Apparatus for observing microscopic biological organisms

Abstract

A microscopic biological organism culture and observation apparatus (1) comprising a support structure (10), one or more chip holders (3) mounted on the support structure, a pump (P), and a valve system (V), each chip holder configured to hold a microfluidic chip (2) having one or more microfluidic channels (54) and a culture chamber (52) therein extending between a pump connection side (44a) of the microfluidic chip and a reservoir side connector (44b) of the microfluidic chip. The support structure includes a reservoir support platform (7) mounted on a movable stage (12), the reservoir support platform (7) configured to hold a plurality of nutrient reservoirs (5) for containing microscopic biological organisms in liquid or nutrients and substances to be tested. The chip holder (3) and / or the microfluidic chip (2) include reservoir-side fluid connectors (26) in the form of hollow tubes extending from the microfluidic chip (2) to tips (26b) at the free ends of the hollow tubes, each tip (26b) being insertable into a corresponding nutrient reservoir (5). The movable stage is coupled to a height actuator mechanism (14) and a vibration mechanism configured to vibrate the reservoir support platform (7) in a horizontal plane with an amplitude smaller than the diameter (D) of the nutrient reservoir (5), thereby actuating a vertical movement of the reservoir support platform by the height actuator mechanism (14).

Description

[Technical field]

[0001] The present invention relates to an apparatus for observing the effects of substances, such as drugs, potentially harmful substances, and potentially beneficial substances, on microscopic biological models, such as parasites, such as the microscopic roundworm C. elegans. [Background technology]

[0002] It is known to observe the growth and behavior of the microscopic roundworm C. elegans, for example to determine the toxicity of substances, for testing pharmaceuticals, for testing pollutants in water, and for various other applications. It is known to use microfluidic chips, for example as described in EP 3209790, in which the microscopic parasites are injected into chambers of the microfluidic chip. A medium containing nutrients and the substance to be tested can be injected at different times in specific concentrations. The growth and activity of the parasites in the microfluidic chambers can be observed under a microscope, the microfluidic chambers being made of a transparent material. A number of channels are provided, each containing a number of successive chambers, each channel being supplied with a similar or separate substance in order to compare the growth and behavior of the parasites in the different chambers. The observation of the growth and activity of the parasites can be carried out over a period of typically 1 to 3 weeks, during which the parasites are supplied with a solution containing nutrients for the growth of the parasites, typically bacteria.

[0003] In conventional systems, the initial loading of the parasites into the microfluidic chip and the subsequent feeding of the parasites are complicated and laborious processes. Moreover, the observation of the parasites' behavior and growth is usually performed in a laboratory with a lot of manual labor, which is not only costly but also reduces the reliability of the monitoring and the conclusions drawn therefrom.

[0004] The observation of other types of parasites, microorganisms and cell-based biological models could also benefit from less tedious and complicated observation and handling procedures.

[0005] US Patent No. 10184104 B2 discloses an automated system for culturing transgenic C. elegans, in which C. elegans are cultured in a well plate cassette with a well insert equipped with a filter so that the medium in the well can be drained. A dispensing system is provided, which aspirates reagents from a reagent assembly and moves to the well plate to dispense the reagents into the wells. An imaging system is then moved to the wells to capture images of C. elegans in the well inserts. One drawback of this system is that the movement from the reagent supply to the well cassette assembly requires bulkier equipment and multiple mechanical movements that can lead to contamination. Another drawback is that imaging of C. elegans in the wells is not ideal in terms of the depth of the wells, as well as nutrients in the medium and other particles therein that affect the quality of the images. The device also requires multiple robotic arms and shakers, increasing the cost, complexity and bulk of the device. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above, it is an object of the present invention to provide a system for more accurately and repeatably monitoring the growth etc. of biological models and their behavior in an economical and reliable manner.

[0007] Biological models may include worms, zebrafish, frog embryos, parasites, bacteria, algae, yeast, organoids, 3D cell models, human and animal tissues.

[0008] In a specific application, it is an object of the present invention to provide a system for more accurately and repeatably monitoring the growth of parasites and their behavior in an economical and reliable manner.

[0009] It would be advantageous to provide an apparatus for monitoring the growth, behavior, and other phenotypes of microscopic parasites or other biological models for the testing of various substances that is easy to set up and operate in an automated procedure.

[0010] It would be advantageous to provide a microfluidic system for the culture and observation of microscopic parasites or other biological models in which the parasites or other models can be easily and reliably observed in multiple chambers in parallel, without air bubbles and other misalignment problems in the microfluidic network. [Means for solving the problem]

[0011] The object of the present invention is achieved by providing a microscopic biological organism culture and observation device as claimed in claim 1.

[0012] The object of the present invention is achieved by providing a method as claimed in claim 11.

[0013] Disclosed herein is a microscopic biological organism culture and observation device, which includes a support structure, one or more chip holders mounted on the support structure, a pump, and a valve system, each chip holder configured to hold a microfluidic chip having one or more microfluidic channels and culture chambers therein extending between a pump-connected side of the microfluidic chip and a reservoir-side connector of the microfluidic chip. The support structure includes a reservoir support platform mounted on a movable platform, the reservoir support platform configured to hold a plurality of nutrient reservoirs for containing microscopic biological organisms or nutrients in a liquid and a substance to be tested. The chip holder and / or the microfluidic chip include a reservoir-side fluid connector in the form of a hollow tube, which extends from the microfluidic chip to a tip at a free end of the hollow tube, each tip being insertable into a corresponding nutrient reservoir.

[0014] According to a first aspect of the present invention, the movable platform is coupled to a height actuator mechanism and a vibration mechanism configured to vibrate the reservoir support platform in a horizontal plane with an amplitude smaller than a diameter of the nutrient reservoir, such that the height actuator mechanism actuates vertical movement of the reservoir support platform.

