Discharge system for cell culture wells and cell culture method

Abstract

The present invention relates to a drainage system for cell culture wells. The system comprises an outlet (1) including a proximal part (10) and a distal part (11, 21), wherein the proximal part (10) comprises a capillary (capillary tube) connecting to the distal part (11, 21), the capillary has an inlet and an outlet, the outlet is connected to the open distal part, the distal part has an outlet opening, and the hydraulic diameter of the outlet opening is larger than the hydraulic diameter of the capillary. The present invention further relates to a method for dynamically culturing cells and / or tissues using the drainage system.

Description

Background Art

[0001] The popularity of large-scale biological assays is increasing. Various tests using cell models that can reproduce as faithfully as possible the microenvironment of tissues from which cells are derived, from the viewpoints of structure, biomechanics, and biochemistry, are increasingly frequently required.

[0002] The ultimate goal is to evaluate how a certain tissue reacts to various stimuli, i.e., physiological stimuli (e.g., growth factors), pathological stimuli (microorganisms, inflammatory factors, etc.) including new aspects of drug resistance, or therapeutic stimuli, and to develop personalized therapies.

[0003] Such tests require the following methodologies and are enhanced by the availability of such methodologies. i) Manipulation of cells / particles, specimens, and reagents in liquid and / or gel phases ii) Controlled seeding of fluorescent / bioluminescent molecules / cells / particles iii) Controlled immobilization of the cells / particles for analysis purposes iv) Maintenance of cell viability and cell function over a period sufficient for analysis v) Controlled exposure to one or more active substances for the purpose of evaluating the impact on the sample

[0004] Techniques for efficiently and reproducibly dispensing liquids in microliter units have made important progress, which has made it possible to increase the number of samples and minimize the required biological samples. This is a particularly important factor when collecting samples from patients.

[0005] According to some studies, static culture has been shown to be inappropriate because it cannot reproduce in vitro the nutritional conditions that occur in vivo and the conditions of exposure to external factors or drugs. Under physiological conditions, blood flow can continuously supply nutrients and remove waste products. Furthermore, the concentration of the added compound is not kept constant over time because it is controlled by metabolic and excretion phenomena.

[0006] U.S. Patent Application Publication No. 2011 / 0020929 discloses a solution applied to a microfluidic system. The microfluidic system generates a flow from a well to a lower compartment and activates the inflow to an upper compartment. This fluid movement is realized using a porous medium.

[0007] There is a need for an apparatus that enables dynamic culturing of biological cells and tissues while allowing for dynamic release of external factors, nutrients, or drugs, with the aim of reproducing the conditions of the physiological microenvironment within a culture well and greatly contributing to the study of drug resistance.

[0008] In particular, there is a need for an apparatus that can manage the controlled discharge of fluid from a well on a large scale and, if necessary, control the recovery of the fluid. SUMMARY OF THE INVENTION

[0009] A discharge system according to one embodiment can regulate the output flow rate of fluid from a cell culture well.

[0010] A well according to one embodiment includes a discharge system and, optionally, an inlet. When the inlet is provided, the inlet and a part of the discharge system include open tubes with capillary sizes, that is, open tubes having a hydraulic diameter of about 0.05 to about 2 mm, respectively enabling the inflow and outflow of fluid to and from the well.

[0011] The multi-well plate according to one embodiment includes the discharge system according to the present invention. The multi-well plate is conveniently used on a microfluidic platform.

[0012] The present invention also relates to a method for perfusing an eluate from a cell culture well and a method for collecting the eluate as needed.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0014] The present invention relates to a discharge system that is conveniently used for cell culture wells.

[0015] In one embodiment, the discharge system includes an outlet 1. Referring to FIG. 1, the outlet 1 includes a proximal portion 10 and distal portions 11, 21. The proximal portion 10 includes a capillary (capillary tube) that connects to the distal portions 11, 21. The capillary has an inlet 30 and an outlet 31, and the outlet 31 connects to the distal portions 11, 21. The distal portion to which the capillary connects is open, and the distal portion has an outlet opening 32, and the hydraulic diameter of the outlet opening is larger than the hydraulic diameter of the capillary.

