KR20160037818A - Novel Process of Preparing Cell Targeting Aptamers - Google Patents

Abstract

The present invention relates to a method of preparing an umbilical cord for binding to a new cell, and more particularly, it relates to a method for culturing a cell in a chip including a single or a plurality of chambers and treating the cell with an elution buffer to elute the cell- The present invention relates to a method of manufacturing an abutment.
Cell SELEX-on-a-chip was developed by applying a microfluidic device for specific elutriation without damaging the cells. By comparing Chip SELEX with conventional SELEX, it is shown that SELEX can be efficiently performed in SELEX-on-a-chip. And it can be applied to various cancer cell-specific platemers having excellent performance by using them.

Description

[0001] The present invention relates to a novel cell targeting method,

The present invention relates to a method of preparing an umbilical cord for binding to a new cell, and more particularly, it relates to a method for culturing a cell in a chip including a single or a plurality of chambers and treating the cell with an elution buffer to elute the cell- The present invention relates to a method of manufacturing an abutment.

Cell SELEX-on-a-chip was developed by applying a microfluidic device for specific elutriation without damaging the cells. By comparing Chip SELEX with conventional SELEX, it is shown that SELEX can be efficiently performed in SELEX-on-a-chip. And it can be applied to various cancer cell-specific platemers having excellent performance by using them.

With the development of drugs, many diseases are treatable, but there are still many diseases that need to be remedied. Above all, cancer is a representative disease that is difficult to completely cure. Most treatments require a combination of surgery and chemotherapy, but they affect whole cells in the human body, so it is difficult to say that there is a proper treatment method.

Recently, aptamer has been in the spotlight for the treatment and diagnosis of cancer. Aptamers are single-stranded oligonucleotides that form specific three-dimensional formation. Thereby allowing the aptamer to have high affinity and specificity for the target molecule. Although aptamers have similar properties to antibodies, they are smaller in molecular weight and can be synthesized more easily in vitro and are more stable at higher temperatures. In addition, toxicity and antigenicity are lower. The aptamer can be separated by an in vitro evolution process such as SELEX (systematic evolution of ligands by exponential enrichment). The target cell comprises 10 ¹⁴ to 10 ¹⁵ random oligonucleotides incubated with the ssDNA or RNA library, and then unbound sequences are washed to collect only the oligonucleotides bound to the cells.

A disadvantage of the conventional cell SELEX method is that cells may be damaged in the elution step, which is caused by the fact that the by-products from the cells can be obtained together with the combined platamer, since the cells are stripped from the plate.

Under these technical backgrounds, the inventors of the present application developed a novel cell SELEX method capable of eluting the platemma without damaging the cells, and completed the present invention.

The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .

And separating the platemers bound to the cells without heating the cells without damaging the cells. Chip SELEX is economical, effective, and can perform faster. Because the chip has a low volume, it can cultivate a variety of different cells without contamination.

The present invention provides a method for manufacturing an umbilical cord that binds to a cell comprising the steps of: culturing a cell in a chip comprising a single or multiple chambers; Binding the cell-specific platemer to the cell; Washing the cells with the aptamer bound thereto; And eluting the platamer bound to the cell with an elution buffer.

According to the present invention, instead of scraping off the cells, only the combined platemor is separated using the elution buffer, so that the platemers bound to the cells can be isolated without damaging the cells and without heating.

Figure 1 shows the shape of a chip according to the invention.
2 is a schematic diagram of a conventional SELEX and a chip SELEX according to the present invention.
Figure 3 shows the results of incubation with cy3-labeled platemer with cells, showing (A): BNL 1 ME A. 7R. 1 cell, (B) after incubation with platemper, BNL 1ME A. 7R. 1 cells. The combined platemor was eluted with elution buffer and (C): BNL 1ME A. 7R. 1 cells, (D) after incubation with elution buffer, BNL 1ME A. 7R. 1 < / RTI > cells.
4 is a graph showing normalized data of the elution amount in the conventional SELEX and the chip SELEX according to the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a method for producing an umbilical cord that binds to a cell comprising the steps of: culturing a cell in a chip comprising a single or a plurality of chambers; Binding the cell-specific platemer to the cell; Washing the cells with the aptamer bound thereto; And eluting the platamer bound to the cell with an elution buffer. This method can separate the platamer bound to cells without damaging the cells and without heating.

