may include 2,2-dimethoxy-2-phenylacetophenone (DMPA). The thioxanthone may include isopropylthioxanthone.

[0328] Once in place, the device can bring two tissues together, and cells can migrate inside or outside the device, interacting locally with other cells to promote healing and tissue remodeling around or inside the cell-containing bioresorbable device. The cell-containing bioresorbable medical device can be a suture 401 of any length or width, a nail 402, a stent 403 of any length or width, a clip 404 that can be locked or compressed, a patch and a graft 405 of any shape and size, and / or a similar structure intended to be used in a living object. Single or multilayer patches and grafts 406 of any shape and size can be produced from a variety of different cell types to promote tissue development, enhance tissue function and / or healing. Patches and grafts can be made of printed stents of porous or tubular structures to allow for delivery of enough nutrients to embedded cells via perfusion. Grid patches can be used to improve the elasticity and structural support to lymphatic vessels, vascular systems, and nerves, thereby improving the function of transplanted tissues. The graft may include, but is not limited to, skin implants, endometrium, neural tissue implants, bladder wall, intestinal tissue, esophageal lining, stomach lining, hair follicle embedded skin, retinal tissue, or any combination thereof. The device may be printed using synthetic or biological materials that may or may not mimic the natural extracellular matrix scaffold. Materials include, but are not limited to, polyethylene glycol diacrylate, collagen, gelatin laminin, fibrin, and / or alginate.

[0329] Fig.25 Bone resorbable screws 407, pins 408, and other implants of various sizes or shapes are shown, the purpose of which is to hold tissue together and the wound heal while remaining implanted in the body. Fig.25 The examples shown in may include cells. The holographically printed grafts and / or patches may include a variety of shapes, such as but not limited to elliptical, rectangular, oval, any other polygonal shape, or any amorphous shape required to repair or reinforce an injured or diseased site. Fig.26 A first hybrid cell seeded, holographically printed patch 410a and a second hybrid cell seeded, holographically printed patch 410b are shown. Fig.26 As shown, the first and second hybrid cell-seeded, holographically printed patches (e.g., 410a and 410b, respectively) may include cardiomyocytes 411 and / or stem cells 412. The cell-seeded printed patches may include accessory cells, including but not limited to monocytes, fibroblasts, endothelial cells in various states of differentiation, or any combination thereof.

[0330] To enhance the structural integrity of some devices, 3D printed materials may be thicker or denser and may or may not contain cells at all locations. These cells are trapped in holes of any size as they are printed to keep the cells in place or allow them to move from where they were originally printed and interact with other cells within their own layer, cells in subsequently or previously printed layers, or with cells in the native tissue in which they are ultimately implanted. Cells are encapsulated ( Fig. 27 ), embedded, captured or contained 400 in a mesh, grid, matrix 414, framework, with any pore size 413 or density that allows cells to move through the pores or be captured in place during development. This forms the basis component of a larger structural framework.

[0331] Three-dimensional photolithography can be used to generate functional local organs or organoids that can serve enhanced or independent physiological functions without having to rely on the implantation site. Such three-dimensional photolithography can be achieved by holographically projecting light using two light modulation systems in series, as disclosed in co-invented U.S. Provisional Patent Application No. 62 / 469,948, entitled MULTI-PHOTON TISSUE PRINTING, which is incorporated herein by reference.

[0332] Non-limiting examples of tissues for augmentation or replacement of function include kidney or generative models of kidney tissue, lung tissue or partial or complete lung lobes and generative models thereof, neural tissue, pancreatic tissue, insulin-producing beta islets and associated tissues, thyroid tissue, spleen tissue, liver tissue, skin tissue, and intestinal tissue. All tissues listed necessarily include all structural components and accessory cells necessary to confer functional capacity, including but not limited to large and small vasculature and lymphatic drainage system and all associated hollow structures, as well as neural and / or immune cells necessary to confer functional capacity.

[0333] In some embodiments, a printed nephrogenesis model is generated by the methods disclosed herein. The basic structural components of the kidney, including but not limited to: the urine collecting duct, the vascularized and dense tissue surrounding the urine collecting duct, and the renal capsule, can be divided into separate computer-aided design (CAD) files and printed sequentially but in any necessary order by an automatic computer control program 1101. Printing can be achieved by sending the computer file to a laser printing system 110, and the structure mimicking the CAD file can be deposited into the biogel and medium chamber 122 sequentially but in the necessary order.

