Instrument for producing cell array and method for producing cell array using the same

Abstract

To provide an instrument for producing a cell array that can array multiple types of cells in one chamber and simultaneously detect multiple odor substances and the like in a solution, and to provide a method for producing a cell array using the instrument.SOLUTION: An instrument for producing a cell array comprises a chamber having a bottom surface and a side surface, and a plurality of microwells provided on the bottom surface of the chamber. Each of the plurality of microwells is composed of a bottom surface that consists of the bottom surface of the chamber and a side wall that stands up from the bottom surface, and one or more openings are formed in the side wall.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an instrument for producing a cell array and a method for producing a cell array using the instrument. [Background technology]

[0002] Olfactory receptors in living organisms are proteins that have been refined through a long history of evolution, and because they recognize the molecular structure of odorants in three dimensions, they are expected to be used as odor sensor elements with high selectivity and excellent sensitivity. One approach to realizing a biohybrid odor sensor is to use cells expressing olfactory receptors and calcium-sensing fluorescent proteins, and to detect the cellular response to odorants by changes in fluorescence intensity.

[0003] Conventional techniques for detecting cellular responses to odorants involve observing cells seeded in small glass-bottom petri dishes with a fluorescence microscope or measuring the fluorescence intensity of cells seeded in microplates (e.g., 96-well plates) with a fluorescence plate reader. In these experiments, each well in a petri dish or microplate is independent, containing only one type of cell and one type of odorant. Consequently, when investigating the interrelationships between multiple types of cells and odorants, increasing the number of cells and odorants required increases the number of measurements required. Furthermore, because odors are composed of multiple odorants, it is important to be able to simultaneously detect the responses of multiple olfactory receptors. For these reasons, there is a need for an assay system that can simultaneously detect the responses of multiple types of cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] M. Termatanasombat et al., J. Chem. Ecol., 42, 716 (2016) [Non-patent document 2] Y. Hirata et al., Lab Chip, 19, 1971 (2019) [Non-patent document 3] Human Olfactory Receptor Sensor for Odor Reconstitution, S. Kuroda et al., Sensors, 23, 6164, (2023) Summary of the Invention [Problem to be solved by the invention]

[0005] In biohybrid odor sensors that use optical methods, conventional detection methods using fluorescence microscopes or plate readers are limited to seeding one type of cell in each well, making it difficult to simultaneously detect multiple odorants contained in samples such as urine.

[0006] Therefore, the object of the present invention is to provide an instrument for producing a cell array, which can array multiple types of cells in a single chamber and enable simultaneous detection of multiple odorous substances, etc. in a solution, and a method for producing a cell array using the instrument. [Means for solving the problem]

[0007] After extensive research, the inventors conceived the idea of ​​forming multiple microwells in a single chamber and inserting individual cells into each. Since the microwells were cylindrical, similar to conventional wells, they fabricated prototypes and conducted experiments. A liquid hydrogel containing cells was pipetted into each microwell and allowed to solidify. However, due to the high viscosity of the cell-containing liquid hydrogel, the microwells were often not completely filled. In particular, air bubbles were often trapped at the bottom of the microwells. In this state, the number and state of cells retained in each microwell varied, making it difficult to perform tests under the same conditions for each microwell.

[0008] As a result of extensive research, the present inventors discovered that by providing openings in the side walls of the microwells, cells can be held in a uniform state in each microwell, and thus completed the present invention.

[0009] That is, the present invention provides the following. (1) a chamber having a bottom and a side; a plurality of microwells disposed on the bottom surface of the chamber; A cell array production device, wherein each of the plurality of microwells is composed of a bottom surface that is the bottom surface of the chamber and a side wall that stands up from the bottom surface, and one or more openings are formed in the side wall. (2) The cell array production tool according to (1), wherein the opening is a slit-shaped notch provided in the side wall of the microwell and extending from the bottom to the top of the side wall. (3) A cell array production device as described in (2), wherein the width of the outer edge of each of the notches is 3% to 25% of the length of the outer edge of the side wall, and when there are multiple notches, the total width of the notches is 6% to 85% of the length of the outer edge of the side wall. (4) The cell array producing instrument according to any one of (1) to (3), wherein the number of the openings is 4 to 8. (5) The cell array producing instrument according to any one of (1) to (4), wherein the microwells are cylindrical. (6) A step of preparing a cell array preparation tool according to any one of (1) to (5); and inserting cells into the microwells. (7) The method according to (6), wherein the cells contained in a hydrogel are inserted into the microwells. [Effects of the Invention]