Biomarkers such as antibodies, platamers, or peptides may be used to diagnose a disease or to detect a given metabolite. As used herein, " platamer " refers to a small single stranded oligonucleotide capable of specifically recognizing a target material with high affinity. The aptamer can be obtained by a specific method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). In contrast to target-purified proteins, Cell SELEX is used to screen platelets targeting cell-specific cells. Cell SELEX may be a better option because it can screen for typing tamper in an in vivo environment. However, the conventional Cell SELEX has a large background problem, and in particular, instead of eluting the platemer, the entire cell was detached, thereby causing a long round of SELEX to obtain the platemer. Therefore, Cell SELEX-on-a-chip has been developed by applying a microfluidic device for specific elutriation without disturbing cells. By comparing Chip SELEX with conventional SELEX, it is shown that SELEX can be efficiently performed in SELEX-on-a-chip. And it can be applied to various cancer cell-specific platemers having excellent performance by using them.

Many diseases are being cured, but cancer and HIV disease still remain a therapeutic area. Significant research is underway for early diagnosis, identification of therapeutic targets, and identification of biomarkers for drug design, but conventional research methods are inefficient and time consuming. The present invention has developed a method for efficiently improving the conventional cell SELEX and cell SELEX-on-a-chip. Compared to conventional methods requiring large quantities of reagents and instrumentation, Ibidi® chips are capable of proceeding with far fewer volumes of reagents and cells. Furthermore, through solution-based elution, DNA contamination with cellular debris produced from the scraping method can be avoided. The efficiency of the new system is improved by more than six times as compared with the conventional Cell-SELEX method, and can be more efficiently used for generating an abdominal marker for a marker such as an cancer biomarker.

The term " Cell SELEX "as used herein refers to a method of selecting SELTEX, which is the most binding to the protein or structure existing on the cell surface, by performing SELEX on the whole living cell, unlike the SELEX method which targets the purified protein. Currently, the development of platamer for most cell surface receptors is carried out using purified proteins through overexpression in E. coli. The reason for using SELEX for live cells is because membrane proteins play an indispensable role in life, especially cell signaling, growth and proliferation, and cell-cell interactions. In cancer cells, cancerous mutations are caused by mutations in the function of cancer receptors that affect expression levels or cancer progression. The purified protein (recombinant protein) contains the same genetic information as the membrane protein on the cell surface, but is transformed or physiologically masked. Under these circumstances, aptamers may not be able to recognize unmodified proto-proteins on the cell surface.

However, in order to use aptamer as a therapeutic agent or as an in vivo diagnostic marker, it is necessary to be able to recognize a prototype of a protein in vivo, which has undergone post-translational modification. However, when cell-SELEX is simply performed, the kinds of receptors and surface proteins located on the cell surface are so complex and diverse that it is difficult to select an electrotamer that binds to a desired target receptor protein. The most challenging aspect of Cell-SELEX is the selection of platemas that bind only to the target cell line. Because the cell surface has more than one receptor, it is difficult to screen for tumors targeting cancer proteins due to the diversity of membrane proteins, and screening takes about 10 rounds or more. Positive cell lines and negative cell lines share a specific group of membrane proteins, which means that there is a high likelihood of selecting an umbilical cord that can bind to both cell lines. The cell concentration and number of passages should be carefully controlled in order to select affaterm with high affinity and specificity for positive cell lines.

In fact, recently, there has been an example of using cell-SELEX as a biomedical device by grafting a fluorescent substance to use as a tumor diagnosis or as a therapeutic agent, or using nanoparticles for drug delivery. In the past, diagnosis of cancer was made by the appearance of cells or tissue changes, but there was an example in which a cell-SELEX method was used to diagnose cancer by selectively applying strong tying of Burkitt's lymphoma cells. In addition, they have combined a platamer with a nanoparticle and a control-release polymer system to form a scaffold for selectively treating cancer cells.

