Systems for storing or culturing organ or tissue models and methods of use thereof

Abstract

The system is designed to verify the functionality and enable handling of excised organ or tissue models, which may be used to culture excised organ or tissue models from donors and / or organ or tissue models made using other techniques such as 3D and / or 4D bioprinting or electrospinning, and comprises a chamber for the organ or tissue model with means for measuring and regulating the temperature of the internal perfusate, a perfusate container with means for measuring and regulating the temperature of the internal perfusate, means for measuring the glucose concentration of the perfusate and administering glucose to the perfusate, means for measuring the pH of the perfusate and administering a substance to adjust the pH of the perfusate, means for non-contact mixing of the perfusate, and means for measuring and controlling flow parameters of the perfusate.

Description

[Technical Field]

[0001] The present invention relates to a system for storing or culturing organ or tissue models with a flow or vascular system and methods for using the system. The system is designed to verify the functionality and enable handling of excised organ or tissue models, but is not limited to this. The system can be used to culture organ or tissue models excised from donors and / or organ or tissue models created using other technologies, such as 3D and / or 4D bioprinting or electrospinning. The system can be used for the flow culture of organs that cannot be excised or cannot be treated in a patient's body. The term "organ flow system" refers to an artificially created organ with formed channels. The term "organ vasculature" refers to a vascular system with endothelial cells excised from a donor or artificially created. The term "storage" refers to preserving an organ or tissue model at a reduced metabolic rate at low temperature or before colonization with cells. The term "cultivation" refers to preserving an organ or tissue model colonized with cells in a normothermic environment that corresponds to in vivo conditions. The term "tissue model" refers to a three-dimensional structure containing living cells, which may additionally contain a vascular system, suspended in a biocompatible material. [Background technology]

[0002] U.S. Patent No. 9,756,851 B2 discloses a concept, method, and apparatus for preserving a harvested organ until transplantation. The organ perfusion apparatus includes a storage chamber for storing the organ. The perfusion circuit includes a first line for supplying oxygenated liquid to the organ and a second line for draining spent liquid from the organ. The perfusion apparatus also includes a device operatively connected to the perfusion circuit for preserving the organ at a generally normothermic temperature. In addition, the device includes a means for controlling the pressure of the perfusion liquid, an oxygenation means for oxygenating at least some of the liquid, a filtering means, and a flow control means for controlling the flow of at least some of the liquid.

[0003] Patent document WO1996029865A1 describes an organ perfusion device capable of perfusing organs at near-normothermic temperatures using blood or other oxygen-carrying substances. The device allows for the assessment of organ viability using online measurements of physiological efficiency. An embodiment uses a computer-controlled blood pump to characterize the physiological state of perfusion. The device includes a section that allows for replacement of lost circulatory volume and a section that allows for the infusion of nutrients, drugs, and components of the perfusion fluid to support organ maintenance or regeneration. The device can also regulate pressure, pH, and temperature; automatically measure the production rates of urine, bile, pancreatic duct secretions, or other physiological exudates; and determine blood or perfusate flow rates, vascular resistance, and organ swelling.

[0004] U.S. Patent Application Publication No. 2017339945A1 discloses an apparatus for perfusion of multiple organs selected from the group including the heart, liver, kidneys, and lungs, comprising a basic unit configured to be detachably connected to a perfusion module for organ perfusion. The basic unit includes tubing connecting a perfusion fluid source to the organ to circulate the perfusion fluid through the organ, first and second pumps connected to the tubing to drive the circulation of the perfusion fluid in the tubing, and a controller connected and configured to control the first and second pumps to control the circulation of the perfusion fluid through the organ. The controller can control the first and second pumps for organ perfusion based on perfusion parameters selected based on the type of organ.

[0005] Document EP1879997A2 discloses a portable organ perfusion device having an organ chamber for supporting an organ immersed in perfusate. A pump circulates the perfusate around a circuit containing the pump, a heat exchanger for cooling the perfusate, an oxygenator for oxygenating the perfusate, and a device for ensuring a constant supply of fluid to the organ vasculature. A bypass channel provides a fluid connection that allows excess fluid to bypass the organ. The device further comprises an oxygen source, a sensor, a power source, and a control unit for receiving information from the sensor and providing control commands to a control means. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 9756851B2 [Patent Document 2] WO1996029865A1 [Patent Document 3] U.S. Patent Application Publication No. 2017339945A1 [Patent Document 4] EP1879997A2 Summary of the Invention [Problem to be solved by the invention]

