Auxiliary components and dispensing method

Abstract

This technology provides a way to stably and accurately dispense hydrogels into cell culture vessels. [Solution] A hydrogel is dropped into a cell culture container 9 which has a recessed receiving section 90 that curves downward, using an auxiliary member 1 that is detachable from the cell culture container 9. The auxiliary member 1 has a main body 20 which has a lower surface that contacts the upper surface of the cell culture container 9 when attached, and an alignment part 30 which aligns the main body 20 with the cell culture container 9 when attached. The main body 20 has a through hole 21 whose vertically extending wall surface 210 overlaps the upper opening of the receiving section 90 when attached to the cell culture container 9 and surrounds the center of the receiving section 90 when viewed in the vertical direction. This allows the position of the micropipette for dropping the hydrogel to be positioned in the receiving section 90 at a desired location, and the hydrogel can be dropped stably and accurately.

Description

Technical Field

[0001] The present invention relates to a technique for dropping a hydrogel into a cell culture container.

Background Art

[0002] Conventionally, when culturing cells, it is known to use a hydrogel as a culture medium. Hereinafter, the hydrogel used as a culture medium is referred to as a gel-like culture medium. In particular, when performing three-dimensional culture such as spheroids or organoids, the need for a gel-like culture medium is high. The hydrogel used as the gel-like culture medium is, for example, an ECM (Extracellular Matrix) such as an agarose gel, agar, collagen gel, or Matrigel. Thus, performing three-dimensional culture using a gel-like culture medium is described in, for example, Patent Document 1.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] When performing three-dimensional culture of cells using a gel-like culture medium, observation may be difficult depending on the shape of the gel-like culture medium. Also in a well plate having a plurality of wells that are cell accommodation parts, when the dropping position of the gel-like culture medium is different for each well, since the position where the gel-like culture medium is formed is different, it takes time for alignment during observation. Furthermore, when the dropping position of the gel-like culture medium is different for each well, there is a risk that the gel-like culture medium contacts the well wall surface and the shape becomes significantly different from that of the gel-like culture medium that does not contact the well wall surface. If the dropping positions of the gel-like culture medium in each well can be stably and accurately aligned, the shape of the gel-like culture medium in the well can be approximated, and thus the observation accuracy is also improved.

[0005] This invention has been made in view of these circumstances, and aims to provide a technology that enables the stable and precise dropping of hydrogel into a cell culture vessel. [Means for solving the problem]

[0006] To solve the above problems, the first invention of the present application is an auxiliary member that is detachable from a cell culture container having a housing portion that is recessed downwards, and comprises a main body portion having a lower surface that contacts the upper surface of the cell culture container when attached to the cell culture container, and an alignment portion that aligns the main body portion and the cell culture container when attached to the cell culture container, wherein the main body portion overlaps the upper opening of the housing portion in the vertical direction when attached to the cell culture container, and has a through hole in which the vertically extending wall surface surrounds the center of the housing portion when viewed in the vertical direction.

[0007] The second invention of this application is an auxiliary member of the first invention, wherein the wall surface of the through hole decreases in diameter as it extends downward.

[0008] The third invention of this application is an auxiliary member of the first invention, further comprising a cylindrical guide portion that extends downward while decreasing in diameter from the lower end of the wall surface of the through hole downward.

[0009] The fourth invention of this application is an auxiliary member of the first invention, wherein the alignment portion abuts against at least two sides of the cell culture vessel.

[0010] The fifth invention of this application is an auxiliary member of the first invention, wherein the alignment portion is a cylindrical portion extending downward from the edge of the main body, and the inner surface of the alignment portion is in contact with the side surface of the cell culture container, or faces it with a small gap between them.

[0011] The sixth invention of this application is an auxiliary member of any one of the first to fifth inventions, wherein the cell culture container has a plurality of the housing sections, and the main body has a plurality of the through holes, and each of the through holes overlaps in the vertical direction with the opening of each of the plurality of the housing sections when attached to the cell culture container, and the wall surface of each of the plurality of through holes surrounds the center of the housing section when viewed in the vertical direction.

