DE102021211875B3 - Bioreactor and method of operating one - Google Patents

Abstract

A bioreactor (2) is specified, which has a reactor chamber (4) for producing a soft tissue (6), which has an analysis unit (8) for measuring a number of tissue parameters (G) of the soft tissue (6), wherein it has a conditioning unit (10) for conditioning the soft tissue (6) according to a number of process parameters (P), wherein it has a control unit (12) which controls the analysis unit (8) and the conditioning unit (10) in such a way that the tissue parameters (G) are measured during production and the process parameters (P) are set depending on the tissue parameters (G), so that the soft tissue (6) is conditioned depending on the tissue parameters (G). A method for operating the bioreactor (2) is also specified.

Description

The invention relates to a bioreactor for producing soft tissue, in particular skeletal muscles, and a method for operating such a bioreactor.

A bioreactor is used to produce tissue, which is then transplanted into a living being after production, for example to replace corresponding diseased, destroyed or lost tissue. In principle, a large number of different types of fabric can be produced, and there are also different types of production. The production of tissue is also referred to as "growth", "maturation" or "tissue engineering".

A part of the production of tissue is the so-called "tissue engineering". This can be divided into two different areas based on the technology used. In the so-called "bottom-up" technique, a tissue framework (also known as a "scaffold") is assembled from individual components, e.g. using 3D printing, cell aggregation, etc. In contrast, in the so-called "top-down" technique a native donor tissue with a corresponding, natural tissue scaffold is used as a starting point and treated and matured as required. The donor tissue is first decellularized to expose the tissue scaffold, and this is then recellularized again. Tissue cultivation according to the "top-down" technique offers several advantages over the "bottom-up" technique, e.g. preservation of the vascularization architecture and preservation of the natural matrix architecture in the tissue scaffold.

In particular, however, the regeneration and/or maturation, i.e. the de- and recellularization, of soft tissue and especially muscle tissue, e.g. 3D skeletal muscles, is very demanding. In order to meet the requirements for clinical usability of the tissue, it is necessary to ensure the highest possible quality of de- and recellularization.

Against this background, it is an object of the invention to generally improve the production of tissue and specifically the regeneration and/or maturation of soft tissue in a bioreactor. To this end, a correspondingly improved bioreactor and an improved method for operating a bioreactor are to be specified.

The object is achieved according to the invention by a bioreactor having the features according to claim 1 and by a method having the features according to claim 16. Advantageous configurations, developments and variants are the subject matter of the dependent claims. The statements made in connection with the bioreactor also apply to the method and vice versa. If steps of the method (i.e. method steps or also process steps) are specified below, advantageous configurations for the bioreactor result from the fact that it is designed to carry out one or more of these steps, in particular by means of a control unit, which is part of the bioreactor.

A bioreactor according to the invention has a reactor chamber for the production, in particular regeneration and/or maturation (i.e. in particular for decellularization and recellularization) of soft tissue, which is accommodated in the reactor chamber for this purpose. The reactor chamber is, for example, a glass cylinder. The soft tissue is also simply referred to below as tissue, but what is meant is a soft tissue, unless something else results from the context. The soft tissue is preferably skeletal musculature, which in particular can be stimulated mechanically and electrically or can also be trained.

The bioreactor also has an analysis unit for measuring a number of tissue parameters of the soft tissue. The tissue parameters indicate in particular the current state or also the actual state of the soft tissue. In addition, the bioreactor has a conditioning unit for conditioning the soft tissue according to a number of process parameters. “A number of” is understood here and also generally to mean “one or more” or “at least one”. In the following it is assumed, without loss of generality, that a number of tissue parameters are measured and a number of process parameters are set, in particular controlled and/or regulated.

In particular, the process parameters control the production and ultimately also influence the tissue parameters. Accordingly, it is possible to influence production by changing the process parameters. Such an influence also occurs in the present case, in other words: by measuring the tissue parameters, the actual progress of the production and the actual quality of the soft tissue are fed back to control the process parameters, so that the actual condition of the soft tissue influences the process parameters and thus also the production. For this purpose in particular, the bioreactor has a control unit which correspondingly controls the analysis unit and the conditioning unit in such a way that the tissue parameters are measured, in particular automatically, in particular repeatedly and/or continuously during production and the process parameters are adjusted in particular in real time depending on the tissue parameters, so that the soft tissue is conditioned in particular automatically depending on the tissue parameters. In this way, the production of the soft tissue (in particular online) is controlled as a function of the actual tissue parameters (ie the current state), which in turn means that the process parameters are in particular continuously and preferably automatically adjusted in such a way that, based on the current state, a predetermined target State is reached, ie in order to achieve the best possible production as a result. An “optimal production” is understood to mean a production that leads to a soft tissue that comes as close as possible to a previously defined specification, eg has specific values for specific tissue parameters. In particular, with the bioreactor described here, non-invasive and continuous analysis of the tissue parameters is advantageously implemented, ie the tissue advantageously remains at all times during the analysis and also during the conditioning in the bioreactor, especially in its reactor chamber, and the conditioning does not have to be stopped or to be interrupted.

A core aspect of the present invention is in particular a bioreactor for the production and specifically for the regeneration and/or maturation of soft tissue, specifically skeletal musculature, using “top-down” technology with multimodal environment and sample parameters. First of all, a donor tissue with a natural tissue framework is used as the starting point and treated and matured as required. In particular, the donor tissue is first decellularized to expose the tissue scaffold, and this is then subsequently recellularized (and thus regenerated altogether). During decellularization, the cells in the donor tissue are removed, leaving a natural organ architecture with a vascular supply network, which is used in the subsequent recellularization to repopulate the tissue framework with cells, e.g. stem cells. However, the details of de- and recellularization are initially irrelevant. In principle, it is also conceivable that only decellularization or recellularization takes place with the bioreactor.

In any case, the tissue in the reactor chamber is washed by a medium which is characterized in particular by one or more process parameters. The medium generally stabilizes tissue maturation during recellularization in terms of osmotic pressure, chemical environment, and nutrient supply, and thus defines the environment around the tissue in the reactor chamber. During production, different media flow into the reactor chamber one after the other, depending on the requirements of the current process step. The respective medium flows into the reactor chamber via a media inlet (also referred to as inlet or flow) and flows out of the reactor chamber again via a media outlet (also referred to as outlet or return). The reactor chamber preferably has an underside with a media inlet for a medium and an upper side with a media outlet for the medium, so that the medium can wash around the soft tissue counter to the force of gravity. The respective medium is in particular a liquid.

Of particular importance in the present bioreactor and the method is that the quality of the tissue is monitored in real time and preferably also without contact, in order to then draw conclusions about the "cell freedom" (the degree of depopulation during decellularization) and the "Regenerative Maturity" (the measure of tissue completion upon recellularization) of the tissue. In other words: the production of the tissue in the bioreactor is monitored online, i.e. during ongoing production, by measuring a number of tissue parameters of the tissue and then controlling a number of process parameters of the bioreactor depending on this, with the aim of optimizing the production . The fabric parameters indicate the current condition (also actual condition) of the fabric and thus also the progress and quality of production. In one possible embodiment, the tissue parameters are also referred to as “morphological and structural parameters”. The tissue parameters preferably include mass, volume, diameter or other dimensions, CIELAB colorimetry or equivalent, tissue stiffness, vessel pressure, vascular pressure, surface texture and/or transparency of the tissue. The process parameters, on the other hand, indicate the environmental modalities of production, i.e. the current state of the bioreactor, and thus characterize the environment of the tissue within the reactor chamber, i.e. the environmental conditions that the bioreactor provides for the production of the tissue, specifically through the medium within the reactor chamber. The process parameters preferably include pH value, pO ₂ value and/or temperature.

Together with the process parameters, the tissue parameters then form the environmental and sample parameters, which overall provide a particularly detailed picture of the quality of the tissue and correspondingly enable particularly optimal feedback. The feedback preferably takes place in two ways: first one, several or all process parameters are suitably adjusted depending on the tissue parameters in order to achieve optimal production. Secondly, one, several or all of the process parameters are suitably also controlled or regulated themselves in order to obtain a defined environment during production with a high level of reliability and to prevent an unwanted change in the process parameters.

