Microfluidic flow cell and system for analyzing or diagnosing biofilms and cell cultures, and the use thereof

Abstract

Microfluidic flow cells for analyzing or diagnosing biofilms and cell cultures. The microfluidic flow cells comprise a support plate with a sample chamber formed therein, which is peripherally limited by chamber walls and a bottom, a cover plate which can be connected to the support plate in a fluid-tight manner, an inlet with an integrated inlet channel, which leads to the sample chamber via an opening, a drain with an integrated drain channel. Holding elements for fixing the support plate to a microscope stage or a holding device are attached to the front sides of the support plate. The invention further relates to systems and their use for analyzing and diagnosing biofilms and cell cultures using these microfluidic flow cells.

Claims

1. A microfluidic flow cell for analyzing or diagnosing biofilms and cell cultures, comprising: a support plate (10) with a sample chamber (20) formed therein, which is peripherally limited by chamber walls (24) and a bottom (22), a cover plate (40), which can be connected with the support plate (10) in a fluid tight manner, an inlet (16) with an integrated inlet channel (17), which leads to the sample chamber (20) via an opening (26), a drain (18) with an integrated drain channel (19), characterized by the following features: holding elements (12) for fixing the support plate (10) to a microscope stage or a holding device (50) are attached to the front sides (11) of the support plate (10).

2. The microfluidic flow cell according to claim 1, characterized in that the cover plate (40) and/or the bottom (22) are attached the support plate (10) in a removable manner.

3. The microfluidic flow cell according to claim 1, characterized in that the holding elements (12) arranged on the front sides (11) of the support plate (10) of the flow cell are strip-shaped, whereas the aspect ratio between the long side and short side is preferably 3:1.

4. The microfluidic flow cell according to claim 1, characterized in that a latching lug (42) and a latching receptacle (44) each are attached to at least one longitudinal side (13) of the support plate, which cooperate with the corresponding latching receptacles (44) and latching lugs (42) of adjacent flow cells.

5. The microfluidic flow cell according to claim 1, characterized in that the diameters of the inlet channel (17) of the inlet (16) and the drain channel (19) of the drain (18) are >1 mm.

6. The microfluidic flow cell according to claim 1, characterized in that a notch (30) for attaching a seal (32) is formed on the top and bottom of the support plate (10).

7. The microfluidic flow cell according to claim 1, characterized in that the cover plate (40) is incorporated in a cover frame (43) and/or the bottom (22) is incorporated in a bottom frame (23).

8. The microfluidic flow cell according to claim 1, characterized in that at least the support plate (10) is entirely made out of polyethylene (PE) or polypropylene (PP).

9. A system for analyzing or diagnosing biofilms and cell cultures, comprising: one or more microfluidic flow cells according to claim 1, a holding device (50) for holding one or more flow cells, with the holding device (50) being designed in a way that one or more flow cells can be pivoted within an angular range of 0 to 180, a pump, which is fluidically connected to the inlet (16) and the drain (18).

10. The system according to claim 9, characterized in that the holding device (50) is a frame with several frame elements (52), with the frame comprising a notch (54) for receiving one or more flow cells, which is cooperating with the holding elements (12) of the support plate (10).

11. The system according to claim 9, characterized in that the holding device (50) is a frame with several frame elements (52), with the frame comprising a notch (54) for receiving one or more flow cells, which is cooperating with the holding elements (12) of the support plate (10).

12. The system according to claim 9, characterized in that the holding device (50) with one or more flow cells can be pivoted within an angular range of 0 to 180 via rotary joint or a stepper motor.

13. The system according to claim 9, characterized in that the flow sensor is configured to determine the flow rate of the incubation medium within the sample chamber (20).

14. The system according to claim 9, characterized in that the temperature sensor is configured to determine the temperature within the sample chamber (20).

15. The system according to claim 9, characterized in that a microcontroller controls the regulation of the flow rate, the temperature, the angular range or the inflow of the incubation medium.

16. The system according to claim 15, characterized in that the microcontroller controls a heating element or fan for temperature control.

17. Use of a microfluidic flow cell according to claim 1 or of a system according to claim 9 for analyzing and diagnosing biofilms or cell cultures.