In the present invention, a novel cell SELEX method capable of eluting the platemer without cell damage has been developed, and a larger amount of platemer can be obtained. For efficiency evaluation, the cell line BNL 1ME A.7R.1, a murine liver cancer cell, was obtained by transforming BNL CL.2 with methylcholanthrene epoxide, and TLS11 aptamer was transformed into BNL 1ME A Targeting .7R.1 cells is known. Experiments were conducted on the conventional method and the new method using these cell lines and compressors, using 100 mm and chips. The chip contains a smaller growth area than the 100 mm plate used in the conventional method, so less volume of reagent is sufficient. Instead of scraping the cells, an elution buffer was used to obtain the combined platamar (Figure 2). This makes it possible to isolate the bound plasters from the cells without cell damage and detachment. This method is expected to result in improved results in various aspects.

The definitions of the main terms used in the description of the present invention and the like are as follows.

As used herein, the term "nucleic acid " refers to single- or double-stranded DNA, RNA, and chemical matrices thereof, which do not interfere with amplification of the selected nucleic acid, including but not limited to, Methylation, an ideal salt phase combination, substitution of 5-bromo-uracil, and the like.

As used herein, the term " microfluidic device " refers to a device capable of controlling or manipulating micro-level microfluidic flow such as ㎕, nL, pL,

In one aspect of the present invention, there is provided a platemer that binds to cells using a multiplex microfluidic device for screening nucleic acid platelets, which can be applied to an improved SELEX (Systematic Evolution of Ligand by Exponential Enrichment) .

Multiplex microfluidic devices used in the present invention have significant advantages over macro scale cultures such as flasks, dishes and well-plates. First, it is highly flexible in the design of microfluidic devices, which allows individual cell types to be tailored to their intended purpose and enables co-culture of cells on the same chip. Moreover, under these microfluidic cell culture conditions, it is possible to mimic the natural microenvironment of the cell. This allows for continuous perfusion cultures and allows for the production and automation of chemical gradients, parallelization, on-chip analysis, or direct connection to a downstream analytical chemistry platform Allowing a small number of cells or single cells to be studied with high aspect and / or spatial resolution. At the same time, microfluidic cell culture allows for reduced consumption of reagents, reduced risk of contamination and high throughput experimentation. Comprehensive, flexible design, single-cell analysis, real-time on-chip analysis via time-lapse microscopy and low reagent consumption make microfluidic cell culture more viable for cell-SELEX applications .

The chip herein may comprise a single or multiple chambers and may be, for example, a--slide (ibidi)), having a 0.6-2.5 cm ² per channel growth area and a 30-100ul channel volume, Lt; / RTI > However, the single chamber may have a problem that a large amount of cells are detached from the washing stage, so that a plurality of chambers is preferable.

In one embodiment, the chamber may comprise 1 X ^{10 5} to 9 X 10 ⁵ cells. As confirmed in the examples, 5x10 < ⁶ > cells were used for the conventional SELEX and 3x10 < ⁵ > cells were used for the Chip SELEX. Since the chamber of the chip is small in volume, the amount of cells required is small. Approximately 10 ⁵ cells per chamber are sufficient.

In addition, the cell culture can be performed at 37 캜. As shown in the examples, when the cells were cultured at 37 ° C, the number of cells to be separated was small, which was appropriate. The elution buffer may be, for example, at least one selected from the group consisting of MgCl ₂ , NaCl, NaOH, and HCl, preferably MgCl ₂ . At this time, the concentration of the buffer may be, for example, 5 mM to 5 M, preferably 100 mM to 3M, and more preferably 500 mM. In addition, the elution buffer can be treated for 10 to 30 minutes, and as confirmed in the examples, 30 minutes is preferred, with the highest concentration of platemer obtained.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

[Example 1]

1-1. Cell culture

BNL.1.ME.7R.1 cells were purchased from American Tissue Culture Collection (ATCC TIB-73), which is a murine liver cancer cell line, and BNL CL.2 was transformed with methylcholanthrene epoxide Lt; / RTI >

The culture medium was DMEM supplemented with 10% serum and incubated at 37 ° C in a 5% CO ₂ incubator. To find out how much seeding amount in the chip should be suitable for the experiment, we looked for the point at which the amount of cells doubled. As a result, it was observed that the cell volume was four times that of the original cell volume and four times that of the cell volume after one day. When the seeding number was ^{1X10 5, 2X10 5, 4X10 4} / 100mm.

