KR20230033303A - Simultaneous separation method of specific cells and exosomes using alginic acid - Google Patents

Abstract

The present invention relates to a method for effectively separating specific cells and exosomes at the same time from a single biological sample, which significantly reduces the time and financial burden on medical staff and patients. More specifically, the method comprises: a step (A) of manufacturing an exosomes separation bead in which an exosome-specific antibody is bound to an alginate bead and a specific cell separation bead in which a specific cell-specific antibody is bound to an alginate bead; a step (B) of centrifuging a biological liquid sample to prepare a part of the supernatant as a sample for exosome separation, and remixing the remainder to prepare a sample for specific cell separation; a step (C) of adding the exosome separation bead to the exosome separation sample, adding the specific cell separation bead to the specific cell separation sample, and settling the liquid samples; a step (D) of separating the exosome separation bead and the specific cell separation bead from the liquid samples, respectively; and a step (E) of separating exosomes or exosome components from the exosome separation bead, and separating specific cells from the specific cell separation bead.

Description

Simultaneous separation method of specific cells and exosomes using alginic acid}

The present invention relates to a method for simultaneously and effectively isolating specific cells and exosomes from a single biological sample.

Exosomes are small-sized (30-150 nm) extracellular vesicles that carry key molecules of the cell, such as proteins, mRNAs and microRNAs. It is understood that exosomes are specifically secreted for the purpose of intercellular signal transmission. Exosomes in eukaryotic cells are involved in erythrocyte differentiation and immune response regulation, and are known to be particularly related to cancer progression, invasion, metastasis, and angiogenesis. there is. Accordingly, attempts have been made to obtain information on the diagnosis and prognosis of various diseases, including cancer, by non-invasively isolating exosomes from bodily fluids such as urine, saliva, and blood, and analyzing proteins, mRNAs, and microRNAs in exosomes. It is being done.

Known exosome isolation methods include ultracentrifugation, density gradientcentrifugation, size exclusion chromatography, exosome precipitation, and immunoaffinity capture. there is. However, this method is uneconomical because the purity and separation efficiency of exosomes to be separated are relatively low, complex equipment is required, and labor is intensive.

On the other hand, by observing and analyzing characteristics such as genetic characteristics, shape, movement, growth, and response to conditions of specific cells, it is possible to increase the understanding of the genetic, physiological, and pathological characteristics of cells and organisms. A variety of academic, medical, and industrial applications can be suggested. Therefore, if exosomes can be isolated from a patient's cell mixture sample at the clinical site and specific cells, such as cancer cells, can be simultaneously isolated and examined from the same sample, furthermore, specific cells among various cell mixtures can be 'alive' without damage. If it can be effectively separated and tested, it will be of great help to a rapid and multifaceted understanding of the patient's disease state.

Conventionally, a method for isolating specific cells from various cell mixtures or a method for isolating exosomes from a mixed sample is known, but a method for effectively separating specific cells and exosomes from a single sample at the same time is a research group including the present inventors. It is not known other than those by (Non-Patent Documents 1 and 2).

Registered Patent No. 10-2107844 (method of separating extracellular vesicles using cations) suggests a method of adding cations to a sample to form a complex between exosomes and cations and separating them by precipitation. However, according to this, since there is a process of removing cells in advance so that cations can be bound only to exosomes, there is nothing to do with isolation of specific cells.

Publication No. 10-2020-0084803 (antibody-coupled nanowire for exosome separation and exosome separation method using the same; withdrawn) discloses a method for separating exosomes by binding antibodies to nanowires made of conductive polymers. there is. However, since the method of this document isolates only exosomes using antibodies, it is not interested in specific cell isolation. Furthermore, since a reducing agent is used for final recovery, there is a concern about damage to exosomes due to the possibility of toxicity, and even if it is used for isolation of specific cells, there is a limit to obtaining cells in a dead or damaged state.

Registered Patent No. 10-1762833 (a method for separating adipose-derived stem cells and exosomes from adipose tissue) treats adipose tissue with collagenase, centrifuges and filters several times, and finally obtains pelleted stem cells and supernatant. It is about how to obtain exosomes. In this case, although cells and exosomes are obtained at the same time, a complicated process is required, and cells in adipose tissue are obtained, not any specific cells.

