Control unit and 3D printer

Abstract

The present application relates to a control unit and a 3D printer comprising such a control unit for controlling a printhead supply unit, a motor for a printer table, a device for applying electromagnetic waves and a droplet printer for producing a simulated high-resolution print, the simulated high resolution print is based on a print model for 3D printing of tissues and organs including vascular structures.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of August 3, 2020, the national application number 202080056040.1 (PCT / EP2020 / 071804), and the invention name “Method for 3D printing vascularized tissues and organs”.

[0002] The present invention relates to a 3D printing method for manufacturing tissues and organs including vascular structures. The novel and innovative core of the method lies in the following points: 1)

[0004] The specialized capillary ink reacts only in the edge region of the printed droplets near the droplets of other ink types, thus producing a single cell layer from which extremely fine capillaries are formed. The uncrosslinked cells in the interior of the droplets are washed away and form the inner cavity of the capillaries. 2)

[0006] A new type of printed model is disclosed, characterized by tissue planes built from orderly applied different cell types and traversed by spatially incised internal hollow vessels (veins, arteries and capillaries).

[0007] The printing model is characterized by the subdivision of larger tissues or organs into individual modules and the simplification of the printing image for an economical printing process. The individual modules printed independently of each other are bonded together to form a higher-level target structure. 3)

[0009] A novel printer table is provided within the 3D printing chamber containing the medium, which ensures that the culture medium is supplied to the printed tissue so that the cells do not die. 4)

[0011] A controllable print head supply unit is provided which mixes the cell concentrate and the ink concentrate and the composition of the ink (eg from different ink tanks) per unit volume for the ink droplets as specified by the printing algorithm and supplies the print head with the mixed ink.

[0012] Preface:

[0013] Due to their specific characteristics and the pros and cons associated therewith, conventional 3D printing systems do not possess the properties required to be able to produce larger, viable tissues and organs.

[0014] In order to produce larger viable tissues or organs by means of 3D printing, it is necessary in particular to ensure the supply of the printed cells. Since the supply of cells can only take place over a distance of about 500 μm by diffusion, a vascular system that is also printed is required. The capillary system is located between the arteries and veins, the individual capillaries of which have a very small diameter of only about 10 μm and are also built up from a cell layer of single cells.

[0015] The methods of 3D printing technology known to date either allow different cell types to be printed simultaneously in one step, but the necessary high printing resolution cannot be achieved for this (e.g. fused deposition modeling or droplet technology), or the required high printing resolution is achieved, but different cell types cannot be selectively printed in one working step (e.g. stereolithography).

[0016] In addition, there are specific 3D printing systems, such as Laser Induced Forward Transfer, in which individual cells are extracted from a matrix by a laser beam and guided to a collector matrix. Here, both high printing resolutions can be achieved and different cell types can be printed in one step. However, the achievable printing speeds are too low, so that this technology can only be used for very small structures.

[0017] Another specific printing system is a modification of 3D stereolithography, in which different cell types can also be printed. However, for this purpose, in particular, the tissue to be printed must be changed between reaction tanks with different cell types. This step also allows only very few cell layers and is a relatively slow process, with which only small structures, such as miniature organs, can be printed.

[0018] Other specific 3D printing methods leave space for subsequent blood vessels in the form of cavities in the printed tissue and first feed the surrounding cells with culture medium. In order to produce real capillaries, these tissues must be transformed into organisms that supply endothelial cells and the muscle cells surrounding them to the cavities. This step can also be done afterwards in an incubator. If in this way, capillaries can eventually be produced in the tissue, the process still takes at least several days and, despite this, the natural complexity of the relevant tissue is still not reached. In addition, using these technologies, only relatively large vessel diameters can be achieved.

[0019] Another possible solution is to 3D print scaffolds, which produce relatively simple tissues from slow-growing cells such as cartilage or bone, and then multiply the cells on the scaffold. In these slow-growing tissue types, due to the low metabolic rate, there is enough time to form capillaries in the body afterwards. However, this operation is limited to tissue types such as bone and cartilage. For simpler cell types that grow more quickly, such as liver cells, it has been shown that very small tissue areas can be successfully transplanted into the liver organ. However, these tissue areas can only consist of very small structures, because otherwise the cells can no longer be fed.

[0020] All previous attempts to use 3D printers to create functioning tissues or organs have resulted in either very small or very slow-growing tissue units, or in larger structures that survived only very briefly due to a lack of significant capillaries.

[0021] Therefore, a 3D printing method is needed, which

[0022] - On the one hand, different cell types can be selectively printed in one working step, and

[0023] - On the other hand, the high printing resolution of the stereolithography method can be achieved.

[0024] - Additionally, the method should have printing speeds that allow for the printing of virtually entire organs.

[0025] Furthermore, the novel printing system should, in particular, already at an early stage during the printing process, thoroughly flush the newly formed tissue with a medium or another liquid suitable for feeding the cells via the capillaries created during the process.

[0026] In particular, the printing method should also provide a novel printing model, by means of which it is possible to form a capillary system between the arterial structure and the venous structure, in which a fluid (eg a medium or blood) can flow from the artery to the vein in the capillary system.

[0027] - In the main embodiment, the realization of the novel printing method requires a novel printer table, since conventional printer tables in the prior art are not able to supply culture medium to the printed tissue during the printing process.

[0028] - Implementation of the novel printing method preferably requires a print head supply unit that mixes the desired cell density and the required ink concentration in the printed droplets as specified by the printing algorithm.

[0029] The object of the invention is achieved by a novel UV-crosslinked droplet method which, due to its low-viscosity ink, allows the use of realistic high-resolution print heads and, in addition, increases the printing resolution to the molecular level by highly specific, spatially confined reactions in the edge region of the individual droplets.

[0030] The droplet method for printing cells to date involves devices in which relatively large droplets consisting of a hydrogel ink and the cells located therein are separated from the print head (thermally or piezoelectrically, see Figure 1 to Figure 2 -7). Usually, there is only one printer nozzle for one cell type ink. This nozzle has a relatively large diameter because the viscosity of the hydrogel in the sol state is still relatively high ( Figure 1 and Figure 2-7 The resulting droplets are relatively large and typically contain a large number of cells (about 2000 cells per droplet).

[0031] Surprisingly, it has been shown that by using a newly formulated photosensitive cell ink instead of the hydrogel used previously, the viscosity of the cell ink droplets can be significantly reduced, making them processable by print heads from the field of high-resolution artistic printing. This was not possible before. As a result, cell ink droplets of several picoliters (each droplet containing about 20 cells) can be printed ( Figure 8-10 ). With this printing resolution, it is already possible to print capillaries with a significantly smaller diameter than the capillaries of previous systems. In addition, it has been shown that the printing resolution can be further increased by configuring the ink so that the crosslinking reaction occurs only in the edge area of ​​the capillary ink droplet. In contrast, the remaining droplet volume is not subjected to the crosslinking reaction and is subsequently rinsed away or removed in other ways in order to be subsequently used as a capillary cavity. Through this novel printing process, the printing resolution can be increased to the molecular level or the order of magnitude of a single cell or cell layer and thus achieve the fineness of a stereolithography system. The capillaries that can be printed with this method can be as fine as human capillaries.

[0032] The crosslinking limited to the edge regions of the capillary droplets can be achieved either by the ink components or by a directed laser beam (or another directed electromagnetic wave). The reaction carried out here can be physical, chemical or biological (see the Examples section).

[0033] During the entire printing process, the produced tissue is preferably thoroughly flushed with culture medium through the capillary structure. To this end, a specific printing model is produced during the printing process, which arranges the formation of capillaries between the future arterial and venous structures.

[0034] If an entire organ is to be printed and later transplanted into the body or patient, the organ can be connected to the patient's blood circulation outside the body in a specially designed device for this purpose for a limited period of time before transplantation. This device can be used to check the physiological values ​​of the organ and determine the optimal time for transplantation. Background technology:

[0035] In Germany, up to 15,000 patients are waiting for a donor organ. Only less than one-third of these patients will find a suitable organ. For research and medicine, about three million animal experiments are carried out in Germany each year, which would become redundant due to alternatives. Another area where 3D printed tissues continue to make great progress is regenerative medicine.

[0036] 3D bioprinting can offer a solution for these and other application areas. With 3D bioprinting, cell structures are produced with a 3D printer, whereby cell planes are stacked in additive manufacturing in order to thereby form complex three-dimensional structures. In particular, techniques such as fused deposition modeling, droplet methods and stereolithography are suitable for bioprinting.

[0037] Fused deposition modeling is a printing method based on extrusion, which processes thermoplastics and composite materials as well as ceramics and metals. With this, cells in alginate or gelatin gel can also be printed. The disadvantage of this method is particularly low printing resolution. The droplet method processes polymer solutions, colloidal suspensions and cell suspensions. Although the resolution of the droplet method is higher than that of fused deposition modeling, it is still not enough to print vascular structures. In contrast, due to the use of a fine laser beam, stereolithography has the highest resolution that can be achieved so far. It processes photohardenable viscous polymer solutions, which are exposed to directional electromagnetic beams to crosslink the solution in space. Its disadvantage is that only a unique cell type can always be printed in the reaction tank. If another cell type is to be added, the reaction bath must preferably be replaced.

[0038] Inkjet bioprinting by market leader Organovo is described in the prior art, which produces ink droplets that each contain 10,000 to 30,000 individual cells (source: Organovo). Organovo uses bioprinting to produce liver tissue in particular. The 3D-printed tissue parts are usually implanted into the damaged organ, which in their way supports the existing liver and thus prolongs the life of the organ until a suitable donor is found.

[0039] In extrusion bioprinting, bioink is ejected from a small needle using mechanical pressure. This allows the entire cell structure to be printed layer by layer. Envisiontec GmbH's bioprinters use this method, for example. It works relatively quickly, but unfortunately the print resolution is very low.

[0040] For example, the companies Cellbricks and TissUse ("organonachip") use stereolithography. Since the hydrogel is crosslinked by means of a laser beam, it offers the highest printing resolution. However, only one cell type can be printed with this method. In order to combine tissues composed of various cell types, the produced tissues must preferably be replaced by different reaction tanks, which slows down the process and the printed tissues can only be of very small sizes.

[0041] In addition to these most commonly used methods, there are a number of methods that are derived from modifications:

[0042] Some techniques aim to spontaneously form very fine capillaries (up to 10 μm in diameter) between larger printed veins and arteries during cell culture (Yu et al., 2014). The problem with this is that it takes longer for the printed tissue to fully mature. The longer the tissue is kept in cell culture, the greater the possibility of artifacts.

[0043] Yet another approach is to print a hose-like structure with a polymer that is then depolymerized and washed away again, and should thereby release a cavity for forming a vascular system (Kolesky et al., 2014; Lee et al., 2014a,b). However, this approach is inadequate and differs from the novel method disclosed here in two ways: 1) it is a hose without endothelial cells and extremely thin branches, requiring longer-term culture (including predictable artifacts), and 2) the printing resolution is too low to produce a realistic capillary system.

[0044] Another solution is dual printing: first, the vascular network is printed with a stereolithography system (which has high resolution, but can only print a single cell type). Subsequently, the printed vascular structure is transferred to another 3D printing system (which has a lower resolution, but can print different cell types), in which the remaining tissue cells are printed (Yu et al., 2014). To this end, in particular, the printed vascular structure must be removed from the reaction vessel of one of the 3D printing systems and transferred to the reaction vessel of the other 3D printing system. The special difference from the method disclosed here is that: 1) single-cell cell layers cannot be printed, and these structures will be too fragile when replaced, and 2) two different printers are required, and the risk of contamination increases when replaced.

[0045] In order to be able to realize the method disclosed herein, in a preferred embodiment, a printer station is particularly required, which can supply culture medium for the printed tissue, and cells will not be insufficiently supplied or die. In addition, the printer station preferably must be located in a printing chamber filled with liquid.

[0046] The printer table and printing chamber of the traditional 3D printing method are not available for this. For example, most 3D printing systems (such as FDN, SLS, BJ, etc.) have a dry printer table in a dry printing chamber and do not have any possibility to contain liquids.