[0007] Solutions are needed that ensure efficient control for maintaining normal temperatures and fluid regulation or pH control for efficient, more automated, and accurate repair or cultivation of organ and tissue models. The objective of the present invention is to provide suitable conditions for the cultivation and storage of organs, such as the pancreas, liver, kidney, lung, heart, small intestine, large intestine, thyroid, skin, and brain, harvested from deceased donors, or tissue models printed with vasculature using other techniques, such as 3D and / or 4D bioprinting. The term "bioengineered organ" refers to both tissue models printed directly with cells using 3D and / or 4D techniques, and to scaffolds (vasculature and tissue models) that subsequently anchor the respective cells. The term "3D bioprinting" refers to the formation of three-dimensional structures containing living cells using additive manufacturing techniques (layer-by-layer material application). In 4D bioprinting, the fourth dimension is time, and the technique results in a band of material that is subsequently formed to transform into a predefined object (shape). [Means for solving the problem]

[0008] In a first aspect of the invention there is provided a system for storing or culturing organ or tissue models preferably removed from a donor and preferably selected from the group consisting of pancreas, liver, kidney, lung, heart, small intestine, large intestine, thyroid, skin, brain or organ or tissue models made using other techniques, preferably using 3D bioprinting techniques, 4D bioprinting techniques and / or electrospinning techniques, preferably in the form of printed organ and / or tissue models with a fluidic or vascular system, comprising: - a chamber for an organ or tissue model, the chamber being provided with means for measuring and regulating the temperature of the perfusate in the chamber during operation of the system, said means preferably comprising a water jacket; and - a perfusate container comprising means for measuring and regulating the temperature of the perfusate in the container during operation of the system, preferably also comprising a heating and cooling plate, the perfusate container being connected to the organ chamber by at least one first line through which perfusate can flow from the perfusate container to the organ chamber during operation, and at least one second line through which perfusate can flow from the organ chamber to the perfusate container during operation; - means for measuring the glucose concentration of the perfusate and administering glucose to the perfusate; - means for measuring the pH of the perfusate and administering substances to adjust the pH of the perfusate; - means for contactless mixing of the perfusate; - means for measuring and controlling perfusate flow parameters, including perfusate pressure and / or perfusate output expressed in "units of volume / units of time"; - optional means for measuring and controlling one or more parameters selected from perfusate lactate concentration, perfusate sodium ion concentration, perfusate chloride ion concentration, perfusate potassium ion concentration, and perfusate oxygenation level; wherein during operation of the system, the system is configured to circulate the perfusion fluid between the organ chamber and the perfusion fluid container.

[0009] The perfusate flow output represents the flow rate of the perfusate expressed in units of volume per unit of time. The term "organ chamber" refers to a chamber suitable for storing or culturing an organ or tissue model.

[0010] Preferably, the system for storing or culturing organ or tissue models includes a system control means adapted to obtain measurements of at least one parameter selected from the group consisting of perfusate temperature in the organ chamber, perfusate temperature in the perfusate container, perfusate glucose concentration, perfusate pH, and measurements of perfusate pressure and / or perfusate flow parameters including perfusate output expressed in "units of volume / units of time", perfusate lactate concentration, perfusate sodium ion concentration, perfusate chloride ion concentration, perfusate potassium ion concentration, and oxygenation. the system control means being further configured and programmed for automatic and / or manual adjustment based on predetermined criteria of at least one parameter selected from the perfusate temperature in the organ chamber, the perfusate temperature in the perfusate container, the perfusate glucose concentration, the perfusate pH, and perfusate flow parameters including perfusate pressure and / or perfusate output, perfusate lactate concentration, perfusate sodium ion concentration, perfusate chloride ion concentration, and perfusate potassium ion concentration, and oxygenation.

[0011] Preferably, the system for storing or culturing organ or tissue models further comprises means for measuring and controlling oxygenation of the perfusate and / or means for removing air from the perfusate, and the system control means is preferably configured and programmed to obtain perfusate oxygenation measurements and to make automatic and / or manual adjustments to the oxygenation of the perfusate based on predetermined criteria.

[0012] The system for storage or cultivation of organ or tissue models preferably further comprises means for measuring loss and replenishment of perfusate, preferably based on readings of the weight of perfusate removed from the system, and the system control means is preferably configured and programmed to automatically and / or manually replenish perfusate based on predetermined criteria.

[0013] Preferably, the means for non-contact mixing of the irrigation fluid is provided as a rocking mechanism for rocking the irrigation fluid container, and the system control means is preferably configured and programmed to automatically and / or manually adjust the rocking of the irrigation fluid container.

[0014] Preferably, oxygenation of the perfusate is carried out using an oxygenator peripherally connected to the perfusate container to form an oxygenator circuit, and the means for mixing the perfusate is provided in the form of a cylindrically designed perfusate container, with the inlet and outlet of the oxygenator circuit being positioned on opposite sides of the perfusate container and tangential to the cylindrical wall of the perfusate container, thereby mixing the perfusate during operation of the system.

[0015] Preferably, the system comprises means for taking a sample of the perfusate.

[0016] Preferably, the organ chamber comprises a rotation mechanism that allows the organ chamber to rotate during operation of the system, and the system control means is preferably configured and programmed to automatically and / or manually regulate the rotation of the organ chamber. Preferably, the system control means is configured and programmed to rotate the organ chamber by 180° every 30 minutes for at least two hours, or by 90° at least four times every 15-60 minutes for at least two hours.