[0334] The volume of the three-dimensional printed structure for implantation can be on the order of 1 micron to tens of centimeters or more. The surface area of ​​a complex tissue structure (such as a lung) occupies several square meters, so the external size of a larger printed organ will necessarily be different from the surface area of ​​a functional unit. Therefore, the methods and systems provided herein can be designed to cover all structural components within the physiological range of functional size and surface area to volume ratio.

[0335] Laser-based holography can be used to polymerize biomatrix materials nearly instantaneously in fixed patterns projected from computer-aided design (CAD) files through spatial light modulators or digital mirror devices. Building a complete generative model may require multiple printing steps and locations.

[0336] Cells may be in any state of genetic or phenotypic differentiation, including undifferentiated, partially differentiated, fully differentiated. Examples of differentiation states include, but are not limited to, pluripotent stem cells, totipotent stem cells. Cells may be autologous; derived from matched donors, umbilical cord blood, cell and tissue banks; or established cell lines. Multiple cell types in the same and / or different differentiation states may be used in a single print layer and / or multiple iterative print layers. Cells may be genetically manipulated before, during, and / or after the printing process by optical switching technology, clustered regularly interspaced short palindromic repeats (CRISPR) technology, viral introduction, or other genetic manipulations. Genetic manipulation is not limited to nuclear DNA, and may include mitochondrial DNA or free-floating plasmids or viral DNA that are not intended to be incorporated into nuclear DNA.

[0337] Printed structures can include cells at high or variable densities (including unbalanced cell densities or controlled cell densities) to promote cell expansion or niche development in specific parts of the device. High or low cell densities can be used depending on the needs of the tissue product. Low cell densities can be as low as 10,000 cells per cubic centimeter of printed material and as high as 1 billion cells per cubic centimeter of printed material. Cells can be of one type or mixed, and printing can be performed in multiple layers.

[0338] Bioprinted materials may contain agents intended to promote the growth of vasculature, including microvasculature, and nerves into the printed structure or into the surrounding native structure. In addition, the printed biomaterials may contain agents intended to promote the differentiation of stem cells or progenitor cells down to a specific lineage. Such agents include, but are not limited to, growth factors, cytokines, chemokines, antibiotics, anticoagulants, anti-inflammatory agents, opioid or non-opioid analgesics, immunosuppressants, immune inducers, monoclonal antibodies, and / or stem cell proliferation agents.

[0339] Methods for printing three-dimensional structures

[0340] The present disclosure provides methods and systems for printing and using three-dimensional (3D) objects. A method for printing and using a three-dimensional object may include generating a 3D projection corresponding to a first portion of an object in a medium containing at least one polymer precursor, while simultaneously generating at least one additional projection corresponding to at least one additional portion of the object in the medium. The combination of the first projection and at least one additional projection may form a 3D object. At least one additional projection may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 75, 100 or more additional projections. At least one additional portion may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 75, 100 or more additional portions. The at least one additional projection may be at most about 100, 75, 50, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less additional projections. The at least one additional portion may be at most about 100, 75, 50, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less additional portions. The first portion may be the same as the at least one additional portion. For example, multiple first portions may be formed. Alternatively, the first portion may be different from the at least one additional portion. For example, a first portion corresponding to the overall shape of an organ may be formed, and a second portion corresponding to the vascular system of the organ may be formed. The first projection and the at least one additional projection may be simultaneously activated in the same print volume. The first projection and the second projection may coexist within the same print volume. For example, the first projection may solidify the rough features of an object, while the second projection may solidify the fine details of the object. The first projection may be activated before the second projection. For example, the first projection may be activated, and the second projection may be activated subsequently. In another example, the first projection may be completed prior to initiation of the second projection.

[0341] The medium may include two or more polymer precursors. The medium may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 75, 100 or more polymer precursors. The medium may include at...

Claims

1. A method for printing a three-dimensional (3D) object, comprising: (a) directing a first light beam into a medium containing a polymer precursor to generate a 3D holographic projection corresponding to a first portion of the 3D object in the medium to solidify a portion of the medium to produce the first portion of the 3D object, wherein the first light beam is a multiphoton light beam; and (b) directing a second light beam into the medium to solidify at least a portion of a remainder of the medium to produce a second portion of the 3D object different from the first portion of the 3D object, wherein the second light beam is a single photon light beam; Wherein, the first light beam and the second light beam are directed into the medium substantially simultaneously. The method of claim 1 , wherein the multiphoton beam is a two-photon beam.