[0015] According to a second aspect of the invention, the support structure includes an enclosure forming a chamber in which one or more chip holders and reservoir support platforms are housed, and a temperature control unit and a temperature sensor for controlling a temperature within the chamber, the enclosure including an openable or removable cover allowing access to the inside of the enclosure.

[0016] In an advantageous embodiment there is a plurality of said chip holders arranged substantially parallel on the support structure.

[0017] In an advantageous embodiment, said microfluidic chips are such that each microfluidic chip comprises a plurality of parallel microfluidic channels, each microfluidic channel fluidly passing through a plurality of successive culture chambers.

[0018] In an advantageous embodiment, each microfluidic channel includes a pump-side connecting orifice and a reservoir-side connecting orifice that are configured to insertably receive the ends of a hollow tube in a sealing manner.

[0019] In an advantageous embodiment, the microfluidic chip is pluggably coupled to the chip holder and fluidically coupled to corresponding fluid tube connectors that sealingly receive the ends of fluid tubes extending to the pump and / or valve system.

[0020] In an advantageous embodiment, the apparatus further includes a tube tip sealing plate including a support frame and an elastomeric pad movable under the tip of the hollow tube and configured to sealingly press said elastomeric pad against said tip during a corresponding operational step of the apparatus.

[0021] In an advantageous embodiment, said corresponding operation step comprises a debubbling operation of a fluid channel in a microfluidic chip.

[0022] In an advantageous embodiment, the reservoir-side connecting orifices of the microfluidic chip and the corresponding hollow tubes are spaced apart in both orthogonal X and Y directions in a horizontal plane, the hollow tubes being spaced apart according to a two-dimensional grid, the hollow tubes being located at the intersections of a substantially square or rectangular grid.

[0023] In an advantageous embodiment, the pump-side fluid coupling includes a number of hollow tubes that are coupled to the chip holder and inserted into corresponding pump-side coupling orifices of the microfluidic chip.

[0024] In an advantageous embodiment, the chip holder includes a base and a cover pivotally connected to the base and movable from an open position in which the microfluidic chip can be placed on the base and to a closed position held by a closing mechanism that clamps the microfluidic chip within the chip holder.

[0025] In an advantageous embodiment, the chip holder includes viewing windows in both the base and the cover, allowing light to shine from the bottom side of the microfluidic chip to the top side that can be viewed with a microscope.

[0026] In an advantageous embodiment, an imaging system including a microscope, an illumination system, and a computing system connected to the valve system and pumps, the actuation mechanism, the illumination system, and the imaging system are configured to automatically manipulate microscopic biological organisms within a culture chamber of a microfluidic chip, and to image the microscopic biological organisms within said culture chamber over a period ranging from days to weeks.

[0027] In an advantageous embodiment, the support structure includes an enclosure forming a chamber in which one or more chip holders and reservoir support platforms are housed, and a temperature control unit and a temperature sensor for controlling the temperature within the chamber, the enclosure including an openable or removable cover allowing access to the inside of the enclosure.

[0028] Also disclosed herein is a method of operating the microscopic biological organism culture and observation device as described above, which includes a parasite feeding operation, comprising operating a pump in a feeding direction to draw liquid from a nutrient reservoir into a culture chamber of the microfluidic chip, and agitating the liquid in the nutrient reservoir by actuating a vibrational and height adjustment movement of the reservoir support platform either during or immediately prior to operation of the pump.

[0029] In an advantageous embodiment, operation of the device includes a chip priming step after loading a new microfluidic chip into the device, which comprises operating a pump in the direction of fluid flow exiting the hollow tube (priming direction) to pump priming fluid through the microfluidic channels and culture chambers of the microfluidic chip until said priming fluid exits the tip of the hollow tube.

[0030] In an advantageous embodiment, the operation of the device includes a tip debubbling operation, which includes pumping liquid in a priming direction and pressing an elastomeric pad against the tip to sealingly close them and generate an overpressure in the microfluidic channel.

[0031] In an advantageous embodiment, the operation of the device includes an on-chip parasite loading operation, which includes positioning a nutrient reservoir containing microscopic biological organisms to be injected into the culture chamber of the microfluidic chip under the tip of the hollow tube, inserting the tip into the liquid, and pulsing the pump to inject the microscopic biological organisms into the culture chamber.

[0032] In an advantageous embodiment, operation of the device includes a reservoir exchange step that includes removing the tips of the hollow tubes from a first set of reservoirs, operating the pump in a priming direction to form droplets at the tips, and then moving the reservoir support platform to insert the tips of the hollow tubes into a new set of nutrient reservoirs.

[0033] Further objects and advantageous aspects of the present invention will become apparent from the appended claims, as well as the following detailed description and the accompanying drawings.

