KR20220130906A - Method for cell block production using alginate-PVA composite beads - Google Patents

Abstract

The present invention relates to a method for producing a cell slice applied to a small object (a cell or a small cell mass), which was previously impossible to produce, and more specifically, to a method for producing a cell slice by using an alginate-PVA composite bead on which the cell or cell mass is fixed as a medium.

Description

Method for cell block production using alginate-PVA composite beads}

The present invention relates to a method for preparing a cell fragment of a cell or a cell mass, and more particularly, to a method for preparing a cell fragment that can be applied to a small object (cell or a small cell mass) that was not previously possible.

For accurate pathology diagnosis, it is necessary to visually confirm the location or existence of tissue lesions, proteins, and genes inside the tissue. A sectioning method is being used to section it so that it can be observed under a microscope.

The section preparation method is largely divided into a freezing method, which involves embedding and freezing in a frozen tissue embedding agent (OCT compound), and a paraffin method using paraffin as an embedding agent.

The freezing method wraps the sample in OCT, freezes it at -10~-40℃, and then micro-cuts it with a cryotome and observes it, so it is advantageous for observation 'as is'. It has the disadvantage that it cannot be stored at room temperature and must be stored below -20℃. In the paraffin method, it is somewhat disadvantageous to observe 'as is' because of the leakage or inactivation of intracellular proteins, lipids, soluble antigenic substances, etc. due to the use of organic solvents and heating, etc. There are advantages.

On the other hand, according to the conventional sectioning method, it is very difficult to distribute the cells suspended in the aqueous solution 'evenly' because paraffin or OCT compounds are oily and highly viscous (they are present in lumps) ㉯ To distribute the cells relatively 'evenly' Even so, it is difficult to specify the location of the cells in the cell block, so it takes a lot of time and effort to accurately find and observe them with an (electron) microscope. It has to go through the process of removing medium → washing → dyeing, etc., but once the embedding agent is removed, there is no medium to hold the cells, so the cells are washed out. Therefore, due to these circumstances, it was practically impossible to section (especially low concentration) cells or microcell masses other than 'tissue' or 'organ'.

In addition, according to these manufacturing methods, since the embedding agent (non-frozen OCT or molten paraffin) has a very high viscosity, it is impossible to section cells with a particularly low concentration (cell number). Due to the overlap, there is a problem that observation is not easy because the 'specific cell' to be observed is masked by other cells.

Patent Laid-Open No. 10-2001-0099854 relates to a method and apparatus for cutting a tissue block, and a method of placing the tissue in a container (mold), injecting an alginic acid polymer-water mixture to embed it, and cooling it to section it with a microtome has been In other words, ㉮ targets relatively macro organs or tissues, ㉯ uses a modified alginate plastic polymer, and ㉰ suggests that the hardened alginate plastic polymer forms a block by enclosing the outside of the organ or tissue. It is not intended for use, and it is not intended for fixation of microscopic cells that are not macroscopic organs or tissues and frozen sections thereof. In addition, according to this, long-term storage of the sample is impossible, and it is not intended for cells in the first place.

Chinese registered patent CN 108956221 (Method for carrying out pathological section production by using sodium alginate) proposes a method of fixing tissue cells on alginate beads and then recovering them and removing the liquid while pressing with gauze to produce a plate shape and then section them. However, according to this, structural damage to the sample (cell) is caused in the process of dehydration under pressure, so it is insufficient to observe the cells 'as they are'. Also, according to this prior art, long-term storage of the sample is impossible.

On the other hand, there is a case in which cells are mixed with additives such as egg albumin and fibrinogen to make them highly viscous, and then cell blocks are manufactured using the paraffin method. However, according to this method, although cell fixation, which was impossible in aqueous solution, is possible, it is difficult to block because it also has to deal with a high-viscosity liquid. In addition, since the cell block produced in this way has a structure in which high-viscosity droplets are contained in solidified paraffin and cells are present in the droplets, it is difficult to obtain precise sections from them, and high-viscosity droplets are easy to separate (flow down) from the sections, making it difficult to obtain cell sections. Not easy.