18. The system according to claim 9 characterized in that the one or no low cells can be pivoted within an angular range of 0 to 90.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows a side view of a first embodiment of a microfluidic cell according to the invention.

[0034] FIG. 2 shows a top view of the embodiment shown in FIG. 1.

[0035] FIG. 3 shows a side view of another embodiment.

[0036] FIG. 4 shows a top view of the embodiment of FIG. 3.

[0037] FIG. 5 shows the both embodiments of FIG. 1/2 and FIG. 3/4 from the front side.

[0038] FIG. 6 shows an exploded drawing of an embodiment of a flow cell with cover plate and bottom.

[0039] FIG. 7 shows an embodiment with removable cover plate and fixed bottom.

[0040] FIG. 8 shows an embodiment with removable cover plate and removable bottom.

[0041] FIG. 9 shows an arrangement of several support plates connected in a row.

[0042] FIG. 10 shows a holding device (frame) with two integrated flow cells.

[0043] FIG. 11 shows individual components of a holding device in form of a frame.

[0044] FIG. 12 shows a system for analyzing or diagnosing biofilms and cell cultures, consisting of a stand, holding device and rotary joints for pivoting one or more flow cells.

[0045] FIG. 13 shows an advanced embodiment with control elements.

[0046] FIG. 14 shows a block diagram of a device controlled via microcontroller.

WAYS TO CARRY OUT THE INVENTION

[0047] FIG. 1 shows a first embodiment of a microfluidic flow cell according to the invention. This comprises a support plate 10 with a sample chamber 20 formed therein, which is peripherally limited by four chamber walls 24. An inlet 16 with an integrated inlet channel 17 and a drain 18 with an integrated drain channel 19 can be seen on the front sides 11 of the support plate 10. The inlet channel 17 or the drain channel 19 lead into the sample chamber 20 via corresponding openings 26 in the chamber wall 24. In the embodiment displayed, the inlet 16 and the drain 18 are embedded in a slot 14 in the support plate 10. The slot facilitates the connection of the pipes to the inlet 16 or from the drain 18 and their stability without disturbing the external geometry of the cell.

[0048] To ensure compatibility with normal and inverted microscopes, holding elements 12 can be found on the front sides 11 of the support plate 10, in order to attach the support plate 10 to a microscope stage or alternatively to a holding device (e.g., for vertical operation in a 90 position). This holding device preferably allows an adjustment within an angular range of 0 to 180, facilitating the colonization of biofilms on both sides of the surfaces. In the embodiment displayed, the holding elements 12 are strip-shaped. i.e. designed as a rectangular web, and preferably have an aspect ratio between the long side and the short side of 3:1. The width of the holding elements 12 are preferably equivalent with the width of the support plate 10. In the embodiment displayed, the long side of the holding elements 12 (i.e., the front side 11 of the support plate 10) is 24 mm long, while the holding elements 12 have a width of 8 mm. The overall length of the support plate 10, including the two holding elements 12, is 75 mm with a width of approximately 24 to 25 mm. Thus, the flow cell according to the invention differs only slightly from the measurements of a conventional microscopy stage, making it compatible with various microscopes. The two holding elements 12 allow the cell to be easily attached to the microscope stage. Preferably, the depth of the sample chamber is between 7 and 8 mm, making the cell compatible with both normal and inverted microscopes. In the preferred embodiment, the sample chamber 20 itself is 40 mm long and 16 mm wide.

[0049] According to the invention, in order to lower the pressure within the sample chamber 20, the diameters of the inlet channel 17 of the inlet 16 and of the drain channel 19 of the drain 18 are extended, in particular to a diameter larger than 1 mm, preferably larger than 1.5 mm.

[0050] The embodiment displayed is designed without a bottom 22 (not shown), but can be equipped with a removable or permanently fixed bottom 22, if required. A cover plate 40 is placed on the top of the support plate 10 and connected to the support plate 10. This provides a sealed sample chamber 20. If the cover plate 40 is transparent, the experimental procedure and the individual parameters can be easily observed. In order to pass large flow rates into the enlarged sample chamber 20 and to prevent connecting tubes from sliding to the inlet 16 or drain 18, the length of the inlet 16 or drain 18 is about 5 mm. This allows large flow rates of up to 100 mi/min.

