Method for producing a perfusable three-dimensional tissue model using 3D bioprinting technology, and tissue model produced by this method

Abstract

A method for producing a perfusable 3D tissue model with channels allowing fluid flow distributed throughout the structure, where a vasculature is bioprinted by extrusion using a bioink, and walls opening and closing the upper channels are printed parallel to the channel axis, where the model body is bioprinted by extrusion using a bioink, the bioink for printing the body being different from the bioink for printing the blood vessels, the resulting system is placed in an incubator to a temperature at which the bioink for printing the vasculature melts, the bioink is removed, and optionally growth is caused in the channels by cells in a medium, the cross section of said channels being the same as the cross section of natural blood vessels present in a living organism.The present invention relates to a bionic model with a perfusable system.

Description

[Technical field]

[0001] The subject of the present invention is a method for producing a perfusable three-dimensional tissue model using 3D bioprinting techniques. The subject of the present invention is also a tissue model produced by this method. The present invention proves to be useful in the field of bioprinting of bionic organs.

[0002] Document US20210041853A1 describes a method for a vascular network model printed by 3D printing, including a stenosed pulmonary artery capable of vascular perfusion. The described method involves obtaining an image of the copied anatomical structure and then generating a geometric tissue model based on this image. The disclosed solution therefore focuses on the method for generating a 3D model of the copied source material, and does not provide any information on subjects related to the printing method or the type of bioink applied.

[0003] Document US20200316254A1 describes the bioprinting of a cardiac patch with a perfusion structure. It consists of a perfusable blood vessel integrally embedded between two layers of anisotropically oriented myocardial fibers. The cardiac patch is created using a dual 3D bioprinting technique, using stereolithography to form the anisotropic structures and extrusion printing to form the perfusion blood vessels. The applied bioink contains, among others, gelatin methacrylate (GelMA) and polyethylene glycol diacrylate (PEGDA). The anisotropic myocardial fibers are formed in layers and perfusable blood vessels are formed at least every fifth layer of anisotropic myocardial fibers.

[0004] Document KR101974716B1 describes the 3D printing of blood vessels: the tissue is printed in two layers, on which bioinks of various compositions can be printed.

[0005] Document US20200047399A1 describes a method for 3D printing of soft polymeric materials, which involves the continuous printing of a solution of a photocurable polymer using extrusion printing of a polymer solution.

[0006] Document US20200024560A1 describes the bioprinting of myocardial tissue. The 3D printing method described in this document involves, inter alia, producing hydrogel scaffolds made of microfibers and directly bioprinting endothelial cells within the scaffolds, the bioprinting step being carried out simultaneously with the step of producing the microfibers. The bioprinting is carried out using an alginate gelatin methacrylate mixture (GeIMA), and the printing process is carried out with controlled anisotropy. The produced hydrogel scaffolds are composed of a number of layers, each layer stacked in a cross configuration, with the main axes of successive layers perpendicular.

[0007] Document WO2019237061A1 discloses a method for producing a biologically printable 3D platform: a coated hydrogel containing gelatin methacrylate, collagen and fibrin, which also contains cells, forms a 3D tissue structure around a 3D network of hollow vascular channels.

[0008] Document CN112891633A describes the fabrication of vascularized myocutaneous flaps by 3D bioprinting. A mixture of gelatin with methacrylic anhydride, sodium alginate, polyethylene glycol diacrylate, and vascular smooth muscle cells is used as the bioink. The printed scaffold is then crosslinked by blue light.

[0009] The document EP3655052A4 describes a method for producing a perfused multi-layered tissue model, which comprises depositing on a substrate one or more fibers that are saturated with cells and that include a plurality of concentric and coaxial layers extending over at least a portion of the length of the fiber. The bioprinting results in a perfused multi-layered structure of tubular tissue.

[0010] In the prior art, no solution is known that describes the bioprinting of models with channels whose cross-sections match those of native blood vessels present in the body. Moreover, no solution exists in which the walls closing the channels are printed according to a pattern and the fibers are printed parallel to the channel axis (Figure 1B), thus making it impossible to eliminate the risk of clotting inside the bionic organ. The aim of the present invention is to develop a method for producing tissue models by 3D printing that eliminates the risk of creating blood clots inside the produced model. In this method, the fibres are printed parallel to the axis of the channels present in the model.

[0011] The subject of the present invention is a method for producing a perfusable three-dimensional tissue model having channels allowing the flow of fluids distributed throughout its structure, comprising the following steps: Bioprinting a vasculature by extrusion using a bioink, where walls opening and closing the upper channel are printed parallel to the axis of the channel, and a model substance is bioprinted using the bioink by extrusion, where the bioink for printing the body is different from the bioink for printing the blood vessels. The resulting system is placed in an incubator to bring it to a temperature that melts the bioink for printing the vasculature. A process for removing the bio-ink. Optionally, causing proliferation within the channel by cells in the medium. The cross section of the channel is the same as that of natural blood vessels present in vivo.