[0034] The present invention will now be described with reference to the accompanying drawings, which show, by way of example, embodiments of the invention. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram of a parasite culture and observation device according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram of the steps in priming and operating a parasite culture and observation device according to one embodiment of the present invention. [Diagram 3] FIG. 1 is a perspective view of a parasite cultivation and observation device according to one embodiment of the present invention with the housing removed (microscope or computing system not shown). [Figure 3a] FIG. 1 is a perspective view of a parasite culture and observation device according to one embodiment of the present invention. [Figure 3b] FIG. 1 is a perspective view of a parasite culture and observation device according to one embodiment of the present invention. [Figure 3c] FIG. 1 is a perspective view of a parasite culture and observation device according to one embodiment of the present invention. [Figure 4] FIG. 4 is a diagram of a portion of the apparatus of FIG. 3, showing a schematic of an illumination system and a microscope for observing the microfluidic chip. [Diagram 5] FIG. 2 is a perspective view of a microfluidic chip in a microfluidic chip holder of an apparatus according to one embodiment of the present invention, showing the cover of the chip holder in an open position. [Figure 5a] 6 is a view similar to FIG. 5, but with the cover in the closed position. [Figure 5b] FIG. 5b is a top view of the device of FIG. 5a. [Figure 5c] 5c is a cross-sectional view taken along line 5c-5c of FIG. 5b. [Figure 5d] FIG. 6 is a perspective view of a portion of the microfluidic chip holder of FIG. 5 without the microfluidic chip and with the cover removed. [Figure 5e] FIG. 6 is a perspective view of a microfluidic chip mounted in the microfluidic chip holder of FIG. 5. [Figure 6] FIG. 1 is a perspective view of multiple microfluidic chip holders in a support structure 10 of an apparatus according to one embodiment of the present invention, further showing fluid tube connectors 4 connected to the chip holders for supplying liquid to the microfluidic chips. [Figure 6a] FIG. 7 is a perspective view of the fluid tube connector of FIG. 6. [Figure 6b] 6b is a perspective view of the base portion of the fluid tube connector of FIG. 6a with the cover portion removed and the fluid tube for supplying liquid removed. FIG. [Figure 7] FIG. 2 is a perspective view showing a tube tip sealing plate of a parasite culturing and observation device according to one embodiment of the present invention. [Figure 8] 4 is a perspective view of a reservoir support platform of the device of FIG. 3 according to one embodiment of the present invention. [Figure 8a] FIG. 9 is a perspective view of a portion of the support platform of FIG. 8 and a chip holder mounted thereon. [Figure 8b]8b is a cross-sectional view taken along line 8b-8b of FIG. 8a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Referring to the figure, a microscopic biological organism culture and observation apparatus 1 according to an embodiment of the present invention includes a support structure 10, which includes a microfluidic cartridge support platform 6 on which one or more chip holders 3 are mounted, each chip holder 3 being configured for mounting a microfluidic chip 2, and an enclosure 15 in which the microfluidic cartridge support platform 6 is housed. The enclosure includes an openable or removable cover 17 that allows access to the inside of the enclosure. The microscopic biological organism culture and observation apparatus 1 further includes a pump P and valve system V coupled to fluidic tubing 36 that fluidly interconnects the microfluidic chip 2 with reagent and waste containers 34.

[0037] The microscopic biological organism culture and observation device further includes a plurality of nutrient reservoirs 5 for initially supplying the microscopic parasites or other microscopic biological organisms into the microfluidic chip, and thereafter supplying nutrients and the substance to be tested to the microscopic parasites or other microscopic biological organisms contained in the chambers 52 of the microfluidic chip 2.

[0038] The support structure 10 further comprises a reservoir support platform 7, on which a number of nutrient reservoirs 5 are positioned and fixed. The microfluidic chip 2 is fluidly connected to the liquid in the nutrient reservoirs 5 via a reservoir-side fluid coupler 26 extending from the base 18 of the chip holder 3. The reservoir-side fluid coupler 26 connects at one end, herein named chip connection section 26a, to a microfluidic channel in the microfluidic chip and extends to an opposite end forming a tip 26b that can be immersed in the liquid in the nutrient reservoir. The reservoir-side fluid coupler 26 is preferably in the form of a hollow tube, preferably a rigid tube, in particular a straight tube similar to a long needle. The hollow tube can be formed, for example, of a metal tube for high rigidity and small diameter and wall thickness. However, the hollow tube can also be made of a plastic material and be semi-rigid, the main aspect being that it has the necessary structural properties to ensure that the tip is positioned to enter the corresponding reservoir.

[0039] The reservoir support platform 7 is mounted on a movable base 12 coupled to a height actuator mechanism 14 for adjusting the height of the reservoir relative to the chip holder 3. The height actuator mechanism 14 includes at least one actuator 16. The height actuator mechanism has a vertical travel greater than the height H of the nutrient reservoir 5, which is sufficient to fully lower the reservoir support platform 7 so that the hollow tube 26 is fully removed and disengaged from the nutrient reservoir 5. The height actuator mechanism 14 allows the hollow tube to be inserted into the nutrient reservoir 5, on the one hand with the tube tip 26b extending to or near the bottom of the nutrient reservoir, and on the other hand with the tube tip 26b fully lifted off the nutrient reservoir 5.

[0040] The reservoir support platform 7 includes a further actuation mechanism configured to oscillate the movable platform 12, or the part of the movable platform to which the reservoir holder is fixed, in a horizontal plane with an amplitude smaller than the diameter D of the nutrient reservoir relative to the chip holder. This motion may be a substantially circular motion, an elliptical motion, a linear oscillatory motion or anything in between such that when the hollow tube 26 is inserted into the reservoir and an oscillatory motion is applied, mixing of the liquid in the reservoir occurs.

[0041] The horizontal oscillatory motion of the reservoir support platform can be actuated simultaneously or asynchronously with the height actuation, and mixing can be performed both with the hollow tubes at a constant height or at varying heights during oscillation.

[0042] Although height actuator mechanism 14 is shown including a pair of scissor-type link arms and a piston 16 which actuates one of the pair of link arms, a variety of other height adjustment mechanisms based, for example, on a linear motor with a screw and nut mechanism, or hydraulic or pneumatic actuation mechanisms may be used without departing from the scope of the invention.

[0043] The oscillatory movement of the reservoir support platform in the horizontal plane can also be achieved by various mechanisms known per se, for example a rotary motor with an eccentre connected to the movable platform 12 .