The bioreactor presented here thus combines an advantageously automated production of tissue, in particular using “top-down” technology, with real-time parametrics, which is controlled by preferably extensive measurements. In other words, the analysis unit generates feedback on the course of the production, so that this production is dynamically and automatically adjusted as required by appropriate control of the process parameters. The bioreactor thus implements automated production. In particular, the control unit connects the tissue parameters and the process parameters to one another and thus also in a physiological context, which enables a particularly ordered tissue maturation and also an optimization of the production to the quality of the tissue. Exactly how this connection and this context are designed is initially not important in the present case and depends heavily on the specific application, the fabric and the required quality.

A particular advantage of the bioreactor described here is in particular a preferably contactless and particularly holistic real-time monitoring of cell freedom (during decellularization) and tissue growth (during recellularization) in the bioreactor, which allows conclusions to be drawn about the quality of the tissue, especially without destroying it or have to be removed from the bioreactor. This is realized in particular by the analysis unit and the conditioning unit, which are integrated into the bioreactor. The bioreactor is therefore particularly suitable for the regeneration of soft tissue and especially muscle tissue, which is particularly difficult to produce. The muscle tissue is preferably skeletal muscle (also "3D skeletal muscle") and not smooth muscle. However, thanks to online monitoring and control by means of an analysis unit, conditioning unit and control unit, high-quality soft tissue can now be produced. The bioreactor does not have to be stopped at any time during production in order to determine the progress and quality of production; this is advantageously done online.

In a preferred embodiment, the conditioning unit has two arms for holding the soft tissue in the reactor chamber. A respective arm is expediently designed as a rod, for example made of plastic or metal. The two arms project into the reactor chamber from outside and are movable relative to each other to apply force, particularly traction, to the soft tissue for mechanical stimulation (i.e., conditioning). In a suitable embodiment, a respective arm is connected to a linear motor for this purpose, so that the arm can be moved back and forth in one direction. The direction is preferably the direction of gravity, which corresponds to a longitudinal direction of the reactor chamber. Conveniently, the arms each end with a holding member, such as a clamp, for holding the tissue and for stretching and expanding (i.e., relaxing) the tissue. A suitable holding element is e.g. a bulldog clamp. The conditioning unit is thus advantageously designed for mechanical conditioning of the tissue during manufacture. Preferably, the arms are arranged vertically to support the soft tissue in the direction of gravity (and in particular also in the longitudinal direction of the reactor chamber) between the arms. In particular, one of the arms is an upper arm and holds the fabric from above and the other arm is a lower arm and holds the fabric from below (in terms of longitudinal direction and gravity).

An embodiment in which the two arms are designed for automatic centering of the soft tissue is particularly expedient. This means in particular that the arms are designed in such a way that the tissue in the reactor chamber is automatically aligned in the longitudinal direction. In a suitable embodiment, the upper arm has a holding element which can be pivoted about a pivot axis perpendicular to the longitudinal direction in order to compensate for a horizontal movement of the tissue. The lower arm preferably has a holding element, which is formed as a hook, with a curved course such that a tip is formed, which is longitudinally arranged below the holding element of the upper arm. The tissue is then expediently prepared at a lower end with a holding loop which is hooked into the hook. The arms are then pulled apart so that the holding loop automatically slips into the tip due to the curved course and the tissue is then automatically centered. Conversely, the tip forms a local minimum for the movement of the tissue, so to speak, more precisely for the movement of the holding strap. The aforementioned configuration for automatic centering is fundamentally independent of the presence of a force sensor and can also be implemented without one. The hand strap itself is ins specifically not part of the bioreactor but is attached to the tissue before the tissue is inserted into the reactor chamber.

The analysis unit preferably has at least one force sensor, which is attached to one of the arms, for the mechanical measurement of the tissue. The force sensor is preferably attached to the upper arm, i.e. to that arm which holds the tissue in the reactor chamber from above and from which the tissue then hangs down, so to speak, in the longitudinal direction of the reactor chamber. The force sensor measures an active or passive soft tissue force or both. This is also known as "mechanical measurement", more precisely as "biomechanical measurement" and is particularly relevant for muscle tissue. The force sensor is preferably used to control or regulate the linear motors and in this way to set a defined force (e.g. for a certain mechanical tension) and/or to obtain a certain force (e.g. contraction force) during tissue growth.

Combined conditioning and analysis is advantageously realized by the arms in combination with the force sensor, because the tissue is analyzed with the force sensor (by mechanical measurement) and conditioned with the arms, in particular depending on the mechanical measurement by the force sensor. For example, a process parameter “force (exercised by the arms)” is linked to a tissue parameter “active and/or passive force (of the tissue)”. Due to the preferred vertical arrangement of the arms, this is also referred to as "vertical analysis and conditioning".

In contrast to the vertical arrangement, a horizontal arrangement, i.e. rotated by 90°, disadvantageously complicates a biomechanical measurement of passive and active forces. During de- and recellularization, the tissue changes both in its volume and in its mass. A biomechanical measurement of the active or passive forces is accordingly subject to a varying resulting mass. The measured force is therefore falsified by a changing systematic error when the mass changes. A vertical, upright measurement, as preferred here, is not subject to this systematic error, since the mass of the tissue acts in the direction of conditioning (e.g. stretching or compression). When starting the measurement, a zero point calibration is expediently carried out first, as a result of which the subsequent measurement can be carried out independently of the mass of the tissue and is also carried out.

For immersion and removal of a tissue from the reactor chamber, the height of the reactor chamber is conveniently adjustable relative to the arms, e.g., by mounting the linear motors and attached arms to a positioning table, e.g., a z-axis positioning table.

A transport lock can expediently be attached to the reactor chamber for removing and transporting the reactor chamber from the bioreactor. The transport lock has in particular a transport clamp which can be fastened to the arms with a fastening element, e.g. screw. First the transport lock is attached, then the arms are detached from the rest of the bioreactor and the reactor chamber with arms and transport lock can be safely removed from the rest of the bioreactor without the tissue slipping in the reactor chamber.

In a preferred embodiment, the conditioning unit for electrical stimulation (i.e. conditioning) of the soft tissue has two electrodes, each of which is designed as a wire. A respective wire runs along one of the two arms into the reactor chamber for direct electrical connection and stimulation of the soft tissue. In this way, a direct stimulation of the tissue is realized. Overall, the conditioning unit is thus designed for electrical conditioning of the tissue. In a particularly expedient embodiment, the two arms each have a channel, e.g. a notch or a cavity, into which the respective wire is inserted, so that it lies overall within a cross-sectional profile of the arm and is guided into the reactor chamber in a particularly protected and space-saving manner. For example, a plastic arm has a lateral notch into which the wire is inserted. In another embodiment, an arm is designed as a tube or hollow cylinder made of metal, e.g. stainless steel, and is coated on the inside with an insulating material, so that a hollow space is formed which is electrically insulated from the metal and through which the wire is guided. The wire is suitably platinum wire.

In a preferred embodiment, the conditioning unit for electrical stimulation (ie conditioning) of the soft tissue has two electrodes (an anode and a cathode) which are arranged along a wall of the reactor chamber. The electrodes are preferably arranged inside the reactor chamber and connected to the medium inside the reactor chamber Contact so that a current flows between the electrodes and through the medium to electrically stimulate the tissue. The electrodes are preferably flat electrodes. In particular, the electrodes follow the course of the wall and are preferably attached to the inside of the wall. The electrodes are used to generate an advantageously (at least in the area of the tissue) homogeneous electrical field within the reactor chamber. In this way, a field stimulation of the tissue is realized. Overall, the conditioning unit is thus designed for electrical conditioning of the tissue. Suitably the reactor chamber has a height and the electrodes extend substantially (ie in particular at least 90%) the full height and/or preferably anyhow the full length of the soft tissue measured in the same direction as the height. In a suitable embodiment, the two electrodes are made from an electrically conductive polymer.

