Microfluidic circulation system, use of a microfluidic circulation system, and device for cultivating biological cells

Abstract

The invention relates to a microfluidic circulation system, to a device for cultivating biological cells and to uses thereof. The circulation system contains a main chamber, a main channel, a pump, a secondary channel fluidically in parallel with the main channel, a cell culture chamber located in the secondary channel, and an element for reducing a volumetric flow in the cell culture chamber, said element having a predefined, unchangeable volumetric-flow resistance and being able to be inserted into and removed from the first secondary channel by means of a releasable connection. By means of the circulation system and the device, it is possible to change or define a volumetric flow through a cell culture chamber simply, quickly, with less susceptibility to error and economically, such that reproducible results can be obtained with minimal risk of downtime.

Description

[0001] FRAUNHOFER SOCIETY...eV

[0002] 249 PCT 2314

[0003] Microfluidic circulation system, use of a microfluidic circulation system and device for cultivating biological cells

[0004] A microfluidic circulation system and a device for cultivating biological cells are provided, and uses are proposed. The circulation system contains a main chamber, a main channel, a pump, a secondary channel arranged fluidically parallel to the main channel, a cell culture chamber arranged in the secondary channel, and an element for reducing a volume flow in the cell culture chamber, which element has a predetermined, unchangeable volume flow resistance and can be inserted into and removed from the first secondary channel via a detachable connection. The circulation system and the device make it possible to change or set a volume flow through a cell culture chamber in a simple, fast, error-free, and cost-effective manner, whereby reproducible results can be obtained with a minimal risk of downtime.In the human body, the supply of blood to the organs and the distribution of medications occur primarily via the cardiovascular system. On the one hand, the heart acts as a pump, and on the other hand, the individual blood flow to the organs is controlled by vasoconstriction or vasodilation. Furthermore, some important organs of the human body are constantly (and independently of vasodilating or vasoconstricting effects) supplied with a large, low-intensity blood flow.

[0005] In microphysiological systems (MPS), which are suitable for cultivating biological cells, in organ-on-chip systems (OOC), and in microscale cell culture analog systems (mCCA), several tissue equivalents must be cultivated together for drug testing and to recreate disease models.

[0006] A critical issue in the co-cultivation of tissue equivalents in an MPS is the different oxygen and nutrient requirements of the different tissue equivalents and, if toxicity is to be investigated, the different distribution of drugs within the system. Different levels of oxygen and nutrient delivery, as well as specific active ingredients, can be achieved in an MPS by specifically adjusting the flow rate of individual cell culture chambers within the MPS to achieve this different delivery. MPSs are known in the state of the art that allow for different flow rates in different cell culture chambers.

[0007] The simplest known microfluidic circulation systems have an internal or external pump that continuously circulates a cell culture medium and one or more cell culture chambers through which the cell culture medium can flow. The channels to the chambers have fixed cross-sections and thus distribute the medium evenly throughout the cell culture chambers (WO 2019 / 122349 A1). These systems are widely established but have several technical disadvantages. On the one hand, the volume flow is fixed due to the channel size, meaning that only a tissue equivalent that requires exactly this volume flow can be cultured in each chamber. However, since the inlet or outlet of the chambers can be obscured by cells or tissue during the culture period, there is a risk that the channel cross-section and thus the volume flow through the culture chamber will change during culture, which can lead to incorrect and unreproducible results.

[0008] Furthermore, more complex microfluidic circulation systems are known that feature actively controllable valves for adjusting the flow into the respective cell culture chambers of these circulation systems. In this context, microfluidic circulation systems are also known that are configured not only to control but also to regulate a volume flow into the individual cell culture chambers (DE 102013011768 A1). The disadvantage of these circulation systems, however, is that they are technically very complex, which makes controlling the valves complex and poses a risk of incorrect use, especially when such circulation systems are used by untrained users. The switching of the valves themselves is also error-prone, which can lead to long downtimes during operation of these circulation systems and even to the loss of (cost-intensive) cultured tissue equivalents.In addition, a dead volume often has to be provided for the technical design of the valves, which can negatively affect the circulation of the medium in the circulation system.

[0009] Apart from that, more complex microfluidic circulation systems are known that feature actively controllable pumps for adjusting the flow into the respective cell culture chambers of these circulation systems (WO 2020 / 041260 A1). This allows an individual volume flow to be set in each cell culture chamber. These circulation systems have the same disadvantages as circulation systems that feature actively controllable valves for adjusting the flow into the respective cell culture chambers. They are technically complex and prone to system-related and user-related errors, which can lead to long downtimes and the loss of expensive tissue equivalents.

[0010] Based on this, the object of the present invention was to provide a microfluidic circulation system and a device for cultivating biological cells that allows a volume flow through at least one cell culture chamber to be changed or determined (in particular, reduced compared to a main volume flow) in a simpler, faster, less error-prone, more reproducible, and / or more cost-effective manner, with minimal risk of downtime. In particular, it should also be possible to prevent channels of the microfluidic circulation system and / or the device from becoming clogged by biological cells, thus ensuring a constant volume flow over long periods of time. Furthermore, uses of the microfluidic circulation system according to the invention should be proposed.

[0011] The object is achieved by the microfluidic circulation system having the features of claim 1, the use having the features of claim 12 and the device having the features of claim 13. The dependent claims show advantageous developments of the subject matter according to the invention.