[0012] Preferably, the temperature of the printhead with the bioink ranges from 10 to 26 °C, the diameter of the printing needles ranges from 100 to 609 nm, the pressure used in the bioprinting process ranges from 5 to 200 kPa, the fiber printing speed ranges from 5 to 40 mm / s, and the length of the printed individual fibers ranges from 150 mm to 5000 mm.

[0013] Preferably, the pressure used in the process of bioprinting the layers surrounding the body and channels of the model is in the range of 5-40 kPa for printing with bioinks containing pancreatic islets, and in the range of 5-200 kPa for printing with bioinks containing cells, in particular endothelial cells or fibroblasts.

[0014] Preferably, the pressure used in the process of bioprinting the vascular system is in the range of 5-200 kPa for printing with bioinks containing cells, in particular endothelial cells or fibroblasts, and at least 5 kPa for printing with bioinks without cells.

[0015] Preferably, printing uses at least one printhead with temperature control.

[0016] Preferably, the bioink for printing blood vessels is a hydrogel containing an extracellular matrix, at a concentration of 5-10% in a phosphate-buffered saline solution, by sonication.

[0017] Preferably, the bioink for printing on vessels is a non-ionic copolymer surfactant, preferably having the formula (CHO CHO) x (x represents 10 to 1000 repetitions).

[0018] Preferably, the bioink used for printing blood vessels is a suspension containing endothelial cells and fibroblasts in a 1:2 ratio with 8 million cells / ml in a hydrogel containing an extracellular matrix at a concentration of 5-10% in phosphate buffered saline.

[0019] Preferably, the bioink used to print the body is a solution of decellularized extracellular matrix that has been treated with an enzyme, preferably pepsin, and comprises at least one crosslinker and a photoinitiator, preferably the crosslinker is gelatin methacrylate, gelatin methacrylamide, and / or hyaluronic acid methacrylate, and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0020] Preferably, step ii) is carried out at a temperature below 37° C. over a period of 24 hours, preferably over a period of 30 to 40 minutes.

[0021] Preferably, the removal of the bio-ink in step iii) is performed by washing the channel with a solution of phosphate buffered saline, cell culture medium, or another liquid at a suitably adjusted temperature at which the bio-ink melts.

[0022] Preferably, the cells grown in step iv) are selected from the group of endothelial cells, fibroblasts, or a mixture thereof in a ratio of 1:2, and the cells grown in the channel have a concentration in the culture medium of 5 to 10 million cells / ml, preferably 8 million cells / ml.

[0023] The present invention also relates to a bionic model equipped with a perfusable system. Here, the model body is printed with a bioink consisting of a solution of decellularized extracellular matrix treated with pepsin and containing at least one crosslinking and photoinitiator, preferably the crosslinking agent is gelatin methacrylate, gelatin methacrylamide, and / or hyaluronic acid methacrylate and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0024] The channel filling is printed with a hydrogel containing a decellularized extracellular matrix, optionally containing endothelial cells and fibroblasts in a 1:2 ratio, with a total number of cells between 5 million and 10 million / ml, and a concentration of 5-10% in phosphate buffered saline, which is generated by sonication or Or, The channel filling is printed with a non-ionic copolymer surfactant.

[0025] Preferably, for each model body fill layer, there are 2-4 outline layers for the model's bionic channels, with the paths of successive layers oriented at angles of up to 90 degrees to each other, and the layers surrounding the channels printed parallel to each other in the same direction.

[0026] Preferably, the layer that opens and closes the model body and the layer that opens and closes the vascular channel are printed in 1 to 10 layers, each layer having a height of 0.1 to 1.2 mm, and the layer of the pancreas body is printed in a sandwich system with a ratio of layer height to channel outer wall height of 1:1.1 to 1:2.