[0012] The seventh invention of this application is a dispensing method for supplying a hydrogel into a cell culture vessel having a recessed portion that is concave downwards, comprising: a) placing the auxiliary member according to any one of the first to fifth inventions along the upper surface of the cell culture vessel; and b) inserting the tip of a micropipette into the through-hole of the auxiliary member and dispensing the hydrogel held in the tip into the containment portion. [Effects of the Invention]

[0013] According to the first to seventh inventions of this application, hydrogel can be stably and accurately dropped into a cell culture vessel.

[0014] According to the second and third inventions of this application, the auxiliary member can be easily and accurately positioned relative to the cell culture vessel.

[0015] According to the seventh invention of this application, in a cell culture vessel having multiple containment compartments, hydrogel can be stably and accurately dropped into each containment compartment such that the dropping position and gel shape are the same. [Brief explanation of the drawing]

[0016] [Figure 1] This is a perspective view of the auxiliary member and well plate according to the first embodiment. [Figure 2] This is a cross-sectional view of the auxiliary member and well plate according to the first embodiment. [Figure 3] This is a partial cross-sectional view showing the process of dropping a gel-like culture medium onto a well plate fitted with an auxiliary member according to the first embodiment. [Figure 4] It is a flowchart showing the process of cell culture treatment using a gel medium. [Figure 5] It is a perspective view of an auxiliary member and a well plate according to the second embodiment. [Figure 6] It is a cross-sectional view of an auxiliary member and a well plate according to the second embodiment. [Figure 7] It is a partial cross-sectional view showing the state of dropping a gel medium onto a well plate with an auxiliary member according to the second embodiment attached thereto.

Mode for Carrying Out the Invention

[0017] Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[0018] <1. First Embodiment> <1-1. Configuration of Well Plate and Auxiliary Member> FIG. 1 is a perspective view of an auxiliary member 1 according to the first embodiment and a well plate 9 to which the auxiliary member 1 is attached. FIG. 2 is a cross-sectional view showing the state of attaching the auxiliary member 1 to the well plate 9.

[0019] The well plate 9 is a container for accommodating and holding a biological sample. The well plate 9 is a substantially plate-shaped container having a plurality of wells (recesses) 90. The well 90 is a housing portion for accommodating and holding a biological sample. As shown in FIG. 1, the plurality of wells 90 are regularly arranged on the upper surface of the well plate 9. This well plate 9 has 96 wells with 8×12 wells.

[0020] In the present embodiment, the shape of the well 90 in a top view is circular. However, the shape of the well 90 in a top view may be other shapes such as a rectangle. The well plate 9 of the present embodiment has the bottom surface and the wall surface of each well 90 formed of a light-transmissive plastic material, but the well plate 9 may be formed of other materials.

[0021] In this embodiment, cell culture is performed using a gel-like medium within the well plate 9. There are roughly two methods for cell culture using a gel-like medium. One method is to culture cells embedded in a gel-like medium using a cell suspension gel in which cells are suspended in the gel-like medium. In this case, a liquid medium is layered over the gel-like medium in which the cells are embedded. The other method is to drop a cell suspension, in which cells are suspended in a liquid medium, onto the upper surface of a gel-like medium laid in a cell culture vessel, and culture the cells so that the settled cells are in contact with the gel-like medium, thereby forming a cell layer on the surface of the gel-like medium.

[0022] The auxiliary member 1 is a member that can be attached to and detached from the well plate 9. The auxiliary member 1 is attached to the well plate 9 by being placed along the upper surface of the well plate 9. The auxiliary member 1 has a main body portion 20 and an alignment portion 30.

[0023] The main body portion 20 is a plate-shaped part. In this embodiment, the main body portion 20 is a rounded rectangle that approximates the shape of the upper surface of the well plate 9. When the auxiliary member 1 is attached to the well plate 9, the lower surface of the main body portion 20 contacts the upper surface of the well plate 9. The main body portion 20 has a plurality of through holes 21 that penetrate vertically. When the auxiliary member 1 is attached to the well plate 9, each of the through holes 21 overlaps vertically with the upper opening of the well 90, and the vertically extending wall surfaces 210 of the through holes 21 surround the center of the well 90 when viewed vertically.