[0012] According to the invention, a microfluidic circulation system is provided, containing or consisting of: a) a main chamber for receiving a liquid, b) a main channel that enters and exits the main chamber; c) a pump that is arranged in the main channel and is suitable for circulating a liquid in a flow direction through the microfluidic circulation system; d) a first cell culture chamber for cultivating biological cells; e) a first secondary channel that branches off from the main channel, enters the first cell culture chamber, exits the first cell culture chamber, and opens into the main channel, wherein the first secondary channel is arranged fluidically parallel to the main channel in the flow direction; and f) a first element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the first cell culture chamber and which has a predetermined, unchangeable volume flow resistance;characterized in that the first element for reducing a volume flow can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom via the detachable connection. The microfluidic circulation system according to the invention allows a volume flow through the first cell culture chamber to be changed or determined in a simpler, faster, less error-prone, and more cost-effective manner than with prior art circulation systems, whereby more reproducible results can be obtained with a lower risk of downtime when using the circulation system and / or the device than with known circulation systems and devices for cultivating biological cells.

[0013] This is due to the fact that the microfluidic circulation system has a first element for reducing a volume flow in the first cell culture chamber, which first element can be inserted into the first secondary channel of the microfluidic circulation system via a detachable connection and removed from the first secondary channel via the detachable connection. The detachable connectability of the first element means that a user can set the volume flow through the first cell culture chamber to a specific value, which is predefined and unchangeable by the first element (i.e., fixed, permanent, or constant), by simply inserting and removing the first element in a quick, easy, and error-free manner. This simple measure leads to a more reproducible setting of the volume flow and to more reproducible results.In addition, adjusting the flow rate through the first cell culture medium can be done more cost-effectively than with known systems, since adjusting the flow rate requires only some mechanical work from the user (for inserting or removing the element), but no electrical energy consumption (as is the case, for example, when adjusting a flow rate using pumps and valves as in state-of-the-art systems). Furthermore, downtimes are reduced, since the first element, due to its predefined, unchangeable flow rate resistance, is less prone to failure than a variable flow rate resistance that can be adjusted via a fault-prone controllable pump and / or a fault-prone controllable valve.

[0014] A further advantage of the microfluidic circulation system according to the invention is its easy reconfigurability, which results from the detachable connection of the first element. In other words, a user has a high degree of flexibility with regard to adjusting the volume flow, as they can flexibly insert various first elements into the first secondary channel, each of which has a different, predetermined, unchangeable volume flow resistance. Recombination of the individual first elements therefore leads to easy reconfigurability of the microfluidic circulation system. The main channel and the main chamber of the microfluidic circulation system also contribute to the ease of adjustment, as they enable circulation of the remaining, unreduced volume flow in the microfluidic circulation system, i.e., the activity of the pump of the microfluidic circulation system does not need to be changed.can be kept constant at a desired, beneficial activity.

[0015] The first cell culture chamber can be inserted into the first secondary channel of the microfluidic circulation system via a detachable connection and removed from it via the detachable connection. This has the advantage that pretreatment of the cell culture chamber (e.g., pre-cultivation of biological cells) can take place outside the microfluidic circulation system, and the cell culture chamber is only inserted into the microfluidic circulation system for use in the system. This simplifies the use of the microfluidic circulation system.

[0016] The first secondary channel of the microfluidic circulation system can have a closable opening suitable for reversibly accommodating the first cell culture chamber via a detachable connection. The advantage of the closable opening is that, when using the microfluidic circulation system, the risk of contamination of a fluid circulating in the microfluidic circulation system is reduced.

[0017] In a preferred embodiment, the first element for reducing a volume flow cannot be controlled electrically, pneumatically, or hydraulically. The advantage is that setting a specific volume flow through the first cell culture chamber requires only mechanical energy from a user, but no additional energy, which makes the operation of the circulation system more economical and environmentally friendly. The first element for reducing a volume flow can contain a hole or a channel that has a smaller cross-sectional area than a cross-sectional area of ​​the first secondary channel. The hole or channel thus represents a mechanical measure with which the first element reduces the volume flow through the first cell culture compartment.

[0018] Furthermore, the first element for reducing a volume flow can be connected to the first cell culture chamber in a form-fitting, force-fitting and / or material-fitting manner, optionally being integral with the first cell culture chamber. One advantage is that the combination of cell culture chamber and the first element for reducing a volume flow is already connected and the user does not have to make the connection themselves. Another advantage is that the combination of the first element for reducing a volume flow and the first cell culture chamber can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom via the detachable connection, i.e.A user can cultivate biological cells in the first cell culture chamber outside the microfluidic circulation system (together with a first element that provides a predetermined, unchangeable volume flow resistance beneficial to the biological cells). This can provide a time advantage and further reduce the susceptibility to errors when using the circulation system.

[0019] The microfluidic circulation system can further comprise a bypass channel and an additional element for reducing a volume flow in the bypass channel. The bypass channel can branch off from a side channel of the microfluidic system (e.g., the first side channel), pass a cell culture chamber, and flow back into the side channel (bypass of the cell culture chamber). The flow direction in the bypass channel is thus parallel to the side channel and thus also parallel to the main channel. The advantage of the bypass channel is that a volume flow through a cell culture chamber (i.e., through the cell culture chamber affected by the bypass) can be adjusted even more finely. The bypass channel and the additional element for reducing a volume flow in the bypass channel can be connected to a cell culture chamber (e.g., the first cell culture chamber) in a form-fitting, force-fitting, and / or material-fitting manner, and can optionally be formed integrally with the cell culture chamber (e.g., the first cell culture chamber).The advantage is that the combination of cell culture chamber, bypass channel, and additional element for reducing a volume flow in the bypass channel is already connected and the user does not have to make the connection himself.

[0020] One idea of ​​the present invention is to equip the first element with a predetermined, unchangeable volume flow resistance that falls into one of three volume flow resistance groups. The background to this is that the volume flows through tissue of the human body can also be roughly divided or simplified into three different volume flow groups, as shown in the following table.