[0047] Although a few 3D printer types are equipped with a printing chamber filled with liquid (e.g. SLA), there is no supply system for the printed tissue that includes additional liquid inlets and outlets, as is required when 3D printing tissues and organs including vascular structures.

[0048] The novel printing system disclosed herein is based on the droplet method and differs from previous droplet systems in a number of aspects listed below:

[0049] 1) New photosensitive bio-inks are used, which are low-viscosity so as not to clog the fine print heads of realistic art printers. Previous ink droplets contained about 2000 to 3000 cells and were composed of hydrogels (Organovo). The hydrogel component is the reason why the droplets are relatively large, because even in the sol state, their viscosity is relatively high. The droplets produced in the method disclosed here are very small, low-viscosity and contain about 20 cells or less. The printed cell clusters are achieved by having a print head for art printing with about 160 or more nozzles, instead of only one nozzle per ink type (as used until now).

[0050] At the same time, the ink has a formulation that allows direct crosslinking and thus prevents the droplets from melting. The photosensitive hydrogels that have been available in the droplet method so far have used a longer hardening time that is triggered by a light source outside the print head and lasts for several minutes. In the novel method disclosed here, the source of electromagnetic waves (e.g. visible light, IR, UV) is located directly in the print head and can be both rigid and directional. 2)

[0052] The specific bio-ink (capillary ink) in the novel printing system disclosed herein is selectively cross-linked only in the edge region of the droplet or in the region adjacent to the droplet of other inks. The selective cross-linking can be triggered by the composition of the ink or by directed electromagnetic radiation. The type of cross-linking can be biological, chemical or physical. The droplet components of the specific bio-ink that are not in the edge region are washed away or otherwise removed, thereby forming the necessary cavities for the generated capillaries.

[0053] The uncrosslinked capillary cells (eg endothelial cells) that are swept into the surrounding tissue during this process become, for example, the starting point for new capillary structures later on or disappear during tissue differentiation.

[0054] Through this process, it is possible to achieve a printing resolution in which only single molecules or single cells are crosslinked in a targeted manner. To date, only high-resolution stereolithography methods have the capability to crosslink single cells. In this case, the droplet size is preferably in the order of magnitude between one femtoliter and 10 microliters.

[0055] The resulting printed images acquire new qualities that were previously unattainable, including

[0056] - On the one hand, it is possible to realistically print different ink types simultaneously (e.g., muscle cells, nerve cells, structural ink, capillary ink), and wherein

[0057] - On the other hand, by selectively limited edge cross-linking of capillary inks, the printing resolution is high enough to form single cell layers (e.g. endothelial cells). 3)

[0059] By forming a load-resistant vascular system as early as during the printing process, it is possible to fully supply the tissues or organs produced as early as during the printing process. The interface of the pump system required for this is first formed by the printer table and the printing plate ( Figure 2-7 ) is provided. In order to produce individual, still more delicate vascular and capillary interfaces for each tissue structure, individual interfaces are printed starting from these interfaces. In this case, in a preferred embodiment, capillary ink and tissue ink can be utilized, or structural ink can be utilized first.

[0060] If a structured ink is used in this case, a gradient transition between the structured ink and the cell ink can be achieved, thereby stabilizing the system. To date, there has not been any droplet technology in which such a supply system for printed cells exists.

[0061] After successful printing, the removable printed foil (on which the finished print is located and which has inlet and outlet connections for liquids) can be further incubated in an incubator. Fig.19 ). 4)

[0063] A new complex printed model is disclosed, which makes it possible to form capillaries between arterial and venous structures, in which a fluid (e.g., a medium or blood) flows from the arterial region into the venous region. Such a printed model is unknown to date and cannot be realized due to the current state of the art.

[0064] The printing model is therefore unique because different cell types can be printed in an orderly manner in a single printing process and structures as large as only a few molecules can be produced in an orderly manner at the same time.

[0065] Preferably, the printed model is characterized by the following properties:

[0066] - Specific algorithms for calculating the printed image for the cell type (ink type) used and for the distances and sizes of capillaries and arterial and venous structures. In this case, the algorithm depends on the requirements for an optimal capillary supply of the module to be printed on the one hand and on the physiological and anatomical requirements of the corresponding tissue structure on the other hand.

[0067] - Tissues / organs consisting of different tissue types and vessels are subdivided into individual modules, which are in turn subdivided into printing planes (slices). The different printed cell types on the printing planes in a module correspond to their vascular position or tissue position in the respective tissue or organ.

[0068] - Preferably, a comprehensive capillary system is produced with an inner diameter of 5 micrometers or more. In each single printing plane, the distance between the capillaries is preferably about 50 micrometers. After the printing is completed, the liquid flowing therein flows from the artery to the vein.

[0069] - The sum of the individual printing planes forms a single module. The module preferably has ascending and descending vessels ( Fig.15 and Fig.16 ), which are interconnected by smaller capillaries that radiate in a fan-shaped pattern between them. The interfaces for arterial input and venous output come from ascending and descending vessels. The resulting microcirculation represents the smallest supply unit in the printed tissue.

[0070] - Assemble the basic modules and glue them into larger tissues or organs. The ascending and descending vessels from the microcirculation merge here into the main vessels of input and output. 5)

[0072] A novel printer table is provided, which can be moved in the Z direction of a printing chamber containing a medium and has supply interfaces, through which the medium can be supplied in an adjustably manner for printed tissue. 6)

[0074] A print head supply unit is provided in which the cell density and ink concentration are mixed in an adjustable manner. The mixed ink for the print head is combined as specified by the printer algorithm. In principle, there are tissue inks, capillary inks and structural inks in the method, wherein other ink types may also occur. Summary of the invention:

[0075] A 3D printing method is needed that

[0076] - Different cell types can be selectively printed in one working step, and

[0077] - Achieve high printing resolutions using stereolithography methods.

[0078] - Additionally, the method should have printing speeds that allow for the printing of virtually entire organs.

[0079] - In addition, the novel printing system should preferably flush the newly formed tissue thoroughly with a medium or other liquid suitable for feeding cells, through the capillaries created during the printing process, as early as possible. Fig.16 ).

[0080] -Preferably, the printing method should also provide a new type of printing model, through which it is possible to form a capillary system between the arterial structure and the venous structure, and the liquid flowing therein (such as medium or blood) can flow from the artery to the vein in the capillary system.

[0081] The printing system should in particular be equipped with a device which ensures that the concentration of cells per unit volume in the ink droplets is sufficiently high without the cells agglomerating in the printing head in the process.

[0082] The preferred process of this method is:

[0083] - providing at least one capillary ink and another bio-ink comprising cells and cross-linked molecules in a droplet printer;

[0084] - applying at least one drop of capillary ink and bio-ink on the reaction surface;

[0085] - bringing electromagnetic waves into contact with the cross-linked molecules within these droplets on the reaction plane;

[0086] - And by means of the non-directional or directional movement of electromagnetic waves in the droplets, the cross-linking molecules are activated, thereby forming a cross-linking structure and thus obtaining a vascular structure.

[0087] - providing at least one capillary ink which is cross-linked only in the edge region of a droplet adjacent to other ink droplets

[0088] - Flushing out or removing the uncrosslinked droplet components of the capillary ink

[0089] - Provide low-viscosity bio-inks that can be cross-linked by electromagnetic waves, which can be processed by the high-resolution print head of a virtual printer and cross-linked directly under the influence of electromagnetic waves

[0090] - a supply system for providing printed cells in a printer including a culture medium (provided via a printer station); Other preferred processes are:

[0091] - Providing a print head supply unit which preferably adjusts the cell density and ink concentration in the ink droplets as specified by the printing algorithm and directs the mixed ink to the print head.

[0092] - A novel printer table inside the 3D printing chamber containing the medium ensures that the printed tissue is supplied with culture medium so that the cells do not die

[0093] - Provide specific algorithms for calculating the printed image for the cell type (ink type) used and for the distances and sizes of capillaries and arterial and venous structures. In this case, the algorithm depends on the requirements for an optimal capillary supply of the module to be printed on the one hand and on the physiological and anatomical requirements of the corresponding tissue structure on the other hand.

[0094] The sum of the individual printing planes preferably forms a single module. The module is provided with at least one ascending and descending vessel, which are interconnected by fan-shaped capillaries. The interfaces for arterial input and venous output are from the ascending and descending vessels. The resulting microcirculation represents the smallest supply unit in the printed tissue.

[0095] - Combining individual modules and gluing them into larger target structures, where the input and output vessels form a common connecting vessel.

[0096] The 3D printing method according to the invention is realized by a high-resolution droplet printer and a device for applying electromagnetic waves (preferably a laser, an ultraviolet lamp or a diode). The ink used here is a low-viscosity ink that can be processed by a high-resolution print head of a realistic art printer and crosslinked directly under the influence of electromagnetic waves, preferably within one second. The volume of the ink droplet can be from 1 femtoliter to 10 microliters.

[0097] The 3D printing method according to the invention also comprises at least one capillary ink, which is selectively cross-linked in the edge region of the droplet or in the region adjacent to the droplet of other inks. The selective cross-linking is either caused by ink components (of the capillary ink and / or the adjacent biological ink) or by directional electromagnetic waves that cross-link individual cells in the edge region of the capillary ink droplet. The uncross-linked regions of the capillary ink are rinsed off or otherwise removed, and cavities for the capillary system and the vascular system are formed.

[0098] By selectively and spatially confined crosslinking in the capillary droplet edge region, not only individual cells but even individual molecules can be activated for crosslinking reactions or for forming additional layers. The printing resolution achieved by this process is even higher than the approximately 20 nm printing resolution of stereolithography methods.

[0099] An exemplary reaction in the edge region of a capillary ink droplet may be:

[0100] - Thiol-ene reaction

[0101] - Key lock reaction

[0102] - Nucleophilic ring opening

[0103] - Self-assembly of molecules and particles

[0104] - Selective factors on cells of capillary inks and / or bio-inks

[0105] - Free radical or cationic polymerization

[0106] - Antibody response

[0107] - Reactions with click chemistry components (e.g. cycloaddition, etc.)

[0108] Other processes in which it is possible for two adjacent edge regions to undergo a spatially restricted reaction or interaction with one another, or in which “self-assembly” processes occur.

[0109] Because the ink composition in the ink droplets required for the corresponding tissue printing is variable according to the cell density and ink concentration and is determined by the printing algorithm,

[0110] Therefore, an object or an aspect of the present invention is to provide a print head supply unit ( Figure 8). In the print head supply unit, the cells cultivated in the cell tank are pumped into the cell concentrator by means of a pump. Here, the cell density is concentrated and excess medium is separated. The defined cell concentration is transferred to the mixing unit via a cell counter. Likewise, a specific volume is introduced from the tank for the ink concentrate into the mixing unit. The mixing unit mixes the ink according to the specifications of the printing algorithm for the desired tissue type and transfers it to the print head. The number of print head supply units depends on the number of inks required and can vary. However, it is also possible not to mix the ink according to the specifications of the printing algorithm.

[0111] In order to keep the printed tissue alive, it may be necessary, for example, to connect it to a supply circuit as early as during the printing process. Furthermore, during the printing process, the printed tissue must preferably be located in a printing chamber containing the medium, so that it does not dry out. Conventional printer stations do not have such a supply system. Therefore, one object or one aspect of the present invention is to provide a printer station that supplies the tissue with culture medium ( Figure 2-7 ).

[0112] Liquids for the inflow and outflow of the medium are, for example, guided from the outside to the printer via an adjustable pump system. The delivery direction of the pump is reversible. In particular, the input is achieved via a hose system, which is connected to the printer table base via an interface connection (e.g. a Luer connection). The medium is guided via a pivot of the printer table base through the printer table to the printing plate. Between the printer table base and the printing plate there is a replaceable silicone diaphragm, which separates the printing chamber containing the medium from the base plate and seals the medium ( Figure 2-7 ). The silicone diaphragm is necessary for sealing because the floor of the print chamber is penetrated by the pins of the micrometer screw and allows the printer table to travel up and down.

