Chip for manufacturing frozen section

Abstract

The present application provides a chip for manufacturing a frozen section, the chip comprising: a base part; a plurality of reservoirs formed to be recessed from the upper surface of the base part; a plurality of accommodating parts formed in the reservoirs and accommodating a material or a solution used for manufacturing a frozen section; and a plurality of discharge holes formed on the lower surfaces of the plurality of accommodating parts and selectively discharging the solution accommodated in the accommodating parts.

Description

Chip for making frozen sections

[0001] The present invention relates to a chip for producing frozen sections. More specifically, the present invention relates to a chip that can easily produce frozen sections using organoids, three-dimensional cell spheroids, tissues, and the like.

[0002] Tissue microarray (TMA) is a technology that neatly arranges tissues from multiple patients onto a single slide. It allows for the comparison and analysis of gene and protein expression across tissues, allowing analysis to be performed under identical conditions, significantly increasing research efficiency. Furthermore, tissue microarray technology significantly reduces reagent, time, and labor costs, and is applicable to most tissue-based techniques, including immunohistochemistry, in situ hybridization, FISH, and in situ PCR.

[0003] The most innovative tissue microarray technology is Frozen Tissue Microarray, which arranges a small number of tissue cores on a single array block, allowing for the simultaneous analysis of multiple different tissue samples. While conventional TMA technology primarily uses formalin-fixed, paraffin-embedded (FFPE) tissue samples, Frozen TMA uses frozen tissue samples. Frozen tissue provides a higher level of RNA and protein preservation than FFPE tissue, making it more suitable for gene expression studies and protein analysis. Due to these characteristics, Frozen TMA is widely used, especially in molecular pathology and bioinformatics studies, and the biopsy method utilizing it is frozen section biopsy.

[0004] However, the Frozen TMA experimental device that has been used in the past (1) has a method of making a silicone mold and using OCT (Compound Optimal Cutting) gel to make an OCT (Compound Optimal Cutting) ice block mold and then using a biopsy punch to plant tissues. In some cases, the OCT (Compound Optimal Cutting) ice block mold may be damaged during the process of planting tissues with a biopsy punch, resulting in failure of the experiment. (2) Since the existing OCT (Compound Optimal Cutting) ice block production mold has holes blocked in the mold, only large tissue samples can be tested, and small organoids and 3D cell spheroids cannot be tested. (3) The existing method of making an OCT (Compound Optimal Cutting) ice block and then planting tissues can be performed at room temperature, but the OCT (Compound Optimal Cutting) ice block mold must be maintained at -10 degrees Celsius so that it does not melt, so it has the disadvantage of being sensitive to temperature.

[0005] The problem that this institute seeks to solve includes providing a chip that can shorten the process of manufacturing an existing Frozen TMA and resolve the shortcomings of the existing Frozen TMA experimental device described above.

[0006] The above tasks are merely examples, and there may be additional new tasks that can be understood by a person skilled in the art through this disclosure.

[0007] In order to solve the above problem, the present invention provides a chip for producing a frozen section, which includes a base portion, a plurality of reservoirs formed to be recessed from an upper surface of the base portion, a plurality of receiving portions formed in the reserves and containing a material or solution used for producing a frozen section, and a plurality of discharge holes formed on the lower surface of the plurality of receiving portions and through which the solution contained in the receiving portions is selectively discharged.

[0008] In addition, the present invention provides a method of utilizing a chip for producing a frozen section according to the present invention, and for example, provides a method of producing a frozen section using a chip according to the present invention, a biopsy method using a chip according to the present invention, etc.

[0009] In addition, the center provides frozen sections produced using a chip for producing frozen sections according to the circle.

[0010] The above means are merely examples, and there may be additional new means that can be understood by a person skilled in the art through this disclosure.

[0011] The benefits of this center include easier production of frozen sections, shortening the time for pathological diagnosis and interpretation through frozen section biopsy.

[0012] In particular, existing Frozen TMAs generally use a tissue core extractor with a diameter of 0.6 mm to 2 mm, and because there is no hole in the ice block production mold, organoids, spheroids, etc. cannot be used for producing Frozen TMA due to size limitations. However, the chip according to the present invention can produce Frozen TMA using all of tissues, organoids, spheroids, etc.

[0013] The above effects are merely examples, and there may be additional new effects that can be understood by a person skilled in the art through this disclosure.

[0014] Figure 1 shows a chip for producing frozen sections according to one embodiment of the present invention.

[0015] Figure 2 shows a reserve according to one implementation example of the present invention.