[0024] Figure 3 is a partial cross-sectional view of the auxiliary member 1 and the well plate 9, showing the process of dropping a hydrogel-like culture medium G onto the well plate 9. As shown in Figures 2 and 3, the wall surface 210 of the through hole 21 narrows in diameter from top to bottom. That is, the wall surface 210 has a frustoconical side shape. As a result, as shown in Figure 3, when the micropipette P is inserted into the through hole 21, the micropipette P is less likely to tilt in the vertical direction. Therefore, the position of the micropipette P relative to the through hole 21 and the well 90 is stable when dropping the gel-like culture medium G. In other words, the position of the micropipette P relative to the well 90 for dropping the hydrogel can be positioned at the desired location, and the hydrogel can be dropped stably and accurately. Consequently, the position and shape of the gel-like culture medium G supplied from the micropipette P within the well 90 are stable.

[0025] The alignment portion 30 is a part used to align the main body portion 20 with the well plate 9 when attaching the auxiliary member 1 to the well plate 9. In this embodiment, the alignment portion 30 has four L-shaped portions 31, each located at one of the four corners of the main body portion 20. When attaching the auxiliary member 1 to the well plate 9, the inner surface of each L-shaped portion 31 is aligned with the side surface of the well plate 9. At this time, the inner surface of one L-shaped portion 31 is brought into contact with two opposing side surfaces of the well plate 9. That is, the alignment portion 30 is brought into contact with at least two surfaces of the side surface of the well plate 9. This allows the auxiliary member 1 to be properly positioned relative to the well plate 9. As a result, the positional relationship between the through hole 21 for positioning the micropipette and the well 90 can be set to the desired positional relationship.

[0026] <1-2. Cell Culture Treatment> Next, we will explain the flow of cell culture processing using gel media, referring to Figure 4. Figure 4 is a flowchart showing the flow of cell culture processing using gel media. As mentioned above, there are two methods for cell culture processing using gel media: one using gel media in which cells are embedded, and another forming a cell layer on the surface of the gel media. In the cell culture processing shown in Figure 4, the method of culturing cells in a cell-embedded gel is used.

[0027] As shown in Figure 4, first, the cell sample is suspended in a gel-like culture medium to obtain a cell-embedded gel (Step S101: Gel-like culture medium preparation step). Meanwhile, the auxiliary member 1 is placed along the upper surface of the well plate 9. This attaches the auxiliary member 1 to the well plate 9 (Step S102: Auxiliary member attachment step). At this time, the inner surface of one L-shaped portion 31 of the auxiliary member 1 is brought into contact with the two sides of the well plate 9 to position the auxiliary member 1 relative to the well plate 9. The order of Step S101 and Step S102 may be reversed, or they may be performed in parallel.

[0028] Next, the tip of the micropipette is inserted into the through-hole 21 of the auxiliary member 1, and the gel-like culture medium (cell-embedded gel) held in the tip is dropped into the well 90. This is done for multiple wells 90, dispensing the gel-like culture medium into multiple wells 90 (Step S103: Gel-like culture medium dispensing step). By using the auxiliary member 1, the gel-like culture medium can be dispensed stably and accurately into multiple wells 90, ensuring that the dispensing position and gel shape are the same.

[0029] Next, liquid culture medium is added to each of the wells 90 into which the gel culture medium has been dropped (Step S104: Liquid culture medium dispensing step). Finally, the auxiliary member 1 is removed from the well plate 9, and the lid of the well plate 9 is attached to the well plate 9 (Step S105: Auxiliary member removal step).

[0030] <2. Second Embodiment> Figure 5 is a perspective view of the auxiliary member 1A and the well plate 9 to which the auxiliary member 1A is attached according to the second embodiment. Figure 6 is a cross-sectional view showing the auxiliary member 1A attached to the well plate 9. Figure 7 is a partial cross-sectional view of the auxiliary member 1A and the well plate 9 showing the process of dropping a gel-like culture medium, which is a hydrogel, onto the well plate 9.

[0031] The well plate 9 shown in Figures 5 to 7 is the same as the well plate 9 shown in Figures 1 to 3. Since many of the components of the auxiliary member 1A are the same as those of the auxiliary member 1 according to the first embodiment, the explanation of the common components will be omitted.

[0032] As shown in Figures 6 and 7, the auxiliary member 1A has a plurality of guide portions 41A extending downward from the lower surface of the main body portion 20A. Each guide portion 41A is a cylindrical part that extends downward from the lower end of the through hole 21A, while decreasing in diameter as it goes downward. Here, the through hole 21A also decreases in diameter as it goes downward, and the wall surface 210A of the through hole 21A and the inner surface of the guide portion 41A are smoothly connected.