[0016] In one embodiment, the hydraulic diameter of the capillary is 0.05 mm to 2 mm, or 0.10 mm to 1.3 mm, or 0.20 mm to 1 mm, and the hydraulic diameter of the outlet opening is 1 mm to 4 mm, or 1.1 mm to 3 mm, or 1.3 mm to 3 mm.

[0017] In one embodiment, referring to FIG. 1A, the distal portion is cylindrical.

[0018] In one embodiment, referring to FIG. 1B, the distal portion tapers upward, that is, the distal portion has an inlet opening and an outlet opening distal to the connection portion at the connection portion with the capillary. The hydraulic diameter of the outlet opening 32 is larger than the hydraulic diameter of the inlet opening, and the hydraulic diameter of the inlet opening is the hydraulic diameter of the capillary.

[0019] In one embodiment, the distal portion 11 has a frustum - of - cone structure shown in FIG. 1C. The upper base of the frustum - of - cone structure, which is the opening of the inlet, has a diameter of 0.05 mm to 2 mm, or 0.10 mm to 1.3 mm, or 0.20 mm to 1 mm. The lower base of the frustum - of - cone structure, which is the outlet opening, has a diameter of 1 to 4 mm, or 1.1 to 3 mm, or 1.3 to 3 mm. Here, the diameter of the outlet opening is always larger than the diameter of the inlet opening.

[0020] The capillary is an empty tube through which the flow of fluid can pass freely and through which particles can also pass, provided that the size of the particles is smaller than the size of the tube itself with a maximum hydraulic diameter of about 2 mm.

[0021] Advantageously, the capillary is an empty tube that can be sterilized and reused.

[0022] The solution proposed in this specification generates a flow from top to bottom as a whole. In fact, the capillary phenomenon intervenes only during the filling process, and then the fluid moves under the action of gravity. Due to the height difference between the free surface and the meniscus of the droplet, a pressure is generated that exceeds the resistance caused by the meniscus of the droplet. This is precisely because the diameter of the outlet opening is larger than the diameter of the inlet opening, so that the pressure is higher than the resistance.

[0023] The present invention further relates to a well having a discharge system and optionally an inlet. The discharge system comprises the outlet described above or is the discharge system described above.

[0024] Referring to FIG. 2, in one embodiment, the well is a well of a multi-well plate, for example, a multi-well plate having 96 wells, 384 wells, or 1536 wells.

[0025] At least one discharge system is conveniently housed within the well. The discharge system comprises outlet 1 according to the present invention, and the inlet of the capillary at the outlet is inside the well. In one embodiment, the capillary is at least partially incorporated into the wall of the well.

[0026] The inlet of the capillary is disposed at an appropriate height within the well. The appropriate height is the maximum height reachable by the fluid volume contained within the well.

[0027] Optionally, a part of the distal portions 11, 21 at the outlet 1 is outside the well and below the bottom of the well.

[0028] In one embodiment, the well further comprises an inlet 12. The inlet 12 is a capillary having an inlet port outside the well and an outlet port inside the well itself. In a preferred form, the inlet capillary is arranged such that the inlet port is outside the well, preferably below the well, and the outlet port is inside the well. In one embodiment, the inlet is at least partially inside the well, and in a further embodiment, the capillary is at least partially incorporated into the wall of the well.

[0029] The well includes the outlet and optionally the inlet, and has any suitable shape. For example, the well has a circular, square, elliptical, or rhomboidal cross-section. For convenience, when the well constitutes one well included in a multi-well, the shape is such that the distance between the centers of adjacent wells is equal to the distance between the centers of adjacent wells in a standard multi-well plate, so that the device can be made compatible with standard measuring instruments.

[0030] In one embodiment, referring to FIG. 2D, the well has a rhomboidal cross-section, and preferably each corner is rounded.

[0031] In this embodiment, the inlet and the discharge system are preferably arranged at diametrically opposite vertices along the maximum diameter of the rhombus. This arrangement promotes particularly advantageous hydrodynamics for culture dynamics. Furthermore, due to this rhomboidal shape, the available space can be utilized to the maximum while maintaining the standard well internal distance, so that a space can be left outside the well itself to accommodate the microfluidic channels required for the device.