[0010] According to the present invention, multiple types of cells can be arrayed in a single chamber, enabling simultaneous detection of multiple odorants in a solution. Furthermore, cells can be held in a uniform state in each microwell, increasing the reliability of test results. Furthermore, the held cells can be in good contact with the sample solution, increasing the sensitivity of the test. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a schematic diagram of a preferred embodiment of the cell array preparation instrument of the present invention. [Figure 2] FIG. 2 is an enlarged perspective view of a microwell in FIG. 1. [Figure 3] FIG. 1 is a diagram illustrating the opening angle of a microwell. [Figure 4] Schematic diagrams of devices fabricated in the following examples: (a) a device with 9 microwells, (b) a device with 25 microwells, (c) a device with 81 microwells, and (d) an enlarged perspective view of a microwell. [Figure 5] These figures show the function of the microwells investigated in the following examples. (a) Dispensing Milli-Q water (trade name) into the microwells by pipetting. (b) Side view of the microwells holding Milli-Q water (trade name). (c) View from above. [Figure 6]These are diagrams showing the chamber portion of the microwell device used in the following examples. Each microwell has a different shape for the opening. The device used was made using a black acrylic plate. Sensor cells encapsulated in hydrogel were dispensed into the microwells (they appear black because the instrument is black). (a) Opening angle 0°. (b) Opening angle 15°. (c) Opening angle 36°. (d) Opening angle 54°. (e) Opening angle 18° - Numerical aperture 0. (f) Opening angle 18° - Numerical aperture 1. (g) Opening angle 18° - Numerical aperture 2. (h) Opening angle 18° - Numerical aperture 4. (i) Opening angle 18° - Numerical aperture 6. (j) Opening angle 18° - Numerical aperture 8. (k) Opening angle 36° - Numerical aperture 1. (l) Opening angle 36° - Numerical aperture 2. (m) Opening angle 36° - Numerical aperture 3. (n) Opening angle 36°- Numerical aperture 4. [Figure 7] This figure shows the response of sensor cells to odorants investigated in the following examples. The shape conditions of each microwell are specified in the graph. (a) Shape condition with a single numerical aperture. (b) Shape conditions with aperture angles of 18° and 36°. au: arbitrary units. [Figure 8] The bar graph summarizes the measurement results of Figure 7. (a) Geometry condition with a numerical aperture of 1. (b) Geometry conditions with aperture angles of 18° and 36°. [Figure 9] These are figures showing an array of sensor cells produced in a microwell device using the collagen gel produced in the example below. (a) An array of sensor cells produced using a device with 25 microwells. Different sensor cells with different fluorescent intensities are used above and below the dotted line. (b) An array of sensor cells produced using a device with 81 microwells. (c) An enlarged view of a microwell holding sensor cells encapsulated in collagen gel. Particulate sensor cells can be seen. DETAILED DESCRIPTION OF THE INVENTION

[0012] A preferred embodiment of the cell array preparation tool of the present invention will now be described with reference to the drawings.

[0013] A preferred embodiment of the cell array production tool of the present invention, shown in FIG. 1, includes a substrate 10. A chamber 12, which is a recess, is formed within the substrate 10. As a recess, the chamber 12 naturally has a bottom and side surfaces. The depth of the chamber 12 is not particularly limited and is typically approximately 2 mm to 10 mm. It is not essential to form a recess within the substrate 10 to form the chamber 12; a chamber having a bottom and side surfaces is sufficient. For example, a dish-shaped container can also be used as the chamber. A plurality of cylindrical microwells 14 are formed on the bottom surface of the chamber 12. In the embodiment shown in FIG. 1, nine microwells 14 are formed. The number of microwells 14 may be any number, but nine or more is preferred, for example, 9 to 196, and preferably 25 to 100. The arrangement of the plurality of microwells is not particularly limited and can be, for example, equally spaced within a square, as shown in the figure.