[0051] FIG. 2 shows a top view of the embodiment displayed in FIG. 1. The dimensions of the support plate 10, the holding elements 12 and the sample chamber 20 can be seen.

[0052] In all embodiments, the length ratio of the front sides 11 to the longitudinal side 13 including holding elements 12 is about 1:3. Lengths of the inlets 16 and the drains 18 between 5 mm and 10 mm are preferred. Preferred diameters of the inlet channel 17 of the inlets 16 and the drain channel 19 of the drains 18 are between 1.5 mm and 2.5 mm.

[0053] FIG. 3 shows a further embodiment in which the sample chamber 20 can be equipped either with or without a bottom 22. The special feature of this embodiment is a circumferential notch 30 on top of the support plate 10 for a seal 32. In the embodiment in which the bottom plate is can be removed, there is also a notch 30 on bottom of the support plate 10 for liquid-tight sealing of the cell. The bottom 22 is either tightly attached to the support plate 10 or, depending on the embodiment, is designed as a removable bottom plate. The seal 32 externally seals the sample chamber 20. On top of the support plate 10, mounting holes 34 can be seen, to which a separate see-through window can be attached. The embodiment displayed can be seen again in FIG. 4 from a top view. The cover plate 40 is preferably transparent. The choice of materials for the cover plate 40 or the bottom 22 can create ideal experimental conditions for analyzing biofilms or cell cultures, in particular for performing macroscopic analyses of biological samples.

[0054] Since the height of the sample chamber 20 is preferably between 8 and 10 mm, it is possible to operate at a significantly reduced pressure, which protects the biofilms to be examined and also averts any possibly occurring air bubbles. In addition, the height of the sample chamber 20 makes it possible to operate in a vertical mode, so that the biofilm can be cultivated on both surfaces of the cover plate 40 and the bottom 22. This does not require an air bubble trap, unlike conventional solutions. The holding elements 12 on the front sides 11 of the support plate 10 allow alternating between a horizontal, vertical or stepless setup in an angular range between 0 and 180. Another advantage of the geometry according to the invention is that several flow cells can be inserted into each other in a modular manner, i.e., a space-saving grouping is possible.

[0055] FIG. 5 shows a side view of the front sides 11 of the support plate 10 of the two embodiments described above. The U-shaped slot 14 with the enclosed inlet 16 or outlet 18 and the integrated inlet channel 17 or drain channel 19 can be seen.

[0056] FIG. 6 shows an embodiment of the flow cell according to the invention in which the cover plate 40 is held by a cover frame 43. The bottom 22 in turn is held by a bottom frame 23. In this embodiment, both surfaces, i.e., the cover plate 40 or the bottom 22, can be exchanged. In doing so, different surfaces can be colonized with biofilms or cell cultures using a flow cell. The biological samples can be used for further experiments outside the flow cell. This enables, for example, the use of special antibodies, fluorescent markers or DNA probes for analyzing or diagnosing, where tiniest amounts of the substances have to be applied to the surface with precise accuracy. At least one latching lug 42 is also attached on the longitudinal side 13 of the support plate 10, which cooperates with a corresponding latching receptacle 44 of an adjacent flow cell. In the embodiment displayed, a latching lug 42 and a latching receptacle 44 can be found on each longitudinal sides 13 of the support plate 10. Corresponding characteristics can also be found on the opposite sides, but shifted so that in case of a serial setup of individual flow cells, the adjacent support plates 10 can each be connected to one another in a modular manner.

[0057] FIG. 7 shows the embodiment in which only the cover plate 40, but not the bottom 22, is removable. Here, the latching lugs 42 and latching receptacles 44 can be seen, too. FIG. 7A illustrates that the cover plate 40 is removable while the bottom 22 is fixed. FIG. 7B shows the combined design. This embodiment is suitable for applications that do not involve microscopy. For example, macroscopic analyses of biological samples can be performed with this embodiment. Experiments can be observed live through a transparent cover plate 40. For follow-up experiments, the samples can be removed from the sample chamber 20 via the cover plate 40.