[0027] Preferably, said bionic model has channels dispersed throughout its structure, the cross-section of the channels being the same as the cross-section of natural blood vessels present in vivo. [Brief description of the drawings]

[0028] The subject matter of the invention is illustrated in the drawings. [Figure 1A] Figure 1A shows the printed pattern of the vasculature, a version printed transversely to the channels of the model. [Figure 1B] Figure 1B shows the printed pattern of the vasculature, which is a version of the model that prints along the channels. [Diagram 2]Figure 2 shows an image of blood clot tissue visible after resection of the bionic pancreas. The blood clot was created by printing the blood vessels of the bionic pancreas. Changing the printing direction to the length direction eliminated this issue (right image). [Diagram 3] Number of washed out cells expressed in percentage points for the tested options of incubation time in the bioreactor / incubator, taking into account the printing technique (X and Y axis channel printing) and the cell introduction method (indirectly using vascular bioink; directly after Pluronic washing out). Error bars correspond to the standard deviation. [Figure 4] Lifespan of washed cells expressed in percentage points for the tested options of incubation time in the bioreactor / incubator, taking into account the printing technique (20 channels printed on the X- and Y-axis) and the cell introduction method (indirectly using so-called vascular bioink, directly after washing with Pluronic). Error bars correspond to the standard deviation. [Diagram 5] Cross-sectional channel scraps stained with hematoxylin (FIG. 5A) and eosin (FIG. 5B). Arrows indicate the nuclei of cells adjacent to the channel wall. [Figure 6] FIG. 6 shows the reaction scheme for the methacrylation of gelatin. [Figure 7] FIG. 7 shows the reaction scheme for the methacrylation of hyaluronic acid. [Figure 8] Figure 8 shows a graph comparing the concentration of secreted insulin in bioconstructs bioprinted with various dECM-based bioinks with the control group (2D culture of INS-1E cells). [Figure 9] FIG. 9 shows a schematic diagram of a bioprinted organ with blood vessels of circular cross-section. [Figure 10] FIG. 10 shows a schematic diagram of a bioprinted organ with blood vessels having channels with a circular cross-section identical to that of natural blood vessels present in vivo. [Figure 11]FIG. 11 shows the actual printed pancreatic body layers, channel layers, and channel fill. [Figure 12] Figure 12 shows a microscopic image of the circular cross-section channels. Surface protrusions and roughness are particularly evident in the upper channel of the pancreas, while the channel surface in the lower part of the pancreas is smoother. [Figure 13] 13 is a microscopic image of a channel showing a cross section of the vasculature (the lumen of a blood vessel). Surface protrusions and roughness are particularly evident in the channel in the upper part of the pancreas, whereas the channel surface in the lower part of the pancreas is smoother. [Figure 14] FIG. 14 is a graph showing the change in absorbance over the course of the hemolysis test. [Figure 15] Figure 15 shows clotting: A-Control, B-Bioink B, C-Pluronic. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The perfusable printing model of the tissue model in the 3D bioprinting technology according to the invention allows the printing of fibers of the bioink disclosed in application number PCT / IB2020 / 056856. The bioink used for the production of the model creates structural conditions in which living cells can function not only within the model but also on its surface. The bioink (containing microorgans such as cells, organoids, pancreatic islets or not containing biological material) constituting the material for the production of the invention is composed of natural components, so as to form a mixture that can be used for printing by extrusion methods. In order to maintain the vital functions of the cells contained in the tissue model, apart from its solid structure, the model is provided with channels in a plane throughout its entirety, with diameters ranging from 1.0 to 3.0 mm. The walls closing the top of the channels are printed according to two fiber placement patterns. In the first pattern, the fibers are placed perpendicular to the channel axis (Figure 1A), while in the second pattern, the fibers are printed parallel to the channel axis (Figure 1B). The optimal and only suitable method is to print in the channels aligned along the X-axis (Figure 1B).

[0030] Regardless of the fiber placement technique adopted, the resulting channels remain tight and free of obstructions. These features allow the flow of a fluid within the lumen of the channel, which may be, for example, a culture medium whose composition corresponds to the culture requirements of the cells contained therein (scientific research), or blood after transplantation of the bioorgan into the recipient's body. Considering blood flow, option B is the only correct solution. Only with such a printing technique is the risk of clotting inside the bionic pancreas significantly reduced, which has been demonstrated in tests on large animals (Figure 2).

[0031] The bioprinting method according to the invention adopts several modifications compared to the methods known from the prior art: The cross-sectional shape of the channels has been changed from circular (Figure 9) to the same cross-section of natural blood vessels present in vivo (Figure 10). The modification of the printing method of the channel walls includes the use of multiple printing heads (head no. 1 for filling the organ, head no. 2 for filling the vessel walls, head no. 3 for filling the channels) and different diameters of the printing needles are used (different diameters for filling the organ, different diameters for printing the channel walls).

[0032] With the introduction of these modifications, the roughness of the upper and lower parts of the channel surface is reduced (Figure 13). A better match of the printed geometry has been achieved. The printing method according to the invention also reduces errors during printing, eliminating poorly adhered bio-ink fibers. The print quality has improved and it is no longer necessary to exchange the bio-ink cartridges of the heads that print the channel walls. The exchange of cartridges always disturbs the printing process (changes in temperature of the bio-ink, mechanical connections of the device, possible loss of calibration).