[0014] FIG. 2 shows an enlarged perspective view of the microwell 14 in FIG. 1. The microwell 14 is composed of a bottom surface 12a of the chamber 12 and a sidewall 14a extending from the bottom surface 12a. The illustrated microwell 14 is cylindrical, with four openings 14b provided in the sidewall 14a. In the illustrated example, the openings 14b are slit-shaped notches extending from the bottom to the top of the sidewall 14a, forming four notches. The shape of the openings 14b is not limited to the notch shown in the figure, and other shapes may be used as long as cells can be inserted uniformly without generating bubbles in the microwell. The number of openings 14b may be one or more, but four to eight is preferred from the viewpoints of maintaining cells under uniform conditions and ensuring good contact with the sample solution. Furthermore, when opening 14b is a slit-shaped notch as shown in the figure, the width of the outer edge of each notch is 3% to 25%, particularly 4% to 20%, of the length of the outer edge of side wall 14a. When there are multiple notches, the total width of the notches is typically 6% to 85%, particularly 10% to 80%, of the length of the outer edge of the side wall. This is preferable from the viewpoint of maintaining cells under uniform conditions and ensuring good contact with the sample solution. That is, when microwell 14 is cylindrical, the central angle (opening angle) formed by the center of the circle viewed from above and both ends of the slit in the circle (see FIG. 3) is typically 11° to 90°, particularly 14° to 72°. When there are multiple openings, the total opening angle is typically 22° to 306°, particularly 36° to 288°. The inner diameter of the microwell is not particularly limited, but is typically about 0.4mm to 1.8mm, preferably about 0.6mm to 1.4mm. The thickness of the sidewall 14a is not particularly limited, but is usually about 0.1 mm to 0.8 mm, and preferably about 0.1 mm to 0.5 mm. The microwell 14 does not necessarily have to be cylindrical, and may have a polygonal or elliptical planar shape when viewed from above.

[0015] The cell array preparation tool described above can be easily prepared by known methods, for example, by processing a plastic plate such as an acrylic plate using a commercially available NC precision processing machine.

[0016] Next, a method for producing a cell array using the cell array production tool of the present invention will be described.

[0017] First, the cells to be arrayed are placed into each microwell using a pipette. Preferably, the cells are suspended in a liquid hydrogel. Examples of hydrogels that can be used include those made by adding a thiol-based crosslinker to a polymer such as polyvinyl alcohol or dextran, and collagen gels. These are commercially available, so commercially available products are preferred (see the Reference Examples below for details). Any cells can be used for the array. For example, when creating an odor sensor, examples include host cells such as insect cells into which genes for olfactory receptors, co-receptors, and calcium-sensing fluorescent proteins have been introduced. Such cells are known and are described, for example, in Non-Patent Document 1.

[0018] When the cell-containing hydrogel solution is poured into each microwell, the presence of opening 14b prevents air bubbles from remaining at the bottom of the microwell, allowing each microwell to be easily filled with the cell-containing hydrogel solution to the bottom, resulting in a uniform filling state in each microwell. Even when the cell-containing hydrogel solution is poured into a microwell, the surface tension of the liquid prevents the hydrogel solution from leaking out of opening 14b and retaining it within the microwell, preventing it from mixing with the hydrogel solution in other microwells. After leaving the microwells, the crosslinking agent solidifies the hydrogel in each microwell. This allows the creation of a cell array. While the cells poured into each microwell are typically different, some of the same cells may be poured into the microwells.

[0019] The fabricated cell array can be used in the same manner as known methods. That is, the sample liquid can be tested by adding the sample liquid to the chamber 12 and measuring signals such as fluorescence from each microwell (see the examples below). In this case, the sample liquid can contact the cells not only through the top of each microwell but also through the openings 14b, improving the sensitivity and accuracy of the test.

[0020] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples.

[0021] Reference Example 1 Preparation of cells for arraying Sensor cells were prepared by the method described in Non-Patent Document 1. Specifically, the gene DNA for the olfactory receptor (OR, Olfactory receptor) and co-receptor (Orco, Olfactory receptor co-receptor) that constitute insect olfactory receptors, as well as the gene DNA for the calcium sensor protein (GCaMP), were introduced into insect cells ExpiSf9 (Thermo Fisher Scientific), and after screening for antibiotic resistance, stable strains in which each gene was stably retained in the genome were generated.

