WO2009132616A2

WO2009132616A2 - Supply system

Info

Publication number: WO2009132616A2
Application number: PCT/DE2009/000528
Authority: WO
Inventors: Claus FÜTTERER; Hans-Ulrich Fried; Arne Brombas
Original assignee: Forschungszentrum Jülich GmbH
Priority date: 2008-04-29
Filing date: 2009-04-17
Publication date: 2009-11-05
Also published as: DE102008021483A1; WO2009132616A3

Abstract

The invention relates to a supply system comprising at least two supply lines that lead to a mixing chamber provided with an outlet line for the mixed fluids. Said supply system comprises a layer system having at least two layers, the first layer (7) being a flow element integrating the mixing chamber (11), at least two channels (10, 10a) that lead into the mixing chamber, and an outlet channel (12) that extends from the mixing chamber (11) to a sample or a substrate, and the second layer (8) is sealingly applied to the first layer (7). The supply system is used to perfuse tissue or cell cultures with fluids.

Description

C o n s e c tio n Supply system

The invention relates to a supply system, in particular a supply system for perfusion.

Prior art supply systems are known for perfusion, consisting of several reservoirs for liquids, each of which is connected via a conduit to a mixing chamber which supplies the mixed liquid to a substrate to be perfused via a drain. These devices are confusing in structure, take up a lot of space, are therefore not easy and a germ-free operation can not be guaranteed. The prior art devices require a high experimental design skill in order to ensure the tightness of the devices. Furthermore lead the supply systems according to the prior art

Cross contamination of liquid volumes from different supply lines. The prior art devices are costly to operate because they require regular cleaning, in particular to ensure sterility, cleanliness and hygiene. A predictability of the parameters for three-dimensional flow conditions is very demanding, so that much computational effort must be operated. An operation of the devices under pressure is not guaranteed safe and leads to leaks. Furthermore, these devices are susceptible to interference from external influences. The temperature of the supply systems is difficult.

It is therefore an object of the invention to provide a supply system for liquids to be supplied to a substrate, which enables a hygienic supply of liquids, easy to manufacture and easy to build and use dense tight and is also still inexpensive , The tightness of the device should allow pressurization of, for example, 7 bar without escaping gas or liquid to the outside. The supply system should be immune to interference and a simple predictability of the flow parameters, such as flow rate

Function of the pressure, mixing efficiency or mixing speed. Cross contamination of liquid volumes of different supply lines should be minimized. The The liquid supplied to the substrate should be easy and as accurate as possible to temper.

Starting from the preamble of claim 1, the object is achieved by the features of the characterizing part of claim 1.

Accordingly, the invention comprises a supply system to a substrate or a sample, comprising at least two feed lines to a mixing chamber and a discharge from the mixing chamber, wherein the feed system is formed by a layer system of at least two layers, of which the first layer is a flow element, into which the mixing chamber, at least two channels leading into the mixing chamber and an exit channel leading from the mixing chamber to a sample or a substrate, are embedded and the second layer is sealingly applied to the first layer.

With the feed system according to the invention, it is now possible to mix liquids from different channels or switch in the supply, for example, alternately or without jerks, easy to perfusion or another attempt to supply the supply of liquids germ-free or clean as required. Of the

Operation is inexpensive and the supply system is easy to manufacture and build. The liquid-carrying flow element is easy to replace. The supply system according to the invention can be applied to higher pressures, such as 7 bar, without affecting the tightness and reliability. Cross-contamination between different channels can be prevented. Furthermore, the feed system according to the invention is robust against external influences, and the flow parameters in each channel can be easily calculated. A temperature control is also possible in a simple manner and very accurately. Through a simple-to-produce flow element of the experimental setup can be customized to the problem or the desired conditions.