[0033] Embodiments: Pluronic®, a non-ionic copolymer surfactant, was used as the supporting bioink.

[0034] cell: As models, we used red fluorescent human dermal fibroblasts (HDFa) and green fluorescent human umbilical vein endothelial cells (HUVEC). HDFa cells with ATCC.

[0035] Example 1: Methacrylation of gelatin in carbonate buffer Add a 250-1000mL three-neck flask equipped with a stir bar to a heat block or water bath (a 5L crystallizer half-filled with water). Using a graduated cylinder, measure out 100-500mL of 1M carbonate buffer (1M sodium bicarbonate to 1M carbonate in a 1:1 V / V ratio) and pour into the flask.

[0036] Secure the necks of the flasks with rubber septa, attach a thermocouple to one side flask so that the sensor is immersed in the solution but does not interfere with mixing, place a vent needle in the center flask, protect the flasks from light with aluminum foil, and heat to a set temperature of 50°C.

[0037] Weigh out 1-1000 g of gelatin on an analytical balance and add it portionwise to the buffer solution to make a 10% solution (mass / volume). Leave the mixture under constant stirring (1000 rpm) until the substrate is completely dissolved (approximately 30 min).

[0038] After the substrate is completely dissolved, measure the pH of the resulting solution and note it in a notebook as pH 1.

[0039] Depending on Table 1 and the intended degree of substitution of methacrylic anhydride, measure the anhydride into a 1-20 ml syringe equipped with a needle according to the following procedure: 1) Weigh the empty syringe with tubing and needle (m1). 2) Measure the target volume of MMA into the syringe and reweigh (m2), ensuring there are no air bubbles in the system. 3) After adding the MMA, reweigh the dispensing system to determine the exact amount of MMA added to the reaction mixture (m3). [Table 1]

[0040] Wrap the syringe with MMA in aluminum foil, place it on the syringe pump, and stick the connected tubing into the septum on the side neck of the flask so that the dispensed anhydride does not flow over the wall directly into the reaction mixture. Set an appropriate flow rate and inject until the solution in the syringe is completely gone, occasionally checking the efficiency of the stirring and the viscosity of the reaction mixture. If necessary, increase the rotation speed of the stirrer.

[0041] After all the weighed MMA has been dropped, weigh the syringe again and calculate the exact amount of MMA added to the reaction (see pt. 5). Continue the reaction at the initial conditions (T=50°C, mixing at 1000-1200 RPM) for approximately 1 hour.

[0042] After this time, measure the pH of the reaction mixture using a pH meter (note it as pH 2) (see pt 4).

[0043] Next, add PBSx1 solution to the mixture in small portions (400-2000 ml) until the reaction mixture is diluted 5-fold (1:4 mixture:PBSx1). The resulting solution is poured into previously prepared dialysis tubing according to the following procedure. The dialysis process is carried out for 3 days at a temperature of 40 ° C, with water change twice a day (usually at 08:00 and 16:00). A total of 6 pours are performed.

[0044] Once the dialysis process is complete, quantitatively transfer the solution from the dialysis tube into a large beaker, then transfer about 600 ml of the solution at a time into a 1000 ml round-bottom flask and concentrate it in a rotary evaporator to about 25-35% of the initial volume (not less than 150 ml). Final concentration parameters: Bath temperature: 45℃ Pressure: 30mbar Rotation: 150 rpm Initial capacitor temperature: -2°C

[0045] Once the concentration process is complete, use a serological pipette to transfer the solution from the flask approximately 12-13 ml at a time (maximum 1 cm in height) into a plastic urine cup with a capacity of 100 ml, described according to the following template: from top left to bottom: 1. Name of sample (e.g. W1GELMA), 2. m1 = mass of container without lid, 3. m2 = mass of container with solution, 4. m3 = mass of container with lyophilisate; bottom right corner - date of sample preparation; top right corner - author's initials.

[0046] The urine cup containing the solution is placed in a -80°C freezer and frozen for at least 1 hour. The frozen samples are then transported in extruded polystyrene foam boxes and freeze-dried. Freeze-drying parameters: Shelf temperature: 0℃ Pressure: 0.100mbar Duration: 24~48 hours

[0047] Weigh the resulting lyophilizates, record the mass in a urine cup (see pt. 14), and determine the percentage of water content of each sample to determine the efficiency of the lyophilization. To do this, enter all mass values ​​into an Excel program file and use the following relationship: M% H2O =m3-m1 / m2-m1*100%

[0048] Collect the resulting product in a collection urine cup and exclude samples whose percentage of moisture content differs significantly from the other samples. Protect the container with parafilm and store at a temperature of -20 °C.