[0044] Advantageously, the combination of height adjustment and horizontal vibration allows for very effective mixing of the liquid in the nutrient reservoir, ensuring that any nutrients and other particles and substances are uniformly distributed in the liquid, during which time the pump P can be activated to suck the liquid and nutrients and other substances from the nutrient reservoir into the microfluidic chip 2. Thus, the nutrients and other particles and substances that fall and collect at the bottom of the nutrient reservoir 5 or float on top of the liquid in the nutrient reservoir are mixed uniformly before sucking the liquid into the microfluidic chamber 52 in the microfluidic chip 2. Mixing may also be performed outside of the suction of the liquid into the microfluidic chamber to ensure homogenous properties of the liquid in the nutrient reservoir, which may be important depending on the liquid and substances contained in the reservoir.

[0045] The enclosure 15 controls, in particular, the temperature, and possibly the humidity and CO within the chamber enclosed by the enclosure and in which the reservoir and the microfluidic chip are located. 2 For control of the concentration, a controlled environment can be provided within the enclosure. The microscopic biological organism culturing and observation device 1 can further comprise a temperature control unit 19, e.g. a heating unit, optionally also a cooling unit, and a temperature sensor 23 for measuring the temperature within the enclosure.

[0046] The microscopic biological organism culture and observation apparatus may further include a computing system 46 connected to one or more actuation mechanisms of the movable stage 12, the valve system V, and the pump P for controlling and coordinating the operation of the pump, the valve system, and the movable stage actuation mechanisms during operation of the apparatus. The computing system is further connected to a temperature sensor.

[0047] The microscopic biological organism culture and observation apparatus 1 further includes an imaging system 42, which may include, among other things, a microscope 44 and a camera system for capturing still or moving images of the microscopic parasites or other microscopic biological organisms within the chamber 52 of the microfluidic chip 2. The imaging system 42 is connected to a computing system 46 that records the captured still or moving images, the computing system including software stored therein for processing and analyzing the images.

[0048] The microscope 44 is mounted on an actuation mechanism (not shown) configured to displace the microscope in the horizontal plane XY to position it over each of the microfluidic chips 2, in particular the section including the culture chambers 52. The microscope may also be adjustable in height Z, and of course includes a focusing mechanism to adjust the focal plane of the microscope at a height within each culture chamber 52. The imaging system 42 further includes an illumination system 38 which generates light below the microfluidic chip that shines through the transparent material of the microfluidic chip to improve image quality of the parasites or other microscopic biological organisms within the chambers 52.

[0049] The illumination system may advantageously include a light source 3a, such as an LED light panel mounted on a support structure 10, which emits light substantially horizontally, which is reflected off a mirror 40 positioned below each of the plurality of chip holders 3. The projection direction of light from the LED panel is thus parallel to the plane XY of the microfluidic chip, which may be in a deported position. This advantageously provides a particularly compact illumination arrangement, in an arrangement that is easy to construct and maintain.

[0050] The chip holder 3 comprises windows 21 in the base 18 and cover 20 to allow light to pass through the portion of the microfluidic chip 2 including the culture chamber 52 for visualization through the microscope 44. The light sources 3a can be positioned along the outer edge of the support structure 10, taking into account the mirror system 40, so that these light sources, in particular the LED panels, can be easily replaced or exchanged, and furthermore, various filters can be inserted between the light sources and the mirrors, depending on the type of light required for observation. For example, lighting of a particular color, fluorescent lighting, or other types of lighting that allow observing certain substances or to better distinguish them from other substances and parasites or other microscopic biological organisms may be desired. For example, fluorescent markers expressed or ingested by parasites or other microscopic biological organisms can be detected.

[0051] The mirror system 40 can be based on mirrors having reflective surfaces or on prisms having internal reflective surfaces depending on the refractive index and the angle of incidence of the light.

[0052] With particular reference to Figures 5-5e and 6-6b, a chip holder 3 according to an embodiment of the present invention includes a base 18 and a cover 20. The chip holder cover 20 may, for example, be hingeably connected to the base 18 and pivotable from an open position as illustrated in Figure 5 to a closed position as illustrated in Figures 5a-5c. In the open position, the microfluidic chip 2 may be positioned on the base 18 and the cover may be closed thereto and locked with a closing mechanism 22, for example in the form of a rotatable latch. Other clamping and closing mechanisms may be provided without departing from the scope of the present invention, for example by a cantilevered elastic beam with a latch shoulder, a bridging element that pivots to span the end of the cover, a screw or bolt mechanism, and various other closing and clamping mechanisms known per se.

[0053] The reservoir-side fluid connector 26 in the form of a hollow tube may be fixed to the base 18 and present a chip connection section 28a protruding above the base 18 to be inserted into a complementary reservoir-side connection orifice 54b extending into the base 50a of the microfluidic chip 2. The chip connection section 26a of the hollow tube 26b is inserted with a tight fit into the corresponding cavity 54b to provide an airtight seal between the tube and the orifice wall. In an embodiment, the chip connection section 26a of the tube may comprise a sealing ring or a sealing coating around the tube to provide a seal between the orifice wall and the tube. Other materials such as adhesives or bonding agents may be provided. The chip connection section 26a of the hollow tube 26 may be welded to the chip, for example by ultrasonic welding. In a first variant, the reservoir-side fluid connector 26 in the form of a hollow tube may be pre-mounted on the microfluidic chip 2 and permanently welded, bonded or attached thereto and discarded together with the microfluidic chip. In another embodiment, the reservoir side fluid coupler 26 in the form of a hollow tube may be permanently fixed to the microfluidic chip holder 3 and reusable for multiple operations with different microfluidic chips.

[0054] In yet another embodiment, the microfluidic chip holder 3 may be a single-use component that is discarded after the operation with the microfluidic chip is completed.