The two electrodes are expediently designed as flat semicircular electrodes, i.e. semicircular in cross section perpendicular to the longitudinal direction, with a narrow gap remaining between the two electrodes in order to achieve electrical separation. The reactor chamber is preferably cylindrical and thus has a lateral surface which forms the wall and along which the two electrodes run. The wall also defines the width of the reactor chamber, which here then corresponds to a diameter of the reactor chamber. Thus, the two electrodes are advantageously arranged overall in such a way that the electric field between the two electrodes is formed in the horizontal direction (i.e. perpendicular to the force of gravity).

In the case of muscle tissue in particular, direct stimulation is not necessarily an advantage, since the course of the electric field along the muscle fibers leads to a linearly decreasing electric field along the muscle cell membranes, and the cells are therefore stimulated inhomogeneously. This can have a significant negative effect on the maturation of the tissue and thus its quality. This is advantageously not the case with field stimulation, specifically perpendicular to the described direct stimulation, i.e. in the direction of a width of the reactor chamber (i.e. running radially).

At least one of the electrodes (preferably each of the two electrodes) expediently has a window through which one or more of the tissue parameters can be measured with the analysis unit from outside the reactor chamber, in particular from different angles around the longitudinal direction. The electrodes therefore do not necessarily cover the reactor chamber completely; rather, a window is left out, through which the analysis unit has optical access into the reactor chamber. Away from the window, however, the electrodes then preferably completely cover the wall. The window preferably extends in the circumferential direction completely along the wall, i.e. around the entire circumference of the reactor chamber (apart from any contact land which may be present, see below). The two electrodes are divided by the window in particular into two sub-electrodes, namely an upper sub-electrode above the window and a lower sub-electrode below the window when viewed in the longitudinal direction. The two sub-electrodes of a single electrode are expediently connected to one another via a contact bridge that is as thin as possible, so that both sub-electrodes have a common potential.

The size of the electrodes and the window is particularly dependent on the size of the tissue. The electrodes are expediently dimensioned and arranged in the reactor chamber in such a way that the window is aligned centrally with respect to the tissue and yet a large part (i.e. in particular at least 30%) of the tissue is covered by the electrodes. This ensures, on the one hand, that the tissue is sufficiently electrically conditioned and, on the other hand, that an optimal analysis is possible. A central area of the tissue is particularly suitable for an analysis, since here the regeneration and/or maturation lags behind the ends of the tissue.

Through the two electrodes in combination with the window for analysis from the outside, a further combined conditioning and analysis is advantageously realized, because the tissue is analyzed through the window in particular with an optical sensor and conditioned with the electrodes, in particular depending on the measurement, which is carried out by the analysis unit through the window. For example, a process parameter "electrical field (between the electrodes)" is linked to a tissue parameter "transparency", "surface texture" or something else (see e.g. above). Due to the preferred horizontal arrangement of the electrodes and in particular the measurement perpendicular to the longitudinal direction, this is also referred to as "horizontal analysis and conditioning".

An embodiment in which the vertical analysis and conditioning are combined with the horizontal analysis and conditioning is particularly preferred. A combination such that the vertical analysis is linked to the horizontal conditioning and/or is also advantageous the reverse the horizontal analysis with the vertical conditioning. In principle, the tissue parameters and the process parameters can be linked to one another as required and as required.

The bioreactor is preferably designed on the one hand for the decellularization, in particular of a donor tissue, and on the other hand also for the recellularization of a tissue framework which was previously obtained in particular from the donor tissue by the decellularization. For this purpose, the bioreactor can expediently be switched between a corresponding decellularization mode, in which decellularization takes place, and a recellularization mode, in which recellularization takes place. Advantageously, the tissue (more precisely, the tissue structure) remains in the reactor chamber when switching over, removal is not necessary. In a suitable configuration for this purpose, the bioreactor has a first switching element, for example a 3-way valve. The first switching element connects the reactor chamber either to a first reservoir or to a second reservoir, both of which are parts of the bioreactor. The first reservoir contains a first, cell-destroying medium for decellularization and the second reservoir contains a second, physiological medium for recellularization, so that the first switching element can be used to set which of the two media flows into the reactor chamber. For the decellularization, the first reservoir is fluidically connected to the reactor chamber, for the recellularization the second reservoir is connected analogously. Both reservoirs are in particular connected to the same media inlet of the reactor chamber, the first switching element is then connected upstream of this media inlet. The bioreactor also expediently has a pump for conveying the respective medium. The pump is suitably arranged between the first switching element and the media inlet.

The first switching element is expediently controlled by the control unit as a function of the tissue parameters and/or process parameters. This is because the tissue parameters and/or the process parameters make it possible to recognize the progress of the decellularization and then to end the decellularization and start the recellularization at the most suitable point in time. A rinsing step (i.e. a process step for rinsing) is expediently carried out in between, in which the tissue framework and the reactor chamber are rinsed with deionized water. In particular, the first switching element is also controlled accordingly here. Overall, this results in a fully automated method in which there is a switchover fully automatically and depending on the tissue and/or process parameters between the various process steps of decellularization and recellularization and optionally a rinsing step. By taking the tissue and/or process parameters into account, switching is advantageously adaptive and takes place at the optimum point in time. For example, one or more tissue parameters indicate that there are no more cells, indicating that decellularization is complete and recellularization can begin.

The media outlet of the reactor chamber is suitably reconnected to the first and the second reservoir so that two circuits are formed, one for decellularization and one for recellularization. Preferably, the bioreactor has a second switching element downstream of the media outlet of the reactor chamber, e.g. a 3-way valve, which selectively returns the medium from the reactor chamber to the first or the second reservoir. In the decellularization mode, the medium is preferably returned to the first reservoir, in the recellularization mode then to the second reservoir. A pump for conveying the medium is again suitably arranged between the medium outlet and the second switching element. On the path of the medium from the reactor chamber to the respective reservoir, an extraction point (or also a residue) is expediently additionally arranged in each case for the discharge of medium from the bioreactor and/or for pressure equalization. Alternatively or additionally, a residue branch branches off downstream of the media outlet and preferably before the pump, for dispensing residues. A pump and also a check valve are expediently also arranged on the residue branch.

The first cell disrupting medium is in a suitable embodiment deionized water or SDS (i.e. sodium lauryl sulphate, e.g. 0.1% SDS) or a combination thereof. An embodiment is preferred in which the first reservoir can be optionally filled with deionized water or with SDS or with a mixture of these two.

Also particularly advantageous is an embodiment in which the bioreactor is designed to flush the reactor chamber after decellularization and before recellularization by filling the first reservoir with deionized water and then flushing the reactor chamber out of the first reservoir. In other words: in a rinsing step (see also above), the reactor chamber is specially cleaned for decellularization tion or generally for cleaning with deionized water, in order to flush out any residues of SDS before recellularization from the reactor chamber and in particular from the tissue framework. For this purpose, in a corresponding process step (ie in the rinsing step), the first reservoir is expediently filled with deionized water and the reactor chamber is then rinsed out of the first reservoir. For this purpose, the bioreactor has one or more switching elements (eg valves) which are suitably controlled by the control unit. The rinsing in the rinsing step is also advantageously automated, without the reactor chamber having to be opened or the tissue having to be removed. The rinsing step is also initiated automatically at the end of the decellularization, since the end of the decellularization is detected by measuring the tissue parameters.

Suitably, the deionized water is supplied from a water supply which is higher than the reactor chamber in terms of gravity, so that the deionized water is supplied purely by gravity and no pump is required to pump it, so that one is also dispensed with.

The second, physiological medium serves in particular to stabilize and supply nutrients to the cells in the recellularization process. In a suitable embodiment, the second medium is a Ringer's solution. However, the precise details of the physiological medium are of secondary importance in the present case and also depend on the tissue that is to be produced with the bioreactor.