3. The method of claim 1, wherein the 3D object is a 3D organoid. The method of claim 1 , wherein the first light beam and the second light beam are directed into the medium through the same light path. The method of claim 1 , wherein the first light beam and the second light beam are directed into the medium through the same optical element.

6. The method of claim 5, wherein the optical element is a phase and / or amplitude modulator.

7. The method of claim 6, wherein the phase and / or amplitude modulator is a digital micromirror device.

8. The method of claim 6, wherein the phase and / or amplitude modulator is a spatial light modulator.

9. A method for printing a three-dimensional (3D) object, comprising: (a) generating, within a medium comprising at least one polymer precursor, a first 3D projection corresponding to a first portion of the 3D object, wherein the first 3D projection comprises a substantially simultaneous holographic array of a plurality of points, wherein the first 3D projection is generated using a first light beam, the first light beam being a multiphoton light beam; and (b) generating at least one additional projection corresponding to at least one additional portion of the 3D object different from the first portion of the 3D object substantially simultaneously with (a), wherein the at least one additional projection is generated by at least one additional light beam comprising a single-photon light beam, and wherein the first projection and the at least one additional projection form the 3D object within the medium.

10. The method of claim 9, wherein the first projection and the at least one additional projection are projected along the same set of components. The method according to claim 10 , wherein the same set of components comprises the same objective lens.

12. The method of claim 9, wherein the first projection and the at least one additional projection are projected along different sets of components. The method of claim 12 , wherein the different sets of components are capable of generating a projection perpendicular to the first 3D projection. The method of claim 12 , wherein the different sets of components are capable of generating a projection parallel to the first 3D projection.

15. The method of claim 12, wherein the different groups of components do not share a common optical axis.

16. The method of claim 12, wherein the different sets of components include one or more objective lenses of different focal lengths.

17. The method of claim 9, wherein the medium is rotating. The method of claim 9 , wherein the at least one additional projection is rotated.

19. The method of claim 18, wherein the at least one additional projection is rotated by rotation of one or more optical elements configured to generate the at least one additional projection.

20. The method of claim 9, wherein the first 3D projection and the at least one additional projection are projected simultaneously.

21. The method of claim 20, wherein the first 3D projection and the at least one additional projection are initially simultaneous.

22. The method of claim 21, wherein the at least one additional projection stops before the first 3D projection.

23. The method of claim 9, wherein the first 3D projection and the at least one additional projection are consecutive.

24. The method of claim 23, wherein the first 3D projection is projected after the at least one additional projection.

25. The method of claim 9, wherein the first 3D projection and the at least one additional projection comprise a combined additive and subtractive manufacturing method.

26. The method of claim 25, wherein the at least one additional projection generates a form of the object in the medium.

27. The method of claim 26, wherein the first 3D projection removes material from the form of the object.

28. The method of claim 9, wherein the at least one additional projection comprises five additional projections.

29. The method of claim 9, wherein the first 3D projection is generated without using a digital micromirror device (DMD).

30. The method of claim 9, wherein the at least one additional projection is generated without the use of a digital micromirror device (DMD).

31. The method of claim 9, wherein: The 3D object is a 3D organoid.

32. The method according to claim 31, wherein: The 3D organoids are lymphoid organoids.

33. The method according to claim 31, wherein: The 3D organoids include B cell germinal centers, thymic developmental niches, lymph nodes, islets of Langerhans, hair follicles, tumors, tumor spheres, nerve bundles or supporting cells, kidney units, liver organoids, intestinal crypts, primary lymphoid organs, and secondary lymphoid organs.

34. The method of claim 31 , further comprising exposing the 3D organoid to an antigen so as to stimulate the production of one or more immune proteins.

35. The method of claim 34, wherein: The antigen is a whole peptide, a partial peptide, a glycopeptide, a whole protein or a protein subunit, a carbohydrate, a nucleic acid, a live virus, a heat-inactivated virus, a virus particle, a membrane-bound or stabilized protein, a phage-displayed antigen, and a whole cell.

36. The method of claim 34, wherein: The one or more immune proteins include antibodies, T cell receptors, and cancer immunotherapeutics.

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