Advantageous embodiments of the invention are specified in the subclaims. The figures show preferred embodiments of the invention in schematic form. It shows:

Fig. 1: A side view of the device according to the invention Fig. 2a: A plan view of the inventive device Fig. 2b: An advantageous embodiment of the reservoir

Figure 1 shows a supply system with storage containers 1, Ia, Ib, which are intended for receiving a liquid. They are each equipped with a pressure source 2, 2a, 2b, which are connected via a pressure regulator 3, 3a, 3b with the reservoirs 1, Ia, Ib. At the storage containers 1, Ia, Ib, there is in each case a supply line 4, 4a, 4b which is equipped with a valve 5, 5a, 5b. The feed lines 4, 4a, 4b equipped with the valves 5, 5a, 5b lead into a layer system 6 with a layer 7 serving as a flow element and a cover layer 8 mounted above. A further layer 9 is attached underneath the layer 7 serving as a flow element the serving as a flow element layer 7 presses against the cover layer. In the serving as a flow element layer 7 channels 10, 1 Oa, 1 Ob are admitted, which connect the leads 4, 4a, 4b with a mixing chamber 11, which is also embedded in the serving as a flow element layer 7. From the mixing chamber 11, a likewise inserted into the layer 7 output channel 12, which is guided to the sample, not shown in the figure in the measuring chamber 14, which is to be supplied to the perfusion or other treatment. Reference numeral 13 refers to temperature control.

In Figure 2a, which shows a plan view of the device according to the invention, the same device features have the same reference numerals.

Figure 2b shows a side view of an alternative embodiment of the reservoir. In this embodiment, the various storage vessels 1, Ia, Ib are stacked on top of each other.

In the following, the invention will be described in its general form.

According to the invention, the supply system comprises at least two layers, of which the first layer is a layer serving as a flow element 7, in which at least two Channels 10, 10a, which open into a mixing chamber 11, and an output channel 12 are recessed.

The fluidizing layer 7 may be a rigid plate which has been cast or into which the channels 10, 10a, the mixing chamber 11 and the outlet conduit 12 have been milled or otherwise recessed.

For the purposes of the invention, it is to be understood that methods have been used with which recesses are introduced into the layer 7 forming the flow element, which channels 10, 10a, the mixing chamber 11 and the outlet channel 12 form. These may be, for example, but not limited to, casting a layer in molds, punches of channels, the mixing chamber and the output line or milling, embossing same. Known methods are photolithography, casting, precision milling or printing.

The materials used are rigid materials such as glass, plastic, e.g. Polymethyl methacrylate (PMMA) PTFE or other plastics and metal, preferably steel, into consideration. On this layer 7, the cover layer 8 is placed, which is liquid-sealingly connected to the flow element forming layer 7.

The seal can be ensured by an elastic intermediate layer of rubber, silicone or grease.

Preferably, the layers 7 and 8 are secured together by means for holding the layers together. These means of holding together can be screwed or clamped.

In a preferred embodiment, the layer 7 serving as a flow element consists of an elastic material, such as rubber or silicone, which is cast. This has the advantage that this layer for each application of fresh, uncontaminated material uncomplicated, inexpensive and can be produced individually without great effort. In this case, the leadership of the channels 10, 10a, 10b can be made according to the desired conditions for each experiment. The preparation can be carried out by simple known methods, in particular, the layer can be cast but also milled. It cleaning operations of the fluidizing layer 7 are saved for reuse.

The use of an elastic casting material results in a seal against the cover layer 8, so that sealing material can be saved.

If the layer 7 serving as a flow element consists of an elastic material, then it can be pressed against the cover layer 8 by a further layer 9, so that it seals off gas-tight or liquid-tight against the cover layer 8. For this purpose, the same means for holding together the layers 7 and 8 can be used, as in the embodiment in which the serving as a flow element layer 7 is formed of rigid material.