[0017] Preferably, the organ chamber, perfusate container, and / or means for measuring and controlling oxygenation of the perfusate have means for regulating the temperature to between 0 and 37°C.

[0018] Preferably, the system further comprises additional means for administering drugs, nutrients and / or irrigation fluid components, and the system control means is preferably configured and programmed to automatically and / or manually administer the drugs, nutrients and / or irrigation fluid components based on predetermined criteria.

[0019] Preferably, air is removed from the perfusion solution by a bubble separator attached to the first line between the perfusion solution container and the organ chamber, and movement of the perfusion solution between the perfusion solution container and the organ chamber and in the opposite direction during operation of the system is performed by at least one pump, preferably a peristaltic pump.

[0020] In a second aspect, there is provided a use of the system according to the invention for storing and / or culturing organ or tissue models extracted from a donor or created by other techniques, preferably using 3D bioprinting, 4D bioprinting and / or electrospinning.

[0021] Preferably, the system according to the invention is used for culturing 3D bioprinted organs, preferably using a rotation mechanism during the stage of colonization of the vasculature with endothelial cells.

[0022] Preferably, the disease state or progression of the organ or tissue model is assessed during storage and / or culture.

[0023] Preferably, the effect of the biologically active agent on the disease state or progression of the organ or tissue model is analyzed during storage and / or culture.

[0024] Preferably, the effect of drug and / or genetic engineering therapy on the state of the organ or tissue model is evaluated during storage and / or culture.

[0025] Maintaining organ or tissue models at normothermic conditions during culture is essential as it provides the organ or tissue model with an optimal metabolic state that is comparable to in vivo conditions, allowing the organ's health to be assessed.

[0026] Perfusate for culturing isolated organs contains, without limitation, a concentration of red blood cells that tend to settle. The use of a non-contact means for mixing the perfusate, preferably in the form of a mechanism for agitating the perfusate container, or a container configuration that allows for the induction of vortex motion within the container, results in a uniform distribution of red blood cells within the perfusate. This also helps maintain sterility in the flow system and mitigates mechanical damage to red blood cells that occurs when using other types of mixers. Mixing the perfusate aids in uniform heating of the perfusate and in the continuous mixing of the liquid's components.

[0027] The rotation mechanism for rotating the organ chamber can be used without limitation when culturing organs created by 3D bioprinting technology and during the colonization of the bioprinted vasculature with endothelial cells. Preferably, the rotation mechanism rotates the organ chamber by 180° every 30 minutes for at least two hours, or by 90° at least four times every 15-60 minutes for at least two hours. The rotation mechanism allows the colonization of the entire vasculature. The rotation is performed so that the cells can "stick" to the channel walls and fall by gravity. The rotation can be performed at a smaller angle, such as 36°, 10 times for at least two hours, or continuously at a speed of, for example, one rotation per hour for at least two hours. The use of this rotation mechanism ensures uniform distribution of the colonized cells across the channel surface.

[0028] The system according to the present invention comprises: - Organs directly from deceased donors and - Organs removed from living donors (e.g., family or crossover transplants); - Organs printed using 3D (and 4D) bioprinting technology (organs with a fluid system including a vascular system) and - 3D (and 4D) bioprinted tissue models with fluid systems (including vascular systems); - Organ and tissue models created by electrospinning technology (organs with fluid systems, including vascular systems); - Organ and tissue models created by combining two methods (bioprinting and electrospinning) and - Organs removed from farmed, laboratory and genetically modified animals; - Genetically engineered organs and - Organs created in an ex vivo (laboratory) setting using tissue engineering and genetic engineering techniques. The method may be used for the cultivation, storage and / or treatment (including regeneration) of the cells.

[0029] Organ and tissue models cultured in the system according to the invention can be used in medicine and for basic research as well as research and development work. Furthermore, the system according to the invention can be used to culture tissue models with fluid systems (including vasculature) created by 3D / 4D bioprinting or other techniques (including electrospinning, which uses an electric field to obtain nanofibers from a molten polymer or its solution) that allow for the creation of models with fluid systems, such as formed cancer-targeted tissue models, induced disease entities, and tissue models suitable for investigating genetic engineering therapies used in both research and personalized medicine.

[0030] The system can be a device used to store and treat organs prior to transplantation, to test the effects, i.e., toxicity, efficacy, of biologically active substances, to assess the severity of specific disease stages, and to evaluate the effectiveness of drug and genetic engineering therapies.

[0031] Advantageously, the system has the capability to precisely dose drugs, active substances, and sample for biological and chemical analysis. These capabilities make the system a multifunctional device capable of conducting advanced research, preparing organs for transplantation, and assessing the functional status of organs, in addition to organ storage / cultivation and treatment. Thus, the system should contribute to improving the effectiveness of transplant procedures and enable advanced research (both at the basic and preclinical stages).