1-2. cell Set-up for SELEX on-a-chip

New cells using μ-slides To set up the optimal experimental conditions for SELEX, the following tests were performed:

First, a temperature test was performed to determine conditions suitable for bonding. In general, cell SELEX binding is at 4 ° C, and when too much cells are isolated at 4 ° C, they are performed at 37 ° C. Both conditions were tested. About 30-40% of the cells were isolated from the chip surface at 4 ° C, and only about 20% of the cells were isolated at 37 ° C.

Second, four DNA elution buffers were tested at different concentrations (HCl: 5 mM, 10 mM, 25 mM, 75 mM, 100 mM, 200 mM, 300 mM, and 400 mM based on HCl, NaOH, MgCl2 or NaCl 50 mM, 100 mM, 500 mM, 1 M, 2 M, 3 M, 4 M; MgCl 2: 100 mM, 500 mM, 1 mM NaCl: 5 mM, 10 mM, 25 mM, 75 mM, M, 2 M, 3 M, 4 M, 5 M).

After checking the performance of the other buffer to elute DNA from the cells, the best performing candidates were obtained: HCl: 200 mM, 300 mM, 400 mM; NaCl: 2M, 3M; MgCl ₂ : 500 mM, 1 M. Finally, 500 mM MgCl ₂ was selected as the elution buffer. The HCl was too acidic to the cells and had a problem. The NaCl concentration was too high for the MgCl ₂ concentration and the DNA elution showed similar performance.

Third, the time required for eluting the DNA without damaging the cells was determined. After binding, the cells were incubated with the elution buffer for 10, 20, or 30 minutes. Then, the eluted DNA was quantified by RT-PCR. 3.34 x 10 ⁶ , 9 x 10 ⁷ , and 2 x ^{10 8} cells were obtained for 10, 20 and 30 min elution times, respectively. Through this, it was confirmed that 30 minutes was the most suitable elution time.

In summary, the elution buffer used for the Chip SELEX was tested by using MgCl ₂ , NaOH, NaCl, HCl, etc. at concentrations of 500 mM MgCl ₂ , .

When cell SELEX binding temperature condition was tested, 37 ℃ was most suitable.

The optimal elutation time was 30 minutes after Q-PCR, when elution time was different after binding to find optimal binding and elution times.

1-3. Cell SELEX

400 pmole of SELEX 5'-Cy3-labeled TLS11 platamer (5'-cy3-ACGCTCGGATGCCACTACAGGCATCCCCATGTGAACAATCGCATTGTGATTGTTACGGTTTCCGCCTCATGGACGTGCTGGTGAC, Bioneer: SEQ ID NO: 1) was prepared. It has already been demonstrated that TLS11 aptamers target BNL 1ME A.7R.1 cells. It was boiled at 95 ° C for 5 minutes to form a secondary structure and cooled on ice for 3 minutes. 5 × 10 ⁶ cells were washed twice with 5 ml of wash buffer [5 mM MgCl ₂ , 4.5 g glucose / L of DPBS] and then washed with 5 ml binding buffer [4.5 g / L glucose, 5 mM MgCl ₂ , 0.1 mg / ml yeast tRNA, and 1 mg / mL bovine serum albumin (BSA) in DPBS) were added to the plates and incubated for 30 minutes at 37 ° C in a 5% CO ₂ incubator. After 30 minutes, the supernatant was removed and washed twice with 5 ml of wash buffer. The cells of the plate were then scraped into the wash buffer and centrifuged at 1000 rpm for 3 minutes. After that, the supernatant was removed and the pellet was loosened with wash buffer.