Antibody-binding-beads in which antibodies against specific pathogens are coupled to conventional magnetic beads have been commercialized (see Non-Patent Documents 3 and 4). However, according to this, 'specific antibody' is determined by the manufacturer, so 'target cell' is limited and diversity is lacking. To overcome this, individual experimenters need 'specific antibody'-coupled-magnetic beads at the experimental site level. There is a limitation that it is difficult to manufacture immediately according to. In addition, according to this, no previous studies have been reported on isolating specific cell populations and exosomes in a well-preserved state .

Registered Patent 10-2107844 Patent Publication 10-2020-0084803 Registered Patent No. 10-1762833

Circulating Tumor Marker Isolation with the Chemically Stable and Instantly Degradable (CSID) Hydrogel ImmunoSpheres (2020.12.18) Tri-modal liquid biopsy: Combinational analysis of circulating tumor cells, exosomes, and cell-free DNA using machine learning algorithm, Clinical and translational medicine (2021.8.4) https://www.thermofisher.com/kr/en/home/life-science/cell-analysis/cell-isolation-and-expansion/cell-isolation.html?SID=fr-dynabeads-2 https://www.qiagen.com/us/search/products?query=magnetic%20beads&page=2

An object of the present invention is to provide a method for simultaneously and effectively isolating specific cells and exosomes from the same biological sample.

The present invention for achieving the above object is

(A) preparing beads for separating exosomes in which an exosome-specific antibody is coupled to alginate beads and beads for separating specific cells in which an antibody specific for specific cells is coupled to alginate beads, respectively; (B) preparing a part of the supernatant as a sample for exosome isolation by centrifuging the biological liquid sample and remixing the remainder as a sample for specific cell isolation; (C) adding the exosome isolation beads to the exosome isolation sample and the specific cell isolation beads to the specific cell isolation sample and leaving them alone; (D) separating the exosome isolation beads and the specific cell isolation beads from the liquid sample; And (E) then separating exosomes or exosome components from the separated beads for separating exosomes, and separating specific cells from the beads for separating specific cells; containing, specific cells and exosomes using alginate It is characterized by a simultaneous separation method.

As described above, according to the present invention, using alginate known to be non-cytotoxic, and without using special equipment or magnetic beads, it is possible to simultaneously select and recover living specific cells and exosomes under mild conditions at room temperature and normal pressure. do.

In addition, according to the present invention, specific cells and exosomes can be simultaneously separated from a single biological liquid sample while the specific cells are alive, so that information on diagnosis and prognosis of various diseases including cancer can be easily obtained in the medical field. do.

In addition, according to the present invention, it is possible to simply isolate specific cells and exosomes from one biological liquid sample at the level of a normal laboratory without requiring expensive equipment or materials or high level of skill, thus saving time and money for medical personnel and patients. This can greatly reduce the burden.

1A and 1B are respectively exemplary flowcharts of a process according to the present invention and exemplary conceptual diagrams showing changing states of beads during the process.
Figure 2 is a photograph of the result of immunocytochemical staining by recovering cancer cells bound to beads.
Figure 3 is a chart showing the efficiency of specific cell isolation according to the method according to the present invention.
Figure 4 is a chart showing that exosomes are well separated by the method according to the present invention.
5 is a diagram showing that nucleic acids of exosomes isolated by the method of the present invention are stable.
6 is an exemplary flow diagram of a variant of the present invention.
7 is an exemplary flow diagram of a variant of the present invention.

Hereinafter, the present invention will be described in more detail with reference to 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 obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical spirit of the present invention based on these examples.

As described above, the present invention,

(A) preparing beads for separating exosomes in which an exosome-specific antibody is coupled to alginate beads and beads for separating specific cells in which an antibody specific for specific cells is coupled to alginate beads, respectively; (B) preparing a part of the supernatant as a sample for exosome isolation by centrifuging the biological liquid sample and remixing the remainder as a sample for specific cell isolation; (C) adding the exosome isolation beads to the exosome isolation sample and the specific cell isolation beads to the specific cell isolation sample and leaving them alone; (D) separating the exosome isolation beads and the specific cell isolation beads from the liquid sample; And (E) then separating exosomes or exosome components from the separated beads for separating exosomes, and separating specific cells from the beads for separating specific cells; containing, specific cells and exosomes using alginate It relates to simultaneous separation methods. A flowchart of the process according to the present invention is exemplarily shown in FIG. 1A and a changing state of beads in the process according to the present invention is shown in FIG. 1B.