[0033] As shown in Figure 7, when the micropipette P is inserted into the through-hole 21A, the wall surface 210A of the through-hole 21A and the inner surface of the guide portion 41A act as guides to prevent the micropipette P from tilting in the vertical direction. By providing the guide portion 41A, the position of the micropipette becomes more stable compared to when only the wall surface 210A of the through-hole 21A acts as a guide for the micropipette P. Therefore, the position and shape of the gel-like culture medium G supplied from the micropipette P within the well 90 become more stable.

[0034] Furthermore, the alignment portion 30A of the auxiliary member 1A is a cylindrical portion that extends downward from the edge of the main body portion 20A. When the auxiliary member 1A is attached to the well plate 9, the inner surface of the alignment portion 30A contacts the side surface of the well plate 9, or faces it with a small gap between them. In the design, the gap between the alignment portion 30A and the side surface of the well plate 9 is set to, for example, 5 micrometers. In this way, when the auxiliary member 1A is attached to the well plate 9, the auxiliary member 1A can be positioned in the desired positional relationship with the well plate 9 with almost no displacement.

[0035] <3. Variant> Although one embodiment of the present invention has been described above, the present invention is not limited to the above-described embodiment.

[0036] In the above embodiment, the positioning portion had a portion facing each of the four sides of the well plate, but the present invention is not limited to this. The positioning portion only needs to be designed to be able to abut against or face at least two of the four sides of the well plate.

[0037] Furthermore, in the above embodiment, an auxiliary member for attachment to a well plate having multiple wells was described. However, the well plate of the present invention may also be attached to a cell culture vessel such as a petri dish having a single containment.

[0038] Furthermore, the elements that appear in the above embodiments and modifications may be combined as appropriate, to the extent that no contradictions arise. [Explanation of Symbols]

[0039] 1,1A: Auxiliary member 9: Well Plate 20,20A: Main unit 21,21A: Through hole 30,30A: Alignment section 31:L-shaped part 41A: Guide section 90: Well 210, 210A: Wall surface G: Gel medium P: Micropipette

Claims

1. An auxiliary member that can be attached to and removed from a cell culture vessel having a recessed receiving section that curves downwards, A main body having a lower surface that contacts the upper surface of the cell culture vessel when attached to the cell culture vessel, A positioning unit for aligning the main body and the cell culture container when attaching it to the cell culture container, It has, The main body is, When installed in the cell culture vessel, the upper opening of the housing section overlaps vertically with the vertically extending wall surface, and the through-hole surrounds the center of the housing section when viewed vertically. An auxiliary member having the following characteristics.

2. An auxiliary member according to claim 1, The wall surface of the through hole is an auxiliary member whose diameter decreases as it extends downwards.

3. An auxiliary member according to claim 1, A cylindrical guide portion extending downward from the lower end of the wall surface of the through hole, with its diameter decreasing as it goes downward. An auxiliary member further having the following.

4. An auxiliary member according to claim 1, The alignment portion is an auxiliary member that contacts at least two sides of the cell culture vessel.

5. An auxiliary member according to claim 1, The alignment portion is a cylindrical part that extends downward from the edge of the main body. The inner surface of the alignment portion is an auxiliary member that abuts against the side of the cell culture vessel, or faces it with a small gap between them.

6. An auxiliary member according to any one of claims 1 to 5, The cell culture vessel has a plurality of the aforementioned storage compartments, The main body portion has a plurality of through holes, Each of the multiple through holes overlaps, in the vertical direction, with the respective openings of the multiple housing sections when the container is attached to the cell culture vessel. Each of the wall surfaces of the multiple through holes is an auxiliary member that surrounds the center of the housing when viewed in the vertical direction.

7. A dispensing method for supplying hydrogel into a cell culture vessel having a receiving section that is recessed downwards, a) A step of placing the auxiliary member according to any one of claims 1 to 5 along the upper surface of the cell culture vessel, b) The step of inserting the tip of a micropipette into the through hole of the auxiliary member and dispensing the hydrogel held in the tip into the containment section, A dispensing method having the following characteristics.

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