[0032] The invention is shown in the embodiment shown in the figures. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is an overall diagram of a system that allows for the cultivation of both organs removed from donors as well as organ or tissue models created using other techniques. [Figure 2] 1 is a graph showing the change in perfusate parameters: a) pressure (mm Hg), b) flow rate (ml / min), c) perfusate loss (g), and d) glucose concentration (mg / ml) during two days of culturing a pig kidney in the system of Example 1. [Figure 3] Graphs showing the changes in perfusate parameters a) pH, b) Na+ ion concentration (mmol / l), c) glucose concentration (mg / dl), d) pCO2 (kPA), e) K+ ion concentration (mmol / l), f) saturation (oxygenation) (%), g) pO2 (kPA), h) Cl- ion concentration (mmol / l) and i) lactate concentration (mmol / l) during the culture of the pig kidneys of Example 1. [Figure 4] FIG. 1 shows the system used to culture the pig kidneys of Example 1. [Figure 5] FIG. 2 is a block diagram showing an electronic system control means. [Figure 6] FIG. 1 shows the geometry of the bioengineered pancreatic organ of Example 2. [Figure 7]Graphs showing monitoring of a) heating / cooling plate temperature (°C) (lower line) and organ chamber temperature (°C) (upper line), b) arterial pressure RR (mm Hg), c) flow rate (ml / min). [Figure 8] 1 is an image of a bioengineered pancreas acquired using a resonance technique. [Figure 9] Graphs showing the changes in perfusate parameters: a) pH, b) glucose concentration (mmol / l), c) lactate concentration (mmol / l), d) pO2 (kPA), e) Na+ ion concentration (mmol / l), f) K+ ion concentration (mmol / l) during 30 hours of culture of the bioengineered pancreas of Example 2. [Figure 10] Figure 1 shows the difference in oxygenation of the perfusate after 30 hours. Sample A is deoxygenated fluid, collected after it left the chamber in which the bioengineered organ was placed. Sample B is oxygen-enriched fluid, collected before it was placed in the chamber. [Figure 11] 1 is a graph showing the difference in insulin concentration following stimulation with varying concentrations of glucose solutions, where the grey line is a control recording of the stimulation in which the islets were inserted with glucose, and the black line is a recording of islets printed in a bioengineered pancreas and maintained in a system according to the invention. [Figure 12] FIG. 1 shows the system used in Example 2 to culture the bioengineered pancreas obtained with the 3D bioprinting process. [Figure 13] FIG. 10 is a cross-sectional view of a dialysis fluid container according to Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0034] Example 1 - Pig kidney culture When optimizing the function of the system according to the invention, the pig kidney was used as a model. - Minimized warm ischemic time and easy to obtain and extract; - Vascularized for easy connection, - A faster metabolism of the organs allows the system to be used to its full potential, - possibility of monitoring organ function based on urinary secretions; It was used for many practical reasons.

[0035] The kidneys of pigs were harvested by interrupting the technological cycle of meat production. The pigs were stunned by electric current, exsanguinated, and then transported directly to the operating room for kidney removal (warm ischemic time <10 min). The harvest was completed when the artery was flushed with UW Ice Solution (University of Wisconsin Solution). During the exsanguination of the pigs, the blood was stored in a sterile manner (heparinized).

[0036] The composition of the perfusion solution and organ culture conditions in the system of the present invention are as follows. The main component of the perfusion solution used during normothermic culture was a low-potassium solution for enhanced mechanical perfusion (Belzer MPS® UW Machine Perfusion Solution) enhanced with red blood cell concentrate in a 7:3 ratio, achieving a hematocrit of approximately 0.15. In addition, the MPS solution was supplemented with CaCl2, amino acid concentrate (Trimel N9-1070 EC), vitamins, sodium bicarbonate (to achieve a pH of 7.35-7.45), heparin, antibiotics (PenStrep), insulin, and dexamethasone. This study compared mean perfusion pressures of 50 mmHg and 75 mmHg. The oxygen partial pressure in the arterial branch of the system was approximately 50 mmHg, and the flow rate through the oxygenator was approximately 3 L / min. Kidneys were cultured for 48 hours and then histopathologically evaluated. Glucose concentrations were maintained at 100-150 mg / L by continuous glucose infusion based on the perfusate concentration. Diuretic losses were automatically replenished with Ringer's solution (at a 1:1 ratio). Viability of the cultured kidneys was assessed based on urine production, glucose consumption, and blood saturation (oxygenation) upstream and downstream of the organ. If perfusion decreased due to increased vascular resistance, the kidneys were treated (as needed) with an infusion of urapidil (Ebrantil). Figures 2 and 3 show the changes in perfusate parameters during the culture of the pig kidneys of Example 1.