[0049] Example 2: Methacrylation of hyaluronic acid. Attach a 100-1000 mL round bottom flask equipped with a magnetic stirrer and measure out 100-500 mL of carbonate buffer using a graduated cylinder. Start the stirrer and set the speed to approximately 1000 rpm (depending on the size of the flask and stirrer).

[0050] Weigh out 0.5-5 g of sodium hyaluronate on an analytical balance and add it in small portions to the flask containing the buffer solution, resulting in a 1% solution (mass / volume). Stir the mixture constantly (1000-1200 rpm) at room temperature until the substrate is completely dissolved (approximately 1 h).

[0051] Once the substrate is dissolved, wrap the flask in aluminum foil and place in a water bath (a crystallizer half-filled with saturated NaCl solution and ice to maintain liquid phase instability). The expected bath temperature is below 0°C. [Table 2]

[0052] Measure the pH of the resulting solution and note it down as a value of pH 1. Use an automatic pipette to evenly distribute the MMA, with at least 15 min between successive drops, so that the total drop time does not exceed 3 h. [Table 3]

[0053] After dropping all the measured MMA, continue the reaction for another hour under the set conditions (T≦4℃, mixing at 1000-1200 RPM), and then measure pH 2. Then, replace the ice and saline in the bath, and continue the reaction for another 24 hours.

[0054] After 24 hours, the pH of the reaction mixture is measured and 1000 ml of cooled demineralized water (T≦5° C.) is added thereto to obtain a 2-fold dilution (1:1 reaction mixture / water). [Table 4]

[0055] The resulting solution is poured into dialysis tubing according to the following procedure. The dialysis process is carried out for 4 days at a temperature of 30°C, with water change twice a day.

[0056] Once the dialysis process is complete, use a serological pipette to transfer the solution from the flask approximately 12-13 ml at a time (maximum 1 cm in height) into a plastic urine cup with a capacity of 100 ml, described according to the following template: from top left to bottom: 1. Name of sample (e.g. T10HAMA), 2. m1 = mass of container excluding lid, 3. m2 = mass of container with solution, 4. m3 = mass of container with lyophilisate; bottom right corner - date of sample preparation; top right corner - author's initials.

[0057] The urine cup containing the solution is placed in a -80°C freezer and frozen for at least 1 hour. The frozen samples are then shipped and freeze-dried. Freeze-drying parameters: Shelf temperature: -5~0℃ Pressure: 0.100~0.500mbar Duration: 24~48 hours

[0058] Weigh the resulting lyophilizates, record the mass in a urine cup (see pt10), and determine the percentage of water content of each sample to determine the efficiency of the lyophilization. To do this, enter all mass values ​​into an Excel program file and use the following relationship: M% H2O =m3-m1 / m2-m1*100%

[0059] Collect the resulting product in a collection urine cup and exclude samples whose percentage of moisture content differs significantly from the other samples. Protect the container with parafilm and store at a temperature of -20 °C.

[0060] Example 3: Preparation of Bioink - General Procedure: Decellularized material was obtained from a local slaughterhouse, and immediately after preparation, the pancreatic tissue was precisely cleaned of fat, large blood vessels, and connective tissue and stored in PBS solution before further handling. The whole process was carried out according to the protocol published by Klak M,Int J Mol Sci 2021;22:1-16.https: / / doi.org / 10.3390 / ijms22137005

[0061] Embodiment 4: Bioink A: The powder form of dECM was dissolved in pepsin (Sigma-Aldrich), and gelatin methacrylate (GelMA; Polbionica Ltd.) or gelatin methacrylamide and hyaluronic acid methacrylate (HAMA; Polbionia Ltd.) were used as crosslinkers. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was used as a photoinitiator under UV-visible light. This mixture was used for extrusive bioprinting.

[0062] Embodiment 5: Bioink B: dECM was dissolved in 1XPBS and stirred at 400 rpm for 5 min. Then, a thermocouple was connected to a Bandelin Sonopuls HD 3100 ultrasonic homogenizer (Bandelin electronic GmbH&Co.KG, Berlin, Germany) to control the temperature of the samples during sonication. The hydrogel samples were placed on ice and treated with a sonotrode, an MS72 probe with an amplitude of 10-65%. The process was carried out for 3-5 min at a temperature not exceeding 37 °C.

[0063] Example 6: Printing of tissue models - general procedure: The tissue models were printed by extrusion, and the temperature of the head of the material used for printing was 18-26 °C. The diameter of the printing needle was 609 nm. The pressure inside the needle oscillated in the range of 5-40 kPa, the fiber printing speed was 5-30 / s, and the length of a single fiber ranged from 150 mm to 5000 mm.