[0016] Figure 3 is a front view of a chip for producing frozen sections according to an embodiment of the present invention.

[0017] Figure 4 is a front view illustrating each part of a chip for producing frozen sections according to an embodiment of the present invention.

[0018] Figure 5 is a cross-sectional view showing each inclination of a chip for producing frozen sections according to one embodiment of the present invention.

[0019] Figure 6 is a drawing illustrating the principle of selective solution discharge from a discharge hole in a chip for producing frozen sections according to one embodiment of the present invention. This drawing should not be construed as limiting the present invention.

[0020] Figure 7 is a drawing illustrating the principle of selective solution discharge from a discharge hole in a chip for producing frozen sections according to one embodiment of the present invention. This drawing should not be construed as limiting the present invention.

[0021] Figure 8 illustrates a method for producing a frozen section according to one embodiment of the present invention.

[0022] Figure 9a is a table comparing the method according to the present invention with the existing TMA manufacturing method.

[0023] Figure 9b compares the process of the method according to the present invention with the existing TMA manufacturing method.

[0024] Fig. 10 shows a small material chip according to embodiment 1 of the present invention.

[0025] Figure 11 shows a large material chip according to embodiment 2 of the present invention.

[0026] Figure 12 shows the results of producing frozen sections according to Example 1.

[0027] Figure 13a shows the process of making frozen sections for materials of various sizes according to Example 2.

[0028] Figure 13b shows the results of producing frozen sections for materials of various sizes according to Example 2.

[0029] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0030] Throughout this specification, whenever a part is said to 'include' a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0031] Throughout this specification, the terms “step of” or “step of” do not mean “step for”.

[0032] Throughout this specification, the term 'combination(s) thereof' included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0033] Throughout this specification, references to 'A and / or B' mean 'A or B, or A and B.'

[0034] Throughout this specification, the term "frozen section production" encompasses the entire process of producing a frozen TMA. While the term "frozen TMA (Frozen Tissue Microarray)" implies the use of a "tissue microarray," this specification uses the term "frozen section" rather than "frozen TMA" to avoid confusion, as not only tissues but also spheroids, organoids, etc. are all possible. In addition, the term "frozen section" in this specification is not limited to "a frozen block mold made by freezing an OCT solution in a chip according to this specification and then cutting it."

[0035] Throughout this specification, the term 'recess' refers to an inwardly recessed shape. It may be expressed as a 'branch shape', a 'concave shape', etc.

[0036] Hereinafter, implementation examples and embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these implementation examples and embodiments and drawings.

[0037] The first aspect of the present invention provides a chip (1000) for producing a frozen section, which includes a base portion (10), a plurality of reserves (100) formed to be recessed from the upper surface of the base portion (10), a plurality of receiving portions (110) formed in the reserves (100) and containing a material or solution used for producing a frozen section, and a plurality of discharge holes (120) formed on the lower surface of the plurality of receiving portions (110) and through which the solution contained in the receiving portions (110) is selectively discharged (Figs. 1 to 4).

[0038] The above base portion (10) can be manufactured using materials generally used in chip manufacturing, and in particular, the chip according to the present invention, including the base portion (10), can also be manufactured through 3D printing.

[0039] Additionally, the base portion (10) may be made of a material with a low strain rate to prevent the frozen section from being damaged due to deformation of the chip during the process of separating the manufactured frozen section from the chip.

[0040] In one specific example of the present invention, the material used for producing the frozen section may be at least one of tissue, organoid, and spheroid.

[0041] As mentioned above, the existing Frozen TMA experimental device produced TMA using only tissue due to size limitations, but the chip according to the present invention can be used to produce frozen sections not only of tissue but also of small-sized materials such as organoids or spheroids.

[0042] In one specific example of the present invention, the solution used for producing the frozen section may include a culture solution or an OCT solution (optimal cutting temperature compound).

[0043] In the present invention, the culture medium may be accommodated in the chip together with the materials used for producing frozen sections, such as tissues, organoids, spheroids, etc., in the chip (1000) for producing frozen sections in an unfrozen state. In the present invention, the OCT solution may be accommodated in the chip during the process of making an ice block frame for producing frozen sections after the materials used for producing frozen sections have been accommodated.

[0044] The reserve (100), which is a component of the main body, may be formed to be recessed from the upper surface of the base (10) or in a branched shape, concave shape, etc. so that materials or solutions used in producing frozen sections can be properly accommodated.