[0032] In one embodiment, the well is made of a hydrophilic material, and grooves for controlling the behavior of the fluid meniscus are provided on the inner wall of the well.

[0033] Advantageously, the system proposed herein can generate a discharge flow using passive components sized to be compatible with a microplate. When the height of the fluid in the well decreases relative to the height of the outlet channel opening, the flow is interrupted. This interrupts the perfusion of the well, keeps the well full, and then the perfusion can be resumed later without disturbing the system.

[0034] If the size of the droplet depends not only on the geometric shape of the distal part, but also on the interaction between the fluid and the materials constituting the system (well, outlet channel, and / or distal part), or on whether the fluid is hydrophobic or hydrophilic, the droplet may become too large. For example, the volume of the droplet becomes approximately equal to the volume of the well that generates the droplet. In this case, droplet detachment causes instability, making prediction difficult, and in addition, the variability of the system increases. For example, in order to appropriately control and collect the eluate, it is desirable for the droplet to reach the bottom of the well, but if the droplet is too large, it may adhere to the side wall of the collection well and thus may not reach the bottom of the well.

[0035] In this regard, the present invention further relates to a discharge system provided with a collection system. The collection system enables the droplet to be detached in a repeatable and controlled manner when the droplet reaches a desired volume. Here, the volume is defined by the distance D between the outlet opening of the outlet and the collection system.

[0036] The droplet contacts the collection system when it reaches the desired volume. Since the collection system is a hollow capillary or, optionally, filled with a porous medium, the droplet can be instantaneously detached and, if a collection well is available, guided to the bottom of the collection well.

[0037] The collection system is initially filled by gravity and capillary action. Once filled, the subsequent dripping by gravity induces a flow. The hydraulic diameter of the collection system is of the same dimension as the hydraulic diameter of the outlet opening of the outlet.

[0038] Referring to FIGS. 8A and 8B, in the well 81, the fluid enters from the inlet 12 and is discharged from the outlet including the proximal portion 10 and the distal portion which is a frustum-shaped distal structure 11. The collection system 80 is disposed expediently at a distance D from the outlet opening of the outlet. The collection system is disposed expediently below the outlet, preferably in a direction parallel to the direction of the capillary forming the proximal portion 10 of the outlet.

[0039] The collection system includes an upper end referred to as a perforated cup 83 and a lower end referred to as an outlet hole 84. The perforated cup interfaces with the droplets generated by the outlet and has dimensions equal to or larger than the droplets themselves.

[0040] In one embodiment, the system further includes a collection well 82.

[0041] The eluate reaches the external environment through the outlet hole 84 or, if there is a collection well 82, reaches the collection well 82.

[0042] In one embodiment, the outlet hole 84 is located on the side wall of the collection system 80, and if there is a collection well 82, at least a part of the outlet hole 84 is disposed in the collection well 82. The outlet hole 84 is placed at a position where it is not invaded by the fluid contained in the collection well itself.

[0043] In a preferred form, referring to FIG. 8B, the collection system is supported by a support. The support is, for example, a cylindrical support 85 and is located at the bottom of the collection well. The cylindrical support includes at least one groove 86, which facilitates the flow of fluid towards the collection well and can prevent the fluid from adhering to the wall of the support.

[0044] The present invention further relates to a method for dynamically culturing cells and / or tissues.

[0045] The method includes - Providing at least one well, wherein the well comprises a drainage system and, optionally, an inlet, the drainage system includes an outlet having a proximal portion and a distal portion, the proximal portion comprises a capillary connecting to the open distal portion, the distal portion has an outlet opening, and the hydraulic diameter of the outlet opening is larger than the hydraulic diameter of the capillary; - Providing at least one cell and / or tissue culture; - Seeding the culture into the at least one well; - When the inlet is provided, perfusing fresh culture medium and / or active substance and / or marker and / or washing buffer into the at least one well at a desired input flow rate through the inlet; - Maintaining a dynamic culture at a desired timing; - Optionally, collecting the effluent discharged from the drainage system for subsequent analysis.