[0037] The system used for the culture of pig kidneys (Figure 4) was equipped with the following: - a sealed organ chamber 2 equipped with means for regulating the temperature of the perfusate in chamber 2 during operation of the system. Chamber 2 contained a heating and cooling circuit 13 in the form of a heating jacket. Chamber 2 was equipped with ports allowing connection of the organ, inflow perfusate, outflow perfusate, and a bubble trap, which is a bubble separator 6 designed to vary the pressure or volume of gas in chamber 2. - a perfusate container 1 provided with means for regulating the temperature of the perfusate in the form of a heating / cooling plate 11, the temperature of which is regulated by a Peltier module 27. The perfusate container 1 is connected to the chamber 2 by at least one first line 17 through which perfusate can flow from the perfusate container 1 to the organ chamber 2 during operation, and by at least one second line 18 through which perfusate can flow from the organ chamber 2 to the perfusate container 1 during operation. - Means for measuring and controlling oxygenation of the perfusate, comprising an oxygenator 5, an oxygenation-saturation sensor 19, and an oxygenation device, which is a gas mixing cylinder assembly and reducer that provides the appropriate pressure and gas flow rate through the oxygenator 5. The oxygenator 5 also comprised a temperature sensor 32 of the oxygenator 5, and a heating and cooling circuit 12 in the form of a heating jacket of the oxygenator 5. The means for measuring and controlling oxygenation of the perfusate is also referred to as the circuit of the oxygenator 5.

[0038] The temperature of the perfusate is regulated by heating and cooling circuits 12 and 13 of the organ chamber 2 and oxygenator 5, allowing the flow of heated or cooled liquid, and the heating and cooling circuit of the perfusate container 1, temperature sensor 30 of the organ chamber 2, temperature sensor 31 of the perfusate container 1, temperature sensor 32 of the oxygenator 5, and regulation system and pump 9 provide a flow of heated or cooled liquid, preferably distilled water, which is used only to transfer heat from the heating device to the heating jacket of the organ chamber 2 and the heating jacket of the oxygenator 5 without coming into contact with the organ or perfusate. - Means 4 for measuring the glucose concentration of the perfusate and administering glucose to the perfusate, comprising an enzyme electrode-based sensor 25, an electronic measurement system and a pump 9 for administering a glucose-containing supplementary fluid. For measurements in direct contact with the perfusate in the flow system, a specially designed disposable enzyme electrode was used. The glucose concentration data was displayed on the user's panel and, based on this reading, the system control means administered a glucose-containing supplementary fluid automatically or when required by the operator. - means 3 for measuring the pH of the perfusate and administering substances for adjusting the pH of the perfusate, said means 3 comprising a pH electrode, an electronic measuring system and a pump for administering the substances for adjusting the pH. - Means for contactless mixing of the irrigation fluid, provided in the form of a rocking mechanism 16 for the irrigation fluid container 1. The rocking mechanism 16 rocks the irrigation fluid container 1. - Means for removing air from the perfusate, comprising two bubble separators 6, i.e., perfusate bubble traps, one of which was placed in the circuit of the oxygenator 5, and the other (described above) was placed just before the organ chamber 2. - Means for measuring and controlling perfusate flow parameters, including the pressure of the perfusate flowing from the perfusate container 1 to the organ chamber 2 during system operation. The means for measuring and controlling the perfusate flow parameters comprised an electromechanical membrane pressure sensor 7, an electronic control system, and a peristaltic pump 9. The pressure was measured in a non-contact manner. The perfusate was separated from the sensor 7 by a flexible membrane. The perfusion pressure was set by the perfusion operator on the user panel using the system control means. The system control means automatically selected the flow rate to achieve the set pressure. The maximum allowable flow rate was also preset on the user panel. - Means 14 for measuring and replenishing the loss of perfusion fluid, comprising a weight based on an extension sensor, an electronic measuring system and a dosing pump 9. The amount of fluid to be added is displayed on the control panel and, depending on the settings, can be filled automatically or manually by the operator of the device using the system control means. - system control means configured and programmed to obtain measurements of parameters, the parameters including at least one parameter selected from the group consisting of the temperature of the perfusate in the organ chamber 2, the temperature of the perfusate in the perfusate container 1, the glucose concentration of the perfusate, the pH of the perfusate, the pressure of the perfusate and / or the output of the perfusate expressed in "units of volume / units of time", the lactate concentration of the perfusate, the sodium ion concentration of the perfusate, the chloride ion concentration of the perfusate, the potassium ion concentration of the perfusate, the oxygenation of the perfusate, and perfusate flow parameters including automatic and / or manual adjustment based on predetermined criteria of at least one parameter selected from perfusate temperature in the organ chamber, perfusate temperature in the perfusate container, perfusate glucose concentration, perfusate pH, and perfusate flow parameters including perfusate pressure and / or perfusate output, perfusate lactate concentration, perfusate sodium ion concentration, perfusate chloride ion concentration, perfusate potassium ion concentration, and perfusate oxygenation.