[0021] Table

[0022] The classification into three volume flow resistance groups reduces the user effort by reducing the complexity of selecting a suitable first element. Furthermore, a simpler and more cost-effective deployment of the microfluidic circulation system is possible.

[0023] The first element for reducing a volume flow can therefore be suitable for reducing a volume flow of a liquid in the first secondary channel to a volume flow that lies in the range of >0 to 5.0%, preferably 1.0 to 4.0%, particularly preferably 1.5 to 3.5%, very particularly preferably 2.0 to 3.0%, in particular 2.5%, of a volume flow of a liquid in the main channel (first element of the 1st group). Alternatively, the first element for reducing the volume flow can be suitable for reducing a volume flow of a liquid in the first secondary channel to a volume flow that lies in the range of >5.0 to 18.0%, preferably 6.0 to 15.0%, particularly preferably 7.0 to 13.0%, very particularly preferably 9.0 to 11.0%, in particular 10%, of a volume flow of a liquid in the main channel (first element of the 2nd group).

[0024] Alternatively, the first element for reducing the volume flow can be suitable for reducing a volume flow of a liquid in the first secondary channel to a volume flow which is in the range of >18.0 to 32.0%, preferably 20.0 to 30.0%, particularly preferably 23.0 to 28.0%, very particularly preferably 24.0 to 26.0%, in particular 25%, of a volume flow of a liquid in the main channel (first element of the 3rd group).

[0025] It is conceivable that the microfluidic circulation system comprises a total of two or three first elements belonging to different groups (e.g., a first element of the 1st group, a first element of the 2nd group, and a first element of the 3rd group). In this case, of course, only one of these first elements can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection. However, the user has flexibility: If they wish to replace the first element with another first element (e.g., from a different group, i.e., with a different, predefined, fixed volume flow resistance), they can do so quickly and easily thanks to the detachable connection to the first secondary channel.

[0026] The main chamber of the microfluidic circulation system, together with the main channel, represents the "rest of the body." If, for example, the first element has a predetermined, unchangeable resistance in the range of >18.0 to 32.0% (3rd group), e.g., specifically 25%, the volume flow through the main channel and the main chamber in this case (i.e., if the microfluidic circulation system does not contain any additional cell culture chambers) is 75%, resulting in a total volume flow of 100%.

[0027] The main chamber can contain at least one component of blood plasma, which is immobilized on an inner wall of the main chamber or separated by a semipermeable membrane from a liquid flowing through the main channel and the main chamber in the direction of flow. The at least one component is preferably albumin, particularly preferably human serum albumin. The advantage of this is that the main chamber in this case simulates blood conditions and the binding of certain components in the liquid flowing through the circulation system to blood components can also be taken into account (e.g., the binding of active substances to albumin). Results can therefore be achieved that better reflect real conditions (i.e., conditions in the human body).

[0028] The main chamber can have an internal volume ranging from 1 to 100 times the internal volume of the first cell culture chamber, optionally also of a second and / or third cell culture chamber of the microfluidic device. The advantage is that real-world conditions are better replicated, since the amount of blood flowing through a specific tissue at a given time (represented by the volume flow through the first cell culture chamber and optionally other cell culture chambers of the circulation system) is smaller than the residual amount of blood in the body (represented by the main channel and the main chamber).

[0029] The microfluidic circulation system may further include a control unit configured to control the pumping activity of the pump. The control unit is preferably configured to control the pump such that a fluid is pumped in a pulsating manner in the microfluidic circulation system, particularly preferably with a pulse frequency in the range of 1 to 2.5 Hz. Pulsating pumping has the advantage of better simulating real-life conditions (pulsating pumping in the human body by the heart as a pump), allowing results to be achieved that are closer to reality.

[0030] The microfluidic circulation system can further contain a first damping channel that branches off from the main channel and does not open into the main channel, except for the end of the first damping channel that branches off from the main channel, and contains a damping element. The damping element is preferably arranged at an end of the first damping channel that is opposite an end of the first damping channel that branches off from the main channel. The first damping channel is particularly preferably arranged downstream of the pump and upstream of the first secondary channel with respect to the flow direction. The first damping channel with the damping element has the advantage that flow and pressure in the microfluidic circulation system are smoothed and reality conditions are better represented when a pump with pulsatile pumping activity is used, because in reality, the pulsatile cardiac activity is dampened or absorbed by the blood vessels.smoothed (temporary dilation of the blood vessels by a heart pulse).

[0031] Optionally, the microfluidic circulation system includes a second damping channel branching off from the main channel, which does not open into the main channel except for one end of the second damping channel branching off from the main channel, and which includes a damping element. The damping element is preferably arranged at an end of the second damping channel opposite an end of the second damping channel branching off from the main channel. The second damping channel is preferably arranged upstream of the pump and downstream of the first secondary channel with respect to the flow direction.The presence of a second damping channel with a damping element can improve the above-mentioned advantage even further, in particular since damping can take place not only near the outlet of the pump (see preferred arrangement of the first damping channel), but also near the inlet of the pump (see preferred arrangement of the second damping channel) and thus fluidically between the first cell culture chamber (optionally also other cell culture chambers of the circulation system).

[0032] The damping element is preferably a semipermeable membrane, wherein the semipermeable membrane particularly preferably has a membrane diameter of at least 3 mm. A membrane diameter in this range has proven particularly effective in making the damping as realistic as possible, i.e. close to a damping of the pulsating volume flow in the human body. The semipermeable membrane is optionally fluidically connected to an oxygen source and / or carbon dioxide source on a side of the semipermeable membrane facing away from the first and / or second damping channel. This configuration has the further advantage that the damping channel can serve as a mass transfer channel for supplying and / or removing oxygen and / or carbon dioxide. This allows specific growth conditions to be set in the first cell culture chamber (and optionally also in other cell culture chambers of the circulation system) in a simple and structurally compact manner.