[0045] In one specific example of the present invention, the side surface of the reserve (100) may be formed at an incline of 45 to 85 degrees with respect to the lower surface of the reserve (100) (Fig. 5, θ1). Such an incline may be set to easily separate the ice block mold produced by freezing the OCT solution contained in the chip during the process of producing a frozen section. Considering the ease of separation, if the incline exceeds 85 degrees, the ice block mold may be separated too easily, which may cause damage to the mold during the experiment, and if the incline is less than 45 degrees, the mold may adhere to the chip during the separation process, making separation difficult.

[0046] The receiving portion (110), which is a component of the main body, is a portion where a material or solution used for producing frozen sections is received within the reserve (100).

[0047] In one specific example of the present invention, the lower surface of the receiving portion (110) may be formed to have a further recessed shape so as to have a slope of 15 to 45 degrees with respect to the lower surface of the reserve (100) (Fig. 5, θ2). This slope is to facilitate the accommodation of materials used in the production of frozen sections during the process of producing frozen sections. For example, when the receiving portion (110) is formed in a funnel shape and the center of the receiving portion (110) forms the lowest lower surface, the materials can be gathered at the center of the receiving portion (110) so that the positions of the materials can be accurately aligned.

[0048] In one specific example of the present invention, the device may further include a fence portion (130) formed to protrude upward from the lower surface of the reserve (100) and surround the receiving portion (110). In addition, in one specific example of the present invention, the fence portion (130) may be a cylindrical shape having a diameter preset to form the receiving portion (110) therein.

[0049] The above fence portion (130) serves as a guide when receiving a material or solution used for producing a frozen section into the reserve (100) using a pipette or the like. That is, the pipette can be brought close to the shape of the fence portion (130), so that the material or solution used for producing a frozen section can be easily received into the reserve (100).

[0050] In addition, the fence section (130) can serve to properly accommodate materials or solutions used in the production of frozen sections in the receiving section (110), while preventing the accommodated materials or solutions from leaking out of the receiving section (110).

[0051] In one specific example of the present invention, the fence portion (130) may include a plurality of side wall (131) portions that extend in the vertical direction and are spaced apart at preset intervals along the circumference centered on the receiving portion (110).

[0052] In one specific example of the present invention, the fence portion (130) may include a slit (132) portion that extends in the vertical direction to connect the reserve (100) and the receiving portion (110) to each other and is spaced apart at a preset interval along the circumference centered on the receiving portion (110).

[0053] The above slit (132) portion can allow a portion of the material or solution used for producing a frozen section to be easily discharged along the slit (132) when an excessive amount of the material or solution used for producing a frozen section is contained in the receiving portion (110) more than necessary, and can maintain the height (water level) of the material or solution between the plurality of reserves (100) and the receiving portion (110) at a constant level.

[0054] In one specific example of the present invention, the fence portion (130) may further include a flange portion (133) formed to protrude outwardly away from the side wall (131) portion and away from the receiving portion (110), and at least a portion of the flange portion (133) may be formed to be connected to the side surface of the reserve (100). The structural stability of the receiving portion (110) may be secured by the flange portion (133).

[0055] The discharge hole (120), which is a component of the main body, is a portion through which the solution used in the production of frozen sections is discharged. The material used in the production of frozen sections may be formed so that only the solution is discharged while remaining in the receiving portion (110). In addition, the solution may be selectively discharged depending on its type.

[0056] In one specific example of the present invention, the discharge hole (120) may have a diameter smaller than that of the receiving portion (110) and may be formed in multiple numbers on the lower surface of one receiving portion (110).

[0057] In one specific example of the present invention, the discharge hole (120) may have a diameter set to allow selective discharge by the combined force of forces including gravity, capillary force, and surface tension of the solution received in the receiving portion (110).

[0058] The selective discharge of the solution as described above may be due to capillary action or Jurin's Law (Figs. 6 and 7). That is, it is due to the interaction between the solution and the discharge hole (120). The detailed principle is described in Fig. 6, but this is only a drawing illustrating the selective discharge according to the present invention and does not limit the present invention.

[0059] Meanwhile, the discharge hole (120) facilitates the discharge of not only the solution but also the bubbles generated when the solution is received. Existing Frozen TMA experimental devices had a problem in that the ice block production mold did not have holes, so the solution was received in the mold and bubbles could not be properly removed even if they were generated. However, the chip according to the present invention solved the above problem by using the discharge hole (120).

[0060] In one specific example of the present invention, the discharge hole (120) may be positioned to avoid the central portion forming the lowest surface of the receiving portion (110). This reduces the probability that the material used for producing frozen sections, collected at the center of the receiving portion (110), will unexpectedly discharge to the outside through the discharge hole (120).