Patent Publication 10-2001-0099854 Chinese registered patent CN 108956221

An object of the present invention is to provide a method capable of blocking and sectioning cells or microtissues (cell masses), not macrotissues.

Another object of the present invention is to provide a method for blocking and sectioning cells or microtissues (cell mass) so that the cells or microtissues are uniformly distributed in a specific region of the block or section.

The present invention for achieving the above object

It is characterized in that it is a method for producing a cell section mediated by alginate-PVA composite beads to which cells or cell masses are immobilized.

The present invention is easy to handle by indirectly embedding cells in the embedding agent instead of embedding them in an alginate bead as a medium to block-fragmentation, and it is possible to homogenize the cell distribution in the gel and improve the cell capture rate, and in particular, it is possible to easily section even low-concentration cells There are advantages to being reconciled.

Therefore, according to the present invention, it is possible to section (especially low concentration) cells or cell masses (microtissues), which were not possible with conventional sectioning methods, so that more various tests can be performed from a small number of samples.

1 is a conceptual diagram and a photograph showing the process according to the method for producing a cell section according to the present invention.
Figure 2 is an exemplary flow chart of the process of manufacturing a cell section for a specific cell according to the present invention.
Figure 3 is a photograph of the aqueous solution and the prepared composite beads used in Examples.
Figure 4 is an electron micrograph of the cell-immobilized beads obtained in Example of the present invention.
FIG. 5 is a fluorescence micrograph in which cells stained with fluorescence are attached to the surface of the AP bead according to the present invention.
6 is a photograph of a paraffin cell block of a specific cell-immobilized bead, prepared in an embodiment of the present invention.
7 is a photomicrograph showing the results of immunocytochemical staining of cell sections, prepared in Examples of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. However, these drawings and embodiments are only examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. It will be natural for those skilled in the art that various modifications and changes can be made within the scope of the technical spirit of the present invention based on these examples.

In the technical field to which the present invention pertains, it is known that blockage or fragmentation by the embedding method is possible at the level of a tissue or organ of 'a certain size or more'. In contrast, in the present invention, the object to be blocked or fragmented is a cell mass composed of a plurality of cells such as cells capable of being suspended in an aqueous solution or fragmented tissue. In the description, claims, and examples of the present invention, only 'cells' are referred to for convenience, but in this case, the cells are a concept including a cell mass of a turbidable size.

On the other hand, the present inventors have developed and applied for a patent (Application No. 10-2021-0006921) for "a method for preparing a cell section mediated by alginate beads" in relation to cell fragmentation. The present invention compensates for the disadvantages of the previous invention (difficulty in handling due to weakening of the strength of the beads) and expands the applicability (fragmentation of specific cells).

As described above, the present invention is based on a cell section production method mediated by alginate-PVA composite beads to which cells or cell masses are fixed, and is divided into those targeting arbitrary cells and targeting specific cells. It will be described in sequence below.

(1) any cell target

The present invention relates to blocking/fragmenting any cell.

More specifically, (A) preparing a cell-fixing bead in which cells or agglomerates are fixed to alginate-PVA composite beads; (B) preparing a cell block by embedding the cell fixing beads in an embedding agent; (C) cutting the cell block to a predetermined thickness to produce a cell fragment; relates to a cell fragment production method using a composite bead comprising the.

In the present invention, the following two methods are possible for step (A).

① (a) a small step of preparing a first solution by turbiding the cells in a mixed aqueous solution of monovalent ionic alginate and PVA; and (b) a small step of preparing cell-immobilized beads by adding the droplets of the first solution to the second solution, which is a polycationic aqueous solution. In this case, the concentration of cells and alginate in the first solution may be appropriately adjusted according to the size or characteristics of the cells or cell mass, the size of the target bead, and the like.

② (a) a small step of preparing a composite bead by adding droplets of a mixed aqueous solution of monovalent ionic alginate and PVA to an aqueous polycation; and (b) adding the composite beads to the cell suspension and allowing the cells to penetrate the beads to prepare a cell-fixed bead; includes.