[0033] The microfluidic circulation system may further include: i) a second cell culture chamber for cultivating biological cells; ii) a second secondary channel branching off from the main channel, entering the second cell culture chamber, exiting the second cell culture chamber, and opening into the main channel, or branching off from the first secondary channel, entering the second cell culture chamber, exiting the second cell culture chamber, and opening into the first secondary channel, wherein the second secondary channel is arranged fluidically parallel to the main channel in the flow direction; and iii) a second volume flow reduction element suitable for reducing a volume flow of a liquid in the second cell culture chamber and having a predetermined, unchangeable volume flow resistance;wherein the second element for reducing a volume flow can be introduced into the second secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom through the detachable connection;

[0034] The advantage of this approach is that cell cultures of two different biological cells (or biological tissues) can be cultivated and examined in the microfluidic cell culture compartment (e.g., kidney cells in the first cell culture chamber and muscle cells in the second cell culture chamber). The examination of the two different biological cells can thus be carried out more quickly and cost-effectively.

[0035] The second cell culture chamber can be inserted into the first secondary channel of the microfluidic circulation system via a detachable connection and removed therefrom via the detachable connection. The second secondary channel can have a closable opening that is suitable for reversibly accommodating the second cell culture chamber via a detachable connection.

[0036] The second element for reducing a volume flow is preferably not electrically controllable, not pneumatically controllable and not hydraulically controllable.

[0037] Furthermore, the second element for reducing a volume flow may contain a hole or a channel having a smaller cross-sectional area than a cross-sectional area of ​​the second secondary channel.

[0038] Apart from that, the second element for reducing a volume flow can be connected to the second cell culture chamber in a form-fitting, force-fitting and / or material-fitting manner, optionally being integral with the first cell culture chamber.

[0039] The second element for reducing a volume flow can be suitable for reducing a volume flow of a liquid in the second secondary channel to a volume flow which is in the range of >0 to 5.0%, preferably 1.0 to 4.0%, particularly preferably 1.5 to 3.5%, very particularly preferably 2.0 to 3.0%, in particular 2.5%, of a volume flow of a liquid in the main channel.

[0040] Alternatively, the second element for reducing a volume flow may be suitable for reducing a volume flow of a liquid in the second secondary channel to a volume flow which is in the range of >5.0 to 18.0%, preferably 6.0 to 15.0%, particularly preferably 7.0 to 13.0%, very particularly preferably 9.0 to 11.0%, in particular 10%, of a volume flow of a liquid in the main channel.

[0041] Alternatively, the second element for reducing a volume flow can be suitable for reducing a volume flow of a liquid in the second secondary channel to a volume flow that is in the range of >18.0 to 32.0%, preferably 20.0 to 30.0%, particularly preferably 23.0 to 28.0%, very particularly preferably 24.0 to 26.0%, in particular 25%, of a volume flow of a liquid in the main channel. Optionally, the second element for reducing a volume flow has a flow reduction property (i.e., a volume flow resistance) that is identical to or different from the first element for reducing a volume flow.

[0042] It is conceivable that the microfluidic circulation system comprises a total of two or three second elements belonging to different groups (e.g., a first element of the first group, a first element of the second group, and a first element of the third group). This provides flexibility for the user: If they wish to replace the second element with another second element (e.g., from a different group, i.e., with a different predefined, fixed volume flow resistance), they can do so quickly and easily thanks to the detachable connection to the second secondary channel.

[0043] Apart from that, the microfluidic circulation system can further comprise: i) n further cell culture chambers for cultivating biological cells, where n is an even number, where n is preferably 1, 2, 3, 4, 5, 7, 8, 9 or 10; ii) n further side channels, each branching off from the main channel, entering one of the n further cell culture chambers, each exiting one of the n further cell culture chambers and opening into the main channel or leading from a side channel of the microfluidic circulation system (e.g.the first or second side channel), enter the nth cell culture chamber, exit the nth cell culture chamber and open into the side channel, wherein the n side channels are each arranged fluidically parallel to the main channel in the flow direction; and iii) n further elements for reducing a volume flow, each of which is suitable for reducing a volume flow of a liquid in one of the n further cell culture chambers and which each have a predetermined, unchangeable volume flow resistance; wherein the n further elements for reducing a volume flow can each be introduced into each of the n further side channels of the microfluidic circulation system via a detachable connection and can be removed from the system via the detachable connection. The advantage here is that cell cultures of n different biological cells (orbiological tissues) can be cultured and examined. The examination of the n different biological cells can thus be carried out more quickly and cost-effectively.

[0044] The n additional cell culture chambers can each be inserted into one of the n additional side channels of the microfluidic circulation system via a detachable connection and can be removed from the system via the detachable connection.

[0045] The n further side channels preferably each have a closable opening which is suitable for reversibly accommodating one of the n further cell culture chambers via a detachable connection.

[0046] The n additional elements for reducing a volume flow are preferably not electrically controllable, not pneumatically controllable and not hydraulically controllable.

[0047] Furthermore, the n further elements for reducing a volume flow can each contain a hole or a channel which has a smaller cross-sectional area than a cross-sectional area of ​​the respective n-th secondary channel.

[0048] In addition, the n further elements for reducing a volume flow can be connected to one of the n further cell culture chambers in a form-fitting, force-fitting and / or material-fitting manner, optionally being integral with one of the n further cell culture chambers.