[0061] In one specific example of the present invention, the chip according to the present invention may further include a discharge portion (140) formed in the reserve (100) and formed on the outside of the receiving portion (110).

[0062] The above discharge unit (140) can allow a portion of the material or solution used for producing a frozen section to flow out of the receiving unit (110) and be discharged outside the chip when the material or solution used for producing a frozen section is excessively contained in the receiving unit (110).

[0063] A second aspect of the present invention provides a method for producing a frozen section using a chip (1000) for producing a frozen section according to the present invention. An example of the production method according to the present invention is shown in FIG. 8.

[0064] In addition, when comparing the existing TMA production method with the method of producing frozen sections according to the present invention, the method according to the present invention has no limitations on the method of obtaining tissues, organoids, spheroids, or such materials compared to the existing method (Fig. 9a).

[0065] In addition, the method according to the present invention significantly reduces the time required to produce frozen sections compared to the existing method (Fig. 9b).

[0066] The third aspect of the present invention provides a frozen section produced using a chip (1000) for producing a frozen section according to the present invention.

[0067] The second and third aspects of the invention share technical characteristics with the first aspect.

[0068] The present invention is described below through implementation examples, examples, drawings, etc. However, the following description does not limit the scope of protection of the present invention.

[0069] Implementation Example 1. Small Material Chip (Fig. 10)

[0070] Although not necessarily limited thereto, when producing a frozen section using a small-sized tissue, spheroid, or organoid (e.g., a size of 500 μm or less; hereinafter, referred to as “material” in Embodiment 1), it may be advantageous to use a frozen section production chip (1000) that does not include a fence portion (130), i.e., includes a base portion (10), a plurality of reservoirs (100), a plurality of receiving portions (110), and a plurality of discharge holes (120).

[0071] When making frozen sections from small-sized materials, the frozen sections can often break when separating the frozen OCT during the process of making the frozen sections using a solution for making frozen sections, such as an OCT solution.

[0072] In this case, in order to prevent the above phenomenon, it may be desirable to use a chip having a shape excluding the fence portion (130). In addition, the diameter of the receiving portion (110) (D in FIG. 10) can be appropriately adjusted according to the size of the material, and preferably, D can be 1 mm to 1.5 mm. The length of D above is only an example, and the chip user can appropriately adjust it according to the size of the material used for making a frozen section. That is, if the length of D is too long, there may be a greater risk that the material will leak out through the discharge hole (120), etc., and if it is too small, it may be difficult to separate the frozen OCT solution without damage, or the material may remain positioned at the lowermost part of the receiving portion (110) and the OCT solution may not properly penetrate between the material and the receiving portion (110), resulting in improper production of a frozen section.

[0073] Implementation Example 2. Large Material Chip (Fig. 11)

[0074] Although not necessarily limited thereto, when producing a frozen section using a large-sized tissue, spheroid, or organoid (e.g., 2 mm to 5 mm in size; referred to as “material” in the following embodiment 2), it may be advantageous to use a chip (1000) for producing a frozen section that includes a fence portion (130), that is, a base portion (10), a plurality of reservoirs (100), a plurality of receiving portions (110), a plurality of discharge holes (120), and a fence portion (130).

[0075] When making a frozen section using a large material, the OCT solution may not properly penetrate between the material and the receiving portion (110) due to the size of the material, resulting in an improperly made frozen section.

[0076] In this case, to prevent the above phenomenon, it may be desirable to use a chip including a fence portion (130). When the fence portion (130) is included, the fence portion (130) can support the material, thereby securing a space for the OCT solution to penetrate between the material and the receiving portion (110). In addition, the diameter of the receiving portion (110) (d in FIG. 11) can be appropriately adjusted according to the size of the material, and preferably, d can be 1 mm to 3 mm. The length of d above is only an example, and the chip user can appropriately adjust it according to the size of the material used for producing a frozen section. That is, if the length of d is too long, the fence portion (130) cannot properly support the material, and if it is too short, it may be difficult to secure a space for the OCT solution to penetrate between the material and the receiving portion (110).

[0077] Example 1. Production of frozen sections

[0078] We conducted an experiment to confirm whether frozen sections were properly produced when using the chip according to our invention.

[0079] To this end, a total of 50,000 cells were cultured in each 96-well plate according to the number of cells presented in the table above under the five conditions in Table 1.