For both embodiments, namely the use of a rigid or elastic layer as a flow element 7 is that these can be made individually for each new application according to the requirements of the experiment to be performed. The newly produced, serving as a flow element layers 7, can be produced germ-free or under sterile conditions. By the arrangements of the layers 7 and 8, the liquid flow is limited to a plane, so that the computational effort for the calculation of the flow behavior is significantly reduced. With straight management of the channels 10, 10a and / or the output channel 12, the computational effort continues to reduce. The flow resistance can therefore be easily calculated. The simple and inexpensive way to recreate a layer serving as a flow element 7 for each use, allows a one-time use of the layer. This eliminates additional cleaning work or the hassle of sterilization. The fluidizing layer 7 is easy to manufacture, especially when cast from an elastomer such as polydimethylsiloxane (PDMS). Due to the interchangeability of layer 7, the delivery system can be adapted to the requirements of each experiment.

The serving as a flow element layer 7 comprises at least two channels 10, 10 a, which are freely selectable in number. Thus, depending on experimental requirements, for example 4, 7, 9 or more channels 10, 10a may be embedded in the layer 7. In a further embodiment of the invention, the channels 10, 10a and / or the output channel 12 may be equipped with means for interrupting the liquid flow. These means for interrupting the liquid flow may be bores in the cover layer 8, which are located above the channels 10, 10a and / or the output channel 12, are embedded in the stamp, which can be lowered to either reduce the liquid flow or completely closed interrupt. For this purpose, snaps or screw connections can be provided which are located in the bores and which are lowered or screwed in the direction of the channels 10, 10a... And / or of the outlet channel 12. The means for interrupting the liquid flow may also be valves which, for example, presses down an elastomer layer over the cover layer 8 and the channel 10, 10a, 12 and / or the mixing chamber in such a way that the channel is closed.

The cover layer 8 can be made of transparent or transparent material, such as PMMA, glass or transparent plastic. This has the advantage that the liquid flowing through the channels 10, 10a, 10b, the mixing chamber 11 and the outlet channel 12 can be observed to detect blistering which could affect the liquid flow. This allows timely intervention in the course of the experiment.

The layer forming the flow element 7 may also be made of transparent or transparent material. In principle, the same materials can be used as in the cover layer, but transparent or transparent silicone is particularly preferred. This also has the consequence that the liquid flowing through the channels 10, 10a, 10b, the mixing chamber 11 and the outlet channel 12 can be observed to be e.g. Bubble formation that affects the flow of liquid.

Similarly, the layer 9 can be transparent or transparent. The advantages are the same as in layers 7 and 8.

In a further preferred embodiment, a further chamber can be located behind the mixing chamber 11 in the outlet channel 12, which receives the sample to be supplied to the perfusion. The device according to the invention may be equipped with detection means which detect certain observables of the sample in the chamber in the outlet conduit 12 which is perfused.

For this purpose, an optical detector may be provided which detects changes in the sample located in the chamber and optionally stores them as a film. In this case, at least the cover layer 8 and / or the lower layer 9 must be transparent, so that an optical observation is possible. In the case of optical observation, all layers 7, 8, 9 can also be transparent.

Furthermore, the chamber located in the exit channel 12 may be equipped with other detectors which allow electrical or magnetic measurements.

As detectors then electrodes, Ampersonden or means for impedance measurement are used. Furthermore, Hall sensors or squids can be used for magnetic measurements.

For non-optical measurements on the sample, the transparent design of the layers 7 and / or 8 and / or 9 is optional.

In an advantageous embodiment of the invention, at least a portion of the channels 10, 10a, and / or the output channel 12 has a cross-sectional geometry, wherein the width of the recessed channels 10, 10a and / or the output channel 12 is greater than the depth. This cross-sectional geometry has the advantage that a particularly good heat exchange with the environment takes place. The cross-section of the channels 10, 10a, and / or the output channel 12 is then, for example, rectangular, wherein the depth is smaller than the width of the cross section or the cross section forms a shallow groove with curves.

In one embodiment, the output region of the output channel 12 may be designed to be wide, so that there is a particularly good heat exchange.