[0049] The n further elements for reducing a volume flow can each be suitable for reducing a volume flow of a liquid in the n further secondary channel to a volume flow which is in the range of >0 to 5.0%, preferably 1.0 to 4.0%, particularly preferably 1.5 to 3.5%, very particularly preferably 2.0 to 3.0%, in particular 2.5%, of a volume flow of a liquid in the main channel. Alternatively, the n further elements for reducing a volume flow can each be suitable for reducing a volume flow of a liquid in the n further secondary channel to a volume flow which is in the range of >5.0 to 18.0%, preferably 6.0 to 15.0%, particularly preferably 7.0 to 13.0%, very particularly preferably 9.0 to 11.0%, in particular 10%, of a volume flow of a liquid in the main channel.

[0050] Alternatively, the n further elements for reducing a volume flow can each be suitable for reducing a volume flow of a liquid in the n further secondary channel to a volume flow which is in the range of >18.0 to 32.0%, preferably 20.0 to 30.0%, particularly preferably 23.0 to 28.0%, very particularly preferably 24.0 to 26.0%, in particular 25%, of a volume flow of a liquid in the main channel.

[0051] Optionally, the n further elements for reducing a volume flow optionally have a flow reduction property that is identical or different to the first element for reducing a volume flow and / or second element for reducing a volume flow.

[0052] It is also conceivable for the microfluidic circulation system to comprise a total of two or three n-th elements belonging to different groups (e.g., a first element of the 1st group, a first element of the 2nd group, and a first element of the 3rd group). This provides flexibility for the user: If they wish to replace the n-th element with another n-th element (e.g., from a different group, i.e., with a different predefined, fixed volumetric flow resistance), they can do so quickly and easily thanks to the detachable connection to the n-th side channel.

[0053] In a preferred embodiment, an inlet and an outlet of the cell culture chamber, optionally of all cell culture chambers of the microfluidic circulation system, are arranged at such a large distance from a surface of the cell culture chamber suitable for cultivating biological cells that biological cells cultivated in the cell culture chamber do not oppose any flow resistance to a fluid flow from the inlet to the outlet of the cell culture chamber. The advantage of this is that clogging of the inlet and outlet of the cell culture chamber(s) can be avoided during use of the microfluidic circulation system, thereby ensuring a constant volume flow over long periods and allowing more reproducible results to be obtained. The mentioned distance is preferably at least 0.5 mm.The surface of the cell culture chamber is optionally a surface of a bottom wall of the cell culture chamber, a surface of a side wall of the cell culture chamber, a surface of an intermediate wall of the cell culture chamber or a surface of a flat substrate inserted into the cell culture chamber for cultivating biological cells.

[0054] The invention further proposes the use of a microfluidic circulation system according to the invention for cultivating biological cells, preferably i) for testing the influence of at least one active ingredient on cultured biological cells; and / or ii) for constructing tissue structures from cultured biological cells, particularly preferably in the presence of at least one growth factor; and / or iii) for investigating the cellular, molecular, and / or vesicular interaction between different biological cell tissue explants and / or biological cell explants; and / or iv) for flushing or applying cryoprotective media or thawing protection media to biological cells or biological tissues for the purpose of preserving cell functions and / or tissue functions before or after cryopreservation.

[0055] According to the invention, a device for cultivating biological cells is further provided, comprising or consisting of: a) a cell culture chamber having a surface suitable for cultivating biological cells, an inlet, and an outlet; b) a channel entering the inlet of the cell culture chamber and exiting the outlet of the cell culture chamber; c) a first element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the channel of the device and which has a predetermined, unchangeable volume flow resistance;characterized in that the device (by its dimensions) is suitable for being introduced into a channel of a microfluidic circulation system via a detachable connection and for being removed from it through the detachable connection, wherein the inlet and the outlet of the cell culture chamber are arranged at a distance of at least 0.5 mm from the surface of the cell culture chamber which is suitable for cultivating biological cells.;

[0056] The device makes it possible to set a volume flow through the cell culture chamber in a simple, fast, error-free, and cost-effective manner. This allows the device to be set or changed within a microfluidic system when used in a microfluidic system. Since the device prevents clogging of the inlet and outlet of the cell culture chamber during use due to the distance of at least 0.5 mm between the inlet and outlet from the surface of the cell culture chamber for cultivating biological cells, a constant volume flow can be ensured over long periods of time. This allows reproducible results to be obtained with minimal downtime when using the device.

[0057] The device can comprise a further element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the channel of the device and which has a predetermined, unchangeable volume flow resistance. Preferably, the first element for reducing a volume flow is arranged at the inlet of the cell culture chamber and the further element for reducing a volume flow is arranged at the outlet of the cell culture chamber. The further element for reducing a volume flow has the advantage that the predetermined and unchangeable volume flow through the cell culture chamber can be adjusted even more precisely to a desired value.

[0058] Furthermore, the device can comprise a bypass channel and an additional element for reducing a volume flow in the bypass channel, wherein the bypass channel branches off from the channel, passes the cell culture chamber, and then flows back into the channel. The bypass channel with the additional element for reducing a volume flow has the advantage that the predetermined and unchangeable volume flow through the cell culture chamber can be adjusted even more precisely to a desired value.

[0059] The subject matter of the invention will be explained in more detail with reference to the following figures, without wishing to restrict it to the specific embodiments shown here.