[0113] According to experience, conventional sealing systems such as sliding ring seals are potential sources of contamination. The new solution to this problem, which is achieved by means of a removable silicone diaphragm, has the advantage of being easy to clean and sterilize. In addition, it can be replaced inexpensively. Due to its high degree of flexibility, the printer table settings can be freely selected. The silicone diaphragm simultaneously serves as a seal between the base plate and the print chamber wall.

[0114] In order to be able to replace the printer table (inside the printing chamber) without any problems, the printer table base (outside the printing chamber) is provided in particular with magnets that attract the printer table. By means of the magnetic force, the connecting pivots of the printer table base are also pressed into the seals of the counter-drilled holes in the printing plate and sealed. This preferred solution according to the invention makes the operation when replacing the printer table extremely simple.

[0115] In order to be able to adequately supply it, the tissue to be printed should be penetrated by a large number of vessels ( Fig.15 ). In order to be able to provide the printing model required for this, a specific algorithm is particularly needed for the cell type (ink type) used and for the distance and size of the capillaries and arterial and venous structures. In this case, in particular, the algorithm depends on the need for an optimal capillary supply of the module to be printed on the one hand, and on the physiological and anatomical requirements of the corresponding tissue structure on the other hand. Tissues / organs composed of different tissue types and blood vessels are specifically subdivided into individual modules, which are further subdivided into printing planes (slices). The different printed cell types on the printing planes in the modules correspond to their vascular positions or tissue positions in the corresponding tissue or organ. In particular, a comprehensive capillary system with an inner diameter of capillaries of about 10 microns or more is generated. On each individual printing plane, the distance between the capillaries is preferably about 50 microns. After the printing is completed, the liquid flowing therein flows from the artery to the vein ( Fig.15 ).

[0116] The sum of the individual printing planes forms a single module. The module preferably has ascending and descending vessels, which are interconnected by smaller capillaries in the form of a fan between them. The interfaces for arterial input and venous output come from the ascending and descending vessels. The resulting microcirculation represents the smallest supply unit in the printed tissue. The individual modules are combined and glued together to form a larger target structure.

[0117] The vascular system, on which the vascular tubes are printed, preferably has the required connection openings located on a printing plate fastened to the printer table. Figure 2-7 ). The function of the printing plate is to distribute the media from the two main interfaces of the printer workbench to the individually required auxiliary interfaces. Because each tissue is constructed anatomically differently, a plate should be created separately for each printing process. Each printing plate is made by 3D printing (such as FDM, SLS, BJ, etc.) in the preparation stage. The data for printing also comes from the system algorithm for arranging the printer. The printing plate corresponds to a working device created separately for printing and is preferably made of a polymer.

[0118] A printing foil, preferably made of a biodegradable or absorbable polymer, is applied to the printing plate and has the same interface as the printing plate. The printing foil facilitates easy and non-destructive removal of the tissue from the printing plate. Due to the biodegradability of the polymer, the printing foil decomposes in the body within a short time ( Figure 2-7 ).

[0119] In order to be able to mature the printed tissue without problems in a universal incubator, the printer table together with the printing plate (optionally only the printing plate) is transferred to an incubator vessel stored in the incubator ( Fig.19 ). The incubator vessel is composed in particular of a trough containing the medium and a cover to be placed. A base is fixedly mounted at the bottom of the trough, on which a printer table base of identical structure is replaceably arranged. The medium hoses leading to the input and output of the printer table base are introduced into the incubator vessel through openings. The printer table with the printing plate, printing foil and printed tissue is placed on the pivot of the printer table base by means of magnets (without silicone diaphragm) ( Fig.19 ).

[0120] Other implementation plans:

[0121] In a preferred embodiment of the invention, the droplet size is in the range of 1 femtoliter to 10 microliters. Advantageously, this droplet size allows an excellent implementation of the invention in the thiol-ene reaction. The thiol-modified molecules inside the droplet do not crosslink under these conditions and are washed away.

[0122] In another preferred embodiment, the capillary ink has thiol-modified molecules, and the surrounding bio-ink has allyl-modified molecules and a photoinitiator for triggering a cross-linking reaction. The cross-linking reaction occurs between the allyl-modified molecules within the bio-ink. The thiol-modified molecules of the capillary ink are selectively cross-linked with the allyl-modified molecules only in the edge region.

[0123] In another preferred embodiment, the copper(I)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes induces a crosslinking reaction in the edge region of the capillary ink, wherein the azide-modified molecules and the alkyne-modified molecules meet only in the boundary region between the capillary ink droplet and the bio-ink.

[0124] In another preferred embodiment, the capillary ink contains epoxy-modified molecules that can cause nucleophilic ring opening with OH groups or NH2 groups of molecules from the bio-ink, or undergo acid-catalyzed or base-catalyzed ring opening due to components from the bio-ink.

[0125] In another preferred embodiment, the capillary ink and the bio-ink contain molecules that form self-assembled structures within the boundary region, for example via ions, aptamers or other structures that tend to self-assemble.

[0126] In a further preferred embodiment of the invention, it is provided that the cells contain selective factors, by means of which they are selectively cross-linked by means of the directed movement of electromagnetic waves within the droplets.

[0127] The new low-viscosity bio-ink and capillary ink that can be cross-linked by electromagnetic waves are composed of the following components: a) the corresponding cell type (optionally including selective factors for the cell type) and cross-linking molecules. In addition, growth factors, signaling molecules, particles for constructing stimulus conduction or other molecules can also be components of the ink.

[0128] By means of a selectively acting energy source, a crosslinking reaction or a polymerization reaction can be triggered from a droplet comprising a mixture of different cells, wherein only the respectively expected cell types are involved. Preferably, uncrosslinked cells can be removed. For example, a small droplet of only 5 picoliters (approximately 20 to 200 cells) can consist of a mixture of vascular endothelial cells and myoblasts. In a preferred embodiment, a ring of fine capillaries is then formed from this mixture by a laser beam with nanometer precision. By a specific energy input of laser 1, preferably only the endothelial cells located on the inner side of the ring are crosslinked in the mixture. In this preferred embodiment, by another specific energy input of laser 2, only adjacent myoblasts ( Fig.13 and Fig.14 ).

[0129] In a further preferred embodiment, it is provided that non-crosslinked cells within the vascular structure are removed by means of a laser.

[0130] Preferably, bioinks consisting of particularly homogeneous cell types are also crosslinked in order, for example, to print larger homogeneous tissue areas.

[0131] In a preferred embodiment, the cells to be cross-linked are included in the forming network.

[0132] In another embodiment, the cells themselves are cross-linked to each other or to the network. For this purpose, they are provided with cross-linking molecules anchored in the cell membrane.

[0133] Surprisingly, by a relatively fast printing process it is possible to relatively quickly transfer the printed tissue to an incubator where further maturation is triggered by environmental factors (eg chemical, molecular biological load or by fluid load).

[0134] Preferably, further cross-linkable inks are printed at specific locations, which inks assume a supporting function for the tissue, carry factors important for the physiological function of the cells, or carry growth factors, markers, signaling molecules, receptors or other binding sites (key-and-lock principle, Schlüssel-Schloss-Prinzip).

[0135] Similar to inkjet printers for realistic art prints, the print head is preferably equipped with a large number of nozzles, which are adapted to the respective rheology of the ink. The printer is also preferably equipped with one or more omnidirectional or directional electromagnetic wave sources, by which the crosslinking reaction is triggered.

[0136] In a preferred embodiment, a print head of a photorealistic art print is utilized which is additionally modified with one or more electromagnetic wave sources.

[0137] In another embodiment, a print head composed of a plurality of modules is utilized, wherein each module corresponds to a print head for realistic art prints including one or more electromagnetic wave sources.

[0138] In another aspect of the present invention, it relates to a new print head configuration (see Figures 9 to 12 ) and involves a new printing process in which different energy sources are used to selectively trigger different reactions ( Figure 9-12 ).

[0139] Fig.13 and Fig.14 It is schematically shown how specific cells can be cross-linked with one another by means of different photoinitiators with the aid of light of a specific wavelength. Lasers, diodes or other energy sources can be used as energy sources (see the Examples section).

[0140] In a preferred embodiment, the printed tissue is fed during the printing process. The interface for the culture medium present on the base plate is extended individually to the tissue to be printed by the structural ink. Starting from the interface on the base plate, a smaller diameter, more delicate interface is printed with the structural ink.

[0141] In another embodiment, the print head is equipped with bubble technology, piezoelectric technology or other print head technology.

[0142] In one embodiment, the source of electromagnetic waves is a non-directional source, such as a diode, laser, ultraviolet lamp, or other source.

[0143] In another embodiment, the electromagnetic wave source is a lens that can be pivoted 180°. The energy source is controllable and movable so that the laser beam can map any pre-programmed shape. In another embodiment, the light is generated in an external light source and transmitted to the exit location through a light-conducting cable.

[0144] In one embodiment, the wavelength of the electromagnetic wave is in the ultraviolet range, the visible range, or the infrared spectrum.

[0145] In a preferred embodiment, a suction device or a compressed air device is provided to remove the flushed-out, uncrosslinked ink material.

[0146] In one embodiment, the vacuum is controllable and is activated only when the suction device is lowered. The distance between the suction device and the object to be printed is controlled by the laser ( Fig.11 and Fig.12 ).

[0147] In another embodiment, no additional device is required to remove the washed-out ink material or cell material, because the washed-out cells form the starting point of new capillaries in the surrounding tissue, or are embedded or disappear together with the surrounding tissue when it differentiates.

[0148] In another embodiment, the uncrosslinked ink material is destroyed by a directed laser beam.

[0149] In another embodiment, uncrosslinked cells, cell debris, or ink are flushed from the device and leak into the reaction chamber.

[0150] In one embodiment, a gas atmosphere (eg, inert gas, CO2, etc.) is generated above the printing plane by a fumigation nozzle.

[0151] In one embodiment, the wavelength and beam width of the electromagnetic wave source are modulatable.The printer is provided with one or more modulatable sources for targeted directed electromagnetic radiation beams.

[0152] In another embodiment, a femtosecond laser is preferably used.

[0153] In one embodiment, during the printing process, the gas content (eg CO2 / O2), pH value and other important cell culture parameters are regulated by the culture medium. Additionally, the gas exchange is also regulated by the gas content in the reaction chamber.

[0154] In one embodiment, the method is performed "upside down" or "top down".

[0155] In one embodiment, the reaction vessel of the printer is optionally loaded into the printer as a disposable insert and can be removed after printing. After printing, the tissue or organ is in a state that can be contacted with a patient or transferred to a cell culture. The reaction vessel has an inflow and outflow interface for the culture medium or blood that feeds the tissue.

[0156] In one embodiment, the reaction vessel is cooled.

[0157] In an application mode in the patient, the reaction vessel is connected to his blood circulation in vitro. Due to the provided patient-typical growth factors and differentiation factors and due to the blood pressure in the still differentiated vascular system, further maturation occurs. In this phase, the sample can optionally be removed from the system in order to monitor the degree of maturation (differentiation) of the tissue or organ and determine the optimal time for transplantation. For this sampling, the reaction vessel has a further interface.

[0158] In conjunction with the method according to the invention, it is advantageous that, before 3D printing, specific cell types are specifically hybridized with molecules so that the cells in the mixture can selectively respond. The connection is composed of a selective factor and an anchoring molecule. The selective factor should preferably trigger lateral crosslinking. The anchoring molecule is preferably used to dock the cell membrane. If its chemical and physical properties allow, the selective factor can also be directly bonded to the cell surface without an anchoring molecule. A spatially controlled electromagnetic wave energy beam specifically directed in terms of its wavelength and its frequency irradiates the selective factor of a specific cell type and activates it, for example, by cleaving a protective group, activating a photoinitiator, activating a reactant, or activating a key-locking component. This crosslinking reaction does not involve other cell types. ( Fig.13 and Fig.14 , see the Examples section).