[0039] The main controller of the system control means was based on a microprocessor 21 and consisted of two parts: a first part that ensured real-time operation by controlling all the peripherals of the device, and a second processor 20 that was responsible for the graphical interface (GUI) that allowed the observation and input of parameters.

[0040] The TEC controller 23 was designed to control the Peltier module 27 to maintain the perfusate temperature between 0 and 37°C, allowing the organ to be treated in both normothermic and hypothermic states. A fan 28 was used to control the radiator temperature to maintain a temperature difference of less than 20°C between the hot and cold sides of the Peltier module.

[0041] The controller 22 recorded the temperature measurements of the Peltier module 27, the temperature measurements of the perfusate heating and cooling plate 11, the temperature measurements in the water jacket of the organ chamber 2, and the temperature measurements in the perfusate container 1.

[0042] Several peristaltic pumps 9 were used, the first one was used for circulating the main perfusion fluid and was controlled to maintain a constant fluid pressure controlled by an expansion pressure sensor 7. Three further pumps 9 were used to administer medicinal substances. It was possible to administer the drugs at a specific flow rate (ml / min).

[0043] The control panel of the system according to the invention can be accessed from the web browser level and can therefore be opened using any device with internet access, such as a mobile phone, a tablet computer or a laptop computer.

[0044] The control panel allowed for setting parameters such as the temperature of the heating and cooling plate 11, the parameters of the PID controller for temperature control, turning the pressure control on and off, the preset perfusate pressure (mmHg), the minimum and maximum perfusate flow rate (output), the preset speed (ml / min) of each step motor 29, turning on the perfusate mixing mechanism, and setting the perfusate mixing speed. The control panel also allowed for monitoring measured parameters such as the perfusate pH, the perfusate glucose concentration, and the temperature of the heating and cooling plate 11. Figure 5 is a block diagram of the electronic system control means.

[0045] Example 2 - Cultivation of organs obtained using bioprinting technology A bioengineered pancreatic organ was used to conduct the studies performed using 3D bioprinting. The organ contained pancreatic islets and included a scaffold made with base bioink and a duct system made with vascular bioink (Figure 6). The duct system consisted of a primary duct that branched into three secondary ducts arranged in a spiral before joining into a single outflow duct. The geometric parameters of the organ were described as follows: - The overall dimensions of the model were 32mm x 40mm x 17.5mm. - The length of one secondary conduit was 340 mm. - The total length of the secondary conduit was 1020 mm. - The diameter of the primary conduit was 1.5 mm. - The diameter of the secondary conduit was 1 mm. - The scaffold volume for the islets was 20.5 ml. - The volume of the tube was approximately 0.9 ml.

[0046] The culture process was carried out under controlled temperature conditions, with the temperature of the organ chamber 2 being 37-39°C and the temperature of the heating and cooling plate 11 for heating the liquid being 36.5-37°C. Flow and pressure levels were also monitored throughout the test (Figure 7).

[0047] The connection of the bioengineered organ to Organ Chamber 2 and monitoring of parameters (pressure, flow, temperature) did not affect the structure and function of the printed vasculature of the bioengineered organ, as confirmed by resonance techniques (Figure 8).

[0048] During incubation of the bioengineered organ in organ chamber 2, perfusate parameters such as oxygenation, pH, glucose concentration, lactate concentration, sodium ions, and potassium ions were monitored in real time (Figure 9).

[0049] Controlling the parameters allowed us to monitor the perfusate composition and improve its quality, if necessary, to maintain the bioengineered organ in optimal culture / incubation conditions. Furthermore, the perfusate used in the system was supplemented with red blood cells, which are biological oxygen carriers. Perfusate samples were taken during the test (after 30 hours) and showed clear differences in perfusate oxygenation (Figure 10). Sample A was the deoxygenated liquid and was taken after it left Chamber 2, where the bioengineered organ was placed. Sample B was the oxygen-enriched liquid and was taken before it entered Chamber 2.

[0050] Tests to evaluate the functionality of the bioengineered organs were also performed, which, in addition to controlling the basic parameters described above, involved taking samples at set time points to evaluate insulin levels after stimulation with glucose solutions of varying concentrations (Figure 11). The results of the bioengineered organs showed no difference between isolated islets printed as bioengineered organs and islets that had not undergone the 3D bioprinting process.