[0064] Embodiment 7: Implantation of cells in the printing process with bioink B - indirect introduction: Tests were carried out to verify the biocompatibility of the materials. One of the key elements was to evaluate the printing technique and its influence on the adhesion of cells. For this purpose, a human cell line model was used (endothelial cells - HUVEC, fibroblasts - aHDF in a ratio of 1:2, total number between 5 and 10 million / ml, the most preferred being: 8 million / ml, present at a concentration and constituting the precursors of the new formation of microvessels). The cells were introduced into the channels in two ways. When printing the described invention, cells in suspension in a so-called vascular bioink (indirect introduction) were delivered by extrusion, filling the channels. The printing of the vascular bioink was carried out with the head temperature set in the range of 15-25 °C. The pressure during printing was 5-30 kPa and the generation of fibers proceeded at a speed of 5-40 mm / s. The bioprinting After the linting process, the system was placed in an incubator to raise the temperature to 37 °C to dissolve and wash away the bio-ink B, resulting in an unobstructed flow system. Removal of the bio-ink is done by washing the channels with a solution of phosphate buffered saline, cell medium, or another liquid with an appropriately adjusted temperature (25-37 °C for bio-ink B). Phosphate buffered saline may be used to wash the channels to clear blockages. When there is already a complete organ containing pancreatic cells / islets, a properly supplemented fluid needs to be used to provide proper nutrition. This is where the term cell medium or fluid comes from.

[0065] Example 8: Implantation of cells with Pluronic® supported bio-ink printing process - direct introduction: Pluronic®, which acts as a support during further printing of the tissue model, was added to the channels located within the tissue model. Once printing was completed, the tissue model was placed at a temperature of 2-8°C where the Pluronic® melts. The tissue model was then flushed with 1xPBS through the channels (recommended speed 5ml / min) to wash out the Pluronic®. Cells in culture medium were then introduced into the unobstructed channels (direct introduction).

[0066] Pluronic® can also be removed by washing the channels with cell media or other fluids at an appropriately regulated temperature (4-8°C for Pluronic®). Phosphate buffered saline may be used to wash the channels to clear blockages. If there is already an intact organ containing pancreatic cells / islets, then a properly supplemented fluid needs to be used to provide adequate nutrition. This is where the term cell media or fluid comes from.

[0067] Example 9: Testing of tissue models printed with Bioink B and Pluronic-supported bioinks: In both cases, the former and the latter cell introduction method, the same procedure was used, specifically placing the tissue model in a bioreactor / incubator as described in PCT / IB2021 / 062190, setting the conditions at 37 °C and CO2 content at 5%. The respective incubation times for the tissue model, HUVEC and aHDF cells were set as follows: 2 h, 5 h, 8 h and 24 h. Once the time for the tested incubation option had elapsed, the channels were washed with 1xPBS at a preferred speed of 5 ml / min for 1 to 10 min (optimally 2 min). The above tests were performed on tissue models printed using both methods of placing the fibers forming the channels. Three replicates were performed for each version presented. Regardless of the channel printing technique chosen, an adhesion phenomenon between the introduced endothelial cells and fibroblasts was observed. The cells showed a morphological change of becoming flattened, indicating favorable culture conditions for growth and proliferation. The number of cells washed out of the channel, taking into account the cell introduction method and expressed as percentage points for each incubation time option tested, is shown in Figure 3.

[0068] Figure 4 shows the lifetime of washed cells in percentage points, taking into account the printing technique (channel printing on the X- and Y-axes) as well as the cell introduction method (indirectly using so-called vascular bioinks and directly after washing with Pluronic) for the tested options of incubation time in the bioreactor / incubator. Error bars correspond to the standard deviation.

[0069] Analysis of the presented results allows the selection of both the most favorable printing technique and the method of introducing cells to form microvessels. Cultivating the tissue model with channels printed from fibers spatially arranged in the Y axis for 5 hours and introducing the cells directly gives the most favorable ratio of cells remaining in the channel and their viability. In the above option, 51.7% of the cells remain in the channel, with a viability of 74% (± 7% SD). The cultivation of the cells that remained in the channel was performed in a closed flow system. The medium flows in the channels of the tissue model at a rate of 0.3 ml / min or more, a value that stimulated the adhesion and proliferation of the cells. The amount of cells from the above options did not decrease over 8 days. The cells did not circulate in the closed system and remained in the channel. After the completion of the 8-day experiment, the tissue model was fixed in 5% formaldehyde, after which immunohistochemical hematoxylin and eosin staining was performed to confirm the presence of cells on the surface of the channel walls, with a photographic record of the channel contents prepared in advance (Figure 5A and 5B).