[0058] FIG. 8 shows the embodiment with removable cover plate 40 and bottom 22 (not shown). Here, the latching lugs 42 and the latching receptacles 44 can be seen, too. Furthermore, the removable cover frame 43 for holding the cover plate 40 and the bottom frame 23 for holding the bottom 22 can also be seen. The cover frame 43 and the bottom frame 23 are designed in a way that they can incorporate the surfaces to be colonized with the biofilm or the cell cultures, i.e., the cover plate 40 or the bottom 22.

[0059] FIG. 9 shows the modular arrangement of three flow cells. The individual support plates 10 are connected to each other via the latching lugs 42 or latching receptacles 44 attached to the longitudinal sides 13.

[0060] FIG. 10 shows a holding device 50 according to the invention, which consists of a frame with several frame elements. In the embodiment displayed, a frame element 52 is placed on another frame element of the frame, closing the frame circumferentially. The support plates 10 of the two flow cells displayed lead into the notch 54 of the frame via the holding elements 12 on the front sides 11, and are thereby held within the holding device 50.

[0061] FIG. 11 again displays the individual frame elements in detail. The frame element 52 (FIG. 11B) is attached to the frame of the holding device 50 (FIG. 11A) via a corresponding lug 56, whereby the lug 56 of the one frame element (52) cooperates with the notch 54 of the frame.

[0062] FIG. 12 shows a system for analyzing or diagnosing biofilms and cell cultures, comprising a stand 60, a stand plate 62, supporting elements 64 and rotary joints 66, between which a holding device 50 according to the invention with the support plates 10 arranged therein is attached in a pivotable manner. This system allows multiple flow cells to be stored together in a pivotable manner, as the frame of the holding device 50 can accommodate multiple flow cells. The rotary joints 66 enable the entire holding device 50 with the flow cells received therein to rotate over an angular range of preferably 180. Alternatively, a stepper motor can be used (not shown). The flow cell is connected to a pump via appropriate lines and connections enabling the inlet 16 and drain 18 to fluidically communicate with each other.

[0063] FIG. 13 shows a system for the analyzing or diagnosing biofilms and cell cultures, which is equipped with one or more microfluidic flow cells. A holding device 50 incorporates a support plate 10 with a sample chamber 20 formed therein. Via rotary joints 66, the support plate 10 is pivotable within an angular range between 0 and 180, preferably an operating range between 0 and 90 is selected. Via supporting elements 64, the holding device 50 is connected to the system. The system comprises a microcontroller (not shown) and is equipped with corresponding sensors in order to receive parameters required for analysis or diagnostics and to perform corresponding control. Thus, the system includes at least one flow sensor and one temperature sensor to determine the flow rate or temperature in the sample chamber. The microcontroller then ensures that the aforementioned parameters are achieved via corresponding control systems. For example, the system includes a heating element and a fan to provide predetermined incubation conditions. Further, the microcontroller also drives one or more drive units, for example stepper motors, to lead an incubation medium into the sample chamber and to pivot the support plate 10 within a predetermined angular range. A display with touchscreen 70 for monitoring and control can be found on the front and be used for programming. The control and regulation units of the system according to the invention are explained in more detail in FIG. 14.

[0064] FIG. 14 shows a block diagram of a system controlled by a microcontroller for the analyzing or diagnosing biofilms and cell cultures. The key feature of the system is a microcontroller that performs various tasks. A power adapter and a voltage regulator as well as appropriate controlling elements are required to operate the system. A real time clock (RTC) is responsible for the timing and sequence of individual process steps, i.e., the RTC is used to determine when and for how long certain process steps are to be carried out. Programming can be carried out according to previously defined parameters. A temperature sensor is responsible for the monitoring of the temperature and ensures the transmission of the data to the microcontroller. If the temperature falls below a certain, previously defined threshold, a heating element is activated via the microcontroller so that the temperature is increased. Vice versa, if the temperature in the sample chamber or the temperature of the incubation medium is above a certain threshold, the microcontroller controls a fan. The microcontroller also controls the drive units, i.e., the motor drivers and the stepper motors. Motor driver I and stepper motor I ensure that the incubation medium is lead into the sample chamber. Motor driver II and stepper motor II ensure that the support plate is rotated within a previously defined angular range or to a defined angle. In doing so, the microcontroller can be programmed to gradually change the angle within a period of time. For example, in the adhesion phase, the angle can be 0, with the plate continuously pivoted up to 90 over time. In this angular range, air bubbles will escape upwards so that the surface of the slide is bubble-free. For connectivity, the system is further equipped, for example, with a USB port and an Internet adapter. A display with touchscreen ensures corresponding operability by the user.