[0055] The chip holder 3 further comprises a pump-side fluid coupler 24 for fluidly interconnecting the microfluidic chip 2 to the fluid tubes 36 connected to the valve system V, the pump P, the reagent and waste containers 34. The pump-side fluid coupler 24 may advantageously comprise a hollow tube having at one end a chip connection section 24a that is inserted into the pump-side connection orifice 54a of the microfluidic chip and extending at the other end to a connector connection section 24b that is inserted into the plug connection portion 32 of the fluid tube connector 4. The pump-side fluid coupler 24 may comprise, for example, an elbow- or L-shaped hollow metal tube embedded in the base 18 of the chip holder 3, or may comprise two separate sections of tube, one forming the chip connection section 24a and the other forming the connector connection section 24b that are fluidly interconnected by a channel formed inside the base 18. Other couplers may be used, for example the tube 24b may be removed and the connection secured by a flexible sealing ring or gasket at the interface of the connector connection section.

[0056] As described above in connection with the reservoir-side fluid connector 26, the pump-side fluid connector 24 may include an external sealing element, may be welded to the microfluidic chip, or may be hermetically connected by a variety of other means.

[0057] Within the microfluidic chip, a microfluidic channel 54 fluidly interconnects a pump-side connecting orifice 54a with a reservoir-side connecting orifice 54b that extends through a plurality of serially arranged microfluidic culture chambers 52. In the illustrated embodiment, there are 16 substantially parallel microfluidic channels 54, each passing through eight serially arranged culture chambers 52, resulting in a total of 128 microfluidic chambers that can be observed on a single microfluidic chip.

[0058] The reservoir-side connecting orifices 54b and corresponding hollow tubes 26 of the microfluidic chip may advantageously be spaced apart in both orthogonal X and Y directions in a horizontal plane, with the hollow tubes spaced according to a two-dimensional grid, the hollow tubes being located at the intersections of a substantially square or rectangular grid, for example, as best shown in FIG. 5e.

[0059] The microfluidic channels 54 can house parasites of the same type and similar age, fed with substantially identical nutrients. This allows observation of parasites in multiple microfluidic chambers for statistical evaluation of parasite behavior, growth, and other phenotypes. This can be further augmented by having similar conditions for each of the multiple microfluidic chips 2 housed in the microscopic biological organism culture and observation device 1. However, the conditions regarding the age of the parasites, the type of nutrients, the substances injected, and the reagents used can be varied from chip to chip, or from channel to channel within the chip 2, or with a particular set of channels, etc. For example, one or some of the multiple channels 54 of the chip may have microscopic parasites or other microscopic biological organisms without the substance to be tested, for control and comparison purposes. Also, the same substance may be provided in different channels 54 or microfluidic chips 2 at different concentrations. It is noted that co-culture of two or more different biological models is also possible in each channel, for example, co-culture of different cell types or different types of parasites and / or bacteria in the same channel.

[0060] The microfluidic chip 2 and the chip holder 3 can be conveniently interconnected to the valve system V and the pump system P, as well as to the reagent and waste containers 34, by means of a fluid tube connector 4. The fluid tube connector 4 includes a chip connection section 24a of the chip holder 3 and a plug connection portion 32 having an orifice for sealingly connecting to the microfluidic chip 2. In an advantageous embodiment, the fluid tube connector 4 can include a bottom portion 28b including a number of grooves 30 for receiving the fluid tubes 36 therein, and a top portion 28a covering the bottom portion and clamping the ends of the fluid tubes 36 therein. The top and bottom portions can be made of various materials, and the clamping together can be performed in a removable or permanent manner, for example, by screws or by bounding or welding. The fluid tubes 36 are preferably in the form of, or at least include portions of, flexible polymer tubes that can be crushed in a peristaltic or shuttle type pump. Thus, the fluid tubes 36 can extend from the fluid tube connector 4 to the valve system V.

[0061] The pump P may be in the form of a peristaltic or shuttle type pump engaging a fluid tube extending between the reagent container 34 and the valve system V. Within the scope of the present invention, the arrangement of the pump P and the valves V may take various forms, for example the pump may be located between the valve system V and the microfluidic chip 2 or a separate, independently controlled pump may be provided between the valve system and the chip. Instead of a peristaltic pump system, a syringe pump system or a membrane type pump system may be provided. Such pump systems are known per se and, like the valve systems, do not need to be described further.

[0062] The support structure 10 of the microscopic biological organism culture and observation device 1 according to an embodiment of the present invention may further include a tube tip sealing plate 9, which may be movably mounted on the support structure and positioned by an actuation mechanism, or alternatively, may be provided below the tip 26b of the hollow tube 26 as a manually insertable plate. The tube tip sealing plate 9 includes a support frame 11 and one or more elastomer pads 13 against which the tip 26b of the hollow tube 26 may be pressed. The tube tip sealing plate 9 is used during a priming operation of the microscopic biological organism culture and observation device 1 to seal the tip 26b of the hollow tube 26 in order to apply pressure in the microfluidic channel 54. The pressure in the microfluidic chip channel and in the fluid tube 36 may be used particularly advantageously during a degassing operation. The tube tip sealing plate 9 may also be used during other steps that require the generation of a certain overpressure in the microfluidic channel.

[0063] In one embodiment (not shown), the tube tip sealing plate 9 may be fixed to the reservoir support platform 7 and movable by a movable mount 12 into a sealing position beneath the tip 26b.

[0064] An example of an operation procedure of the microscopic biological organism culturing and observation device 1 according to one embodiment of the present invention will be described with reference to FIG.

[0065] In a first step, one or more microfluidic chips are mounted in corresponding chip holders 3 fluidly connected to corresponding fluid tube connectors 4. A pump P is operated to aspirate liquid, in particular constituting a priming liquid, e.g. an aqueous solution such as DI water, 70% ethanol, or Direct S medium (per se a typical medium used for parasite liquid culture and maintenance), from a container of reagents 34 and fill the fluid tubes 36, the microfluidic channels 54 and the hollow tubes 26 with the priming liquid, thus removing air from the fluid channel system.