This cross-sectional geometry has the advantage that a particularly good heat exchange with the environment takes place, which favors a targeted and efficient temperature control. Deep channels 10, 10a, and / or a deep outlet channel 12 have the advantage that a particularly good flow is possible. For example, with a deep channel or channel, the depth-to-width ratio is 1 or greater. The cross section may also have curves. Between large heat exchange and small flow resistance must be weighed according to the application.

For tempering, a heating plate or a cooling element, for example a Peltier element or other tempering means, such as tempered liquid streams, can be introduced, which provide the desired temperature. The tempering means may for example be applied or embedded between the layer forming the flow element 7 and the layer 9, but also at other locations, such as in the cover layer 8 or the lower layer 9. The temperature control means can be arranged as desired in the region of the channels 10, 10a, 10b and / or in the region of the mixing chamber 11 and / or in the region of the outlet line 12, for example between the layer 9 and the layer 7 forming the flow element. It is also possible to introduce additional lines into the layer forming the flow element 7 and / or the cover layer 8 and / or the layer 9, which allow a circulation of Temperierflüssigkeiten. Different channels 10, 10a, 10b can also be kept at different temperatures. Desirable temperatures are physiological temperatures which are generally higher than 32 ° C, for example 37 ° C. However, all temperature ranges of interest can be adjusted. For example, temperatures below 0 ° C, but also high temperatures, eg. B. 8O ⁰ C can be selected. If liquids, for example living cells, are supplied as substrate, extreme temperatures can also be selected for the respective cells in order to investigate their reaction.

The channels 10, 10a, 10b are connected via supply lines 4, 4a, 4b with the storage containers 1, Ia, Ib in connection.

The supply lines 4, 4a, 4b can be shut off by the valves 5, 5a, 5b from the channels 10, 10a, 10b .... As valves solenoid valves are preferably used.

The storage container 1, Ia, Ib can be arranged side by side or substantially side by side. In a preferred embodiment, the storage containers 1, Ia, Ib are stacked on top of one another and furthermore preferably of a flat geometry. The result of the flat geometry is that the liquid surface forming in the reservoir is comparatively large compared to a geometry with a greater depth in relation to the bottom surface. As a result, the liquid forms a large surface, which is in optimal exchange with the surrounding gas phase. As a result, a maximum saturation of the liquid can be achieved with the surrounding gas. This is particularly advantageous if the liquid is to contain defined dissolved gas fractions. Examples of gas compositions are mixtures with defined oxygen content and / or defined CO ₂ content for pH buffering. Other gases include hydrogen sulfide, sulfur dioxide, ammonia, nitrogen or artificial air.

The valves 5, 5a, 5b may be designed so that an optional return of the liquid to be conveyed to the storage containers 1, Ia, Ib is possible.

The storage container 1, Ia, Ib are connected to a pressure source 2. This pressure source is preferably a pressurized gas supply or a pump with pressure vessel which is filled with gas. In this case, each storage container 1 can be connected to a respective pressure source or at least two storage containers are connected to a pressure source. The connection of the reservoir to a gas source allows the supply of liquid for a channel 10 with the respective desired gas, with which the liquid is to be acted upon. Different liquid streams corresponding to different channels 10, 10a, 10b can be supplied with different gases and / or pressures. For this purpose gas reservoirs of different filling are provided as pressure sources. As gases are in particular CO ₂ , or oxygen, nitrogen but also other gases into consideration.

For pressure regulation between the pressure source 2 and the storage containers 1, 1 a valves 3, 3 a, 3 b are provided. The valves 3, 3a, 3b are preferably solenoid valves. In particular, by pressure regulating modules, in particular solenoid valves, a particularly accurate pressure regulation can be realized. Controlled pressure regulation can minimize the stress and thus the impairment of tissue during the experiment. Fluctuations in flow rates, especially when switching from In this way, gases or gases can be excluded so that continuous experimental conditions can be maintained. If at least two storage containers 1, Ia are connected to a pressure source, the channels 10, 10a assigned to these storage containers I ₅ Ia can be charged with the same gas and the same pressure.