[0046] The method according to the present invention preferably uses an input flow rate of 0 to 100 μl / min, 0.1 to 60 μl / min, 0.5 to 50 μl / min, or 1 to 25 μl / min.

[0047] In an embodiment where the drainage system comprises an outlet and a collection system, when the droplet discharged from the outlet reaches a desired volume defined by the distance D between the outlet opening and the perforated cup of the collection system, the droplet contacts the collection system, whereby the droplet is instantaneously detached and guided through the outlet hole into the external environment or into a collection well.

[0048] By changing the distance D, the flow can be controlled. When the distance D is smaller than the diameter of the outlet opening of the outlet, a continuous flow is discharged. The input flow rate may vary during the execution of the method. As a mere example, the method is a step in which the input flow rate is 0, a step in which the culture is incubated with the amount of fluid present in the well, a further step in which the input flow rate is about 10 μl / min, a further step in which the amount of fluid contained in the well is rapidly replaced, a further step in which the input flow rate is about 5 μl / min, and a further step in which the amount of fluid contained in the well is slowly replaced. Each of the above steps can be repeated periodically.

[0049] In one embodiment, for example, for each of the outlets, a vacuum or pressure is applied downward or upward, respectively, to pre-fill the fluid. In this embodiment, after the outlet is filled and the first droplet is generated, the system operates passively.

[0050] Referring to FIG. 3, in the first step, the fluid is introduced from the inlet 12 into the well 81 (FIG. 3A). In a subsequent step, when the fluid in the well reaches the height at which the inlet of the capillary 10 of the outlet is located, the fluid enters the outlet and is drawn toward the outlet by capillary action and gravity due to the presence of the frustum-shaped distal structure 11 (FIG. 3B). When a droplet is formed, when the force exceeds the critical mass, a part of the fluid contained in the well can be discharged from the discharge system and collected (FIG. 3C). That is, when the gravity acting on the droplet exceeds the surface tension, the droplet detaches from the distal portion 11.

[0051] In one embodiment, the system according to the present invention is used in a microfluidic platform. In this case, referring to FIG. 5, the platform 1 includes two layers, namely an upper layer 51 and a lower layer 52.

[0052] In one embodiment, the microfluidic platform is the microfluidic platform described in International Publication No. WO 2021 / 220173.

[0053] In one embodiment, the upper layer 51 functions to generate a matrix "microdroplet" containing organoids. For example, the upper layer is a "microdroplet" plate as described in International Publication No. WO 2021 / 220173, and the droplets are suspended in wells contained in the lower layer 52. The lower layer 52 is, for example, a multi-well plate.

[0054] As an example, in this embodiment, the method of the present invention includes the following steps regarding the operation steps outlined in FIG. 4.

[0055] - Step 1, filling by microdroplet formation (FIG. 4A) Place the "microdroplet" plate with the movable element 3 in the stationary position upward (upper panel of FIG. 4A) and fill the integrated microfluidic circuit with fluid. The fluid is SOL. After filling, the fluid can flow freely and occupies the hydrophilic regions present in the integrated microfluidic circuit, namely the microchannels and the hydrophilic surface 12 with relief elements. Due to the hydrophilic surface 12 with relief elements, the fluid expands to form a SOL cap 17 protruding from the "microdroplet" plate. When the integrated microfluidic circuit is conveniently filled with the fluid, the fluid gels to obtain a "microdroplet" plate, and the hydrophilic regions of the integrated microfluidic circuit are uniformly occupied by the gel. In particular, the fluid that forms the SOL cap 17 located at each of the hydrophilic surfaces 12 with relief elements forms "microdroplets" of the gel 17.

[0056] In one embodiment, for the gelation process, turn the "microdroplet" plate downward (lower panel of FIG. 4A), where it has been found that downward gelation is advantageous for obtaining a gel of microdroplets with a larger volume than the gel of microdroplets obtained by gelation with the upward "microdroplet" plate.