[0159] In one embodiment, the selectivity factor comprises a lipophilic molecule, such as a perfluorocarbon or a lipid, as an anchor molecule. In another embodiment, the selectivity factor comprises a cationic molecular region or a molecular region with a positive partial charge as an anchor molecule. In another embodiment, the selectivity factor comprises a peptide compound, such as a cell penetrating peptide or a polynucleotide, as an anchor molecule.

[0160] In combination with the teachings of the present invention, a new type of bio-ink is used:

[0161] Unlike the high viscosity bio-inks of the conventional droplet method previously utilized, which include long chain macromolecules contained therein, the novel bio-inks of the methods disclosed herein are very low viscosity and contain photoinitiators. They contain very short chain, smaller cross-linking molecules, which cross-link directly after contact with electromagnetic waves. In the preferred embodiment, these molecules have allyl modification or thiol modification.

[0162] In another embodiment, the printing chamber is fumigated.

[0163] In another embodiment, instead of being at an angle of 10-90° to the printing plate, the capillaries are printed parallel to it (0-10°).

[0164] In another embodiment, the capillaries have been flushed prior to looping.

[0165] The general process of printing - theoretical basis and preferred implementation scheme:

[0166] The invention disclosed herein describes a method for 3D printing organs and tissues. A larger printing area is divided into individual modules, which are printed independently of each other (on the same or different printers) and then combined and glued (for example using fibrin adhesive) to form a target organ or target tissue. In this case, the input and output vessels of the individual modules are combined into an input or output main vessel. The individual modules are preferably cubic structures with defined side lengths.

[0167] Tissues or organs are not identical in structure. Even within the same species, each individual is slightly different from the others. This is not only reflected in size, but also in individual development. For example, the muscles of an athlete have significantly more developed capillaries due to the higher oxygen demand than those of a non-athlete. The body adapts to environmental conditions, is never completely identical, and is constantly being built up and broken down.

[0168] Therefore, the function of the tissue is decisive, not the exact anatomical replication of the patient's tissue. Therefore, a modular system is disclosed here, which realizes the function of the tissue, but is simplified and systematized as much as possible, so that the 3D printing of the tissue can be carried out economically.

[0169] The building blocks of a tissue or organ are cubic individual modules with defined side lengths (1)( Figure 15-18 ). To supply the tissue, which can be composed of one or more cell types and can therefore also form functional tissues such as organs, on one side the ascending vessels (2) are supplied, which later form arteries. On the opposite side, the descending vessels (3) form veins. Smaller vessels (4) fan out from these vessels, extending on the other side to the height of the opposite vessel and extending parallel to each other ( Figure 2 The smaller vessels (4) are interconnected by bridging vessels (6), which later form capillaries, closing the "blood loop" and forming the microcirculation ( Figure 15-18 ).

[0170] Microcirculation ( Fig.15 ) presents the smallest supply unit in the printed tissue. Here, the medium is guided through the ascending vessel (artery) (2) to the small vessel (4) belonging to the ascending branch. From the smaller vessel, the medium flows through the bridging vessel (6) into the smaller vessel (4) located below it, belonging to the descending branch and connected to the descending vessel (3).

[0171] Because when printing vessels with capillary ink, many cells are printed per drop, and only cells in the edge area can cross-link with the surrounding tissue, it is necessary to flush out the excess cells. For this reason and to supply the printed cells, each microcirculation is thoroughly flushed with the medium in a short time after each completion. During each flushing process, the microcirculation located below the last microcirculation is also flushed and supplied with cells. The medium flow flushes out the uncross-linked cells. Horizontally elongated vessels are not optimal for this, so the printed vessels are all elongated at an angle of 10 to 90 degrees to the printing plate.

[0172] Ascending and descending vessels Fig.15 , Fig.16 )(2 and 3) are raised at an angle of 90°. The vessel fan (5) can be raised at an angle of 10-90°. Printing is performed in such a way that the center of the individual modules is raised and then lowered, so that the smaller vessels are always stretched upward relative to the printing plate. In this way, for the unprinted vessels, excess cells can be washed out well. If the microcirculation (7) is closed, the medium can flow away through the descending vessels ( Fig.15 and Fig.16 ).

[0173] Prints vein fans at defined intervals ( Fig.15 and Fig.16 ), wherein the vascular fans of the ascending and descending vessels extend alternately below each other. Each vascular fan of the ascending vessel (2) and the descending vessel (3) is connected to each other through a bridging vessel (6) and forms a microcirculation (7) ( Fig.15 and Fig.16 ). Repeat this process for a period of time until the single module is filled. In order to print tissues or organs, print a tissue module consisting of multiple single modules ( Fig.17 )(8). In its orientation, staggered ( Figure 7 ), print the individual modules flush and side by side on the printing plate, thus obtaining a tissue module with a defined side length and thickness. Organ modules can be printed on different printers in parallel and connected to form larger tissue units or organ units ( Figure 5 ).

[0174] In order to be able to connect the tissue module to the body's circulation, preferably the tissue closure module (8) must be printed ( Fig.15 and Fig.17 ) and tissue connection module (9)( Fig.15 and Fig.17 ).

[0175] The tissue closing module (8) is composed of a tissue module in which the ascending and descending vessels taper upward and are thereby closed in a single module and are closed by a plurality of cell layers (10).

[0176] The connecting module (9) must preferably aggregate the individual vessels of the individual modules into a larger vessel, taking into account anatomical and surgical regulations in the process. In the process, the input vessels and output vessels extend to the connecting vessel (9) in different planes. The ascending and descending vessels are in turn connected to each other via bridging vessels to supply the tissue ( Fig.15 , Fig.17 ).

[0177] General description of the printing process ( Figure 1-Figure 12 ):

[0178] In order to realize the economical printing process, the tissue to be printed is preferably subdivided into individual tissue modules, which are in turn composed of individual modules. The tissue modules composed of individual modules and the individual modules form a cross section consisting of a defined plane of the entire tissue or organ together with its different cell types and vessels. This subdivision is achieved by means of a specific algorithm. By printing a single plane (tissue profile), a three-dimensional tissue is produced.

[0179] Corresponding to the provisions of the tissue module, a printing plate comprising supply openings is printed from a polymer.

[0180] Likewise, a printing foil is printed from a biodegradable polymer, which is fixed together with the printing plate on the printer table and inserted into the printing chamber.

[0181] Fill and connect the tanks of capillary ink, tissue ink and structural ink.

[0182] The necessary cell types are grown in external laboratories that have been equipped and certified for this purpose, and they are pre-concentrated for the printing process and transported to the printer in tanks.

[0183] The containers for the medium inflow and outflow are filled and the inlet and outlet hoses are connected to the corresponding connections on the base of the printer table. The inlet and outlet are supplied via a pump system with supply and return flow. The printer chamber is also filled with medium, which can be kept constant via the inflow and outflow system. The printing process can now be started and the printing plate moves into its starting position.

[0184] First, the vascular interface is printed onto a printing foil on a printing plate using structural ink. Afterwards, the printer prints plane by plane, and in the process forms individual tissue regions and vessels layer by layer, and in the process sinks into the medium of the printer chamber until the tissue module is completed. In this case, the printing process is programmed so that in a cycle that is always repeated, after reaching a specific height of the tissue, a short flushing process is entered to flush out excess and uncrosslinked cells. This flushing procedure can be present in different embodiments. Preferably, after a defined number of printing processes, the ascending branch is flushed once, and then after another defined number of printing processes, the descending branch is flushed. Always alternate until the tissue module is printed. By alternating flushing processes, the inflowing and outflowing vessels can be flushed without overloading the vessels.

[0185] When the individual modules have been printed, they can be easily removed from the printer table with the help of the printing foil. The printed individual modules are combined into complete tissues or organs by means of adhesives (e.g. fibrin adhesives). The input and output vessels of the individual modules are combined into larger output and output vessels.

[0186] The tissue can continue to be supplied with medium through the arterial and venous circulation printed in the tissue and mature in the incubator for many more days before being shipped.

[0187] A single module taking the liver as an example ( Fig.15 , Fig.16 , Fig.17 , Fig.18 ):

[0188] The liver is composed of approximately one to one and a half million lobules, each 1-2 mm in diameter. Two vessels terminate in the liver, the hepatic artery, which supplies the tissues with oxygen and nutrients, and the portal vein, which carries blood from the stomach and intestines along with ingested nutrients and toxins. Veins that merge into the vena cava and the common bile duct arise from the liver.

[0189] Here, a single module consists of a single hexagonal liver lobule (1) with a diameter of 2 mm ( Fig.17 a) formation. The liver lobules are arranged side by side in 5 rows and each row is 5 rows high ( Fig.15 d) By arranging the hepatic lobules in a plurality of rows, each offset by half a hepatic lobule every second row, they can mesh with one another when forming a tissue module.

[0190] A single hexagonal liver lobule has three vessels at each corner (artery (2), portal vein (4), common bile duct) and a vein (5) in the middle. All four vessels form a microcirculation ( Figure 2 ).

[0191] For each angular vessel, the hepatic lobule is subdivided into six segments (6), which are traversed by microcirculation and form vascular fans (7) ( Figure 4). Two afferent vessels (i.e., arteries and portal veins) lead to the veins in parallel in the microcirculation. The veins extend from the middle in the opposite direction and extend below the afferent vessels, so that the three vessels form a triangle. The arteries and portal veins are connected to the veins via vascular bridges and form the microcirculation. The common bile duct is located in the center of the vascular triangle.

[0192] Printing process of liver module ( Fig.17 , Fig.18 ):

[0193] In order to achieve an economical printing process, the tissue to be printed is subdivided into individual tissue modules, which in turn consist of individual modules. The tissue modules and individual modules formed by individual modules form a cross-section consisting of a defined plane of the entire tissue or organ with its different cell types and vessels.

[0194] This single module is further subdivided into multiple continuous planes (slices) through an algorithm. Each plane then forms a two-dimensional printed image with different cell types and patterns. By printing these planes (slices), three-dimensional tissues are generated.

[0195] Corresponding to the provisions of the liver module, a printing plate including supply openings is printed from a polymer. The supply openings are the input and output vessels (artery, portal vein, vein, common bile duct) of the individual liver lobules.

[0196] Likewise, a printing foil is printed from a biodegradable polymer (preferably polyhydroxybutyrate, PHB), comprising a supply opening, which is fastened together with the printing plate to the printer table and is inserted into the printing chamber.

[0197] Fill the containers for capillary ink, tissue ink, and structural ink. Connect the canisters for hepatocytes, endothelial cells, cholangiocytes, and epithelial cells.

[0198] In a preparation phase, the cell types required for the respective printing are propagated by an external laboratory that is equipped and certified for this purpose, pre-concentrated for the printing process and transported to the printer in corresponding tanks.

[0199] The containers for the medium inflow and outflow are filled and the inlet and outlet hoses are connected to the corresponding connections on the printer table base. The inlet and outlet are supplied via a pump system with feed and return flow.

[0200] The printer chamber also contains media, which can be kept constant by flowing in and out of the system.

[0201] The printing process can now be started and the printing plate is moved into its initial position. All processes are arranged by algorithms programmed for this purpose, which in turn also receive the control signals of the printer and process them further.

[0202] The printer now prints plane by plane and in the process forms individual tissue areas layer by layer and in the process sinks into the medium in the printer chamber until the tissue module is completed. The printing process is programmed so that after reaching a certain printing height of the tissue, a short flushing process begins in order to flush out excess and uncrosslinked cells. The printing is programmed in this case so that the flushing process is prolonged as the number of completed printing planes increases in order to transport the flushed-out cells into the printing chamber.

[0203] This flushing procedure can take place in different embodiments. In a preferred embodiment, the ascending limb is flushed once and then, after another defined number of printing processes, the descending limb is flushed. This is always alternated until the tissue module is printed. By means of alternating flushing processes, the inflowing and outflowing vessels can be flushed without overloading the vessels.

[0204] Once the liver module has been printed, it can be easily removed from the printer table with the help of the printing foil.