The method of fixing the cells to the alginate beads of step (A) and the method of preparing and sectioning the cell block of step (B) (C) are in accordance with methods well known in the art, so detailed and detailed descriptions thereof are omitted. do.

The present invention can be applied not only to the paraffin method but also to the freezing method. Accordingly, the embedding agent may be a paraffin or an OCT compound.

Hereinafter, in comparison with the conventional sectioning method, the cell section fabrication process according to the present invention will be described in detail with reference to FIG. 1 .

First, according to the present invention, cells or microcellular masses are not directly embedded in the embedding agent, but are fixed to alginate-PVA composite beads (fixed according to method ② above in the drawing). At this time, the volume of the composite bead is very small compared to the volume of the final embedding agent, and since the cells are evenly suspended in the aqueous solution during the immobilization process or the cells penetrate the beads evenly, the cells are evenly distributed over the entire space of the bead, and directly into the embedding agent. It is fixed at a high concentration compared to being buried. In this way, cell-immobilized beads of a size and quantity sufficient to handle in a conventional sectioning method are prepared. (Step (A))

Next, the prepared cell fixing beads are embedded in a predetermined embedding agent according to the conventionally known freezing method or paraffin method and solidified to obtain a cell block (step (B)), and a cell section of a predetermined thickness is obtained using a slicer (step (C)) ).

In this way, even if the cell section prepared according to the manufacturing method according to the present invention is seated on a slide or hold and then the embedding agent is removed, as can be seen from the conceptual diagram of the cell section shown in step (C) of FIG. Since the cells are in a state of being fixed by the part expressed as

When the final cell section prepared by the present invention is observed with an (electron) microscope, it would be good if the cell fixing beads were stained with a predetermined color so that the position of the cells, that is, the position of the beads, could be easily identified.

The present invention is characterized in that single cells or fine cell masses that cannot be sectioned can be sectioned. In particular, the advantages of the present invention can be further exhibited when targeting a low concentration of single cells or microcellular masses.

(2) target specific cells

Another present invention relates to a method of blocking/fragmenting only desired specific cells in a cell mixture in which various types are mixed. An exemplary flow chart of the procedure of the method for producing a cell section according to the present invention is shown in FIG. 2 .

Specifically, (A) preparing alginate-PVA composite beads; (B) preparing an antibody-binding bead by attaching an antibody specific to a specific cell to the surface of the composite bead; (C) preparing a specific cell-immobilized bead in which a specific cell is bound to the antibody-binding bead; (D) preparing a cell block by embedding the specific cell fixing beads in an embedding agent; and (E) cutting the cell block to a predetermined thickness to produce a cell fragment. The specific process will be described in detail in the following examples.

Compared to the above-mentioned (1) invention, there is a difference in that a composite bead is prepared and an antibody is attached to the surface to prepare and use an antibody-binding bead. Although an EpCAM (epithelial cell adhesion molecule; epithelial cell binding molecule) antibody was used in the Examples, it is natural that an appropriate antibody specific for a cell to be isolated can be selected. The method for preparing the composite bead, the method for binding cells, the method for embedding and sectioning the cell-fixed composite bead, the type of embedding agent, the addition of dye, etc. are the same as in (1) described above, and thus a repeated description will be omitted.

In the present invention, when attaching an antibody specific to a specific cell to the surface of the composite bead, it is preferable to modify the surface of the composite bead by a predetermined method in order to increase the adhesion of the antibody. Specific modification examples are described in the Examples.

Meanwhile, in the specific cell-immobilized bead obtained in step (C), as can be conceptually confirmed in the enlarged view of the specific cell-immobilized bead in FIG. 2 , the specific cell is exposed on the surface of the bead. Cells exposed to the surface of the bead may be detached from the bead in the subsequent cell block manufacturing step. Accordingly, in the present invention, there may be a small step of coating a specific cell-fixed bead with an alginate gel in order to prevent the detachment of cells once bound to the beads. This is also described in detail in the following examples.