In the present invention, alginate beads, which are widely used in recognition of safety and stability, were applied as a support for physically binding antibodies, cells, and exosomes. Alginate beads can be prepared by adding droplets of an aqueous solution of monovalent ionic bonded alginate to an aqueous solution of polycations. In the present invention, predetermined antibodies (exosome-specific antibody and specific cell-specific antibody) are conjugated to the alginate beads prepared in this way to separate beads for exosome isolation and beads for specific cell isolation, respectively. Before binding the antibody to the alginate beads, it is preferable to modify the surface of the beads by a predetermined method in order to increase the strength of the alginate beads and increase the binding efficiency of the antibody. Although the surface modification method of alginate beads has been described in the following examples, in addition, various surface modification methods known in the art can be applied (step (A)).

According to the present invention, apart from the preparation of beads, a biological liquid sample is centrifuged to prepare a part of the supernatant as a sample for exosome isolation, and the remainder as a sample for specific cell isolation by remixing (step (B)).

Subsequently, beads for exosome isolation were added to the sample for isolation of exosomes, and beads for isolation of specific cells were added to the sample for isolation of specific cells, respectively, and the antibody-specific exosomes and specific cells were bound to each antibody for a predetermined time Leave it alone (step (C)).

Then, the beads for separating the exosomes and the beads for separating the specific cells were separated from the liquid sample (step (D)), and then the exosomes or exosome components were separated from the separated beads for separating the exosomes, and the It is to separate specific cells from beads for specific cell isolation (step (E)). 'Separating exosomes or components of exosomes' means separating intact exosomes or lysing exosomes to separate substances contained in exosomes, such as proteins, mRNAs, or microRNAs, if necessary. The step (E) may include (a) a substep of liquefying the separated beads or treating the beads with a reducing agent. In the following examples, the beads were gelated by treating the beads with EDTA, but the beads were might be dismantled.

The following is an example according to the method for simultaneously separating specific cells and exosomes according to the present invention.

[Example]

In this example, whole blood was used as a biological liquid sample, and epithelial cell adhesion molecule (EpCAM) was used to specifically separate epithelial cells and circulating tumor cells (CTC) in whole blood. Anti-EpCAM specific antibody was used, and an antibody anti-CD63 specific to CD63 protein present in the vesicle membrane was used to isolate exosomes.

However, using a biological liquid sample other than whole blood in the same way, using a different type of cell-specific antibody to isolate the same cell, and using an antibody specific to that cell to isolate a different type of cell It will be natural that it is possible to use antibodies specific for other antigens present in the vesicle membrane to isolate exosomes.

EpCAM is an epithelial cell adhesion factor that is also expressed in epithelial cells, but has the characteristic of being overexpressed in hematopoietic cancer cells. Therefore, cell separation using this antibody is considered to have a much higher probability of binding cancer cells than normal cells, so it is a cancer cell trapping factor. used as

Whole blood samples were obtained from 13, 14, and 72 healthy people (Healthy), benign tumor patients (Benign), and cancer patients (Malignant), respectively.

1. Preparation of beads for specific cell isolation and exosome isolation

Add calcium chloride dropwise to 5% (w/v) alginate solution and stir for 1 hour at room temperature. The obtained spherical alginate beads are reacted with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-Hydroxy-Succinimide (NHS) solutions at room temperature for 1 hour to modify the surface.

For example, the surface-modified beads obtained are separated in half, reacted with anti-EpCAM (1:100) and anti-CD63 (1:100) antibodies for 1 hour, and then washed with PBS to separate beads for specific cells. (anti-EpCAM alginate beads) and exosome separation beads (anti-CD63 alginate beads) are prepared.

2. Simultaneous isolation of specific cells and exosomes from biological liquid samples

(1) Separation of supernatant from liquid sample

The whole blood sample is centrifuged at 3,000 rpm for 10 minutes, and a portion of the supernatant (plasma or serum) is taken for exosome isolation, and the rest is mixed evenly and used for specific cell isolation.