[0205] When all tissues necessary for the liver module as well as the tissue closure module and the tissue connection module are printed, an adhesive (such as fibrin adhesive) is used to combine them into a complete tissue or organ.

[0206] The tissue can continue to be supplied with medium through the arterial and venous circulation printed in the tissue and mature in the incubator for many more days before being shipped ( Fig.19 ).

[0207] Example:

[0208] Examples 1) to 4) correspond to the examples described in the "Summary of the Invention" section for illustrative purposes. Next, they are supplemented by more embodiments:

[0209] The reference numerals listed in the examples relate specifically to a specific drawing.

[0210] 1) General process of printing - theoretical basis and preferred implementation scheme:

[0211] Fig.15 :

[0212] The invention disclosed herein describes a method for 3D printing organs and tissues. A larger printing area is divided into individual modules, which are printed independently of each other (on the same or different printers) and then combined and glued (for example using fibrin adhesive) to form a target organ or target tissue. In this case, the input and output vessels of the individual modules are combined into an input or output main vessel. The individual modules are preferably cubic structures with defined side lengths.

[0213] Tissues or organs, while identical in structure, are not completely identical. Even within the same species, each individual is slightly different from the others. This is not only reflected in size, but also in individual development. For example, the muscles of an athlete have significantly more developed capillaries due to the higher oxygen demand than those of a non-athlete. The body adapts to environmental conditions, is therefore never identical, and undergoes a constant process of construction and breakdown.

[0214] Therefore, the function of the tissue is decisive, not the exact anatomical replication of the patient's tissue. Therefore, a modular system is disclosed here, which realizes the function of the tissue, but is simplified and systematized as much as possible, so that the 3D printing of the tissue can be carried out economically.

[0215] The building blocks of a tissue or organ are cubic individual modules with defined side lengths (1)( Fig.15 a). To supply the tissue, which can be composed of one or more cell types and can therefore also form functional tissues such as organs, on one side the ascending vessels (2) are supplied, which later form arteries. On the opposite side, the descending vessels (3) form veins. Smaller vessels (4) fan out from these vessels, extending on the other side to the level of the opposite vessel and extending parallel to each other ( Fig.15 b). The smaller vessels (4) are interconnected by bridging vessels (6), which later form capillaries, closing the "blood loop" and forming the microcirculation (7) ( Fig.15 c and Fig.15 d).

[0216] The microcirculation represents the smallest supply unit in the printed tissue. Here, the medium is guided through the ascending vessels (arteries) (2) to the small vessels (4) belonging to the ascending limb. From the smaller vessels, the medium flows through the bridging vessels (6) into the smaller vessels (4) located below them, belonging to the descending limb and connected to the descending vessels (3).

[0217] Because when printing vessels with capillary ink, many cells are printed per drop, and only cells in the edge area can cross-link with the surrounding tissue, it is necessary to flush out the excess cells. For this reason and to supply the printed cells, each microcirculation is thoroughly flushed with the medium in a short time after each completion. During each flushing process, the microcirculation located below the last microcirculation is also flushed and supplied with cells. The medium flow flushes out the uncross-linked cells. Horizontally elongated vessels are not optimal for this, so the printed vessels are all elongated at an angle of 10 to 90 degrees to the printing plate.

[0218] The ascending and descending vessels (2 and 3) are raised at an angle of 90°. The vessel fan (5) can be raised at an angle of 10-90°. Printing is performed in such a way that the center of the individual modules is raised and then lowered, so that the smaller vessels are always stretched upward relative to the printing plate. In this way, the excess cells can be washed out of the unprinted vessels very well. If the microcirculation (7) is closed, the medium can flow away through the descending vessels ( Fig.15 f).

[0219] The vascular fans are printed at defined intervals, wherein the vascular fans of the ascending and descending vessels extend alternately below each other. Each vascular fan of the ascending (2) and descending (3) vessels is connected to each other via a bridging vessel (6) and forms a microcirculation (7) ( Fig.15 d). Repeat this process for a period of time until the individual modules are filled. In order to print tissues or organs, a tissue module (8) consisting of a plurality of individual modules is printed. Fig.15 g) Printing the individual modules flush and side by side on the printing plate, thus obtaining a tissue module with defined side length and thickness. Organ modules can be printed on different printers in parallel and connected to form larger tissue units or organ units ( Fig.15 e).

[0220] In order to be able to connect the tissue module to the body's circulation, in particular, a tissue closure module (8) must be printed ( Fig.15 g) and tissue connection module (9) ( Fig.15 h).

[0221] The tissue closing module (8) is composed of a tissue module in which the ascending and descending vessels taper upward and are thereby closed in a single module and are closed by a plurality of cell layers (10).

[0222] The connecting module (9) must preferably aggregate the individual vessels of the individual modules into a larger vessel, taking into account anatomical and surgical regulations in the process. In the process, the input vessels and output vessels extend to the connecting vessel (11) in different planes. The ascending and descending vessels are in turn connected to each other via bridging vessels to supply the tissue.

[0223] 2) General description of the printing process:

[0224] In order to achieve an economical printing process, the tissue to be printed is subdivided into individual tissue modules, which in turn consist of individual modules. The tissue modules consisting of individual modules and the individual modules form a cross-section consisting of a defined plane of the entire tissue or organ together with its different cell types and vessels. This subdivision is achieved with the help of specific algorithms. By printing individual planes (tissue sections), a three-dimensional tissue is generated.

[0225] Corresponding to the provisions of the tissue module, a printing plate comprising supply openings is printed from a polymer.

[0226] Likewise, a printing foil is printed from a biodegradable polymer, which is fixed together with the printing plate on the printer table and inserted into the printing chamber.

[0227] Fill and connect the tanks for capillary ink, tissue ink, and structural ink.

[0228] The necessary cell types are grown in external laboratories that have been equipped and certified for this purpose, and they are pre-concentrated for the printing process and transported to the printer in tanks.

[0229] The containers for the medium inflow and outflow are filled and the inlet and outlet hoses are connected to the corresponding connections on the base of the printer table. The inlet and outlet are supplied via a pump system with supply and return flow. The printer chamber is also filled with medium, which can be kept constant via the inflow and outflow system. The printing process can now be started and the printing plate moves into its starting position.

[0230] First, the vascular interface is printed onto a printing foil on a printing plate using structural ink. Afterwards, the printer prints plane by plane, and in the process forms individual tissue regions and vessels layer by layer, and in the process sinks into the medium of the printer chamber until the tissue module is completed. In this case, the printing process is programmed so that in a cycle that is always repeated, after reaching a specific height of the tissue, a short flushing process is entered to flush out excess and uncrosslinked cells. This flushing procedure can be present in different embodiments. Preferably, after a defined number of printing processes, the ascending branch is flushed once, and then after another defined number of printing processes, the descending branch is flushed. Always alternate until the tissue module is printed. By alternating flushing processes, the inflowing and outflowing vessels can be flushed without overloading the vessels.

[0231] When the individual modules have been printed, they can be easily removed from the printer table with the help of the printing foil. The printed individual modules are combined into complete tissues or organs by means of adhesives (e.g. fibrin adhesives). The input and output vessels of the individual modules are combined into larger output and output vessels.

[0232] The tissue can continue to be supplied with medium through the arterial and venous circulation printed in the tissue and mature in the incubator for many more days before being shipped.

[0233] 3) A single module using the liver as an example ( Fig.17 )

[0234] The liver is composed of approximately one to one and a half million lobules, each 1-2 mm in diameter. Two vessels terminate in the liver, the hepatic artery, which supplies the tissues with oxygen and nutrients, and the portal vein, which carries blood from the stomach and intestines along with ingested nutrients and toxins. Veins that unite to form the vena cava, as well as the common bile duct, arise from the liver.

[0235] Here, a single module consists of a single hexagonal liver lobule (1) with a diameter of 2 mm ( Fig.17 a) formation. The liver lobules are arranged side by side in 5 rows and each row is 5 rows high ( Fig.17 d) By arranging the hepatic lobules in a plurality of rows, each offset by half a hepatic lobule every second row, they can mesh with one another when forming a tissue module.

[0236] A single hexagonal liver lobule has three vessels at each corner (artery (2), portal vein (4), common bile duct) and a vein (5) in the middle. All four vessels form a microcirculation ( Fig.17 b).

[0237] For each angular vessel, the hepatic lobule is subdivided into six segments (6), which are traversed by microcirculation and form vascular fans (7) ( Fig.17 d). Two afferent vessels (i.e., arteries and portal veins) lead to the veins in parallel in the microcirculation. The veins extend from the middle in the opposite direction and extend below the afferent vessels, so that the three vessels form a triangle. The arteries and portal veins are connected to the veins via vascular bridges and form the microcirculation. The common bile duct is located in the center of the vascular triangle.

[0238] 4) Example printing process of liver module ( Fig.17 , Fig.18 ):

[0239] In order to achieve an economical printing process, the tissue to be printed is subdivided into individual tissue modules, which in turn consist of individual modules. The tissue modules and individual modules formed by individual modules form a cross-section consisting of a defined plane of the entire tissue or organ with its different cell types and vessels.

[0240] This single module is further subdivided into multiple continuous planes (slices) through an algorithm. Each plane then forms a two-dimensional printed image with different cell types and patterns. By printing these planes (slices), three-dimensional tissues are generated.

[0241] Corresponding to the provisions of the liver module, a printing plate including supply openings is printed from a polymer. The supply openings are the input and output vessels (artery, portal vein, vein, common bile duct) of the individual liver lobules.

[0242] Likewise, a printing foil is printed from a biodegradable polymer (preferably polyhydroxybutyrate, PHB), comprising a supply opening, which is fastened together with the printing plate to the printer table and is inserted into the printing chamber.

[0243] Fill the containers for capillary ink, tissue ink, and structural ink. Connect the canisters for hepatocytes, endothelial cells, cholangiocytes, and epithelial cells.

[0244] In a preparation phase, the cell types required for the respective printing are propagated by an external laboratory that is equipped and certified for this purpose, pre-concentrated for the printing process and transported to the printer in corresponding tanks.

[0245] The containers for the medium inflow and outflow are filled and the inlet and outlet hoses are connected to the corresponding connections on the printer table base. The inlet and outlet are supplied via a pump system with feed and return flow.

[0246] The printer chamber also contains media, which can be kept constant by flowing in and out of the system.

[0247] The printing process can now be started and the printing plate is moved into its initial position. All processes are arranged by algorithms programmed for this purpose, which in turn also receive the control signals of the printer and process them further.

[0248] The printer now prints plane by plane and in the process forms individual tissue areas layer by layer and in the process sinks into the medium in the printer chamber until the tissue module is completed. The printing process is programmed so that after reaching a certain printing height of the tissue, a short flushing process begins in order to flush out excess and uncrosslinked cells. The printing is programmed in this case so that the flushing process is prolonged as the number of completed printing planes increases in order to transport the flushed-out cells into the printing chamber.

[0249] This flushing procedure can take place in different embodiments. In a preferred embodiment, the ascending limb is flushed once and then, after another defined number of printing processes, the descending limb is flushed. This is always alternated until the tissue module is printed. By means of alternating flushing processes, the inflowing and outflowing vessels can be flushed without overloading the vessels.

[0250] Once the liver module has been printed, it can be easily removed from the printer table with the help of the printing foil.

[0251] When all tissues necessary for the liver module as well as the tissue closure module and the tissue connection module are printed, an adhesive (such as fibrin adhesive) is used to combine them into a complete tissue or organ.

[0252] The tissue can continue to be supplied with medium through the arterial and venous circulation printed in the tissue and mature in the incubator for many more days before being shipped.