[0022] Reference Example 2 Preparation of cell-containing hydrogel solution The hydrogel used was TrueGel3D (True7, product name, Sigma-Aldrich). The detailed protocol and solution composition are shown below. 1) Mix 41.5 μL of water, 6 μL of TrueGel3D (trade name) buffer, and 5 μL of SLO-DEXTRAN (trade name) included in the TrueGel3D (trade name) kit in a microtube by pipetting. 2) Add the mixed hydrogel solution to the required amount of sensor cell pellet and suspend gently by pipetting. 3) Add 7.5 μL of the crosslinker included in the kit to the hydrogel solution in which the sensor cells are suspended, and mix gently by pipetting. 4) After mixing, gelation begins immediately. Pipetting becomes difficult after about 10 minutes, so dispense the hydrogel quickly into the microwell device (1 μL / microwell). This volume of hydrogel is equivalent to dispensing sensor cells encapsulated in the hydrogel into approximately 60 microwells.

[0023] Reference Example 3 Preparation of cell-containing hydrogel solution (part 2) A cell-containing collagen gel solution was prepared in the same manner as in Reference Example 2, except that a commercially available collagen gel was used as the hydrogel.

[0024] Example 1: Preparation of the device We fabricated devices with microwells within a chamber, as shown in Figures 4(a), (b), and (c). A perspective view of each microwell is shown in Figure 4(d). Fabrication was performed using an NC precision machining tool by cutting a 5 mm-thick acrylic plate. Transparent or black acrylic plates were used to fabricate the devices. The devices fabricated in this example had either 9 (Figure 4(a)), 25 (Figure 4(b)), or 81 (Figure 4(c)) microwells within the chamber. The sidewalls of the microwells were formed by small protrusions extending from the bottom of the chamber. The microwells were cylindrical structures with an outer diameter of 1.6 mm, an inner diameter of 1.2 mm, and a height of 1.0 mm. The chamber in which the microwells were located was 3.0 mm deep. The microwells were spaced 0.4 mm apart, with a 2.0 mm separation between the microwells and the chamber wall. The microwell walls had openings in some areas, allowing solutes and solvents in the chamber to access the interior of the microwells through the top and side of the microwells. We also confirmed that microwells with this structure can stably retain water dispensed by pipetting (Figure 5).

[0025] Example 2 Relationship between openings in the wall of the microwell and the response of the sensor cells 1. Examination of the opening of the microwell by measuring the response of the sensor cells We investigated the effective opening angle and number of openings in the walls of the microprotrusions that form the microwells. Hydrogel-encapsulated sensor cells were pipetted into the microwells, and after filling the chamber with assay solution, odorants were added and the response of the sensor cells was measured using a fluorescence imager.

[0026] The combinations of aperture angle and number of apertures (hereinafter referred to as "numerical aperture") that were considered are shown below. The number in parentheses indicates the total value of the aperture angle. 1) Opening angle 0° - Numerical aperture 0 2) Opening angle 18° - Opening numbers 1 (18°), 2 (36°), 4 (72°), 6 (108°), 8 (144°) 3) Opening angle 36° - Opening numbers 1 (36°), 2 (72°), 3 (108°), 4 (144°) 4) Opening angle 54° - Numerical aperture 1

[0027] 2. Experimental Methods and Conditions 1) A hydrogel solution, excluding the crosslinking agent, was prepared in a microtube (see Reference Example 2). 2) After centrifuging the sensor cell culture solution (300 × g, 5 minutes), the supernatant was removed with an aspirator and the precipitated sensor cells were collected. 3) The prepared hydrogel solution was added to the sensor cell precipitate and suspended by pipetting. Next, the crosslinker was added and gently mixed by pipetting (the final concentration of sensor cells was 1 × 10 8 cells / mL). 4) The sensor cells encapsulated in the hydrogel solution were dispensed in 1 μL aliquots into the microwells of the microwell device. Here, a device with nine microwells made from black acrylic was used. The microwell device was stored in a plastic dish with a Bemcot immersed in Milli-Q water (trade name) to prevent the sample from drying out. 5) The microwell device was stored in the plastic dish and left to stand in an incubator at 27°C for 25 minutes to allow the hydrogel solution to gel. 6) After confirming gelation of the hydrogel solution, the chamber of the microwell device was filled with 200 μL of assay solution (HBSS (Hank's Balanced Salt Solution (containing Ca and Mg, but not phenol red)) / 20 mM HEPES pH 7.2 / 0.1% BSA (Bovine Serum Albumin)). 7) The microwell device was placed in a fluorescence imager (DP-T130z, Biotools). 8) Green fluorescence (530 nm) was detected under blue light (485 nm) excitation, and fluorescence image recording was initiated (exposure time 8 ms, 1 image every 3 seconds). After 30 seconds, the lid of the fluorescence imager was opened, and 50 μL of a ligand solution (HBSS / 20 mM HEPES pH 7.2) containing the target odorant (acetophenone) was pipetted into the chamber (final odorant concentration 100 μM). 9) The recording lasted a total of 330 seconds. 10) The recorded green fluorescent images were used to measure and quantify the changes in fluorescent brightness in the microwell area using ImageJ (NIH). 11) The fluorescence intensity immediately after the addition of the ligand solution was defined as F0, and the change in fluorescence intensity was normalized using this value ((F-F0) / F0) and graphed.