[0066] The pump P injects liquid up to the tip 26b, and when a certain amount of liquid has flowed out of all of the tips 26b of one or more microfluidic chips, the tube tip sealing plate 9 can be pressed against the tip 26b to seal the tip. The pump P can be operated to create an overpressure in the fluid channel in a debubbling operation. Optionally, the debubbling operation can include an initial compression stage followed by the release of the tube tip sealing plate from the tip 26b and the operation of the pump to inject more priming fluid through the tube tip 26b. Resealing the tip, pumping to create an overpressure in the microfluidic channel, and then releasing can be performed one or more more times. This procedure effectively removes air bubbles in the microfluidic channel.

[0067] Parasites or other microscopic biological organisms can then be loaded into the culture chamber 52 of the microfluidic chip 2. This can be done, for example, by placing early-stage parasites, small enough in size to be pumped into the microfluidic chamber 52, into the liquid in the nutrient reservoir 5 located below the hollow tube, immersing the hollow tube in the nutrient reservoir, and operating the pump in the opposite direction to the priming direction. The valve system V can be actuated to direct the pump liquid into a waste container, taking into account that some of the parasites are pumped through all of the culture chamber 52 into the fluid tube 36.

[0068] The nutrient reservoir 5 can then be removed after the parasites or other microscopic biological organisms have been loaded into the microfluidic chip and a slight forward pumping is applied to ensure that a droplet forms at the tip 26b of the hollow tube 26. This ensures that the tube is completely filled with liquid and has no air inside when the tip is subsequently inserted into the nutrient reservoir 5 containing the nutrients and substances to be tested. These substances are then drawn into the microfluidic channels 54 and chambers 52 of the microfluidic chip 2 by operating the pump in the opposite direction to the priming direction.

[0069] During the loading operation of the parasites or other microscopic biological organisms on the chip, it is noted that the pumps may be operated in a pulsed manner to force the parasites or other microscopic biological organisms through filters between the microfluidic chambers 52, which are configured and sized to prevent the parasites or other microscopic biological organisms from swimming or flowing out of the chambers under conditions of lower pressure fluid flow. Thus, once loaded into the microfluidic chambers 52, nutrients and substances to be tested can flow through the microfluidic chambers under low pressure, low flow rate conditions, where the microfluidic parasites or other microscopic biological organisms remain within the microfluidic chambers, providing nutrients and substances to be tested in a process that may typically last from days to weeks. However, some tests may only last a few hours or minutes (e.g., acute toxicity). Temperature and possibly other environmental conditions may be advantageously controlled within the enclosure chambers to promote optimal growth of the parasites or other microscopic biological organisms for the duration of the test. Temperature cycling can also be performed, if desired, to test temperature-related factors of the microscopic biological organisms.

[0070] During that time, the microscope 44 may be positioned successively over the various microfluidic chips 2 to capture still or moving images, and multiple microscopes may be provided on the system to simultaneously capture images of the microfluidic chambers and the parasites or other microscopic biological organisms therein.

[0071] [List of feature references] Microscopic biological organism culture and observation device 1 (e.g. parasite culture and observation equipment) Support structure 10 Enclosure 15 Cover 17 Temperature control (heating / cooling) unit 19 Temperature Sensor 23 Microfluidic Cartridge Support Platform 6 Reservoir Support Platform 7 Movable stand 12 Height Actuator Mechanism 14 Actuator 16 Reservoir Support Plate 8 Tube tip sealing plate 9 Support frame 11 Elastomer Pad 13 Chip Holder 3 Base 18 Pump side fluid connector 24 Chip connection section 24a Connector joint section 24b Reservoir side fluid coupler 26 A hollow (e.g., rigid) tube (e.g., a needle) Chip connection section 26a Tip part 26b Tip Support Section 18a Connector Support Section 18b Cover 20 Viewing window 21 Closure Mechanisms / Clamps / Latches22 Fluid Tube Connector 4 Upper 28a bottom 28b groove 30 Plug connection part 32 Nutrient Reservoir 5 Pump P Reagent and waste containers34 Valve System V Fluid tube 36 Lighting System 38 light source LED Panel 39 Mirror 40 Imaging system 42 Microscope 44 Computing Systems 46 Microfluidic Chip 2 Base 50a Culture chamber 52 Microfluidic Channel 54 Pump side coupler 54a Reservoir side connector 54b Cover 50b Reservoir tube diameter D Reservoir tube height H

[0072] [Embodiment] (1) A microscopic biological organism culture and observation device (1), comprising: A support structure (10), one or more chip holders (3) mounted on the support structure, a pump (P), and a valve system (V), Each chip holder is configured to hold a microfluidic chip (2) having one or more microfluidic channels (54) and a culture chamber (52) therein that extend between a pump connection side (44a) of the microfluidic chip and a reservoir side connection (44b) of the microfluidic chip; The support structure includes a reservoir support platform (7) mounted on a movable bed (12); the reservoir support platform (7) is configured to hold a plurality of nutrient reservoirs (5) for containing microscopic biological organisms or nutrients in a liquid and a substance to be tested; the chip holder (3) and / or the microfluidic chip (2) comprise reservoir-side fluid connectors (26) in the form of hollow tubes extending from the microfluidic chip (2) to tips (26b) at the free ends of the hollow tubes, each tip (26b) being insertable into a corresponding nutrient reservoir (5); The movable platform is coupled to a height actuator mechanism (14) for actuating a vertical movement of the reservoir support platform (7) by the height actuator mechanism (14) and a vibration mechanism configured to vibrate the reservoir support platform (7) in a horizontal plane with an amplitude smaller than a diameter (D) of the nutrient reservoir (5) when the free end of the hollow tube is inserted into the nutrient reservoir. (2) The apparatus of embodiment 1, wherein there are multiple chip holders arranged substantially parallel on the support structure. (3) The device of embodiment 1, associated with said microfluidic chip (2), wherein each microfluidic chip comprises a plurality of parallel microfluidic channels (54), each microfluidic channel fluidly passing through a plurality of successive culture chambers (52). (4) The device of embodiment 3, wherein each microfluidic channel (54) includes a pump-side connecting orifice (54a) and a reservoir-side connecting orifice (54b), the orifices being configured to insertably receive the ends of hollow tubes in a sealing manner. (5) The device of embodiment 1, wherein the microfluidic chip is pluggably connected to the chip holder (3) and fluidically connected to a corresponding fluid tube connector (4) that sealingly receives an end of a fluid tube (36) extending to the pump and / or the valve system.