[0060] Figure 1 schematically shows a microfluidic circulation system according to the invention. The microfluidic circulation system contains a main chamber 1 for receiving a liquid and a main channel 2 that enters and exits the main chamber 1. The microfluidic circulation system further contains a pump 3, which is arranged in the main channel 2 and is suitable for circulating a liquid in a flow direction (see arrow in Figure 1) through the microfluidic circulation system. Furthermore, the microfluidic circulation system contains a first cell culture chamber 4 for cultivating biological cells and a first secondary channel 5, which branches off from the main channel 2, enters the first cell culture chamber 4, exits the first cell culture chamber 4, and opens into the main channel 2. The first secondary channel 5 is arranged fluidically parallel to the main channel 2 in the flow direction.Furthermore, the microfluidic circulation system contains a first flow-reducing element 6, which is suitable for reducing the flow of a liquid through the first cell culture chamber and has a predetermined, unchangeable flow resistance. The microfluidic circulation system is characterized in that the first flow-reducing element 6 can be inserted into the first secondary channel 5 of the microfluidic circulation system via a detachable connection and can be removed therefrom via the detachable connection.

[0061] Figure 2 schematically shows another microfluidic circulation system according to the invention. The microfluidic circulation system has the same features as the microfluidic circulation system shown in Figure 1, with the following additional features: It further contains a bypass channel 19 with an additional element 20 for reducing a volume flow in the bypass channel 19, wherein the bypass channel is arranged fluidically parallel to the main channel 2 (here also fluidically parallel to the secondary channel 5) and branches off from the main channel 2 (here also from the first secondary channel 5) and opens into the main channel 2 (here also into the first secondary channel 5). The bypass channel allows a flow in the first secondary channel 5 that bypasses the first cell culture chamber 4 and thus a finer adjustment of the volume flow passing through the first cell culture chamber 4.

[0062] Figure 3 shows another microfluidic circulation system according to the invention. The microfluidic circulation system has the same features as the microfluidic circulation system shown in Figure 1, with the following additional features: It contains a one-piece combination of a first cell culture chamber 4 for cultivating biological cells and a first element 6 and a further element 7 for reducing a volume flow through the first cell culture chamber 4. In this combination, a surface 8 for cultivating tissue (e.g., a mesh with a mesh size of 500 pm) is illustrated above the volume flow.Furthermore, the microfluidic circulation system contains a one-piece combination of a second cell culture chamber 9 for cultivating biological cells and a second element 11 for reducing a volume flow through the second cell culture chamber 9, and a second secondary channel 10, wherein the second secondary channel 10 is arranged fluidically parallel to the main channel 2 and branches off from the main channel 2, enters the second cell culture chamber 9, exits the second cell culture chamber 9 and opens into the main channel 2.Furthermore, the microfluidic circulation system contains a one-piece combination of a third cell culture chamber 12 for cultivating biological cells and a third element 14 for reducing a volume flow through the third cell culture chamber 12, and a third secondary channel 13, wherein the third secondary channel 13 is arranged fluidically parallel to the main channel 2 and branches off from the main channel 2, enters the third cell culture chamber 12, exits the third cell culture chamber 12, and opens into the main channel 2. Figure 4 schematically shows another microfluidic circulation system according to the invention.The microfluidic circulation system has the same features as the microfluidic circulation system shown in Figure 1, with the following additional features: It further contains a second cell culture chamber 9 for cultivating biological cells, a second element 11 for reducing a volume flow through the second cell culture chamber 9 and a second secondary channel 10, wherein the second secondary channel 10 is arranged fluidically parallel to the main channel 2 and branches off from the main channel 2, enters the second cell culture chamber 9, exits the second cell culture chamber 9 and opens into the main channel 2.In addition, the microfluidic circulation system contains a third cell culture chamber 12 for cultivating biological cells, a third element 14 for reducing a volume flow through the third cell culture chamber 12 and a third secondary channel 13, wherein the third secondary channel 13 is arranged fluidically parallel to the main channel 2 and branches off from the main channel 2, enters the third cell culture chamber 12, exits the third cell culture chamber 12 and opens into the main channel 2.In addition, the device includes a first damping channel 15 which branches off from the main channel 2 and does not open into the main channel 2 except for one end of the first damping channel which branches off from the main channel 2, and a first damping element 16, wherein the first damping element 16 is arranged at an end of the first damping channel 15 which is opposite to an end of the first damping channel 15 which branches off from the main channel 2, wherein the first damping channel 15 is arranged downstream of the pump 3 and upstream of the first sub-channel 5 with respect to the flow direction (see arrow).Furthermore, the device contains a second damping channel 17, which branches off from the main channel 2 and does not open into the main channel 2, except for one end of the first damping channel 17, which branches off from the main channel 2, and a second damping element 18, wherein the second damping element 18 is arranged at an end of the second damping channel 17, which is opposite an end of the second damping channel 17, which branches off from the main channel 2, wherein the second damping channel 17 is arranged, with respect to the flow direction (see arrow), upstream of the pump 3 and downstream of the first secondary channel 5. Figures 5A-5E schematically show various views of devices according to the invention for cultivating biological cells.These are one-piece combinations of a cell culture chamber 4 with a first element 6 for reducing a volume flow and a further element 7 for reducing the volume flow, wherein the elements 6, 7 for reducing the volume flow are suitable for reducing a volume flow of a liquid in the cell culture chamber and each have a predetermined, unchangeable volume flow resistance. The dimensions of the combinations shown here make them suitable for detachable insertion into the circulation system according to the invention. The device according to the invention also has a surface 8 for cultivating biological cells (e.g. in the form of a net with a mesh size of 500 μm) spatially above the volume flow, which surface is suitable for allowing a volume flow to flow past the biological cells.Furthermore, Figures 2C-2E show embodiments of the combinations for the structural design of the first element 6 for reducing a volume flow in order to reduce the volume flow through the first cell culture chamber 4. Figure 5C shows a configuration for reducing the volume flow to 2.5% of a volume flow of a liquid in the main channel, Figure 5D shows a configuration for reducing the volume flow to 10% of a volume flow of a liquid in the main channel, and Figure 5E shows a configuration for reducing the volume flow to 25% of a volume flow of a liquid in the main channel.