The solenoid valves can be controlled so that a constant flow or a targeted variation is possible.

The feed system according to the invention can be used for example for the following applications.

- Perfusion for olfactory tissue, where quick and precise between 8 or more different odorous substances contained solutions to be switched. The Sandwich

Structure of the supply system allows fast switching and easy and very fast replacement of the components in contact with the solution (contamination).

- Patch clamp on retina tissue requires at least 32 degrees Celsius, whereby CO ₂ and O ₂ can no longer be sufficiently kept in solution. Increased pressure would allow this.

- Intestinal tissue for the study of carcinogenesis.

- cell culture in microsystems.

All substances of interest, in particular biological material, can be used as examination objects, depending on the problem.

Examples of examination objects are cell cultures, even under unusual pressure and temperature conditions, biological tissue, surfaces to be treated, different organic or inorganic materials, for example in crystal growth.

The device according to the invention advantageously comprises only two or three elements, namely the layers 7, 8 and 9, instead of a plurality of tubes, which are cumbersome to handle and which have the disadvantages listed in the prior art. In particular, the device according to the invention is compact and has good operational reliability even at elevated pressures of, for example, 7 bar. The dimensions may vary as needed, however, the present invention joined layers have dimensions in the order of millimeters or centimeters in terms of side lengths.

The flow element 7 can allow flow rates of typically a few μl / min to 100 ml / min, depending on the channel geometry and pressure. .

Example:

The first part consists of several storage tanks that contain the liquids (nutrient solution, buffer, pharmaceuticals, etc.). The second part consists of a very compact layered three-component system. It combines electrically controllable valves, a miniaturized mixing chamber and a temperature control and can be easily integrated into a wide variety of already existing experimental setups (patch-clamp setup, confocal

Microscopes, two-photon microscopes). Liquids can be selected from the storage tanks via the valves and directed into the perfusion chamber.

Pressure regulated storage tanks

The liquids are pressed out of the storage containers by pneumatic pressure. A pneumatic pressure system allows a very precise flow regulation and simultaneous fumigation with CO ₂ -O ₂ at 37 degrees (physiological conditions) at elevated levels

Pressure (increased gas absorption capacity). In addition, one is thereby independent of the elevated placed voluminous arrangement of traditional hydrostatic flow control.

perfusion

Design criteria: simplicity, compactness, speed, flexibility

- The system is made up of only 3 components layered: control element, flow element, heating element. The assembly is easy.

- The flat arrangement of the channels allows fast and easy realization of complex channel systems. Only one component has to be produced: the flow element.

- The flat arrangement allows direct integration of perfusion system, perfusion chamber and other elements.

- The flat arrangement allows efficient simulation of the flow and correct Design before realization.

- The elastomer layer has dual function: Carrier of the channel system and seal.

- The flow element is easily replaceable in case of contamination or desired channel system change. Its production is simple and can be performed by non-experts.

- The channel system can be designed as desired and manufactured using standard techniques.

- To ensure the fastest and artifact-free switching and mixing, miniaturized and electrically controllable valves are used. - Due to the compactness and the small amounts of liquid in the microchannels, the heat transfer to the flowing solution is considerably better. Temperature control therefore becomes more efficient and stable.

- The compactness simplifies the integration z. B. in microscope assemblies.

- Small amounts of liquid can be switched faster and more precisely. - The temperature control is therefore more efficient and stable.

- The compactness simplifies the integration z. B. in microscope assemblies.