[0057] - Step 2, separation of microdroplets (Fig. 4B) Turn the microdroplet plate in which the hydrophilic region of the integrated microfluidic circuit is uniformly occupied by the gel-phase fluid downward, and place it conveniently on the cell culture plate 52 containing the culture medium 19. The movable elements 3 filled with the gel droplets 17 also reach the operating positions independently of each other or emerge from the fixed element 2 toward its upper surface.

[0058] Advantageously, the cell culture plate 20 is a multi-well plate, and at least one well of the cell culture plate is conveniently arranged below the movable element 3 filled with the gel droplet 17.

[0059] - Step 3, immersion (Fig. 4C) Immerse the gel microdroplets 17 on the movable element 3 in the culture medium 19. In one embodiment, the gel microdroplets 17 are immersed in the culture medium while remaining suspended from the movable element 3 throughout the entire treatment period.

[0060] In another embodiment, the movable element 3 retreats from the operating position to the rest position and releases the gel "microdroplets" 17 into the culture medium 19.

[0061] The "microdroplets" 17 contain cell substances, such as organoids, and are retained in the culture medium throughout the entire treatment period.

[0062] - Step 4, treatment The well provided with the culture medium 19 is provided with an inlet and an outlet according to the present invention. Through the inlet, the culture medium and / or the active substance and / or the marker and / or the washing buffer solution will be newly introduced at a desired time throughout the entire treatment period. The perfusion is possible without disturbing the equilibrium of the culture, and the fluid discharge is regulated by the outlet.

[0063] The liquid discharged from the outlet is collected, if necessary, onto a desired medium for subsequent use. Advantageously, by collecting the liquid drop by drop, it can be collected separately in individual steps of the process.

[0064] By providing a distal portion having a hydraulic outlet opening diameter larger than the hydraulic diameter of the capillary, droplets for a system with capillary inlet and outlet can be reliably formed within a well of standard height. The hydraulic outlet opening diameter is expediently selected based on the surface tension of the fluid used, the manufacturing / coating material of the discharge system itself, and the input flow rate.

[0065] The adjustment of the discharge system is a passive adjustment, and once perfusion is started, the perfusion proceeds autonomously and passively.

[0066] Advantageously, the system described herein enables high reproducibility of the method, versatility and uniformity of the output liquid volume.

[0067] The system described herein is modular and compatible with devices commonly used in cell biology laboratories.

[0068] Advantageously, with this system, the eluate can be collected from each well over time and separated from each other.

[0069] The following examples are for the sole purpose of illustrating the solution and are not intended to limit the solution in any way, the scope of which is determined by the following claims.

Examples

[0070] Example 1: Calculation of the volume and ejection time of ejected droplets The experiment was conducted in the wells according to the present invention having an inlet and an outlet. The outlet is provided with a frustoconical distal portion, and under different experimental conditions, three distal portions with different outlet opening diameters were tested. The inlet opening diameters of the outlets with distal portions having an outlet opening diameter of less than 1.5 mm, 2 mm, or 2.5 mm were tested with the same 0.13 mm.

[0071] The wells were filled with a controlled input flow rate of 10 μl / min. At the stationary stage, the liquid level in the wells is stable. The extra liquid entering from the inlet is discharged from the outlet and collected in the Eppendorf below. Every time 10 drops are collected, the weight of the Eppendorf is measured so that the average weight estimated for each individual droplet is equal to one-tenth of the measured weight. By knowing the density of the liquid under the same temperature conditions, the volume of each individual droplet is calculated. The experiment was repeated three times for each configuration.

[0072] The experiment was repeated using three different liquids: distilled water, PBS, and culture medium. The results obtained are shown in FIGS. 6A, 6B, and 6C respectively, from which it can be seen how the discharge system functions for all the liquids tested and how the volume changes as a function of the liquid due to different surface tensions. As the outlet opening diameter of the distal portion of the outlet increases, the volume of the droplet further increases.

[0073] In a similar experiment, the release times of individual droplets under different experimental conditions were measured. From the results shown in FIG. 7, it can be seen that when the flow rate of the fluid entering the well is the same, the release time of an individual droplet, that is, the elapsed time from the release of one droplet to the release of the next droplet, becomes longer as the outlet opening diameter of the distal portion increases and shorter as the surface tension decreases.