[0073] (6) The apparatus of embodiment 1, further comprising a tube tip sealing plate (9) including a support frame (11) and an elastomeric pad (13) movable under the tip (26b) of the hollow tube and configured to sealingly press the elastomeric pad (13) against the tip during a corresponding operational step of the apparatus. (7) The apparatus of embodiment 6, wherein the corresponding operation step includes a debubbling operation of the fluid channel in the microfluidic chip. (8) The apparatus of embodiment 1, wherein the reservoir-side connecting orifices (54b) and corresponding hollow tubes (26) of the microfluidic chip are spaced apart in both orthogonal X and Y directions in a horizontal plane, the hollow tubes are spaced apart according to a two-dimensional grid, and the hollow tubes are disposed at the intersections of a substantially square or rectangular grid. (9) The device of embodiment 1, wherein the pump-side fluid connector (24) comprises a plurality of hollow tubes connected to the chip holder (3) and inserted into corresponding pump-side connector orifices (54a) of the microfluidic chip (2). (10) The apparatus of embodiment 1, wherein the chip holder comprises a base (18) and a cover (20) pivotably connected to the base and movable from an open position in which the microfluidic chip (2) can be placed on the base (18) to a closed position held by a closing mechanism (22) that clamps the microfluidic chip within the chip holder.

[0074] (11) The apparatus of embodiment 1, wherein the chip holder includes viewing windows (21) in both the base (18) and the cover (20) allowing light to penetrate from the bottom of the microfluidic chip to the top side that can be viewed with a microscope (44). (12) The apparatus of embodiment 1, wherein an imaging system (42, 44) including a microscope (44), an illumination system (38), and a computing system (46) connected to the valve system (V) and the pump (P), the actuation mechanisms (14, 16), the illumination system (38), and the imaging system (42, 44) are configured to automatically manipulate microscopic biological organisms in the culture chamber (52) of the microfluidic chip (2) and to image the microscopic biological organisms in the culture chamber over a period ranging from several days to several weeks. (13) The apparatus of embodiment 1, wherein the support structure includes an enclosure (15) forming a chamber in which the one or more chip holders and the reservoir support platform are housed, and a temperature control unit (19) and a temperature sensor (23) for controlling a temperature within the chamber, the enclosure including an openable or removable cover (17) allowing access to the inside of the enclosure. (14) A method for operating the microscopic biological organism culture and observation device (1) according to any one of the first to thirteenth embodiments, comprising: 2. A method comprising a parasite feeding operation, comprising: operating the pump (P) in a feeding direction to draw liquid from the nutrient reservoir (5) into the culture chamber (52) of the microfluidic chip; and agitating the liquid in the nutrient reservoir (5) by actuating a vibrational and height adjustment movement of the reservoir support platform (7) either during operation of the pump or immediately prior to operation of the pump. (15) The method according to embodiment 14, wherein the operation of the device includes a chip priming step after loading a new microfluidic chip into the device, the chip priming step including operating the pump in the direction of fluid flow exiting the hollow tube (priming direction) to pump the priming solution through the microfluidic channel (54) and the culture chamber (52) of the microfluidic chip until the priming solution exits the tip (26b) of the hollow tube (26).

[0075] (16) The method of embodiment 14, wherein the operation of the device includes a tip debubbling operation, the debubbling operation including pumping liquid in a priming direction and pressing an elastomeric pad (13) against the tip to sealingly close them and generate an overpressure in the microfluidic channel. (17) The method of embodiment 14, wherein the operation of the apparatus includes an on-chip parasite loading operation, comprising positioning a nutrient reservoir in the nutrient reservoir (5) containing microscopic biological organisms to be injected into the culture chamber (52) of the microfluidic chip (2), placing the nutrient reservoir under the tip of the hollow tube and inserting the tip into the liquid, and operating the pump in pulses to inject the microscopic biological organisms into the culture chamber (52). (18) The method of embodiment 14, wherein operation of the apparatus includes a reservoir exchange step, which includes removing the tip of the hollow tube from a first set of reservoirs, operating the pump in a priming direction to form droplets at the tip, and then moving the reservoir support platform (7) to insert the tip of the hollow tube into a new set of nutrient reservoirs (5).

Claims

1. A microscopic biological organism culture and observation device (1), comprising: A support structure (10), one or more chip holders (3) mounted on said support structure, a pump (P), and a valve system (V), Each chip holder is configured to hold a microfluidic chip (2) having one or more microfluidic channels (54) and a culture chamber (52) therein, the microfluidic chip extending between a pump-side connector (54a) of the microfluidic chip and a reservoir-side connector (54b) of the microfluidic chip; The support structure includes a reservoir support platform (7) mounted on a movable bed (12); said reservoir support platform (7) configured to hold a plurality of nutrient reservoirs (5) for containing microscopic biological organisms or nutrients in a liquid and a substance to be tested; the chip holder (3) and / or the microfluidic chip (2) comprise reservoir-side fluid connectors (26) in the form of hollow tubes extending from the microfluidic chip (2) to tips (26b) at the free ends of the hollow tubes, each tip (26b) being insertable into a corresponding nutrient reservoir (5); The movable stage is coupled to a height actuator mechanism (14) for actuating a vertical movement of the reservoir support platform by the height actuator mechanism (14) and a vibration mechanism configured to vibrate the reservoir support platform (7) in a horizontal plane with an amplitude smaller than a diameter (D) of the nutrient reservoir (5) when the free end of the hollow tube is inserted into the nutrient reservoir.