REFERENCES

[0065] AbuZineh K, Joudeh L I, Al Alwan B, Hamdan S M, Merzaban J S, Habuchi S (2018). Microfluidics-based super-resolution microscopy enables nanoscopic characterization of blood stem cell rolling. Sci Adv. 4(7):eaat5304. doi: 10.1126/sciadv.aat5304. [0066] Alessandri, K., u.a.: All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide. Sci.Rep. (2017) 7:42378. [0067] Babic J, Griscom L, Cramer J. Coudreuse D (2018). An easy-to-build and re-usable microfluidic system for live-cell imaging. BMC Cell Biol. 19(1):8. doi: 10.1186/s12860-018-0158-z. [0068] Bryers J D (2008). Medical biofilms. Biotechnol Bioeng. 1; 100(1):1-18. doi:10.1002/bit.21838. [0069] de Carvalho C (2018). Marine Biofilms: A successful microbial strategy with economic implications. Frontiers in Marine Science. 5. 10.3389/fmars.2018.00126. [0070] Epshteyn, A. A., u.a.: Membrane-integrated microfluidic device for high-resolution live cell imaging. Biomicrofluidics (2011) 5(4): 046501-046501-6. [0071] Flemming H C, Wingender J, Szewzyk U, Steinberg P, Rice S A, Kjelleberg S (2016). Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 11; 14(9):563-75. doi: 10.1038/nrmicro.2016.94. [0072] Fleming D, Rumbaugh K P (2017). Approaches to Dispersing Medical Biofilms. Microorganisms. 1; 5(2). pii: E15. doi: 10.3390/microorganisms5020015. [0073] Gale B, Jafek A, Lambert C, Goenner B, Moghimifam H, Nze U, Kamarapu S (2018). A review of current methods in microfluidic device fabrication and future commercialization prospects. Inventions. 3.60. 10.3390/inventions3030060. [0074] Hall-Stoodley L, Costerton J W, Stoodley P (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol. 2(2):95-108. [0075] Little B, Lee J, Ray, R (2008). The influence of marine biofilms on corrosion: A concise review. Electrochimica Acta. 54. 2-7. 10.1016/j.electacta.2008.02.071. [0076] Niaounakis M (2017). Degradation of plastics in the marine environment. 10.1016/B978-0-323-44354-8.00003-3. [0077] Paie P, Martinez Vzquez R, Osellame R, Bragheri F, Bassi A (2018). Microfluidic Based Optical Microscopes on Chip. Cytometry A. 93(10):987-996. doi:10.1002/cyto.a.23589. [0078] Pinto M, Langer T M, Hffer T, Hofmann T, Hemdl G J (2019). The composition of bacterial communities associated with plastic biofilms differs between different polymers and stages of biofilm succession. PLoS One. 14(6):e0217165. doi: 10.1371/journal.pone.0217165. [0079] Sakai K, Chariot F, Le Saux T, Bonhomme S. Nogue F. Palauqui J C. Fattaccioli J (2019). Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology. Plant Methods. 15:79. doi: 10.1186/s13007-019-0459-z. [0080] Tolker-Nielsen T, Steinberg C (2014). Methods for studying biofilm formation: flow cells and confocal laser scanning microscopy. Methods Mol Biol. 1149:615-29. doi: 10.1007/978-1-4939-0473-0_47. [0081] Wolfaardt G M, Lawrence J R, Robarts R D, Caldwell S J, Caldwell D E (1994). Multicellular organization in a degradative biofilm community. Appl Environ Microbiol. 60(2):434-46.