[0070] Embodiment 10: Bioprinting of a Bionic Pancreas - (BT1) Model Description:The pancreas body was printed with bioink A and the channel fill was printed with bioink B / Pluronic. Pancreas models (pancreas body and channel fill) printed by extrusion printing using nozzles with diameters ranging from 100 μm to 650 μm. The height of each layer ranges from 0.1 to 1.2 mm. The layers of the model were filled from 10 to 100% and included double contours of the pancreas body and channels, improving the precision and tightness of the printed channels. The paths of successive layers were placed at a 90° angle to each other. The channels were positioned in the model so that below and above the channels there are pancreas body layers. The layers surrounding the channels are always printed parallel to each other in the same direction.

[0071] Printing Pattern: To print the model, two printheads with temperature control are required.

[0072] The layers that open and close the pancreas body each have 1 to 10 layers and are 0.1 to 1.2 mm high.

[0073] Layers of the actual pancreatic body (including cells / islets) and blood vessel contours are printed while maintaining the same layer height.

[0074] Filling of the channels with Bioink B or Pluronic proceeds without cross-linking.

[0075] Example 11: Bioprinting of a Bionic Pancreas - (BT2) Layers of Varying Heights Model Description:The pancreas body is printed with bioink A, the channel filling is printed with bioink B and / or Pluronic. Pancreas model printed using layers of two heights. The pancreas body and channel filling are printed by extrusion printing using nozzles with diameters of 100 μm to 650 μm, and the channel contours are printed with needles with diameters of 50 to 500 μm. The rule is followed that there are 2 to 4 layers of pancreatic duct contours per layer of pancreatic body filling. The layers of the model are filled 10 to 100% and the paths of successive layers are arranged at an angle of 90° to each other. The channels are arranged in the model so that below and above the channels there are pancreas body layers. The layers surrounding the channels are always printed parallel to each other in the same direction.

[0076] Printing Pattern: To print the model, three printheads with temperature control are required.

[0077] The layers that open and close the pancreas body each have 1 to 10 layers and are 0.1 to 1.2 mm high.

[0078] The layers of the actual pancreatic body (containing cells / islets) are printed in a sandwich system with a layer-height ratio of 1:1.1 to 1:2.0 relative to the outline of the channel (flow system).

[0079] Example 12: Description of the shape and function of printed organs (models): The axis of the blood vessels lies in a single plane: a plane parallel to the plane of the printed layer of the organ. The cross-section of the vessels is the same shape as that of natural blood vessels present in the body, ensuring the smoothest possible surface of the 3D printed vessels. Functional tests of beta INS-1E cells were performed. For this purpose, pieces were printed from four main types of bioinks, with the central part filled with cells. The specific types of bioinks were diversified with regard to the amount of dECM powder.

[0080] Types of dECM-based bioinks: A) Sonicated 5% hydrogel, powder content 0–50% B) Sonicated 10% hydrogel, powder content 0–50% C) Non-sonicated 5% hydrogels (prepared by enzymatic digestion), with powder content of 0–50%. D) Non-sonicated 10% hydrogels (prepared by enzymatic digestion), with powder content of 0–50%.

[0081] Beta cell functionality was demonstrated for each bioink; however, after 7 days of observation (culture at 37 °C; 5% CO2) and performing a glucose stimulation test, it was demonstrated that the best longevity was observed for the sonicated dECM-based bioinks with dECM contents ranging from 1% to 50%. The first diffusion signals were observed at the 2nd to 3rd minute of the experiment (graph in Figure 10).

[0082] Example 13: Coagulation test-hemolysis test flow: In the first step of the experiment, sterile biomaterials (dECM-based bioink (containing bioink B) and Pluronic) (200 µl) were poured onto the surface of the pits (16 mm diameter) of a 24-pit plate.

[0083] Fresh whole blood without anticoagulants was then drawn from the large animals that had been receiving anticoagulants and from animals that had not received these drugs. 200 μl of whole blood was applied to each biomaterial tested.

[0084] After the incubation period, 1000 μl of deionized water was added to the samples (to cover the entire test sample), and the plate was then shaken at 100-300 rpm for 30 seconds. In the next step, the plate was incubated motionless for 5 min to release the free hemoglobin, after which time 200 μl samples were collected to read the absorbance at a wavelength of 540 nm.

[0085] For each blood type, a positive control (characterized by maximal hemolysis) was also prepared, along with water and test substance controls (Figure 15).

[0086] The absorbance value is proportional to the concentration of free hemoglobin in deionized water due to lysis of red blood cells. This method indirectly correlates with the degree of blood clotting on the surface of the biomaterial (Figure 14).