[0028] 3.Results Figure 6 shows bright-field images of the microdevice used in the experiment, in which sensor cells encapsulated in hydrogel were dispensed and observed using a stereomicroscope. In the bright-field image, the area corresponding to the hydrogel is observed as black. It was confirmed that under all shape conditions, the sensor cells encapsulated in the hydrogel were retained without flowing out of the microwells and did not mix with the sensor cells in adjacent microwells.

[0029] Figure 7 shows the time course of normalized fluorescence intensity changes in the sensor cell response to odorants using each microdevice. The curves in the graph represent the average values ​​for nine microwells in one microwell device. First, for the geometry condition with a numerical aperture of 1, the sensor cell response increased with increasing aperture angle, reaching a maximum at 36°. 54° showed a response equivalent to that of 36°. Next, we investigated the effective numerical aperture for the geometry condition with an 18° aperture angle. The sensor cell response increased with increasing numerical aperture, reaching a maximum at a numerical aperture of 4. Numerical apertures 6 and 8 showed a response equivalent to that of numerical aperture 4. We also investigated the numerical aperture for the geometry condition with an aperture angle of 36°. The sensor cell response increased with increasing numerical aperture, reaching a maximum at a numerical aperture of 4.

[0030] Figure 8 shows a bar graph summarizing the responsiveness results from Figure 7. The graph shows the average and standard deviation of the maximum change in fluorescence intensity of the sensor cells for nine wells under each shape condition. First, for the shape condition with a numerical aperture of 1, it was confirmed that the responsiveness of the sensor cells increased as the aperture angle increased, as was the case with the graph of fluorescence intensity change over time shown in Figure 4. Next, comparing the results for aperture angles of 18° and 36°, the responsiveness of the sensor cells was greatest at a numerical aperture of 4, both at aperture angles of 18° and 36°. Furthermore, both (18° aperture angle - numerical aperture 4, 36° aperture angle - numerical aperture 4) showed equivalent responsiveness.

[0031] From the above results, it was confirmed that providing openings in the walls of the microwells is useful for improving the responsiveness of the sensor cells, and that providing four or more openings is effective regardless of the opening angle.

[0032] Example 3: Collagen gel used as hydrogel A transparent acrylic plate was processed to produce a device similar to that in Example 1. The same procedures as in Example 2 were carried out, except that the cell-containing hydrogel solution containing cells in a collagen gel prepared in Reference Example 3 was used. The results are shown in Figure 9.

[0033] As shown in FIG. 9, a cell sensor could be fabricated even when collagen gel was used as the hydrogel. [Explanation of symbols]

[0034] 10 Substrate 12 Chambers 12a Bottom of chamber 14 microwells 14a Microwell sidewall 14b Microwell opening

Claims

1. a chamber having a bottom and a side; a plurality of microwells disposed on the bottom surface of the chamber; A cell array production device, wherein each of the plurality of microwells is composed of a bottom surface that is the bottom surface of the chamber and a side wall that stands up from the bottom surface, and one or more openings are formed in the side wall.

2. 2. The cell array producing instrument according to claim 1, wherein the opening is a slit-shaped cutout provided in the side wall of the microwell and extending from the bottom to the top of the side wall.

3. The cell array production device of claim 2, wherein the width of the outer edge of each of the notches is 3% to 25% of the length of the outer edge of the side wall, and when there are multiple notches, the total width of the notches is 6% to 85% of the length of the outer edge of the side wall.

4. The cell array producing instrument according to claim 3 , wherein the number of the openings is 4 to 8.

5. The cell array preparation tool according to any one of claims 1 to 4, wherein the microwell is cylindrical.

6. A step of preparing a cell array preparation tool according to any one of claims 1 to 4; and inserting cells into the microwells.

7. The method of claim 6 , wherein the cells contained in a hydrogel are inserted into the microwells.

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