[0063] Figures 6A-6E schematically show various views of further devices according to the invention for cultivating biological cells, which have the same features as the devices shown in Figure 5, with the following additional features: The devices each have a bypass channel 19 and an additional element 20 for reducing a volume flow in the bypass channel 19. The bypass channel 19, together with the additional element 20, is suitable for guiding a portion of the volume flow past the cell culture chamber, i.e., the volume flow through the cell culture chamber and the volume flow through the bypass channel 19 add up to a total volume flow. The bypass channel 19 and the additional element 20 for reducing a volume flow in the bypass channel 19 make it possible to adjust the volume flow flowing through the cell culture chamber even more precisely.

[0064] 1: Main chamber;

[0065] 2: Main channel;

[0066] 3: pump;

[0067] 4: first cell culture chamber for cultivating biological cells;

[0068] 5: first secondary channel;

[0069] 6: first element for reducing a volume flow;

[0070] 7: additional element for reducing the volume flow (downstream of the first cell culture chamber);

[0071] 8: Surface for positioning tissue above the volume flow;

[0072] 9: second cell culture chamber for cultivating biological cells;

[0073] 10: second secondary channel;

[0074] 11: second element for reducing a volume flow;

[0075] 12: third cell culture chamber for cultivating biological cells;

[0076] 13: third secondary channel;

[0077] 14: third element for reducing a volume flow;

[0078] 15: first damping channel;

[0079] 16: first damping element;

[0080] 17: second damping channel;

[0081] 18: second damping element;

[0082] 19: Bypass channel;

[0083] 20: additional element to reduce the volume flow.

Claims

Patent claims 1. A microfluidic circulation system, containing or consisting of: a) a main chamber for receiving a liquid, b) a main channel that enters and exits the main chamber; c) a pump that is arranged in the main channel and is suitable for circulating a liquid in a flow direction through the microfluidic circulation system; d) a first cell culture chamber for cultivating biological cells; e) a first secondary channel that branches off from the main channel, enters the first cell culture chamber, exits the first cell culture chamber, and opens into the main channel, wherein the first secondary channel is arranged fluidically parallel to the main channel in the flow direction; and f) a first element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the first cell culture chamber and which has a predetermined, unchangeable volume flow resistance;characterized in that the first element for reducing a volume flow can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom via the detachable connection; 2. Microfluidic circulation system according to the preceding claim, characterized in that the first cell culture chamber can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom via the detachable connection, wherein the first secondary channel preferably has a closable opening which is suitable for reversibly accommodating the first cell culture chamber via a detachable connection.

3. Microfluidic circulation system according to one of the preceding claims, characterized in that the first element for reducing a volume flow i) is not electrically controllable, is not pneumatically controllable, and is not hydraulically controllable; and / or ii) contains a hole or a channel that has a smaller cross-sectional area than a cross-sectional area of the first secondary channel; and / or iii) is connected to the first cell culture chamber in a form-fitting, force-fitting, and / or material-fitting manner, optionally being integral with the first cell culture chamber.

4. Microfluidic circulation system according to one of the preceding claims, characterized in that the first element for reducing a volume flow is suitable for reducing a volume flow of a liquid in the first secondary channel to a volume flow which is in the range of i) >0 to 5.0%, preferably 1.0 to 4.0%, particularly preferably 1.5 to 3.5%, very particularly preferably 2.0 to 3.0%, in particular 2.5%, of a volume flow of a liquid in the main channel; or ii) >5.0 to 18.0%, preferably 6.0 to 15.0%, particularly preferably 7.0 to 13.0%, very particularly preferably 9.0 to 11.0%, in particular 10%, of a volume flow of a liquid in the main channel; or iii) >18.0 to 32.0%, preferably 20.0 to 30.0%, particularly preferably 23.0 to 28.0%, very particularly preferably 24.0 to 26.0%, in particular 25%, of a volume flow of a liquid in the main channel.

5. Microfluidic circulation system according to one of the preceding claims, characterized in that the main chamber i) contains at least one component of blood plasma which is immobilized on an inner wall of the main chamber or is separated by a semipermeable membrane from a liquid which flows through the main channel and the main chamber in the flow direction, wherein the at least one component is preferably albumin, particularly preferably human serum albumin, ii) has an internal volume which is in the range of 1 to 100 times an internal volume of the first cell culture chamber, optionally also a second and / or third cell culture chamber of the microfluidic device.

6. Microfluidic circulation system according to one of the preceding claims, characterized in that the microfluidic circulation system further includes a control unit configured to control a pumping activity of the pump, wherein the control unit is preferably configured to control the pump such that a liquid in the microfluidic circulation system is pumped in a pulsating manner, particularly preferably with a pulse frequency in the range of 1 to 2.5 Hz.