[0087] Higher absorbance values ​​indicate higher hemoglobin concentration and less blood clotting on the surface of the biomaterial.

Claims

1. A method for manufacturing a perfusible three-dimensional tissue model in which channels enabling fluid flow are distributed throughout the structure, i) Bioprinting the vascular system by extrusion, the bioink for printing blood vessels is a mixture containing a nonionic copolymer surfactant and a hydrogel containing extracellular matrix, endothelial cells, and fibroblasts. The bio-ink fibers that form the wall opening and closing the top of the channel are printed parallel to the channel axis. The model body is bioprinted using an extrusion method, and the bioink used to print the body contains a dECM solution in pepsin. The vascular bio-ink is printed on fibers that are printed in the same direction as the main bio-ink fibers, which are printed in parallel positions. Here, the temperature of the printhead equipped with bio-ink is in the range of 10–26°C, the diameter of the printing needle is in the range of 100–609 nm, the pressure used in the bio-printing process is in the range of 5–200 kPa, the fiber printing speed is in the range of 5–40 mm / s, and the length of the individual printed fibers is in the range of 150 mm–5000 mm. ii) The process of placing the obtained system in an incubator and bringing it to a temperature at which the bio-ink for printing the vascular system melts, iii) A step of washing the channel with phosphate-buffered saline, iv) A step of selectively inducing proliferation within the channel by cells in the culture medium, Includes, A method for manufacturing the channel, wherein the cross-section of the channel is the same as the cross-section of a natural blood vessel present in the body.

2. A method according to claim 1, characterized in that the pressure used in the process of bioprinting the body and the layer surrounding the channels of the model is in the range of 5 to 40 kPa in the case of printing with a bioink containing pancreatic islets, and in the range of 5 to 200 kPa in the case of printing with a bioink containing cells, particularly endothelial cells or fibroblasts.

3. A method according to claim 1, characterized in that the pressure used in the process of bioprinting a vascular system is in the range of 5 to 200 kPa in the case of printing with a bioink containing cells, particularly endothelial cells or fibroblasts, and in the case of printing with a bioink that does not contain cells, the pressure is in the range of at least 5 kPa.

4. A method according to any one of claims 1 to 3, characterized in that at least one print head equipped with a temperature control function is used for printing.

5. A method according to any one of claims 1 to 3, wherein the bioink for printing blood vessels is a hydrogel composition containing an extracellular matrix, which is present in a solution of phosphate-buffered saline at a concentration of 5 to 10% by sonication, and contains endothelial cells and fibroblasts and formula (C ３ H ６ O.C. ２ H ４ O) ｘ A method characterized by comprising a compound in which x represents a compound that repeats 10 to 1000 times.

6. A method according to any one of claims 1 to 3, characterized in that the bioink used for printing blood vessels comprises a suspension containing endothelial cells and fibroblasts in a 1:2 ratio in a hydrogel containing an extracellular matrix at a concentration of 5 to 10% in phosphate-buffered saline, wherein the cell count is 8 million / ml.

7. A method according to any one of claims 1 to 3, wherein the bioink used to print on a body comprises at least one crosslinking agent and a photoinitiator, preferably the crosslinking agent being gelatin methacrylate, gelatin methacrylamide, and / or hyaluronic acid methacrylate, and the photoinitiator being lithium phenyl-2,4,6-trimethylbenzoyl phosphinate.

8. A method according to any one of claims 1 to 3, characterized in that step ii) is carried out at a temperature of 37°C or lower over a period of 24 hours, preferably over a period of 30 to 40 minutes.

9. A method according to any one of claims 1 to 3, characterized in that the cells proliferating in step iv) are selected from the group consisting of endothelial cells, fibroblasts, or a mixture thereof in a 1:2 ratio, and the cells proliferating in the channel have a concentration of 5 million to 10 million cells / ml, preferably 8 million cells / ml, in the culture medium.

10. A bionic model comprising a perfusable system obtained by the method described in claim 1, Each model body-filled layer has 2 to 4 outline layers for the model's bionic channels, with the paths of consecutive layers positioned at angles of up to 90 degrees to each other, and the layers surrounding the channels being parallel to each other in the same direction. The layers that open and close the model body and the layers that open and close the blood vessel flow consist of 1 to 10 layers, and the height of each layer is 0.1 to 1.2 mm. A bionic model characterized by a body layer structure that is a sandwich system, with the ratio of layer height to channel outer wall height being in the range of 1:1.1 to 1:

2.

11. A bionic model according to claim 10, characterized in that channels are distributed throughout the entire structure of the bionic model, and the cross-section of the channels is the same as the cross-section of natural blood vessels present in living organisms.

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