[0051] The system used for the cultivation of bioengineered organs (Figure 12) included: - a sealed, movable organ chamber 2 equipped with means for regulating the temperature of the perfusate in chamber 2 during operation of the device. Chamber 2 contained a heating and cooling circuit 13 in the form of a heating jacket, a rotation mechanism 15 for rotating organ chamber 2, and a rotation drive mechanism for chamber 2. Chamber 2 was equipped with ports allowing connection of the organ, inflow perfusate, outflow perfusate, and a bubble separator 6 designed to vary the pressure or volume of gas in chamber 2. - a perfusate container 1 provided with means for regulating the temperature of the perfusate in the form of a heating / cooling plate 11, the temperature of which is regulated by a Peltier module 27. The perfusate container 1 is connected to the chamber 2 by at least one first line 17 through which perfusate can flow from the perfusate container 1 to the organ chamber 2 during operation, and by at least one second line 18 through which perfusate can flow from the organ chamber 2 to the perfusate container 1 during operation.

[0052] The temperature of the perfusate was regulated by a heating and cooling circuit in the organ chamber 2 and the perfusate container 1, a temperature sensor 30 in the organ chamber 2, a temperature sensor 31 in the perfusate container 1, and a control system and pump 9 that allowed for the flow of heated or cooled liquid. The organ chamber 2 had a heating and cooling circuit 13 that allowed for the flow of heated or cooled liquid. - Means 4 for measuring the glucose concentration of the perfusate and administering glucose to the perfusate, comprising an enzyme electrode-based sensor 25, an electronic measurement system and a pump 9 for administering glucose-containing fluid. For measurements in direct contact with the perfusate in the flow system, a specially designed disposable enzyme electrode was used. Glucose concentration data was displayed on the user's panel and, based on this reading, additional fluid was administered by the system control means, either automatically or when required by the operator. - means 3 for measuring the pH of the perfusate and administering a substance for adjusting the pH of the perfusate, comprising a pH electrode, an electronic measuring system and a pump 9 for administering a substance for adjusting the pH; - Means for contactless mixing of the irrigation fluid, provided in the form of a rocking mechanism 16 for the irrigation fluid container 1. The rocking mechanism 16 rocks the irrigation fluid container 1. - Means for removing air from the perfusate, comprising a perfusate bubble trap, which is a bubble separator 6 placed just before the organ chamber 2. - Means for measuring and controlling perfusate flow parameters, including the pressure of the perfusate flowing from the perfusate container 1 to the organ chamber 2 during system operation. The means for measuring and controlling the perfusate flow parameters comprised an electromechanical membrane pressure sensor 7, an electronic control system, and a peristaltic pump 9. The pressure was measured in a non-contact manner. The perfusate was separated from the sensor 7 by a flexible membrane. The perfusion pressure was set by the perfusion operator on the user panel using the system control means. The system control means automatically selected the flow rate to achieve the set pressure. The maximum allowable flow rate was also preset on the user panel. - A system control system corresponding to that of example 1. This system control means furthermore allowed for control of the rotation of the organ chamber 2.

[0053] Example 3 The perfusate container 1 of the system used to culture the porcine kidney in Example 1 was not equipped with a heating mechanism using a rocking mechanism 16 or a Peltier module 27. The container 1 was cylindrical with a port located at the bottom (Figure 13). The perfusate inlet and outlet of the oxygenator 5 circuit were positioned on opposite sides of the perfusate container 1, tangential to the cylindrical wall, to introduce vortex motion into the container 1, achieving non-contact mixing of the perfusate. The liquid flow rate at the inlet and outlet of the oxygenator 5 circuit was set to 0–2 L / min. Heating or cooling of the perfusate was performed using a water jacket 33. The perfusate container 1 was fabricated using 3D printing technology. [Explanation of symbols]

[0054] 1. Perfusion fluid container 2. Organ Chamber 3. Means for measuring pH 4. Means for measuring glucose concentration 5. Oxygen supply 6. Bubble separator 7. Pressure sensor for perfusion fluid 8. Means for taking samples of the perfusate 9 Peristaltic Pump 10 Containers for drugs, nutrients and / or irrigation fluid components 11 Heating and cooling plate 12 Oxygen supply heating and cooling circuit 13 Organ chamber heating and cooling circuit 14. Means for measuring perfusate loss 15 Rotation mechanism for rotating the organ chamber 16. Irrigation fluid container rocking mechanism 17 First Line 18 Second Line 19 Oxygen addition / saturation sensor 20 GUI Processor 21 Real-time Microprocessor 22 TEC controller 23 Step Motor Controller 24 Temperature Measurement System 25 Glucose Sensor 26 Liquid Level Sensors 27 Peltier module 28 fans 29 Step Motor 30 Organ chamber temperature sensor 31 Temperature sensor for irrigation fluid container 32 Oxygen supply temperature sensor 33 Water jacket of the irrigation fluid container of Example 3