7. Microfluidic circulation system according to one of the preceding claims, characterized in that the microfluidic circulation system further comprises i) a first damping channel branching off from the main channel, apart from one end of the first damping channel branching off from the main channel, does not open into the main channel, and a damping element, wherein the damping element is preferably arranged at an end of the first damping channel opposite an end of the first damping channel branching off from the main channel, wherein the first damping channel is particularly preferably, with respect to is arranged downstream of the pump and upstream of the first secondary channel, with respect to the flow direction; and ii) optionally contains a second damping channel which branches off from the main channel, does not open into the main channel except for one end of the second damping channel which branches off from the main channel, and contains a damping element, wherein the damping element is preferably arranged at an end of the second damping channel which is opposite an end of the second damping channel which branches off from the main channel, wherein the second damping channel is preferably arranged upstream of the pump and downstream of the first secondary channel with respect to the flow direction;wherein the damping element is preferably a semipermeable membrane, wherein the semipermeable membrane particularly preferably has a membrane diameter of at least 3 mm, wherein the semipermeable membrane is optionally fluidically connected to an oxygen source and / or carbon dioxide source on a side of the semipermeable membrane facing away from the first and / or second damping channel; 8. Microfluidic circulation system according to one of the preceding claims, characterized in that the microfluidic circulation system further comprises: i) a second cell culture chamber for cultivating biological cells; ii) a second secondary channel branching off from the main channel, entering the second cell culture chamber, exiting the second cell culture chamber and opening into the main channel or branching off from the first secondary channel, entering the second cell culture chamber, exiting the second cell culture chamber and opening into the first secondary channel, wherein the second secondary channel is arranged fluidically parallel to the main channel in the flow direction; and iii) a second element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the second cell culture chamber and which has a predetermined, unchangeable volume flow resistance; wherein the second element for reducing a volume flow can be introduced into the second secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom through the detachable connection, wherein preferably the second cell culture chamber can be introduced into the first secondary channel of the microfluidic circulation system via a detachable connection and can be removed therefrom through the detachable connection, wherein the second secondary channel preferably has a closable opening which is suitable for reversibly receiving the second cell culture chamber via a detachable connection.

9. Microfluidic circulation system according to claim 8, characterized in that the second element for reducing a volume flow i) is not electrically controllable, is not pneumatically controllable, and is not hydraulically controllable; and / or ii) contains a hole or a channel that has a smaller cross-sectional area than a cross-sectional area of the second secondary channel; and / or iii) is connected to the second cell culture chamber in a form-fitting, force-fitting, and / or material-fitting manner, optionally being integral with the first cell culture chamber.

10. Microfluidic circulation system according to claim 8 or 9, characterized in that the second element for reducing a volume flow is suitable for reducing a volume flow of a liquid in the second secondary channel to a volume flow which is in the range of i) >0 to 5.0%, preferably 1.0 to 4.0%, particularly preferably 1.5 to 3.5%, very particularly preferably 2.0 to 3.0%, in particular 2.5%, of a volume flow of a liquid in the main channel; or ii) >5.0 to 18.0%, preferably 6.0 to 15.0%, particularly preferably 7.0 to 13.0%, very particularly preferably 9.0 to 11.0%, in particular 10%, of a volume flow of a liquid in the main channel; or iii) >18.0 to 32.0%, preferably 20.0 to 30.0%, particularly preferably 23.0 to 28.0%, very particularly preferably 24.0 to 26.0%, in particular 25%, of a volume flow of a liquid in the main channel; wherein the second volume flow reduction element optionally has a flow reduction property that is identical or different to the first volume flow reduction element.

11. Microfluidic circulation system according to one of the preceding claims, characterized in that an inlet and an outlet of the cell culture chamber, optionally of all cell culture chambers of the microfluidic circulation system, are arranged at such a large distance from a surface of the cell culture chamber suitable for the cultivation of biological cells that biological cells cultivated in the cell culture chamber do not oppose any flow resistance to a liquid flow from the inlet to the outlet of the cell culture chamber, wherein the distance is preferably at least 0.5 mm, wherein the surface of the cell culture chamber is optionally a surface of a bottom wall of the cell culture chamber, a surface of a side wall of the cell culture chamber, a surface of an intermediate wall of the cell culture chamber or a surface of a flat substrate inserted into the cell culture chamber for the cultivation of biological cells.

12. Use of a microfluidic circulation system according to one of the preceding claims for cultivating biological cells, preferably i) for testing the influence of at least one active ingredient on cultured biological cells; and / or ii) for constructing tissue structures from cultured biological cells, particularly preferably in the presence of at least one growth factor; and / or iii) for investigating the cellular, molecular, and / or vesicular interaction between different biological cell tissue explants and / or biological cell explants; and / or iv) for flushing or applying cryoprotective media or thawing protection media to biological cells or biological tissues for the purpose of preserving cell functions and / or tissue functions before or after cryopreservation.

13. A device for cultivating biological cells, comprising or consisting of: a) a cell culture chamber having a surface suitable for cultivating biological cells, an inlet and an outlet; b) a channel entering the inlet of the cell culture chamber and exiting the outlet of the cell culture chamber; c) a first element for reducing a volume flow, which is suitable for reducing a volume flow of a liquid in the channel of the device and which has a predetermined, unchangeable volume flow resistance; characterized in that the device is suitable for being introduced into a channel of a microfluidic circulation system via a detachable connection and for being removed therefrom through the detachable connection, wherein the inlet and the Outlet of the cell culture chamber is arranged at a distance of at least 0.5 mm from the surface of the cell culture chamber which is suitable for cultivating biological cells.

14. Device according to claim 13, characterized in that the device comprises a further element for reducing a Volume flow which is suitable for reducing a volume flow of a liquid in the channel of the device and which has a predetermined, unchangeable volume flow resistance, wherein preferably the first element for reducing a volume flow is arranged at the inlet of the cell culture chamber and the further element for reducing a volume flow is arranged at the outlet of the cell culture chamber.

15. Device according to one of claims 13 or 14, characterized in that the device comprises a bypass channel and an additional element for reducing a volume flow in the bypass channel, with the bypass channel branching off from the channel, passing the cell culture chamber and then flowing back into the channel.