[0253] 5) Extended variant of printer construction:

[0254] The printer is built from the following components:

[0255] - a plurality of print heads comprising a plurality of nozzles for the droplet method and one or more spatially controllable electromagnetic wave sources causing an energy input modulatable in terms of wavelength, intensity and frequency,

[0256] - Among them, the modulated electromagnetic waves are beneficial to selectively activate the cross-linking reaction,

[0257] - a closable reaction tank containing a medium, which includes interfaces for medium exchange, sampling and an input path for the medium or patient blood that can be optionally changed after printing, which can be removed from the printer and used as an incubator,

[0258] a base plate located in the reaction tank, which comprises an interface for the generated vascular system, through which the medium flows continuously or intermittently and is used to supply the printed tissue,

[0259] - multiple tanks for the required inks,

[0260] - Different capillary inks, bio-inks and structural inks,

[0261] - structural regions (e.g. supporting structures, extracellular matrix or other non-cellular components, such as polyhydroxyalkanoates, fibrinogen, collagen, myelin, glucosamine, glycolipids, peptides, sugar molecules, fatty acids, key molecules, organic and inorganic molecules for stimulus conduction, etc.),

[0262] - characteristic molecules (e.g. molecules with different physiological properties, such as growth factors, transcription factors, receptor molecules, signaling molecules, RNA, DNA, differentiation molecules, etc.),

[0263] - Homogeneous cell types (e.g. myocytes, hepatocytes, neurons, etc.),

[0264] - a mixture of heterogeneous cell types (e.g. endothelial cells, myocytes) for printing vasculature,

[0265] - During the printing process, the printer table can move up and down, and optionally, the print head can move up and down instead of it or both can move up and down together.

[0266] 6) Example of aging printed tissue in an incubator

[0267] The incubator facilitates differentiation of the printed tissue or organ. After printing, although a tissue population with a high cell density has been produced, the cells have not yet reached their final tissue structure and degree of differentiation. The tissue or organ is printed directly into a chamber that will later be used as an incubator. Alternatively, the tissue or organ can also be transferred to another incubator. The incubator provides important differentiation parameters necessary for differentiation, such as chemical or molecular biological substances or flow parameters.

[0268] Alternatively, the incubator can be run with culture medium and at a later time point connected to the patient's blood circulation in vitro or immediately after printing. Upon contact with the patient's blood, the necessary differentiation and growth factors reach the printed structure and the cells can adapt to the body.

[0269] 7) Exemplary configuration of print head ( Fig.19 )

[0270] The print head consists of a first sub-zone for droplet printing of bio-ink, capillary ink and structural ink. The droplet method is usually carried out by means of a piezo nozzle or a bubble nozzle. In terms of the flow and the arrangement of the generated droplets, the printing process is similar to the cyan / magenta / yellow / black / grey / light grey / light magenta / light cyan system of a photo printer. Instead of the pigment ink, the required amount of bio-ink, capillary ink and structural ink is used.

[0271] In the second subarea, the print head is provided with one or more omnidirectional or directional electromagnetic wave sources. The directional sources as a whole generate a selective energy source, which is modulatable in terms of wavelength, intensity and frequency. Ultraviolet lamps, light-emitting diodes, lasers or screens are usually used as electromagnetic wave sources. Commonly used electromagnetic waves are wavelengths in the ultraviolet, visible and infrared spectra.

[0272] The functional units of the print head for the droplet method and for the method of providing electromagnetic waves may also be arranged separately from each other. The print head may also be constructed from a series of modules of such units.

[0273] 8) Other alternative structures of the reaction tank including the bottom plate, the medium input ( Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 ,picture 7)

[0274] A printer worktable that can be moved up and down is arranged in the printing chamber of the printer and includes a plurality of interfaces for the medium to flow through the printed blood vessel structure.

[0275] Outside the printing chamber, an interface is connected to an external system for maintaining specific cell culture parameters. These parameters mainly include temperature, pH value, CO2 content, O2 content, nutrients, growth factors, etc. Since the uncrosslinked cells in the printed vascular structure are also washed out in the form of cell fragments or as whole cells, the medium flowing into the printing chamber must preferably be controlled in terms of its parameters and, if necessary, purified or replaced.

[0276] During the printing process of the vascularized tissue, the medium is introduced into the vascular structure printed thereon via the interface. This is advantageous on the one hand for feeding the produced tissue and on the other hand for free flushing of the hollow structures of the vascular system. The medium enters the vascular structure via the interface of the printer table, flows through the printed vascularized tissue and seeps out again on the still open cavity of the vascular system above the side facing the print head. From there the medium either flows passively back into the printer chamber or is actively removed from the printing surface during the printing process (e.g. flushed or sucked away).

[0277] The printer table continues to sink into the printing chamber during the printing process, so that the printed tissue is surrounded and flushed by the medium in the printing chamber. The uppermost area of ​​the tissue, about 1-5 mm, protrudes from the medium. Since the thiol-ene reaction is not sensitive to O2, it is usually not necessary to fill the air-filled area of ​​the reaction tank with an inert gas (e.g., nitrogen, argon).

[0278] 9) Provide necessary digital information for 3D printer software

[0279] According to medical imaging methods, digital data about the cell structure of tissues and organs are obtained and programmed into 3D printing data. These methods include, for example: MRT method or "DISCO transparency" method. Methods for determining these data are known to those skilled in the art. Based on these data, with the help of special algorithms, the number of modules required for economically printing the required cell types, sizes and distances of capillaries, veins and arteries is calculated. For economical printing, it is not necessary to replicate the biological structure in its original form. The construction of a single module has been described in 1) to 4).

[0280] 10) New printer software

[0281] The printer software processes the information about the tissue area to be printed and forwards it to the print head and the printer table for execution. Unlike conventional 3D printer programming, droplet technology (printing pattern and droplet size of a specific ink) and modulated laser technology in the case of a directional electromagnetic wave source (required wavelength, energy intensity and frequency, spatial control) are combined and matched to each other here. Here, the release of a specific ink type into the reaction tank containing the tissue and the droplets on the printing plate plays a central role in the spatial and temporal coordination of the electromagnetic waves provided in space and time at the wavelength, frequency and intensity that vary from ink to ink for crosslinking. For vascular structures, the focus is on the selective crosslinking of endothelial cells and muscle cells in the ink droplets from the cell mixture (section "Protective groups and photoinitiators used").

[0282] 11) Maturation of printed tissues and organs until transplantation:

[0283] The incubator containing the tissue or organ is provided with an interface for in vitro connection to the patient's blood circulation or to a system providing a medium. By incubation starting from a few hours to a few days, the 3D printed cell structure is further differentiated. In particular, by connecting the printed vascular circulation to the patient's blood circulation, important development factors are provided to the cells. In addition, for differentiation, the endothelial cells and muscle cells of the printed vascular structure require a specific intravascular pressure and a specific flow velocity caused by the blood or the medium.

[0284] During the maturation phase, it is possible to check specific physiological values ​​in an incubator via an interface and thus monitor the maturation of the organ or tissue. If the physiological values ​​of the printed organ or tissue reach the physiological values ​​of the natural organ within a certain range, it can be transplanted into the patient.

[0285] 12) Exemplary construction of the new low viscosity ink:

[0286] The ink is composed of short-chain cross-linked molecules with low viscosity, photoinitiators and cells. The short-chain cross-linked molecules have at least one (usually two or more) functional group, which is usually composed of an allyl group or a thiol group and undergoes a polymerization reaction or an addition reaction.

[0287] Commonly used crosslinking molecules are methacrylate-modified and thiol-modified molecules, such as methacrylate-modified or thiol-modified peptide chains, polyhydroxyalkanoates or hyaluronic acid. Bio-inks and structural inks are either mainly constructed from methacrylate-modified molecules (free radical polymerization or addition) or also from a mixture of methacrylate-modified molecules and thiol-modified molecules (thiol-ene reaction). In contrast, capillary inks are usually constructed from pure thiol-modified crosslinking molecules, which only crosslink in the edge area of ​​the droplet under the interaction with the allyl-modified crosslinking molecules of the bio-ink and structural ink.

[0288] The photoinitiators commonly used either work in the range of about 380 nm or together with photoinitiators in the infrared range. Photoinitiators in the infrared range are stereo sources of energy similar to multiphoton stereolithography (nonlinear multiphoton absorption), where different energy sources must be superimposed to trigger the reaction.

[0289] When printing cells that include selective factors (eg, cleavable protecting groups for thiol groups), additional photoinitiators below 380 nm are used.

[0290] Examples of photoinitiators used are: DAROCUR 1173, IRGACURE 2959 and 369, (Ru(phen)3 / Iod / (TMS)3Si-HPIS) based on "P. Xiao et al. / Progress in Polymer Science, 41 (2015) 32-66, S. 46", and photoinitiator 183. Photoinitiators used for infrared excitation are: H-Nu-IR 780 and H-Nu-IR 815. It has been demonstrated that a range of photoinitiators from the visible range can also be activated by multiphotons.

[0291] 13) Provide cells required for printing:

[0292] Cells for the novel 3D printing method disclosed herein can be provided in various ways. In this case, the origin of the cells depends on the desired use purpose of the tissue or organ printed therewith.

[0293] The simplest way to generate cells is to use commercially available cell lines. Using such cells, it is possible to optimize printing and culture parameters, as there is no need to use relatively expensive induced stem cells (iPS) or cells isolated from organs and tissues for testing.

[0294] Another cell source is the isolation of specific cell types from organs and tissues. In addition to animal cells for research, this method has also been used in a variety of ways in regenerative medicine, such as isolating intervertebral disc chondrocytes from prolapsed material in intervertebral disc surgery. Dissolve the prolapsed material, and isolate and propagate cells from it and also use them in 3D printing of tissues.

[0295] The most demanding cell source is the generation of induced stem cells (iPS). There are different established working groups' modes of operation for this purpose. iPS cells are necessary for the printing of human tissues and organs that are subsequently transplanted into the human body. However, these cells and the structures printed thereby can also be used as test materials for personalized medicine in cell culture. iPS cells are usually cultivated from the skin cells of donors. Skin cells are reverse differentiated into iPS cells, propagated, and differentiated into the cell types required for printing. These processes are not yet mature for a wide range of applications, but represent the prospects for the manufacture of human autologous transplants for organs and tissues.

[0296] Other sources of cell material are cell lines bred for specific purposes, which are often modified in some way (eg DNA-based, RNA-based or protein-based).

[0297] In principle, every cell type (primary or cell line) can be grown and printed by the methods disclosed here.

[0298] 13) Exemplary inks for printing with cells containing selective factors ( Fig.13 , Fig.14 ):

[0299] A possible solution for cross-linking capillary ink only in the edge region is to use a cell mixture consisting of endothelial cells and myocytes that selectively cross-link each other in the droplet. In order to be selectively activated by a directed laser beam, these cell types require specific selectivity factors. This selectivity factor is, for example, a cleavable protective group on a functional thiol group, which is located on the cell membrane of the relevant cell type. By using different protective groups that can be cracked at different wavelengths, different cell types can be separated from each other in their cross-linking reactions. Usually, in this application, the capillary ink is composed of cross-linked molecules with thiol modifications that are anchored on the cell membrane, and is composed of free thiol-modified, protected molecules in the ink. Additionally, there are allyl-modified molecules in the ink to make thiol cross-linking reactions possible.

[0300] 14) Exemplary configurations and selectivity factors of inks ( Fig.13 , Fig.14 ):

[0301] By means of selectivity factors, light-dependent thiol-ene reactions are selectively achieved within the spatial crosslinking range determined by the laser. These selectivity factors consist, for example, of photocleavable protective groups that block the thiol groups. Due to their different molecular structures, these protective groups can be cleaved by different wavelengths of light. Those protective groups are selected that are not correlated within their excitation spectrum.

[0302] In order to selectively crosslink only specific cell types from the mixture, but not other cell types, within the range irradiated by the laser, the protective groups on the thiol groups must be cleaved on the relevant cells. Thus, only these cells and these crosslinking molecules can undergo the thiol-ene reaction. Next, in this example, the thiol crosslinking reaction is initiated by a photoinitiator whose excitation spectrum is higher than the wavelength of all the protective groups used so as not to erroneously cleave them (see examples below).

[0303] 15) Printing vascular systems with cells containing selective factors:

[0304] Since the diameter of the fine capillaries between arteries and veins is only a few micrometers, but the droplets in the droplet method are larger, the inner monolayer ring composed of endothelial cells from the cell mixture droplet is selectively crosslinked. Similarly, the ring surrounding this ring, composed of myocytes from the target mixture, is selectively crosslinked. For this purpose, the endothelial cells or myocytes carry protective groups that can be cleaved at different wavelengths as selectivity factors (see examples below).