Claims

1. A system for storing or culturing organ or tissue models created using one or more technologies selected from 3D bioprinting technology, 4D bioprinting technology, and electrospinning technology, comprising: an organ chamber (2) for the organ or tissue model, equipped with means for measuring and regulating the temperature of the perfusate in the organ chamber (2) during operation of the system; a perfusion fluid container (1) equipped with means for measuring and regulating the temperature of the perfusion fluid in the container (1) during operation of the system, the perfusion fluid container (1) being connected to the organ chamber (2) by at least one first line (17) allowing the perfusion fluid to flow from the perfusion fluid container (1) to the organ chamber (2) during operation, and by at least one second line (18) allowing the perfusion fluid to flow from the organ chamber (2) to the perfusion fluid container (1) during operation; - means (4) for measuring the glucose concentration of said perfusate and administering glucose to said perfusate; means (3) for measuring the pH of the perfusate and administering substances to adjust the pH of the perfusate; - means for contactless mixing of said perfusion fluid; means for measuring and controlling flow parameters of said perfusate, including the perfusate pressure and / or the perfusate output expressed in "units of volume / units of time"; - means for measuring and controlling one or more parameters selected from the lactate concentration of the perfusate, the sodium ion concentration of the perfusate, the chloride ion concentration of the perfusate, the potassium ion concentration of the perfusate, the oxygenation level of the perfusate; A system for storing or culturing an organ or tissue model, comprising: configured to circulate the perfusion fluid between the organ chamber (2) and the perfusion fluid container (1) during operation; The organ chamber (2) comprises a rotation mechanism (15) that allows rotation of the organ chamber (2) during operation of the system. A system for storing or culturing organ or tissue models.

2. 2. A system for storing or culturing organ or tissue models, comprising a system control means configured and programmed to acquire measurements of at least one parameter selected from the group consisting of the temperature of the perfusate in the organ chamber (2), the temperature of the perfusate in the perfusate container (1), the glucose concentration of the perfusate, and the pH of the perfusate, and measurements of flow parameters of the perfusate, including the pressure of the perfusate and / or the output of the perfusate expressed in "units of volume / unit of time", the lactate concentration of the perfusate, the sodium ion concentration of the perfusate, the chloride ion concentration of the perfusate, the potassium ion concentration of the perfusate, and the oxygenation level of the perfusate, wherein the system control means is further configured and programmed for automatic and / or manual adjustment of the at least one parameter based on predetermined criteria.

3. 3. The system for storing or culturing organ or tissue models of claim 2, further comprising means for measuring and controlling oxygenation of the perfusate and / or means for removing air from the perfusate.

4. A system for storing or culturing organ or tissue models as described in claim 1, 2 or 3, further comprising means (14) for measuring and replenishing the loss of perfusion fluid.

5. 5. A system for storing or culturing organ or tissue models according to claim 1, 2, 3 or 4, wherein the means for non-contact mixing of the perfusate is provided as a rocking mechanism (16) for rocking the perfusate container (1).

6. 5. A system for storing or culturing organ or tissue models according to claim 3 or 4, wherein oxygenation of the perfusate is carried out by means of an oxygenator (5) connected peripherally to the perfusate container (1) to form an oxygenator circuit, and wherein the means for non-contact mixing of the perfusate is provided in the form of a cylindrically designed perfusate container (1), the inlet and outlet of the oxygenator circuit of the perfusate container (1) being arranged on opposite sides of the perfusate container (1) tangentially to the cylindrical wall of the perfusate container (1), thereby mixing the perfusate during operation of the system.

7. 7. A system for storing or culturing organ or tissue models according to any one of claims 1 to 6, comprising means (8) for taking a sample of the perfusate.

8. A system for storing or culturing organ or tissue models as described in claim 2 or 3, wherein the system control means is configured to automatically and / or manually adjust the rotation of the organ chamber (2).

9. 9. A system for storing or culturing organ or tissue models according to any one of claims 1 to 8, wherein the organ chamber (2), the perfusate container (1) and / or the means for measuring and controlling the oxygenation of the perfusate comprise means for regulating the temperature between 0 and 37°C.

10. 10. A system for storing or culturing organ or tissue models according to any one of claims 1 to 9, further comprising additional means for administering drugs, nutrients and / or perfusate components.

11. 11. A system for storing or culturing organ or tissue models according to any one of claims 3 to 10, wherein air is removed from the perfusion solution by a bubble separator (6) attached to the first line (17) between the perfusion solution container (1) and the organ chamber (2), and wherein movement of the perfusion solution between the perfusion solution container (1) and the organ chamber (2) and in the opposite direction during operation of the system is carried out by at least one pump (9).

12. 12. Use of the system according to any one of claims 1 to 11 for storing and / or culturing organ or tissue models extracted from a donor or created by other techniques.

13. 13. The use of claim 12, wherein the system of claim 8 is used to cultivate a 3D bioprinted organ.

14. 13. The use of claim 12, wherein the disease state or progression of the organ or tissue model is assessed during storage and / or culture.

15. 15. The use according to claim 14, wherein the effect of the biologically active substance on the disease state or progression of said organ or tissue model is analyzed during storage and / or culture.

16. The use according to claim 14, wherein the effect of drug therapy and / or genetic engineering therapy on the condition of said organ or tissue model is evaluated during storage and / or culture.

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