The actual cells for recellularization are in particular supplied separately, i.e. separately from the media which are supplied from the first and second reservoir. The cells are thus not fed into the reactor chamber via the media inlet, but preferably directly into the tissue (more precisely: tissue scaffold) via an arterial access. For this purpose, the reactor chamber suitably has a corresponding arterial access, via which a cell culture medium from a third reservoir together with cells can be supplied to the soft tissue for recellularization. The cells are thus first added to a cell culture medium and this mixture is then fed into the reactor chamber via the arterial access. The arterial access is e.g. a line that leads into the tissue. The cell culture medium is also referred to as a nutrient solution and is in particular a liquid. Accordingly, the bioreactor has a supply for the cell culture medium, which leads into the third reservoir. On the way to the reactor chamber, the cells from a cell supply are then mixed into the cell culture medium, whereby "mixing in" does not necessarily mean "mixing", but more generally that the cell culture medium and the cells are either together or sequentially and advantageously via the arterial access Line are guided into the reactor chamber.

An embodiment in which the third reservoir is connected to the reactor chamber via a line is particularly advantageous, with a loading valve, in particular an “HPLC” valve (HPLC=high-performance liquid chromatography), being arranged along the line for supplying the cells. The loading valve is designed in such a way that by switching it over, a fluid section made of cells is inserted between two fluid sections made of cell culture medium. In other words: with the loading valve, two streams are, as it were, interwoven, namely a first stream of cell culture medium and a second stream of cells. Along the line there are then a number of fluid sections which follow one another in the flow direction and consist of different media, namely cell culture medium on the one hand and cells on the other. The loading valve is suitably a 6-way valve and accordingly has six ports which are connected to one another in pairs, resulting in three pairs, with two adjacent ports being connected to one another so that one can be used as an input and the other as an output. Other numbers of inputs are also suitable, for example eight, but preferably at least six. With an increasing number of inputs, there is in particular the possibility of supplying cells from different sources without having to rebuild the bioreactor and specifically the cell supply.

The loading valve is commonly used to introduce cells into the soft tissue from the outside. In a first switching position of the loading valve, the cells are added from the outside and flow through the loading valve, but not yet into the line, until a stream of cells is visible in a residue downstream of the loading valve. The loading valve is then switched over to a second switching position to feed the cells into the line. This will feed the cells directly into the tissue while now the cell medium will flow directly into the residue without stopping the flow of cell culture medium. After the cells have been fed in, the loading valve is switched over again, in particular to the first switch position, so that cell culture medium flows into the reactor chamber again, but no more cells; the cell feed is connected to the residue again. This results in the advantage that the cells are not subject to any shear forces when they are supplied, because the two Flows of cell culture medium on the one hand and cells on the other hand are not mixed with each other, but partially exchanged, i.e. a fluid section of the cell culture medium is discarded (i.e. conveyed into the residue) and replaced by a fluid section made of cells, so that viewed along the line three fluid sections result, namely a Fluid segment of cells, which is arranged between two fluid segments of cell culture medium. Another advantage is that a particularly suitable part of the cells is also fed in, because the loading valve prevents a top flow with any air bubbles or a residual flow with any cell debris or dead cell material from being mixed in, these are instead discarded in the residue. Only a mid-section will ultimately flow into the reactor chamber.

The loading valve expediently has a temperature sensor which is connected to a temperature control element, for example also in the loading valve and/or in the third reservoir. A temperature of the cells and/or the cell culture medium is measured with the temperature sensor and is thus monitored and the temperature control element is controlled as a function thereof in order to compensate for any temperature fluctuations. A temperature fluctuation results, for example, from the fact that the cells have a different temperature than the cell medium. A temperature shock that is disadvantageous for the cells is then prevented by controlling the temperature control element.

The use of the loading valve is fundamentally independent of the supply via the arterial access and is also generally expedient for the supply of the cells.

In an advantageous embodiment, the first and the second medium can also flow into the reactor chamber via the arterial access. This has the advantage that the tissue can also be rinsed directly with the first and the second medium in a corresponding process step. In a suitable embodiment, a third switching element, e.g. A selection of the first or second medium is suitably made by means of a further, fourth switching element, e.g. again a 3-way valve, which is arranged upstream of the aforementioned third switching valve and connects it optionally to the first or second reservoir. In this way, extensive rinsing with either a physiological medium or a cell-destroying medium can also be carried out automatically, in particular, without having to convert the bioreactor. For this purpose, the switching elements are suitably controlled by the control unit in accordance with the process control of the bioreactor.

In a suitable embodiment, one of the process parameters is a partial oxygen pressure, ie pO ₂ value, for example in the reactor chamber or the medium on the way there. In this case, the conditioning unit has a gas supply for supplying oxygen to the second reservoir, ie to the physiological medium. The gas feed supplies air, for example, which contains oxygen, or a gas mixture with oxygen and at least one other gas, with the proportion of oxygen generally being preferably adjustable. The control unit then controls the gas supply (ie the total supply of gas and/or the proportion of oxygen, if this is adjustable) as a function of the partial oxygen pressure. The conditioning unit is thus designed to condition the tissue with a specific pO ₂ value. The gas supply enables a supply of oxygen. The pO ₂ value is then set by supplying oxygen to the second reservoir. Suitably, an oxygen sensor is placed along the path of the second medium from the second reservoir to the reactor chamber for measuring the pO ₂ value of the medium for the purpose of controlling the gas supply.

Alternatively or additionally, in a suitable embodiment, one of the process parameters is a pH value, for example in the reactor chamber or in the medium on the way there. In this case, the conditioning unit has a CO ₂ supply for supplying CO ₂ into the second reservoir, and the control unit controls the CO ₂ supply as a function of the pH value. The conditioning unit is thus designed to condition the tissue with a specific pH value. A pH sensor is suitably placed along the path of the second medium from the second reservoir to the reactor chamber for measuring the pH of the medium for the purpose of controlling the CO ₂ supply. In addition, the comments on the pO ₂ value also apply analogously to the pH value.

Controlling the pH value and/or the pO ₂ value as described above for the second medium is alternatively or additionally also advantageous for the cell culture medium from the third reservoir and implemented analogously in a suitable embodiment.

In an advantageous embodiment, one of the process parameters is a temperature, for example in the reactor chamber or of the medium on the way there. In this case, the conditioning unit has a temperature control element, for example a heater, for temperature control of the respective medium. suitable Alternatively, a respective temperature control element is arranged in the first reservoir, in the second reservoir and/or in the third reservoir for the purpose of temperature control of the corresponding medium. One or more temperature sensors then measure the temperature of the respective medium and the control unit then controls the temperature control elements accordingly in order to set a specific temperature in the reactor chamber. For example, a first temperature sensor measures the temperature in the reactor chamber and depending on this the control unit controls the temperature control elements in the first and second reservoir, while a second temperature sensor measures the temperature of the cell culture medium on the way from the third reservoir to the reactor chamber and the control unit controls the temperature control element in the third depending on this reservoir controls.

The electrical and mechanical conditioning of the tissue as well as the measurement and control of process parameters (pH value, pO ₂ value, temperature) were primarily described above. A particular advantage of the bioreactor described here is the analysis unit, which measures the tissue parameters and thus enables a concrete assessment of the quality of the tissue and the progress of production and thus direct intervention in the production in the form of a control of the process parameters depending the tissue parameter. The analysis unit and the measurement of tissue parameters should therefore be discussed briefly below.

The analysis unit preferably has an optical sensor for the non-invasive measurement of at least one tissue parameter during production. An optical measurement has the advantage that it is non-invasive, i.e. it is carried out from outside the reactor chamber, so that the measurement does not have to be interrupted during production or the tissue removed or transferred from the reactor chamber. In addition, the measurement is advantageously carried out online. The optical sensor is suitably a reflected light camera or a spectroscope or the like or a combination thereof. A combination of several optical sensors is also advantageous. The wall of the reactor chamber is particularly transparent to light, which is then detected with the optical sensor. Also possible and suitable is an embodiment in which light is radiated into the reactor chamber for measurement in order to then, for example, measure its scattering, reflection or transmission on the tissue. If, as described above, the bioreactor has two electrodes for field stimulation, the measurement is expediently carried out through the window already described, which is suitably dimensioned for this purpose. An embodiment is also advantageous in which the tissue can be moved relative to the optical sensor (e.g. by means of the arms mentioned or a positioning table), so that the tissue can be examined from different positions.