[0305] After selective cleavage of the protective groups on the corresponding cell types, the thiol-ene reaction can be carried out at the cells where the protective groups have been removed. Thus, firstly, by laser beam 1, the protective groups are removed for about 5 to 7 endothelial cells and the protected, thiol-modified crosslinking molecules located therebetween in the droplet to form a ring structure, and then by laser beam 2, they are annularly crosslinked with each other. Laser beam 2 is favorable for exciting the photoinitiator and has a higher wavelength than laser beams 1 and 3.

[0306] This results in a monolayer endothelial ring with an inner diameter of about 10 micrometers, the inner surface of which consists of uncrosslinked, occluded cells. Next, the outer myocytes and protected, thiol-modified crosslinking molecules adjacent to the endothelial ring are freed of their protective groups by laser beam 3 and crosslinked with one another by laser beam 2 to form an outer ring. Laser beams 1 and 3 cleave the corresponding protective groups at different wavelengths, while laser beam 2 causes the activation of the photoinitiator, which initiates the thiol-ene reaction.

[0307] The non-crosslinked cells inside the capillaries are destroyed by the higher energy laser beam (laser beam 4) and the resulting cell debris is washed away by the inflowing medium.

[0308] 16) In the case where the ink includes a selective factor, the protective groups and photoinitiators used ( Fig.13 ,picture 14)：

[0309] While for crosslinking of inks with homogeneous cell types, the reaction type can be freely chosen and performed non-selectively, different cell types comprising heterogeneous inks have thiol-ene reactants comprising specific protecting groups that are cleaved for the corresponding wavelengths. In this case, the curves for the necessary activation energies must not overlap ( Fig.13 ).

[0310] For crosslinking of endothelial cells or the surrounding myocytes, the following UV-cleavable protecting groups were used:

[0311] 325nm: (7,8-bis(carboxymethyloxy)coumarin-4-yl)methoxycarbonyl for myocytes

[0312] 400nm: α-carboxy-4-methoxy-2-nitro

[0313] (Source: Wavelength-selectively cleavable, photolabile protecting groups for thiols, Nico Kotzur, 2009)

[0314] First, the higher wavelength required is used to cleave the protecting groups so as not to activate other protecting groups by mistake. For the original thiol-ene reaction, a photoinitiator (Ru(phen)3 / Iod / (TMS)3Si-HPIS) with an excitation spectrum of 532 nm is used so as not to cleave the protecting groups located below by mistake. The photoinitiator 183 (P. Xiao et al. / Progress in Polymer Science 41 (2015) 32–66, S. 46) excited at 473 nm can also be used.

[0315] In the first step, the protective groups on the thiol groups of the endothelial cells are cleaved at 400 nm (laser beam 1). In this range, the thiol groups of the myocytes still have protective groups. Subsequently, the photoinitiator is activated at 532 nm and a thiol-ene reaction is triggered on the groups that have been freed of the protective groups (laser beam 2). By spatially directing two consecutive laser beams (first 400 nm and then 532 nm), 5 to 12 endothelial cells are typically crosslinked with each other in a ring.

[0316] In the second step, the protective groups on the thiol groups of the myocytes are cleaved at 325 nm (laser beam 3). Since the laser beams directed spatially for the endothelial cell range (400 nm and 532 nm) do not reach the range of the myocircle surrounding the endothelial cells, the endothelial cells of the cell mixture are present here, which still have protective groups and cannot participate in the thiol-ene reaction of the thiol groups of the myocytes that remove the protective groups. Subsequently, by activating the photoinitiator at 532 nm, the cross-linking of the myocytes located in the ring is achieved (laser beam 2).

[0317] In this way, it can be ensured that substantially only desired cell types are embedded in the corresponding structure.

[0318] For other necessary selectivity factors, additional protecting groups for thiol groups are available ( Fig.13 ), for example, a group ((7-bis(carboxymethyl)amino)coumarin-4-yl)methoxycarbonyl) that can be cleaved at 436 nm. The cross-linking process is similar to that described above. (Source: Wavelength-selectively cleavable, photolabile protecting groups for thiols, Nico Kotzur, 2009)

[0319] By means of a higher energy laser beam 4, the non-crosslinked cells inside the capillaries are destroyed and the debris is washed away with a medium.

[0320] 17) Exemplary preparation of autologous patient cells using selective factors

[0321] Cells extracted from the patient (e.g. skin cells) are transformed into induced stem cells (iPS), propagated, dispersed into individual categories and subsequently differentiated into the desired cell types. This preparation process is independent of the printing program and is only mentioned here as an example to describe the approach to achieving a true 3D organ. Depending on the complexity of the organ to be printed, the process can last from weeks to months. After the desired cell line is produced, it can be stored frozen until it is printed. The frozen cells can be thawed, hybridized with selective factors and printed. It is safer for the cells to be cultured in cell culture for 24 hours after thawing and then hybridized with their specific selective factors.

[0322] Usually, in order to anchor in the cell membrane, the selective factor is connected to an anchor molecule. The anchor molecule used is generally universal for all cell types. Surprisingly, it has been shown that perfluorinated anchor molecules are the most effective. According to experience, these molecules bind to 100% of the participating cells within 2 minutes. However, lipophilic molecules, peptides, ions or antibodies can also be used as anchor molecules.

[0323] 18) Prepare cells containing selective factors for printing:

[0324] Immediately prior to printing, the cells of the capillary system are hybridized with their specific selective factors: First, the endothelial cells and myocytes used are prepared separately for printing in cell culture and hybridized with their corresponding selective factors. They are then aggregated in the capillary ink.

[0325] More explanation about the attached pictures:

[0326] Figure 1 ：

[0327] Figure 1 : Exemplary, schematic printer structure and working mode:

[0328] The basis of the present invention is a conventional realistic piezoelectric printer.

[0329] Newly disclosed are variations of ink input (1-5) and configurations of printer workstations (12, 13, 14, 15).

[0330] The printer is constructed by an ink supply unit (1-5), a piezoelectric print head (15), a printer table (13), a printing chamber (14) containing a medium and a motor (12) of the printer table. In addition, a medium tank (8) and a waste tank (11) for supplying tissue including an input hose (9) and a hose (10) for outputting the medium are located on the printer.

[0331] The individual components are surrounded by a housing (6). A central control unit / computer / software (7) is located outside the housing and communicates with components 1-5, 15 and 12 (dashed lines).

[0332] Figure 2-7 ：

[0333] Figure 2-7 : Exemplary construction and function of the printer workbench:

[0334] In the following, a new printer workbench system is disclosed. The printer workbench is mainly composed of five parts:

[0335] The printing plate (7), the printer table (6) including the supply interface, can be lowered into the printing chamber by means of a stepper motor (3) and a micrometer screw (2).

[0336] With the aid of a 3D printer (FDM, SLS, SLM, SLA or other printer), the printing plates (7) are printed individually for the organs or organoids, since the openings for supplying the tissue may be different. The differences are caused by positioning, number and diameter.

[0337] The printing plate (7) made of a biocompatible polymer has connection openings for the inflow and outflow of the medium (1), from which a conduction system extends in the plate for the tissue alone and into further openings on the surface for supplying the tissue. The openings can be positioned differently and can also have different diameters. Vessels for supplying the tissue can then be printed on these openings.

[0338] The printing plate is screwed onto a printer table (6), which is made of a magnetic material and has a borehole in which a connection piece (1) for the inflow and outflow of the medium is inserted. Below the printing plate, there is a printer table base (5) into which the pin of the screw micrometer is assembled with low friction, and an interface (e.g., a Luer interface) for the inflow and outflow of the medium (1). In a preferred embodiment, the inflow and outflow connection piece on the upper side of the printer table base (5) is designed as a pivot and precisely matches the borehole of the printer table. A magnetic plate is arranged on the upper side of the printer table base. The printer table and the printer table base are connected to each other by the force of the magnet, thereby establishing a connection from the interface for inflow and outflow to the printing plate. The sealing ring in the borehole prevents the medium from flowing out. By means of the magnetic connection, the printed tissue together with the printing plate and the printer table can also be separated from other fixedly installed components without any problem and inserted into other supply units.

[0339] The printer table together with the printing plate can be lowered into the printing chamber (space above the silicone pad) by means of a screw micrometer (2) and a stepper motor (3). The printing chamber consists of a base plate (8) and a wall (9) made of, for example, stainless steel, and thereby forms a space into which the medium can be loaded via an input device. The printed tissue is lowered into the medium in order to feed it or protect it from drying out. By lowering the printed area, the printing plane remains at a constant height. Overflow due to squeezing of the medium is prevented by an overflow opening.

[0340] In a particular embodiment, a silicone pad (10) for sealing is provided above the printer workbench base (5).

[0341] It is tensioned on the printer table base (5), wherein the pivots for the inflow and outflow remain free and reach the edge of the print chamber floor. By screwing the print chamber wall (9) onto the print chamber (8) floor, the print chamber is sealed on the one hand, and the underside of the printer table base is separated from the print chamber by the pins of the micrometer screw. Due to the high flexibility of the silicone pad, the upward and downward movement of the printer table can also be carried out without any problems.

[0342] In another embodiment, in order to seal the printing chamber, instead of using a silicone gasket, a seal (eg a sliding ring seal) is arranged between the pin of the micrometer screw (2) and the bottom plate of the printing chamber (8).

[0343] In other embodiments, other structures may be used to seal the print chamber.

[0344] Figure 8 ：

[0345] Figure 8: Exemplary construction and mode of operation of the print head supply unit:

[0346] In the newly disclosed print head supply unit, cells cultivated in a cell tank (2) are pumped via a pump (4) into a cell concentrator (6), where the cell concentration is increased and excess medium is removed (9). The defined cell concentration is transferred via a cell counter (8) to a mixing unit (7), into which ink concentrate can also be introduced. The ink concentrate is conveyed in defined volumes from a storage container (1) to the mixing unit (7) via a pump including a measuring unit (5). In the mixing unit (7), the ink for the print head is mixed as specified in the printing program and introduced into a hose leading to a piezoelectric print head (12) by controlling the cell counter.

[0347] The number of print head supply units depends on the amount of bio-ink required and can vary.

[0348] The print head supply unit is on the one hand controlled by the printer algorithm to mix the required ink composition and on the other hand is provided with a series of sensors which provide the printer algorithm with information about the properties of the currently mixed ink, to which the algorithm reacts.

[0349] Fig. 9 and Fig.10

[0350] Fig. 9 and Fig.10 : Preferred structural embodiment of a cell concentrator (6) for concentrating cells by a decanter

[0351] For the enrichment of the cells, the following methods are preferably used as cell concentrators (6), which are distinguished by their long-term operation. The enrichment of the cells is achieved by reducing the liquid phase, the medium. On the one hand, the separation of the solid phase and the liquid phase can be achieved by centrifugal force or by filter properties.

[0352] In these structural embodiments, a fluid centrifuge or a decanter is used to enrich the cells. Both systems work according to the same principle, with the help of centrifugal force.

[0353] In a centrifuge, the separation of solid and liquid phases is achieved with the help of centrifugal acceleration. In the rotating bowl of the centrifuge, solid particles, which have a higher density and are therefore heavier than the liquid, are moved outwards with the help of centrifugal force. They form a sediment on the inner wall of the centrifuge bowl.

[0354] Decanter drumIt has a cylindrical cone-shaped outer shape and its rotation speed is adapted to the respective separation task. The product reaches the full peripheral speed in the drum and rests against the drum cover of the centrifuge as a cylindrical ring. The solids contained in the product, due to the higher density, are deposited on the inner wall of the drum under the influence of the centrifugal force. The length of the cylindrical drum part and the cone angle of the conical drum part can be adapted to the respective separation task when manufacturing the decanter.