(2) Isolation of specific cells and confirmation of viability

① The prepared anti-EpCAM alginate beads are reacted with a 1:1 diluted solution of PBS and the liquid sample (whole blood) for specific cell isolation obtained in 2(1) above for 1 hour. After the reaction, it is washed with sterile distilled water to remove non-specific reactive cells.

The beads are reacted in EDTA (0.5 M, pH 8.0) for 10 minutes and adhered to the slide. Fix the cell-attached slide with 4% paraformaldehyde solution for 10 minutes. Rinse with tap water, add a 90 degree or higher antigen retrieval solution, and react for 1 hour. React with sodium citrate solution and antibody EpCAM for 1 hour at room temperature. After washing with washing buffer, the secondary antibody is reacted at room temperature for 40 minutes. Wash with water, stain with DAB reagent at room temperature for 3 minutes, and react with Mayer's hematoxylin for 1 minute at room temperature for nuclear staining. In FIG. 2, cancer cells selected with anti-EpCAM-coated alginate beads were recovered and a photomicrograph of the result of EpCAM immunocytochemical staining was attached. The brown-stained portion is the EpCAM expression portion.

② Although not in whole blood, when cells were sorted by the method according to the present invention, the selection rate, recovery rate, and survival rate were confirmed using the colorectal cancer cell line SW480 (see FIG. 3). As can be seen in the figure, when the method according to the present invention was applied, very good results were obtained with a selection rate of 73.1 ± 5.0%, a recovery rate of 93.4 ± 3.6%, and a survival rate of 87%.

(3) Isolation of exosomes

Anti-CD63 alginate beads are reacted with 250 μl of liquid sample (plasma or serum) for exosome isolation obtained in 2(1) above for 1 hour. After the reaction, it is washed with PBS to remove non-specific reactants.

The beads are reacted in EDTA (0.5 M, pH 8.0) for 10 minutes and transferred to a sterile 2ml tube. Add 500 μL of Trizol® to a 2ml tube, mix thoroughly for 3 minutes, and then stand upright for 5 minutes. Add 100 μL chloroform, mix for 15 seconds, and stand upright for 10 minutes. Centrifuge at 13000 rpm at 4℃ for 10 minutes, transfer the supernatant to a new tube, and add 500 μL isopropanol. After thoroughly mixing, stand upright for 10 minutes at room temperature. After 10 minutes of reaction, react for 10 minutes at 4° C. at 13000 rpm and discard the supernatant. Add 600 μL of 70% ethanol and centrifuge at 13000 rpm at 4℃ for 5 minutes. After centrifugation and air drying for 5 minutes, 50 μL of distilled water is added to the pellet that has settled to the bottom. (RNA extraction method)

Whether exosomes are well separated by the present invention was compared with a commercially available kit (ExoQuick) for exosome isolation. Efficient separation of exosomes can be confirmed by whether or not the nucleic acid concentration of exosomes is separated (see FIG. 4). As can be seen in FIG. 4, even if exosomes are isolated with anti-CD63-coated alginate beads as in the present invention in all types of samples (normal subjects, benign tumor patients, and malignant tumor patients), nucleic acids are almost similar to those of commercially available kits. was extracted, which shows that exosomes are sufficiently separated by the method according to the present invention.

In order to confirm the stability of the nucleic acid obtained from the exosomes isolated by the present invention, a nucleic acid stability measuring instrument was used and compared with a control (separated by ExoQuick) (see FIG. 5). It can be seen in the figure that the stability of the control and the nucleic acids obtained by the examples according to the present invention are almost similar, and these stability results (RIN value range is integrity value from 0 to 10, RQI = RIN value: 7.9, 7.4, 8.9, 7.4).

As can be seen in the above examples, specific cells and exosomes were efficiently separated from one liquid sample according to the method according to the present invention.

On the other hand, in the above description, as an example, the liquid sample was separated into two and separately applied beads for exosome isolation and beads for specific cell isolation, respectively. However, if it is a mixed solution of only specific cells and exosomes, it will be very easy to separate specific cells and exosomes by a simple method such as centrifugation. Therefore, the present invention is capable of the following two modifications.