[0080] Culture conditions (unit: 1,000 cells) Condition Cancer Lung Fibroblast Total 1 HT 2 9 2 5 2 5 5 0 2 HCT 1 1 6 2 5 2 5 5 0 3 U 8 7 MG 2 5 2 5 0 4 HCT 1 1 6 5 0-5 0 5 A 5 4 9 2 5 2 5 5 0

[0081] And after growth for 5 days in a 96 well plate, they were transplanted into a chip according to the present invention to produce frozen sections, and the results are shown in Fig. 12. As a result of the experiment, it was confirmed that spheroids under different conditions could be frozen and arranged accurately in a 5 x 5 array using the chip according to the present invention, and the EpCAM and DAPI staining results also support the above results.

[0082] Additionally, it was confirmed that frozen sections could be produced even though the size of this spheroid was very small, 300 to 500 μm.

[0083] Example 2. Production of frozen sections using spheroids and tissues

[0084] To verify whether the chip according to the present invention can produce frozen sections regardless of the size of the material used for the frozen section, frozen sections were produced using spheroids or mouse tissues using the chips according to Embodiment 1 and Embodiment 2 above. The production process is shown in FIGS. 13a and 13b.

[0085] The results also confirmed that frozen sections were properly produced when using large spheroids and mouse tissues, as in 13c, as well as when using small spheroids.

[0086] [Explanation of symbols]

[0087] 1000: Chip for making frozen sections

[0088] 10: Bass section

[0089] 100: Reserve

[0090] 110: Reception area

[0091] 120: Exhaust hole

[0092] 130: Fence Department

[0093] 131: Side wall

[0094] 132: Slit

[0095] 133: Flange

[0096] 140: Exhaust

Claims

1. Base section; A plurality of reserves formed to be recessed from the upper surface of the base portion; A plurality of receiving portions formed in the above reserve and containing a material or solution used for producing a frozen section; A chip for producing frozen sections, comprising a plurality of discharge holes formed on the lower surface of the plurality of receiving portions and through which a solution received in the receiving portions is selectively discharged.

2. In paragraph 1, A chip for producing frozen sections, wherein the material used for producing the frozen sections is at least one of tissue, organoid, and spheroid.

3. In paragraph 1, A chip for producing frozen sections, wherein the solution used for producing the frozen sections includes a culture solution or an OCT solution (optimal cutting temperature compound).

4. In paragraph 1, A chip for producing frozen sections, wherein the side surface of the above reserve is formed at an incline of 45 to 85 degrees with respect to the lower surface of the above reserve.

5. In paragraph 1, A chip for producing frozen sections, wherein the lower surface of the above-mentioned receiving portion is formed to be further recessed so as to have an incline of 15 to 45 degrees with respect to the lower surface of the above-mentioned reserve.

6. In paragraph 1, A chip for producing frozen sections, further comprising a fence portion formed to protrude upward from the lower surface of the reserve and surround the receiving portion.

7. In paragraph 6, A chip for producing frozen sections, wherein the above-mentioned fence portion is a cylindrical shape having a diameter set to form the receiving portion therein.

8. In paragraph 6, A chip for producing frozen sections, wherein the fence portion includes a plurality of side wall portions extending in the vertical direction and spaced apart at preset intervals along the circumference centered on the receiving portion.

9. In paragraph 6, A chip for producing frozen sections, wherein the fence portion extends in the vertical direction to connect the reserve and the receiving portion with each other and includes a slit portion spaced apart at a preset interval along the circumference centered on the receiving portion.

10. In paragraph 8, A chip for producing frozen sections, wherein the fence portion further includes a flange portion formed to protrude outwardly away from the receiving portion from the side wall portion.

11. In paragraph 10, A chip for producing frozen sections, wherein at least a portion of the flange portion is formed to be connected to a side surface of the reserve.

12. In paragraph 1, A chip for producing frozen sections, wherein the discharge holes have a diameter smaller than that of the receiving portion and are formed in multiple numbers on the lower surface of one receiving portion.

13. In paragraph 1, A chip for producing frozen sections, wherein the discharge hole has a diameter set to allow selective discharge by the combined force of forces including gravity, capillary force, and surface tension of the solution received in the receiving portion.

14. In paragraph 1, A chip for producing frozen sections, wherein the discharge hole is positioned to avoid the center portion forming the lowest surface of the receiving portion.

15. In paragraph 1, A chip for producing frozen sections, which further comprises a discharge portion formed in the above reserve and formed outside the receiving portion.

16. A method for producing a frozen section using a chip for producing a frozen section according to Article 1.

17. A frozen section produced using a chip for producing a frozen section according to Article 1.

18. A biopsy method using a chip for producing frozen sections according to Article 1.

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