In an advantageous embodiment, the optical sensor has an optical input for receiving light from the reactor chamber, and the optical input can be rotated around the reactor chamber for optical measurement from different directions. An angle-dependent measurement of the tissue parameters is thus implemented. The optical input can preferably be rotated completely around the reactor chamber, so that a 360° all-round measurement is possible and preferably also implemented. For this purpose, the optical input is suitably mounted on a turntable, which is in particular ring-shaped and runs around the reactor chamber. In particular, the turntable is driven by a motor so that the optical input can be rotated relative to the reactor chamber, in particular about a longitudinal axis of the reactor chamber extending in the longitudinal direction. Alternatively or additionally, the height of the optical input can be adjusted for moving in the longitudinal direction. Advantageously, the optical input is repeatedly rotated around the reactor chamber in order to repeatedly carry out a 360° all-round measurement.

Which tissue parameters are specifically measured with the analysis unit using the optical sensor is of secondary importance; what is more important is that this is done online and non-invasively and that there is direct feedback to the process parameters in order to achieve optimal production.

The bioreactor expediently has a housing in order specifically to protect the reactor chamber and the optical entrance from scattered light. Lighting for maintenance is suitably installed within the housing and, alternatively or additionally, UV lighting for sterilization. The switching elements, pumps, sensors and reservoirs described are preferably arranged outside the housing, with the exception of the oxygen sensor and the pH sensor, which are preferably each designed as optical sensors and are then suitably placed inside the housing. The switching elements, pumps, reservoirs and corresponding lines for the media together form a fluidic system of the bioreactor. The lines are suitably made of a plastic with the lowest possible Shore value of 40, for example. The fluidic system is thus arranged largely outside the housing.

In the method according to the invention for, in particular, automated operation of a bioreactor, the bioreactor has a reactor chamber in which soft tissue is produced. The bioreactor also has an analysis unit for measuring a number of tissue parameters of the soft tissue, a conditioning unit for conditioning the soft tissue according to a number of process parameters, and a control unit which controls the analysis unit and the conditioning unit in such a way that the tissue parameters are measured during production and the process parameters are set depending on the tissue parameters, so that the soft tissue is conditioned depending on the tissue parameters. Advantages and advantageous configurations of the method result from what has already been said.

If parts, components, devices and elements were described above which were not explicitly identified as parts of the bioreactor, these parts, components, devices and elements are preferably parts of the bioreactor.

“Control”, “controlled” or a “control” was mentioned above in various contexts. Advantageous variants result from the fact that there is even regulation, ie a particular part or a particular parameter regulates or is regulated.

• 1 einen Bioreaktor,
• 2 teilweise den Bioreaktor aus 1 in einer perspektivischen Ansicht,
• 3 den Bioreaktor aus 2 in einer Seitenansicht,
• 4 eine Reaktorkammer und Arme des Bioreaktors aus 1,
• 5 eine Transportsicherung für den Bioreaktor aus 1,
• 6 eine Reaktorkammer und Elektroden des Bioreaktors aus 1 in einer Ansicht von oben,
• 7 die Reaktorkammer und Elektroden aus 6 in einer perspektivischen Ansicht von der Seite,
• 8 den Bioreaktor aus 1 als Fluidikschaltbild,
• 9 einen Ausschnitt einer Leitung des Bioreaktors aus 1.

Exemplary embodiments of the invention are explained in more detail below with reference to a drawing. They each show schematically:

• 1 a bioreactor,
• 2 partially off the bioreactor 1 in a perspective view,
• 3 off the bioreactor 2 in a side view,
• 4 a reactor chamber and arms of the bioreactor 1 ,
• 5 a transport lock for the bioreactor 1 ,
• 6 a reactor chamber and electrodes of the bioreactor 1 in a view from above,
• 7 the reactor chamber and electrodes 6 in a perspective view from the side,
• 8th off the bioreactor 1 as fluid circuit diagram,
• 9 a section of a line of the bioreactor 1 .

In 1 a highly simplified bioreactor 2 according to the invention is shown with its functional parts. Show for illustration 2 and 3 then some mechanical and electrical parts of the bioreactor 2. The 4 until 7 show various details of the bioreactor 2 from different views. 8th Finally, as a fluidic circuit diagram, FIG.

The bioreactor 2 has a reactor chamber 4 for producing a soft tissue 6, which is accommodated in the reactor chamber 4 for this purpose, here a glass cylinder. In the exemplary embodiment shown, the production is a regeneration and maturation with decellularization and subsequent recellularization. The soft tissue 6 is also simply referred to as tissue 6 below.

Furthermore, the bioreactor 2 has an analysis unit 8 for measuring a number of tissue parameters G of the soft tissue 6. The tissue parameters G indicate the current state or actual state of the soft tissue 6. In addition, the bioreactor 2 has a conditioning unit 10 for conditioning the soft tissue 6 according to a number of process parameters P. “A number of” is understood here and also generally to mean “one or more” or “at least one”. In the exemplary embodiment shown here, multiple tissue parameters G are measured and multiple process parameters P are set without loss of generality.

The process parameters P control the production and thus ultimately also influence the tissue parameters G. Correspondingly, a change in the process parameters P influences the production in the present case. For this purpose, the bioreactor 2 has a control unit 12, which correspondingly controls the analysis unit 8 and the conditioning unit 10 in such a way that the tissue parameters G are measured repeatedly and/or continuously during production and the process parameters P are set in real time as a function of the tissue parameters G. so that the soft tissue 6 is conditioned depending on the tissue parameters G. In this way, the production of the soft tissue 6 is controlled online depending on the actual tissue parameters G (i.e. the current state), which in turn continuously and automatically adjusts the process parameters P in such a way that, based on the current state, a specified target state is achieved will

The bioreactor 2 shown here is suitable specifically for the regeneration of soft tissue 6, specifically muscle tissue, in a "top-down" technique with a multimodal environment and sample parametrics. First, a donor tissue with a scaffold is used as a starting point and treated and matured as needed. The donor tissue is first decellularized to expose the tissue scaffold, which is then subsequently recellularized (and thus regenerated altogether). In this case, the tissue 6 in the reactor chamber 4 is surrounded by a medium which is characterized by one or more process parameters P. The medium generally stabilizes the maturation of the tissue 6 during recellularization in terms of osmotic pressure, chemical environment and nutrient supply. During production, different media flow into the reactor chamber 4 one after the other, depending on the requirements of the current process step, as specifically described below in connection with FIG 8th will be explained in more detail. The respective medium flows into the reactor chamber 4 via a media inlet 14 and flows out of the reactor chamber 4 again via a media outlet 16 . In the present case, the reactor chamber 4 has an underside 18 with a media inlet 14 and an upper side 20 with a media outlet 16, so that the medium can wash around the soft tissue 6 against the force of gravity.

In the present bioreactor 2 and the associated method for its operation, the quality of the tissue 6 is monitored in real time and also without contact in order to then draw conclusions about the "freedom from cells" (the degree of depopulation during decellularization) and the " regeneration maturity” (the measure of the completion of the tissue 6 in the recellularization) of the tissue 6 to draw. In other words: the production of the tissue 6 in the bioreactor 2 is monitored online by measuring a number of tissue parameters G and then controlling a number of process parameters P depending on this, with the aim of optimizing the production. The fabric parameters G indicate the current state of the fabric 6 and thus also the progress and the quality of production. The tissue parameters G are also referred to herein as "morphological and structural parameters" and include, for example, mass, volume, diameter or other dimensions, CIELAB colorimetry or equivalent, tissue stiffness, vessel pressure, vascular pressure and/or transparency and surface texture of the tissue 6. The process parameters P on the other hand, indicate the environmental modalities of production, i.e. the current state of the bioreactor 2, and thus characterize the environment of the tissue 6 within the reactor chamber 4, i.e. the environmental conditions which the bioreactor 2 provides. The process parameters P include, for example, pH value, pO2 value and/or temperature.