[Example]

1. Preparation of specific cell immobilization beads

(1) Preparation of cell immobilization beads

① Each 2% (w/v) sodium alginate and PVA aqueous solution were mixed in the same volume, boiled at 85°C with constant stirring, and then cooled to room temperature to prepare the first solution. Using an extruder, the first solution was dropped into a 100 mM calcium chloride (CaCl ₂ ) solution (second solution) in the form of spherical beads and stirred at room temperature for 1 hour to induce gelation (hardening) of the beads to obtain composite beads . The composite beads were washed several times with deionized water, and then stored in PBS buffer at room temperature and used when necessary. A photograph of alginate solution (①), PVA solution (②), alginate + PVA mixed solution (③), EDC/NHS solution (④) and the prepared composite bead (⑤) are attached to FIG. 3, respectively.

② The composite bead was immersed in a mixture of 200 mM 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and 200 mM N-hydroxysulfosuccinimide (Sulfo-NHS) at 4°C and stirred at room temperature for 1 hour. was subjected to surface modification to activate EDC-NHS coupling. The surface-modified composite beads were washed with PBS buffer solution.

Then, the surface-modified composite beads were added to a 4°C EpCAM antibody solution (0.5 mg/mL, 1: 100 diluted in PBS) and stirred for 1 hour to induce antibody binding to the surface of the composite beads. Then, the beads were washed with PBS buffer to prepare antibody-binding beads .

Separately, the lung cancer cell line H358 was fluorescently stained with 10 μM of CellTracker (Invitrogen) for 30 minutes, and then washed with a buffer solution to prepare a fluorescently stained lung cancer cell line H358.

③ EpCAM antibody-binding beads were mixed with lung cancer cell lines A549 (fluorescent staining) and H358 solution, respectively, and stirred at room temperature for 1 hour to obtain two types of specific cell-immobilized beads bound to each lung cancer cell line and beads. The cell fixing beads were washed three times in PBS buffer.

Lung cancer cell lines A549 and H358 carcinoma are adenocarcinomas, A549 is an EGFR-expressing cell line, and H358 is a cell line with low EGFR expression.

(2) Observation of the surface of the cell-immobilized beads

① The surface morphology of the cell-immobilized beads obtained above was observed using SEM/EDS equipment (SU8200; Hitachi, Tokyo, Japan). A comparative example is a composite bead prepared in (1)① above in which an antibody and a cell are not bound.

Phenomorphisms were obtained through SEM mode at an acceleration voltage of 2 kV and a working distance of 4.7 mm. All images were magnified at a magnification of 1K. The prepared bead samples were mounted on an SEM stub and attached to a double-sided adhesive tape coated with osmium (3.0 nm thick) to prevent degradation or filling effects.

An electron micrograph of the surface of the cell-immobilized bead is shown in FIG. 4 . As can be seen from the photo, the surface of the composite bead (Comparative Example) to which the antibody and cells are not bound is generally flat, whereas the cell immobilized bead (Example) to which the antibody and cells are bound has cells bound to the surface (Picture) You can see the part marked with a light blue rectangle). It is thought that the small messy part in the Example is only bound to the antibody.

② A fluorescence microscopy photograph of the cell-immobilized beads bound to the lung cancer cell line H358 fluorescence stained is attached to FIG. 5 . Fluorescently-stained cells are indicated by thick dots of yellow-green fluorescence, indicating that specific cells are well fixed to the beads.

2. Fabrication of cell blocks and cell fragments

The cell-immobilized beads were immersed in 1% (w/v) sodium alginate aqueous solution, taken out, coated with alginate, and then immersed in 100 mM calcium chloride (CaCl ₂ ) solution and left for 1 hour to add a protective layer.

Paraffin cell blocks were obtained as follows according to the standard procedure for preparing FFPE (Formalin Fixed Paraffin Embedded) cell blocks from the specific cell-fixed beads with a protective layer formed in this way.