(1) Transformation 1

This is a method in which beads for exosome isolation and beads for specific cell isolation are separately manufactured and then reacted with one liquid sample. That is, (A) preparing beads for separating exosomes in which an exosome-specific antibody is bound to alginate beads and beads for separating specific cells in which an antibody specific for a specific cell is bound to alginate beads, respectively; (B) adding the beads for exosome isolation and the beads for specific cell isolation to the biological liquid sample at the same time and leaving it alone; (C) then separating the beads from the liquid sample; (D) then liquefying the separated beads or treating the beads with a reducing agent; and (E) separating cells and exosomes or exosome components from the liquefied or reducing agent-treated solution. A flow chart thereby is shown in FIG. 6 .

Since specific details for each step in the modified example may be the same as those described above, additional description is omitted.

(2) Transformation 2

This is a method in which an exosome-specific antibody and a specific cell-specific antibody are coupled to a bead for simultaneous separation of exosome-specific cells, and then reacted with one liquid sample. That is, (A) preparing a bead for separation in which an exosome-specific antibody is bound to alginate beads and an antibody specific to a specific cell is bound to alginate beads; (B) simultaneously adding the beads for separation to the biological liquid sample and leaving it alone; (C) then separating the beads from the liquid sample; (D) then liquefying the separated beads or treating the beads with a reducing agent; and (E) separating cells and exosomes or exosome components from the liquefied or reducing agent-treated solution. A flow chart thereby is shown in FIG. 7 .

Since specific details for each step in the modified example may be the same as those described above, additional description is omitted.

Claims (6)

(A) preparing beads for separating exosomes in which an exosome-specific antibody is bound to alginate beads and beads for separating specific cells in which a specific cell-specific antibody is bound to alginate beads, respectively;
(B) preparing a part of the supernatant as a sample for exosome isolation by centrifuging the biological liquid sample and remixing the remainder as a sample for specific cell isolation;
(C) adding the exosome isolation beads to the exosome isolation sample and the specific cell isolation beads to the specific cell isolation sample and leaving them alone;
(D) separating the exosome isolation beads and the specific cell isolation beads from the liquid sample;
(E) then separating exosomes or exosome components from the separated beads for separating exosomes, and separating specific cells from the beads for separating specific cells;
A method for simultaneously separating specific cells and exosomes using alginate, characterized in that it comprises a.
The method of claim 1,
In the step (A),
(a) a small step of preparing alginate beads by adding droplets of an aqueous solution of monovalent ionic bonded alginate to an aqueous solution of polycations;
(b) a small step of mixing the alginate beads with a specific cell-specific antibody or an exosome-specific antibody and allowing them to bind;
A method for simultaneously separating specific cells and exosomes using alginate, characterized in that it comprises a.
The method of claim 2,
Between the substeps (a) and (b),
(a') a substep of modifying the surface of the alginate beads;
Method for simultaneously separating specific cells and exosomes using alginate, characterized in that it further comprises.
The method of claim 1,
In the step (E),
(a) a substep of liquefying the separated beads or treating the beads with a reducing agent;
A method for simultaneously separating specific cells and exosomes using alginate, characterized in that it comprises a.
(A) preparing beads for separating exosomes in which an exosome-specific antibody is coupled to alginate beads and beads for separating specific cells in which an antibody specific for specific cells is coupled to alginate beads, respectively;
(B) adding the beads for exosome isolation and the beads for specific cell isolation to the biological liquid sample at the same time and leaving it alone;
(C) then separating the beads from the liquid sample;
(D) then liquefying the separated beads or treating the beads with a reducing agent;
(E) separating cells and exosomes or exosome components from the liquefied or reducing agent-treated solution;
A method for simultaneously separating specific cells and exosomes using alginate, characterized in that it comprises a.
(A) preparing beads for separation in which an exosome-specific antibody to alginate beads and a cell-specific antibody to alginate beads are combined together;
(B) simultaneously adding the beads for separation to the biological liquid sample and leaving it alone;
(C) then separating the beads from the liquid sample;
(D) then liquefying the separated beads or treating the beads with a reducing agent;
(E) separating cells and exosomes or exosome components from the liquefied or reducing agent-treated solution;
A method for simultaneously separating specific cells and exosomes using alginate, characterized in that it comprises a.

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