The tissue parameters G together with the process parameters P then form the environmental and sample parameters, which overall provide a particularly detailed picture of the quality of the tissue 6 and accordingly enable feedback, which takes place here in two ways: first, the process parameters P are dependent on the Tissue parameters G adjusted to achieve optimal production. Secondly, the process parameters P themselves are also controlled or regulated in order to maintain a defined environment during production with a high level of reliability and to prevent the process parameters P from being changed unintentionally.

The bioreactor 2 presented here thus combines automated production of tissue 6 in "top-down" technology with real-time parametrics, which is controlled by extensive measurements. In other words: the analysis unit 8 generates feedback on the course of the production, so that this production is dynamically and automatically adjusted as required by appropriate control of the process parameters P. For this purpose, the control unit 12 places the tissue parameters G and the process parameters P in connection with one another and thus also in a physiological context, which enables orderly tissue maturation and also optimization of the production to the quality of the tissue 6.

How special in 2 and 3 As can be seen, the conditioning unit 10 in the embodiment shown here has two arms 22 for holding the soft tissue 6 in the reactor chamber 4. A respective arm 22 is designed here as a plastic rod. The two arms 22 protrude into the reactor chamber 4 from outside and are movable relative to one another in order to exert a force on the soft tissue 6 for mechanical stimulation. In the exemplary embodiment shown, a respective arm 22 is connected to a linear motor 24 for this purpose, so that the arm 22 can be moved back and forth in a direction, which is the direction of gravity here, which corresponds to a longitudinal direction L of the reactor chamber 4. The arms 22 are here also arranged vertically to support the soft tissue 6 in the direction of gravity. Furthermore, the analysis unit 8 here has at least one force sensor 26 which is attached to one of the arms 22 and measures an active or passive force of the soft tissue 6 or both. The force sensor 26 is also used, for example, to control or regulate the linear motors 24 and in this way to set a defined force (eg for a specific mechanical tension) and/or to maintain a specific force during the growth of the tissue 6 .

Furthermore, in the exemplary embodiment shown, the two arms 22 are designed for the automatic centering of the soft tissue 6. This means that the arms 22 are designed in such a way that the tissue 6 in the reactor chamber 4 is automatically aligned in the longitudinal direction L. This is in detail in 4 1 is shown showing reactor chamber 4 and arms 22. FIG. For automatic centering, the upper arm 22 has a holding element 25 (here a bulldog clamp), which can be pivoted about a pivot axis perpendicular to the longitudinal direction L in order to compensate for a horizontal movement of the tissue 6, this is shown in 4 illustrated by a horizontal double arrow. The lower arm 22, on the other hand, has a holding element 27, which is designed as a hook, with a curved course such that a tip 29 is formed, which is arranged in the longitudinal direction L below the holding element 25 of the upper arm 22. The tissue 6 is then prepared at a lower end with a holding loop 31 which is hooked into the hook. The arms 22 are then pulled apart so that the holding loop 31 then automatically slips into the tip 29 due to the curved course and the tissue 6 is then automatically centered as a result. this is in 4 illustrated by two oblique arrows pointing towards tip 29. The tip 29 forms a local minimum for the movement of the hand strap 31.

In order to immerse and remove a tissue 6 from the reactor chamber 4, the latter can be adjusted in height relative to the arms 22, here by the linear motors 24 and the arms 22 attached thereto being attached to a positioning table 28. To remove and transport the reactor chamber 4 from the bioreactor 2, a transport lock can be attached to the reactor chamber 4, which 5 is shown in a top view. The transport lock has a transport clamp 30 which can be fastened to the arms 22 with a fastening element 32 . First, the transport lock is attached, then the arms 22 are detached from the rest of the bioreactor 2 and the reactor chamber 4 with arms 22 and transport lock can be removed from the bioreactor 2.

In an embodiment that is not explicitly shown, the conditioning unit 10 has two electrodes for electrical stimulation of the soft tissue 6, each of which is designed as a wire, with a respective wire running along (e.g. inside) the two arms 22 into the reactor chamber 4 for direct electrical connection and stimulation of the soft tissue 6. For example, the two arms 22 each have a channel, e.g. a notch, into which the respective wire is inserted, so that it lies overall within a cross-sectional profile of the arm 22 and is particularly protected and space-saving in the reactor chamber 4 to be led.

In the embodiment shown here, the conditioning unit 10 for the electrical stimulation of the soft tissue 6 has two flat electrodes 34 which are arranged along a wall 36 of the reactor chamber 4 and are thereby arranged inside the same. The two electrodes 34 are each designed as semicircular electrodes. A gap extends in the longitudinal direction L between the electrodes 34 in order to prevent direct contact of the electrodes 34 with one another. This is particularly evident in 6 recognizable, which in a view from above as in 5 which shows the reactor chamber 4 and the two electrodes 34 . 7 then shows a different view from the side. The electrodes 34 serve to generate an electric field within the reactor chamber 4. The reactor chamber 4 has a height H, and the electrodes 34 extend essentially over the entire height H and/or at least over the entire length of the soft tissue 6, in the same way Direction measured as the height H. The two electrodes 34 are made of an electrically conductive polymer, for example. As can be seen from the figures, the reactor chamber 4 shown here is cylindrical and thus has a lateral surface which forms the wall 36 and along which the two electrodes 34 run. The wall 36 also defines a width B of the reactor chamber 4, which then corresponds to a diameter of the reactor chamber 4 here.

In the present case, at least one of the electrodes 34 has a window 38 through which one or more of the tissue parameters G can be measured with the analysis unit 8 from outside the reactor chamber 4 . The electrodes 34 therefore do not necessarily cover the reactor chamber 4 completely; rather, a window 38 is left out, through which the analysis unit 8 obtains optical access into the reactor chamber 4 . In the exemplary embodiment shown, the window 38 extends in the circumferential direction U completely along the wall 36. The two electrodes 34 are each divided into two sub-electrodes by the window 38, namely, viewed in the longitudinal direction L, an upper sub-electrode above the window 38 and a lower sub-electrode below the Window 38. This is especially in 7 recognizable, in which one of the two electrodes 34 is visible and is divided through the window 38 into upper and lower sub-electrodes. The two sub-electrodes of an individual electrode 34 are then connected to one another via a contact web 39 that is as thin as possible, so that both sub-electrodes have a common potential.

The bioreactor 2 shown here is designed on the one hand for the decellularization of a donor tissue and on the other hand also for the recellularization of a tissue framework which was previously obtained from the donor tissue by the decellularization. How out 6 can be seen, for this purpose the bioreactor 2 can be switched between a corresponding decellularization operation and a recellularization operation. When switching over, the tissue 6 remains in the reactor chamber 4, removal is not necessary. For this purpose, the bioreactor 2 has a first switching element 40 , here a 3-way valve, which connects the reactor chamber 4 either to a first reservoir 42 or to a second reservoir 44 . The first reservoir 42 contains a first, cell-destroying medium 46 for decellularization and the second reservoir 44 contains a second, physiological medium 48 for recellularization, so that the first switching element 40 can be used to set which of the two media 46, 48 flows into the reactor chamber 4 . For the decellularization, the first reservoir 42 is fluidically connected to the reactor chamber 4, for the recellularization the second reservoir 44 is analogous. The bioreactor 2 also has a pump 50 for conveying the respective medium 46, 48.