[0074] By increasing the flow rate of the fluid entering the well, the release time of each droplet in the three experimental models decreases and reaches a flow rate threshold, which is higher for the experimental model with the largest outlet opening diameter. A discharge system with a release time longer than the flow rate threshold cannot compensate for the inflow, and as shown in the graph of FIG. 7, the fluid level in the well rises.

Claims

1. A cell culture well drainage system comprising an outlet (1) including a proximal portion (10) and distal portions (11, 21), The proximal portion (10) is equipped with a capillary tube connected to the distal portions (11, 21), The aforementioned capillary tube has an inlet and an outlet, A discharge system in which the outlet is connected to the open distal portion, the distal portion has an outlet opening, and the hydraulic diameter of the outlet opening is larger than the hydraulic diameter of the capillary tube.

2. The discharge system according to claim 1, wherein the hydraulic diameter of the opening of the inlet of the capillary is 0.05 mm to 2 mm, or 0.10 mm to 1.3 mm, or 0.20 mm to 1 mm, and the hydraulic diameter of the outlet opening is 1 mm to 4 mm, or 1.1 mm to 3 mm, or 1.3 mm to 3 mm.

3. The discharge system according to claim 1 or 2, wherein the distal portion tapers upward from the bottom.

4. The discharge system according to claim 3, wherein the distal portion has a frustoconical structure, the upper base of the frustoconical structure, which serves as the inlet opening, has a diameter of 0.05 mm to 2 mm, or 0.10 mm to 1.3 mm, or 0.20 mm to 1 mm, and the lower base of the frustoconical structure, which serves as the outlet opening, has a diameter of 1 to 4 mm, or 1.1 to 3 mm, or 1.3 to 3 mm.

5. The discharge system according to claim 1 or 2, further comprising a collection system (80), the collection system being a tube having an upper end called a perforated cup (83) and a lower end called an outlet hole (84), wherein the perforated cup is positioned at a distance D from the outlet opening of the outlet, and the volume of the droplet is defined by the distance D.

6. The discharge system according to claim 1 or 2, wherein the well is a well of a multiwell plate.

7. A cell culture well comprising the discharge system according to claim 1 or 2.

8. The well according to claim 7, further comprising an inlet which is a capillary tube having an inlet port housed outside the volume defined by the well and an outlet port housed inside the volume defined by the well.

9. A well according to claim 7, wherein the cross-section of the well is rhomboid, and the well comprises an inlet and an outlet located at opposite vertices along the large diameter portion of the rhomboid.

10. The well according to claim 7, wherein the outlet and the inlet are optionally located within or incorporated into the wall of the space defined by the well.

11. - An outlet according to claim 1 or 2, - Equipped with a collection system (80), The collection system is a tube including an upper end called a perforated cup (83) and a lower end called an outlet hole (84), wherein the perforated cup is positioned at a distance D from the outlet opening of the outlet, and the volume of the droplet is defined by the distance D, as described in claim 7.

12. The well according to claim 7, further comprising a collection well (82) for fluid discharged from the collection system.

13. A multiwell plate comprising a plurality of wells as described in claim 7.

14. A method for dynamically culturing cells and / or tissues, - To provide at least one well, the well comprising a discharge system including an outlet, the outlet comprising a proximal and distal portion, the proximal portion comprising a capillary connected to the open distal portion, the distal portion having an outlet opening, the hydraulic diameter of the outlet opening being greater than the hydraulic diameter of the capillary; - Prepare at least one cell and / or tissue culture; - The culture is seeded into at least one of the wells; --Perfussing at least one well with fresh culture medium and / or active substance and / or marker and / or wash buffer at a desired input flow rate; - Maintaining the dynamic culture until the desired timing; -Optionally, the discharged liquid from the outlet may be collected for subsequent analysis. A method that includes this.

15. The method according to claim 14, wherein the well further comprises an inlet, and the perfusion is performed through the inlet at an input flow rate of 0 to 100 μl / min, 1 to 60 μl / min, 0.1 to 60 μl / min, 0.15 to 50 μl / min, or 1 to 25 μl / min.

16. The method according to claim 14 or 15, further comprising a collection system for the discharge system.

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