2. The apparatus of claim 1 , wherein there are a plurality of said chip holders arranged substantially parallel on said support structure.

3. 2. The apparatus of claim 1, associated with said microfluidic chip (2), each microfluidic chip including a plurality of parallel microfluidic channels (54), each microfluidic channel fluidly passing through a plurality of successive culture chambers (52).

4. 4. The device of claim 3, wherein each microfluidic channel (54) includes a pump-side connection orifice (54a) that is the pump-side connector (54a) and a reservoir-side connection orifice (54b) that is the reservoir-side connector (54b), the pump-side connection orifice (54a) and the reservoir-side connection orifice (54b) configured to insertably receive ends of hollow tubes in a sealing manner.

5. 2. The apparatus of claim 1, wherein the microfluidic chip is pluggably connected to the chip holder (3) and fluidically connected to a corresponding fluid tube connector (4) that sealingly receives an end of a fluid tube (36) extending to the pump and / or the valve system.

6. 2. The apparatus of claim 1, further comprising a tube tip sealing plate (9) including a support frame (11) and an elastomeric pad (13) movable under the tip (26b) of the hollow tube and configured to sealingly press the elastomeric pad (13) against the tip during a corresponding operation step of the apparatus.

7. The apparatus of claim 6 , wherein the corresponding operation step includes a debubbling operation of the microfluidic channel in the microfluidic chip.

8. The apparatus of claim 1, wherein the reservoir side connector (54b), the reservoir side connecting orifice (54b) and the corresponding hollow tube (26), are spaced apart in both orthogonal X and Y directions in a horizontal plane, the hollow tubes being spaced apart according to a two-dimensional grid, the hollow tubes being disposed at the intersections of a substantially square or rectangular grid.

9. Further comprising a pump-side fluid connector (24), The device of claim 1, wherein the pump-side fluid connector (24) includes a plurality of hollow tubes connected to the chip holder (3) and inserted into pump-side connector orifices (54a) of the corresponding pump-side connectors (54a) of the microfluidic chip (2).

10. 2. The apparatus of claim 1, wherein the chip holder comprises a base (18) and a cover (20) pivotably connected to the base and movable from an open position in which the microfluidic chip (2) can be placed on the base (18) to a closed position held by a closing mechanism (22) that clamps the microfluidic chip within the chip holder.

11. 2. The apparatus of claim 1, wherein the chip holder includes viewing windows (21) in both the base (18) and the cover (20) to allow light to penetrate from the bottom of the microfluidic chip to a top side that can be viewed with a microscope (44).

12. 2. The apparatus of claim 1, wherein an imaging system (42, 44) including a microscope (44), an illumination system (38), and a computing system (46) connected to the valve system (V) and the pump (P), the actuation mechanism (14, 16), the illumination system (38) and the imaging system (42, 44) are configured to automatically manipulate microscopic biological organisms in the incubation chamber (52) of the microfluidic chip (2) and to image the microscopic biological organisms in the incubation chamber over a period ranging from several days to several weeks.

13. 2. The apparatus of claim 1, wherein the support structure includes an enclosure (15) forming a chamber in which the one or more chip holders and the reservoir support platform are housed, and a temperature control unit (19) and a temperature sensor (23) for controlling a temperature within the chamber, the enclosure including an openable or removable cover (17) allowing access to the inside of the enclosure.

14. A method for operating a microscopic biological organism culture and observation device (1) according to any one of claims 1 to 13, comprising the steps of: A method comprising a parasite feeding operation, comprising operating the pump (P) in a feeding direction to draw liquid from the nutrient reservoir (5) into the culture chamber (52) of the microfluidic chip, and agitating the liquid in the nutrient reservoir (5) by actuating a vibrational and height adjustment movement of the reservoir support platform (7) either during operation of the pump or immediately prior to operation of the pump.

15. 15. The method of claim 14, wherein operation of the device includes a chip priming step after loading a new microfluidic chip into the device, the chip priming step including operating the pump in the direction of fluid flow exiting the hollow tube (priming direction) to pump the priming fluid through the microfluidic channel (54) and the culture chamber (52) of the microfluidic chip until the priming fluid exits the tip (26b) of the hollow tube (26).

16. 15. The method of claim 14, wherein the operation of the device includes a tip debubbling operation, the tip debubbling operation including pumping liquid in a priming direction and pressing an elastomeric pad (13) against the tips to sealingly close them and generate an overpressure in the microfluidic channel.

17. 15. The method of claim 14, wherein the operation of the device includes an on-chip parasite loading operation, comprising placing a nutrient reservoir containing microscopic biological organisms to be injected into a culture chamber (52) of the microfluidic chip (2) under the tip of the hollow tube and inserting the tip into the liquid in the nutrient reservoir, and operating the pump in pulses to inject microscopic biological organisms into the culture chamber (52).

18. 15. The method of claim 14, wherein the operation of the device includes a reservoir exchange step, comprising removing the tip of the hollow tube from a first set of nutrient reservoirs (5), operating the pump in a priming direction to form a droplet at the tip, and then moving the reservoir support platform (7) to insert the tip of the hollow tube into a new set of nutrient reservoirs (5).

Images