In the present case, the media outlet 16 is again connected to the first and the second reservoir 42, 44, so that two circuits are formed, one for the decellularization and one for the recellularization. Downstream of the media outlet 16, the bioreactor 2 analogously has a second switching element 52, again a 3-way valve, which returns the medium from the reactor chamber 4 to the first or the second reservoir 42, 44 as desired. A pump 54 for conveying the medium is in turn arranged between the media outlet 16 and the second switching element 52 . On the path of the medium from the reactor chamber 4 to the respective reservoir 42, 44, in the exemplary embodiment shown, a removal point 56 is additionally arranged in each case for discharging medium from the bioreactor 2 and/or for pressure equalization. In addition, a residue branch 58 branches off downstream of the media outlet 16 and before the pump 54 for dispensing residues. A pump 60 and a check valve 62 are also arranged on the residue branch 58 .

The first, cell disrupting medium 46 is presently deionized water or SDS (i.e. sodium lauryl sulfate, e.g. 0.1% SDS) or a combination thereof. For this purpose, the first reservoir 42 can optionally be filled with deionized water or with SDS or with a mixture of these two.

The second, physiological medium 48 serves to stabilize and supply nutrients to the cells in recellularization mode. The second medium 48 is a Ringer solution, for example.

The actual cells for recellularization are supplied separately to the media 46, 48, i.e. not via the media inlet 14 of the reactor chamber 4, but via an arterial access 64 directly into the tissue 6 (more precisely: tissue framework). For this purpose, the reactor chamber 4 has a corresponding arterial access 64, via which a cell culture medium 66 from a third reservoir 68 can be supplied together with cells to the soft tissue 6 for recellularization. The cells are thus first added to a cell culture medium 66 and this mixture is then fed into the reactor chamber 4 via the arterial access 64 . The bioreactor 2 accordingly has a supply 70 for the cell culture medium 66 which leads into the third reservoir 68 . On the way to the reactor chamber 4 , the cells from a cell feed 72 are then added to the cell culture medium 66 .

In the embodiment shown here, the reactor chamber 4 can also be rinsed with deionized water specifically after the decellularization or also generally for cleaning, in order to rinse out any residues of SDS from the reactor chamber 4 and also from the tissue framework before the recellularization. For this purpose, the first reservoir 42 is filled with deionized water and then the reactor chamber 4 is flushed out of the first reservoir 42 . For this purpose, the first switchover element 40 is actuated accordingly by the control unit and, if necessary, also the switchover valves 74, 76 if flushing is to be carried out alternatively or additionally via the arterial access 64.

In the embodiment shown here, the third reservoir 68 is connected to the reactor chamber 4 via a line 65, with a loading valve 67, here an “HPLC” valve (HPLC = high-performance liquid chromatography), being arranged along the line 65 for supplying the cells. The loading valve 67 here has six connections, which are connected to one another in pairs, resulting in three pairs, with two adjacent connections being connected to one another in each case, so that one can be used as an input and the other as an output. The loading valve 67 is designed in such a way that by switching it over, a fluid section F1 made of cells is inserted between two fluid sections F2 made of cell culture medium 66 . In other words: with the loading valve 67, two streams are interwoven, so to speak, namely a first stream out cell culture medium 66 and a second stream of cells. Along the line 65 there are then several fluid sections F1, F2, which follow one another in the direction of flow and consist of different media, namely cell culture medium 66 on the one hand and cells on the other. this is in 9 shown, which shows a section of the line 65, with fluid sections F1, F2 as described. The loading valve 67 is generally used to introduce cells into the soft tissue 6 from the outside. In a first switching position of the loading valve 67, the cells are added from the outside and flow through the loading valve 67, but not yet into the line 65, until a stream of cells is visible in a residue 69 downstream of the loading valve 67. The loading valve 67 is then switched to a second switching position to feed the cells into the line 65. As a result, the cells are pumped directly into the tissue 4, while the cell medium 66 now flows directly into the residue 69 without the flow of cell culture medium 66 to stop. After the cells have been fed in, the loading valve 67 is switched over again, so that cell culture medium 66 flows into the reactor chamber 4 again, but no more cells; instead, the cell feed 72 is connected to the residue 69 again.

In the present case, the loading valve 67 has a temperature sensor 71 which is connected to a temperature control element 86 in the third reservoir 68 here. A temperature of the cells and/or the cell culture medium 66 is measured with the temperature sensor 71 and is thus monitored and the temperature control element 86 is controlled as a function thereof in order to compensate for any temperature fluctuations.

In the exemplary embodiment shown, the first and the second medium 46 , 48 can also flow into the reactor chamber 4 via the arterial access 64 . In a corresponding process step, the tissue 6 can also be rinsed directly with the first and the second medium 46, 48. For this purpose, a third switching element 74, here a 3-way valve, is arranged downstream of the third reservoir 68, which can be switched in such a way that either the cell culture medium 66 or one of the two media 46, 48 from the first and second reservoir 42, 44 can be fed to the arterial access 64 arrived. The first or second medium 46, 48 is selected by means of a further, fourth switching element 76, here again a 3-way valve, which is arranged upstream of the aforementioned third switching valve 74 and connects it either to the first or second reservoir 42, 44. In this way, extensive rinsing can also be carried out automatically, optionally with physiological medium 48 or cell-destroying medium 46, without having to convert the bioreactor 2. For this purpose, the switching elements 74, 76 are suitably controlled by the control unit 12 in accordance with the process control of the bioreactor 2.

In the embodiment shown here, one of the process parameters P is a partial oxygen pressure, ie pO ₂ value. In this case, the conditioning unit 12 has a gas supply 78 for supplying oxygen into the second reservoir 44. In the present case, for example, air containing oxygen, optionally even with an adjustable proportion of oxygen, is supplied via the gas supply. The control unit 12 then controls the gas supply 78 depending on the partial oxygen pressure. The conditioning unit 10 is thus designed to condition the tissue 6 with a specific pO ₂ value. The gas supply 78 enables oxygen to be supplied, and the pO ₂ value is then set by supplying air to the second reservoir 44 . An oxygen sensor 80 is also arranged here along the path of the second medium 48 from the second reservoir 44 to the reactor chamber 4, for measuring the pO ₂ value of the medium for the purpose of controlling the gas supply 78.

In addition, one of the process parameters P is a pH value. In this case, the conditioning unit 10 has a CO ₂ supply 82 for supplying CO ₂ into the second reservoir 44 and the control unit 12 controls the CO ₂ supply 82 depending on the pH value. The conditioning unit 10 is thus designed to condition the tissue 6 with a specific pH value. A pH sensor 84 is arranged along the path of the second medium 48 from the second reservoir 44 to the reactor chamber 4 for measuring the pH value of the medium for the purpose of controlling the CO ₂ supply 82. The statements on the pO ₂ value also apply analogously for pH.

In a variant that is not explicitly shown, a control of the pH value and/or the pO ₂ value as described above for the second medium is alternatively or additionally implemented analogously for the cell culture medium 66 from the third reservoir 68 .

Another process parameter P is a temperature. The conditioning unit 10 has a temperature control element 86 for temperature control of the respective medium 46, 48, 66. In the exemplary embodiment shown, a respective temperature control element 86 is arranged in the first reservoir 42, in the second reservoir 44 and in the third reservoir 68, for temperature control of the corresponding medium 46, 48, 66. A plurality of temperature sensors 88 then measure the temperature of the respective medium 46, 48, 66 and the control unit 12 controls the temperature control elements 86 accordingly in order to in the reactor chamber 4 a set a specific temperature. In the present case, a first temperature sensor 88 measures the temperature in the reactor chamber 4 and, depending on this, the control unit 12 controls the temperature control elements 86 in the first and second reservoirs 42, 44, while a second temperature sensor 88 controls the temperature of the cell culture medium 66 on the way from the third reservoir 68 to the reactor chamber 4 and the control unit 12 controls the temperature control element 86 in the third reservoir 68 as a function of this.