Due to the compactness and the small amounts of liquid in the microchannels, the heat transfer to the flowing solution is better. The first part consists of several storage tanks that contain the liquids (nutrient solution, buffer, pharmaceuticals, etc.). The second part consists of a very compact, layered, three-component system. It combines electrically controllable valves, a miniaturized mixing chamber and a temperature control and can be easily integrated into a wide variety of existing experimental setups (patch-clamp setup, confocal microscopes, two-photon microscopes). Liquids can be selected from the storage tanks via the valves and directed into the perfusion chamber.

Claims

Patent claims

A supply system, comprising at least two supply lines to a mixing chamber, which has an output line, characterized in that the supply system is formed by a layer system of at least two layers, of which the first layer (7) is a flow element, in which the

Mixing chamber (11), at least two channels (10, 10 a) leading into the mixing chamber (11), and an output channel (12) leading from the mixing chamber (11) to a sample or a substrate, and the second Layer (8) is sealingly applied to the first layer (7).

2. Supply system according to claim 1, characterized in that the serving as a flow element layer (7) consists of a rigid material and is fastened in a liquid-tight manner with fastening means to the cover layer 8.

3. supply system according to claim 2, characterized in that serving as a flow element layer (7) consists of metal, stainless steel, glass, plastic or PMMA.

4. supply system according to claim 1, characterized in that serving as a flow element layer (7) consists of an elastic material, and by a further layer (9) by means of fastening means to the cover layer (8) is pressed sealingly.

5. supply system according to claim 4, characterized in that the serving as a flow element layer (7) consists of rubber, silicone or PDMS.

6. supply system according to one of claims 1 to 5, characterized that at least one of the layers from the group

serving as a flow element layer (7),

- cover layer (8)

- Another layer (9), with which serving as a flow element layer (7) is pressed by fastening means to the cover layer (8) sealing, made of transparent or transparent material.

7. Supply system according to one of claims 1 to 6, characterized in that the cross-sections of at least a portion of the channels (10, 10 a) and / or the output channel in the width have a larger dimension than in the depth.

8. Supply system according to one of claims 1 to 7, characterized in that the cross sections of at least a portion of the channels (10, 10 a) and / or the output channel in the depth have a larger dimension than in the width.

9. supply system according to one of claims 1 to 8, characterized in that it is equipped with means for controlling the temperature of liquid streams, at least part of the channels (10, 10 a) and / or the output channel (12) and / or the mixing chamber 11 temper.

10. supply system according to claim 9, characterized in that the temperature control is at least one component from the group consisting of Peltier element, hot plate, liquid-carrying channels or lines.

11. Supply system according to one of claims 1 to 10, characterized in that at least a part of the channels (10, 10 a) and / or the output channel (12) is equipped with means for interrupting the liquid flow.

12. Supply system according to one of claims 1 to 11, characterized in that at least a part of the channels (10, 10a) is connected via supply lines (4, 4a) to a storage container (1, 1a).

13. Supply system according to claim 12, characterized in that the supply lines (4, 4 a) with valves (5, 5 a) are equipped.

14. Supply system according to claim 13, characterized in that the valves (5, 5a) allow a return of a liquid flow from the reservoir (1, Ia) to the supply system back into the reservoir.

15. Supply system according to one of claims 12 to 14, characterized in that the storage container (1, Ia, Ib) have a flat geometry and are stacked.

16. Supply system according to one of claims 12 to 15, characterized in that the storage container (1, Ia) to a pressure source (2, 2a) are connected.

17. Supply system according to claim 16, characterized in that the pressure source (2, 2a) comprises a reservoir (1, Ia) with a gas or liquid reservoir.

18. Device according to claim 16 or 17, characterized in that all storage containers (1, Ia) are connected to a single pressure source (2).

19. The apparatus of claim 16 or 17, characterized in that at least two storage containers (1, Ia) to different pressure sources (2, 2a) are connected.

20. Device according to one of claims 1 to 19, characterized that in the output line (12) is a chamber which can receive the sample.

21. The device according to claim 20, characterized in that it has detectors which enable optical and / or electrical and / or magnetic measurements on the sample in the chamber according to claim 19.