[0355] Fig.11 and Fig.12

[0356] Fig.11 and Fig.12 : Preferred structural embodiment of a cell concentrator (6) for concentrating cells by dynamic filtration

[0357] In cross-flow microfiltration, the filter membrane is overflowed by the medium containing cells during the filtration process, so that two main flow directions orthogonal to each other are generated. The main flow direction is the filtration flow through the filter membrane and the overflow parallel to the filter membrane. The overflow of the suspension reacts on the formation of the filter cake during the filtration process, so that the cell concentrate can flow away. The efficiency of the filter characteristics is variable by the number and pore size of the holes in the filter membrane. Particularly suitable for this are hollow fibers (capillary membranes or also known as hollow ribs), whose effective power is also enhanced by the pinch effect. The inner diameter of common hollow fibers is about 1.5 mm (3.0 mm to 0.1 μm is also possible), and its pore size is 200 to 5 nm (2000 nm to 1.0 nm is also possible). Depending on the application, hundreds to thousands of capillaries are summarized in the module and cast (hollow fiber module). With the help of a circulation pump, the unfiltered product circulates through the capillaries until the sediment in the retentate is concentrated, so that it needs to be emptied and purified. (Ripperger 1992), (Melin&Rautenbach 2007).

[0358] Fig.13

[0359] Selectivity factor - Protective groups for thiol groups cleavable at wavelengths of 325 nm, 400 nm and 436 nm (Source: Wavelength-selectively cleavable, photolabile protecting groups for thiols, Nico Kotzur, 2009)

[0360] Fig.14

[0361] Cells including selective factors. Light-colored cells represent endothelial cells and dark-colored cells represent muscle cells. Both cell types are present as a mixture in the capillary droplet and can be selectively activated by two laser beams with different wavelengths to undergo a cross-linking reaction.

[0362] Fig.15

[0363] Fig.15 : Organization module structure:

[0364] The building blocks of a tissue or organ are cubic individual modules with defined side lengths (1)( Fig.15 a). To supply the tissue, which can be composed of one or more cell types and can therefore also form functional tissues such as organs, on one side the ascending vessels (2) are supplied, which later form arteries. On the opposite side, the descending vessels (3) form veins. Smaller vessels (4) fan out from these vessels, extending on the other side to the level of the opposite vessel and extending parallel to each other ( Fig.15 b). The smaller vessels (4) are interconnected by bridging vessels (6), which later form capillaries, closing the "blood loop" and forming the microcirculation (7) ( Fig.15 c and Fig.15 d).

[0365] The microcirculation represents the smallest supply unit in the printed tissue. Here, the medium is guided through the ascending vessels (arteries) (2) to the small vessels (4) belonging to the ascending limb. From the smaller vessels, the medium flows through the bridging vessels (6) into the smaller vessels (4) located below them, belonging to the descending limb and connected to the descending vessels (3).

[0366] Because when printing vessels with capillary ink, many cells are printed per drop, and only cells in the edge area can cross-link with the surrounding tissue, it is necessary to flush out the excess cells. For this reason and to supply the printed cells, each microcirculation is thoroughly flushed with the medium in a short time after each completion. During each flushing process, the microcirculation located below the last microcirculation is also flushed and supplied with cells. The medium flow flushes out the uncross-linked cells. Horizontally elongated vessels are not optimal for this, so the printed vessels are all elongated at an angle of 10 to 90 degrees to the printing plate.

[0367] The ascending and descending vessels (2 and 3) are raised at an angle of 90°. The vessel fan (5) can be raised at an angle of 10-90°. Printing is performed in such a way that the center of the individual modules is raised and then lowered, so that the smaller vessels are always stretched upward relative to the printing plate. In this way, the excess cells can be washed out of the unprinted vessels very well. If the microcirculation (7) is closed, the medium can flow away through the descending vessels ( Fig.15 f).

[0368] The vascular fans are printed at defined intervals, wherein the vascular fans of the ascending and descending vessels extend alternately below each other. Each vascular fan of the ascending (2) and descending (3) vessels is connected to each other via a bridging vessel (6) and forms a microcirculation (7) ( Fig.15d). Repeat this process for a period of time until the individual modules are filled. In order to print tissues or organs, a tissue module (8) consisting of a plurality of individual modules is printed. Fig.15 g) Printing the individual modules flush and side by side on the printing plate, thus obtaining a tissue module with defined side length and thickness. Organ modules can be printed on different printers in parallel and connected to form larger tissue units or organ units ( Fig.15 e).

[0369] In order to be able to connect the tissue module to the body's circulation, in particular, a tissue closure module (8) must be printed ( Fig.15 g) and tissue connection module (9) ( Fig.15 h).

[0370] The tissue closing module (8) is composed of a tissue module in which the ascending and descending vessels taper upward and are thereby closed in a single module and are closed by a plurality of cell layers (10).

[0371] The connecting module (9) must preferably aggregate the individual vessels of the individual modules into a larger vessel, taking into account anatomical and surgical regulations in the process. In the process, the input vessels and output vessels extend to the connecting vessel (11) in different planes. The ascending and descending vessels are in turn connected to each other via bridging vessels to supply the tissue.

[0372] Fig.16

[0373] Fig.16 : Rinse the resulting capillaries thoroughly with medium:

[0374] In this case, the printing process is programmed so that in a cycle that is always repeated, after reaching a certain height of the tissue, a short flushing process is entered to flush out excess and non-crosslinked cells. This flushing program can be present in different embodiments. Preferably, after a defined number of printing processes, the ascending branch is flushed once, and then after another defined number of printing processes, the descending branch is flushed. This is always alternated until the tissue module is printed. By means of the alternating flushing processes, the inflowing and outflowing vessels can be flushed without overloading the vessels.

[0375] Fig.17

[0376] Fig.17 :Liver Organ Module

[0377] A single module using the liver as an example:

[0378] The liver is composed of approximately one to one and a half million lobules, each 1-2 mm in diameter. Two vessels terminate in the liver, the hepatic artery, which supplies the tissues with oxygen and nutrients, and the portal vein, which carries blood from the stomach and intestines along with ingested nutrients and toxins. Veins that unite to form the vena cava, as well as the common bile duct, arise from the liver.

[0379] Here, a single module consists of a single hexagonal liver lobule (1) with a diameter of 2 mm ( Fig.17 a) formation. The liver lobules are arranged side by side in 5 rows and each row is 5 rows high ( Fig.17 c) By arranging the hepatic lobules in a plurality of rows, each offset by half a hepatic lobule every second row, they can mesh with one another when forming a tissue module.

[0380] A single hexagonal liver lobule has three vessels at each corner (artery (2), portal vein (4), common bile duct) and a vein (5) in the middle. All four vessels form a microcirculation ( Fig.17 b).

[0381] For each angular vessel, the hepatic lobule is subdivided into six segments (6), which are traversed by microcirculation and form vascular fans (7) ( Fig.17 d). Two afferent vessels (i.e., arteries and portal veins) lead to the veins in parallel in the microcirculation. The veins extend from the middle in the opposite direction and extend below the afferent vessels, so that the three vessels form a triangle. The arteries and portal veins are connected to the veins via vascular bridges and form the microcirculation. The common bile duct is located in the center of the vascular triangle.

[0382] Fig.18

[0383] The individual printed liver organ modules are superimposed on each other:

[0384] Fig.19

[0385] Fig.19 : Example configuration of an incubator:

[0386] The tissue culture incubator consists of a tank (1) containing a medium and a cover plate (2) to be placed. A base (3) is fixedly mounted at the bottom of the tank, on which a printer table base (5) of the same structure is replaceably mounted. The medium hoses (4) leading to the input and output of the printer table base are introduced into the incubator vessel through openings. The printer table together with the printing plate, printing foil and printed tissue are placed on the pivot of the printer table base by means of magnetic force.

[0387] Fig. 20 and Fig.21 :

[0388] Fig. 20 and Fig.21: Exemplary configurations of printheads including non-directional and directional electromagnetic radiation sources.

Claims

1. A control unit (7) for controlling a print head supply unit (1, 2, 3, 4, 5), a motor (12) for a printer table (13), a device for applying electromagnetic waves and a droplet printer (15) for producing realistic high-resolution prints based on a printing model for 3D printing of tissues and organs including vascular structures, characterized in that The following features exist in the printed model: - a tissue plane of the printed model consisting of different ink types, characterized in that the tissue plane can be printed by means of the droplet printer for realistic high-resolution prints and can be cross-linked by means of the device for applying electromagnetic waves, wherein some of the different ink types undergo cross-linking or polymerization only in the edge region of the droplets near other droplet types, - a tissue plane composed of orderly applied different cell types, which is crossed by an incised internal hollow vessel (152, 153, 154), - Larger tissues or organs are subdivided into individual modules (151) and the printed image is simplified for an economical printing process, - printing the individual modules (151) independently of one another on the same droplet printer (213) or on different printers and subsequently bonding the individual modules (151) into the desired configuration of the tissue or organ, - a single module (151) comprising at least one ascending branch (152) and at least one descending branch (153) for medium input and medium output and capillaries located therebetween in a fan-shaped manner, The individual modules form a microcirculation, which forms the smallest vascular unit. - when building a larger structure from a single module (151), merging the microcirculation into a main vessel for medium inlet and medium outlet, The printed tissue is stabilized by a biodegradable or absorbable printing foil which defines two printing sides in the Z direction and has a secondary or primary interface for the vessels.

2. A control unit (7) according to claim 1, comprising a specific algorithm for calculating the printing model and for scheduling the printing process, characterized in that The calculation of the printing model is carried out for the cell types used and for the distances and sizes of capillaries and arterial and venous structures, and in a common, modular and economically structured printing model, on the one hand, the optimal capillary supply and on the other hand, the anatomical conditions of the corresponding tissue structures are simplified, and The algorithm decomposes the larger target structure into smaller individual modules (151) that can be printed independently of each other, and The algorithm provides data information for 3D printing printing plates (27) and printing foils, and can be exported from the computer program for use by other 3D printers, and The algorithm for scheduling the printing process receives and processes the following information: - information about the anatomical and physiological data and dimensions of the tissues and organs to be printed, - from the print head supply unit (1, 2, 3, 4, 5) about the tank (8, 82, 191), the cell concentration in the reactor and in the cell concentrator (86) and at its output, information on the volumes mixed in the mixer (87), and information on the processes in the print head (811), - information about the position of the printer table (13), the medium level in the printing chamber (14), the medium volume flow per unit time in the inlet and outlet sections in the area of ​​the printer table, and information about the conveying direction of the medium volume flow, - information about the current size of the printed tissue, - information about the instantaneous power of the printer table motor (12), and the algorithm receives information about the activity of the source of electromagnetic waves (202, 203), And the algorithm sends the following signals to the corresponding printer areas: - signals to the individual components of the print head supply unit (1, 2, 3, 4, 5) in order to mix the required ink components, - a signal to a pump (84, 85) for a specific volumetric transport of the ink into the mixer and into the print head (811), - arranging the inlet and outlet of the medium on the path from the printer table base (25) to the tissue vessels and for arranging the signal for the flushing process in the capillaries, - a signal of the desired movement of the printer table (13), - a signal sent by the print head (811) for selectively controlling the printer nozzle (213) and for the movement of the print head slider, - a signal sent to a device for applying electromagnetic waves (202, 203), - Signal for optional printing breaks during capillary flushing.

3. A 3D printer comprising a control unit according to claim 2 and a print head supply unit for 3D printing tissues and organs including vascular structures, characterized in that: The print head supply unit is constructed from the following components: - a cell concentrator in which the cells cultivated in the cell tank are concentrated to a specific cell concentration, - ink concentrate tank, a mixing unit including a cell counter, in which the desired number of cells per unit volume and the desired ink concentration are mixed and directed to the print head as specified by the printing algorithm, wherein the number of print head supply units depends on the amount of necessary ink and can vary, and The print head supply unit is on the one hand controlled by the printer algorithm to mix the required ink composition and on the other hand has a series of sensors which provide the printer algorithm with information about the properties of the ink currently mixed, to which information the algorithm reacts.

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