In the bioreactor 2 shown here, the analysis unit 8 has an optical sensor 90 for the non-invasive measurement of at least one tissue parameter G. The optical sensor 90 is, for example, a front-light camera or a spectroscope or the like. A combination of several optical sensors 90 is also possible. The wall 36 of the reactor chamber 4 is transparent to light, which is then detected by the optical sensor 90 . An embodiment is also possible in which light is radiated into the reactor chamber 4 for measurement in order to then measure its scattering, reflection or transmission on the tissue 6, for example. If, as described above, the bioreactor 2 has two electrodes 34 for field stimulation, the measurement is carried out through the window 38 already described, which is suitably dimensioned for this purpose. An embodiment is also possible in which the tissue 6 can be moved relative to the optical sensor 90 (e.g. by means of the mentioned arms 22 or the positioning table 28), so that the tissue 6 can be examined from different positions. The window 38 is then correspondingly higher or several partial windows are placed one above the other in the longitudinal direction L and form interruptions in the electrodes 34.

In the exemplary embodiment shown, the bioreactor 2 has two optical sensors 90, namely a reflected-light camera and a spectroscope (in 1 only one sensor 90 is shown for simplicity). The spectroscope has an optical entrance 92 for receiving light from the reactor chamber 4, and the optical entrance 92 is rotatable around the reactor chamber 4 for optical measurement from different directions. The reflected-light camera also has an optical input 92 and, in contrast to the spectroscope, can be rotated overall. An angle-dependent measurement of the tissue parameters G is thus implemented. The optical input 92 and the incident light camera can even be rotated completely around the reactor chamber 4 here, so that a 360° all-round measurement is possible. For this purpose, the optical input 92 on the one hand and the incident-light camera on the other hand are mounted on a turntable 94 which, for example, is ring-shaped here and encircles the reactor chamber 4 . The turntable 94 is driven by a motor, not explicitly shown, so that the optical input 92 and the incident light camera can be rotated relative to the reactor chamber 4 , namely around a longitudinal axis of the reactor chamber 4 extending in the longitudinal direction L.

Reference List

2

bioreactor

4

reactor chamber

6

soft tissue, tissue

8th

analysis unit

10

conditioning unit

12

control unit

14

media input

16

media exit

18

bottom

20

top

22

poor

24

linear motor

25

holding element (upper arm)

26

force sensor

27

holding element (lower arm)

28

positioning table

29

Top

30

transport clamp

31

hand strap

32

fastener

34

electrode

36

wall

38

window

39

contact bar

40

first switching element

42

first reservoir

44

second reservoir

46

first, cell-destroying medium

48

second, physiological medium

50

pump

52

second switching element

54

pump

56

sampling point

58

backlog branch

60

pump

62

check valve

64

arterial access

65

Management

66

cell culture medium

67

loading valve

68

third reservoir

69

Residue

70

Feeder (for cell culture medium)

71

temperature sensor

72

cell delivery

74

third switching element

76

fourth switching element

78

air supply

80

oxygen sensor

82

CO ₂ supply

84

pH sensor

86

tempering element

88

temperature sensor

90

optical sensor

92

optical input

94

turntable

B

Broad

F1

fluid section (from cells)

F2

Fluid section (from cell culture medium)

G

tissue parameters

H

Height

L

longitudinal direction

P

process parameters

u

circumferential direction

Claims (16)

bioreactor (2), - this having a reactor chamber (4) for the production of a soft tissue (6), - this having an analysis unit (8) for measuring a number of tissue parameters (G) of the soft tissue (6), - wherein this has a conditioning unit (10) for conditioning the soft tissue (6) according to a number of process parameters (P), - wherein this has a control unit (12) which controls the analysis unit (8) and the conditioning unit (10) in such a way that the tissue parameters (G) are measured during production and the process parameters (P) are set depending on the tissue parameters (G). be so that the soft tissue (6) depending on the tissue parameters (G) is conditioned. Bioreactor (2) after claim 1 wherein the conditioning unit (10) has two arms (22) for holding the soft tissue (6) in the reactor chamber (4), the two arms (22) projecting into the reactor chamber (4) from outside and being movable relative to one another in order to exert a force on the soft tissue (6) for mechanical stimulation, the analysis unit (8) having at least one force sensor (22) which is attached to one of the arms (22) for the mechanical measurement of the tissue. Bioreactor (2) after claim 2 , wherein the two arms (22) are designed for automatic centering of the soft tissue (6). Bioreactor (2) according to one of Claims 1 until 3 , wherein the conditioning unit (10) for electrical stimulation of the soft tissue (6) has two electrodes (34) which are arranged along a wall (36) of the reactor chamber (4) for generating an electric field within the reactor chamber (4). Bioreactor (2) after claim 4 wherein at least one of the electrodes (34) has a window (38) through which one or more of the tissue parameters (G) can be measured with the analysis unit (8) from outside the reactor chamber (4). Bioreactor (2) according to one of Claims 1 until 5 , wherein the analysis unit (8) has an optical sensor (90) for the non-invasive measurement of at least one tissue parameter (G) during production. Bioreactor (2) after claim 6 , wherein the optical sensor (90) has an optical input (92) for receiving light from the reactor chamber (4), wherein the optical input (92) can be rotated around the reactor chamber (4) for optical measurement from different directions . Bioreactor (2) according to one of Claims 1 until 7 , wherein this has a first switching element (50) which connects the reactor chamber (4) optionally with a first reservoir (42) or a second reservoir (44), the first reservoir (42) containing a first, cell-destroying medium (46) for contains decellularization and the second reservoir (44) contains a second, physiological medium (48) for recellularization, so that the first switching element (50) can be used to set which of the two media (46, 48) flows into the reactor chamber (4). Bioreactor (2) after claim 8 , This being designed in such a way for flushing the reactor chamber after decellularization and before recellularization by filling the first reservoir with deionized water and then flushing the reactor chamber out of the first reservoir. Bioreactor (2) according to one of Claims 1 until 9 , wherein one of the process parameters (P) is a partial oxygen pressure, wherein the conditioning unit (10) has a gas supply (78) for supplying oxygen into the second reservoir (44), the control unit (12) depending on the gas supply (78). controlled by the oxygen partial pressure. Bioreactor (2) according to one of Claims 1 until 10 , wherein one of the process parameters (P) is a pH value, wherein the conditioning unit (10) has a CO ₂ supply (82) for supplying CO ₂ into the second reservoir (44), wherein the control unit (12) the CO ₂ supply (82) controls depending on the pH. Bioreactor (2) according to one of Claims 1 until 11 , wherein the reactor chamber (4) has an arterial access (64) via which a cell culture medium (66) from a third reservoir (68) together with cells can be supplied to the soft tissue (6) for recellularization. bioreactor after claim 12 , wherein the third reservoir (68) is connected to the reactor chamber (4) via a line (65), a loading valve (67) being arranged along the line (65) for supplying the cells, the loading valve (67) being such is designed such that a fluid section (F1) made of cells is inserted between two fluid sections (F2) made of cell culture medium (66) by switching the same. Bioreactor (2) according to one of Claims 1 until 13 , wherein the reactor chamber (4) has a bottom (18) with a media inlet (14) for a medium, and a top (20) with a media outlet (20) for the medium, so that the soft tissue (6) against gravity can be washed around by the medium. Bioreactor (2) according to one of Claims 1 until 14 , wherein the conditioning unit (10) for electrical stimulation of the soft tissue (6) has two electrodes, each of which is designed as a wire, with a respective wire running along one of the two arms (22) into the reactor chamber (4), for direct electrical connection and stimulation of the soft tissue (6). Process for operating a bioreactor (2), a. wherein the bioreactor (2) has a reactor chamber (4) in which a soft tissue (6) is produced, b. wherein the bioreactor (2) has an analysis unit (8) for measuring a number of tissue parameters (G) of the soft tissue (6), c. wherein the bioreactor (2) has a conditioning unit (10) for conditioning the soft tissue (6) according to a number of process parameters (P), i.e. wherein the bioreactor (2) has a control unit (12) which controls the analysis unit (8) and the conditioning unit (10) in such a way that the tissue parameters (G) are measured during production and the process parameters (P) are measured as a function of the tissue parameters ( G) are adjusted so that the soft tissue (6) is conditioned depending on the tissue parameters (G).

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