Cell immobilization beads were carefully fixed/dehydrated in the order of 4% paraformaldehyde, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol and 100% ethanol. Immersion of the dehydrated beads in Histogel solution as an intermediate solvent for 20 minutes was repeated twice. Then, the beads were mounted on the cassette, and the cassette was immersed in liquefied paraffin wax (55-60° C.) for 6 hours, then taken out and solidified at about 4° C. to prepare a cell block . A photograph of the prepared cell block is attached to FIG. 6 , and as can be seen from the photograph, the cell fixing beads (indicated by the red dotted line in the photograph) are inserted into the paraffin block.

Then, the obtained cell block was sliced with a conventional sectioning machine to obtain a cell section with a thickness of 5 to 10 μm.

3. Immunocytochemical staining of prepared cell sections

In histological pathology diagnosis, after H&E staining, various additional staining (ex special staining, immune cell/histochemical staining, fluorescent staining, etc.) carried out. As antibodies for immunostaining, p63, Napsin A, and EGFR were exemplarily selected.

Attach the cell sections of lung cancer cell lines A549 and H358 obtained in the above method to a glass slide, and dip 10 in 4% paraformaldehyde solution. Rinse with tap water, add antigen recovery solution at 90°C or higher, and react for 1 hour. React with sodium citrate solution and react with antibody TTF-anti1 for 1 hour at room temperature. Wash with wash buffer and react with secondary antibody at room temperature for 40 minutes. Wash with water, stain with DAB reagent at room temperature for 3 minutes, and nuclear stain with mayer's hematoxylin for 1 minute at room temperature. A micrograph of the stained sample is shown in FIG. 7 .

As can be seen from the picture shown, it can be confirmed that the expression of TTF-1 was stained with a brown color by the DAB staining agent. Lung cancer cell lines A549 and H358 are adenocarcinoma cell lines. A549 is an EGFR-expressing cell line, and H358 is a low-EGFR-expressing cell line.

Claims (9)

A method for producing a cell section using alginate-PVA composite beads to which cells or cell masses are immobilized.
The method according to claim 1,
(A) preparing a cell-fixing bead in which cells or agglomerates are fixed to alginate-PVA composite beads;
(B) preparing a cell block by embedding the cell fixing beads in an embedding agent;
(C) cutting the cell block to a predetermined thickness to produce a cell fragment;
A method for producing a cell fragment mediated by a composite bead, characterized in that it comprises.
The method according to claim 1,
The composite bead is a method for producing a cell fragment using a composite bead, characterized in that a dye of a predetermined color to be distinguished from the embedding agent is added.
The method according to claim 1,
(A) preparing alginate-PVA composite beads;
(B) preparing an antibody-binding bead by attaching an antibody specific to a specific cell to the surface of the composite bead;
(C) preparing a specific cell-immobilized bead in which a specific cell is bound to the antibody-binding bead;
(D) preparing a cell block by embedding the specific cell fixing beads in an embedding agent;
(E) cutting the cell block to a predetermined thickness to produce a cell fragment;
A method for producing a cell fragment mediated by a composite bead, characterized in that it comprises.
5. The method according to claim 4,
The step (A) is,
(a) a substep of preparing a mixed aqueous solution of monovalent ion-bonded alginate and PVA at a predetermined concentration, and (b) adding droplets of the mixed aqueous solution to a polycation aqueous solution to prepare alginate-PVA composite beads A method for producing a cell fragment using a composite bead as a medium.
5. The method according to claim 4,
In the step (B),
A method for producing a cell fragment mediated by a composite bead, characterized in that the antibody is attached after the surface of the composite bead is modified.
5. The method according to claim 4,
After step (C),
A method for producing a cell fragment mediated by a complex bead, characterized in that the specific cell fixing beads are coated with an alginate gel.
5. The method according to claim 4,
The embedding agent is a method for producing a cell section through a composite bead, characterized in that paraffin or OCT compound.
5. The method according to claim 4,
Alginate bead-mediated cell fragment production method, characterized in that the dye of a predetermined color is added in the step (A).

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