Heat was applied to the cells for 5 minutes at 95 ° C to remove the platamer. Then return the Sentry for 5 minutes at 13200 rpm. At this time, only the supernatant was obtained separately, and the solution was subjected to PCI phenol: chloroform: isoamyl alchohol (PCI,

1-4. Chip cell SELEX μ-slide chip contains six chambers. 3 pmole of cy3-labeled TLS11 aptamer was required for each chamber, so a total of 18 pmoles of the platamer was prepared. It was boiled at 95 ° C for 5 minutes to form a secondary structure and cooled on ice for 3 minutes. And each chamber contains the 5x10 ⁴ of each cell, using a wash buffer of 30㎕ was washed two times prior. Then cytoplasmic (cy3-labeled TLS11 aptamer) was incubated with binding buffer dissolved at 37 ° C for 30 min. At the end of the incubation, the unbound aptamer was washed twice with 30 μl wash buffer, and the supernatant was collected in another tube. At this time, fluorescence microscopy confirmed whether aptamer was bound to each cell.

30 μl of elution buffer [500 mM MgCl ₂ ] was added to detach the urethromer from the cells and allowed to stand at room temperature for 30 minutes. The cells were then washed twice with 30 μl of elution buffer and the chip was confirmed by fluorescence microscopy to see if the aptamer was well removed from the cells. The aptamers obtained from the elution process were prepared with ethanol through the process of PCI phenol: chloroform: isoamyl alchohol (PCI, sigma).

1-5. Realtime-PCR for quantification.

To carry out real-time PCR (RT-PCR), 20 μl of a mixture was prepared using Power SYBR Green PCR Mastermix (Applied Biosystems). 1 μl of forward primer (ACG CTC GGA TGC CAC TAC AG, Bioneer: SEQ ID NO: 2) and reverse primer (GTC ACC AGC ACG TCC ATG AG, Bioneer: SEQ ID NO: 3) Template pressure tamer.

The heating cycling protocol is as follows. Denaturation was carried out at 95 ° C for 10 minutes, and 95 cycles of 30 seconds → 52 ° C for 1 minute → 72 ° C for 1 minute were repeated for 40 cycles. After amplification, the PCR product was inactivated for 15 seconds at 95 ° C. The melting curve was analyzed at 52 ° C and 95 ° C with a temperature gradient of 0.3 ° C / s. And amplified only specific products.

[Results and Analysis]

The aptamer was successfully eluted without separation of the chip from the surface (Fig. 3). 3A and 3C, it was confirmed by an optical microscope that a large number of cells were attached to the surface of the chip after elution.

In addition, fluorescence signals were observed when the cells were observed by fluorescence microscopy after binding, confirming that the cyto-3 labeled aptamer was attached to the surface of the cells. However, after elution, the fluorescence signal was greatly reduced, thus confirming that the cyto-3 labeled aptamer was successfully desorbed from the surface of the cells (Fig. 3b, 3d).

The difference in efficiency between chip cell SELEX and conventional cell SELEX was measured by quantitative analysis using RT-PCR. As shown in Table 1, 18 pmole (1.08 × ^{10 13} ) aptamers were used for Chip SELEX and 1.11 × ^{10 11} aptamers were eluted. In the conventional SELEX process, 2.4 × ^{10 14} aptamers were used and 4.07 × ^{10 11} aptamers were eluted. Therefore, it was confirmed that Chip SELEX according to the present invention uses about 22 times smaller amount of platamer. Regarding the plumma extracting rate, it proves that the Chip SELEX according to the present invention is more efficient than the conventional SELEX method.

[Table 1]

Conventional Cell SELEX and Chip Cell SELEX use different amounts of aptamers and are normalized for more accurate calculations (FIG. 4). After normalization, it was confirmed that Chip Cell SELEX according to the present invention obtained about 6.07 times more elute than the conventional Cell SELEX method, which proves that the Chip Cell SELEX method according to the present invention is more efficient than the conventional method.

In experiments demonstrating the efficiency of the new method of SELEX using the chip, 5x10 ⁶ cells were used in the conventional method, 40 pmol of aptamer was used, and a total of 30.0 ml of buffer was used. On the other hand, Chip SELEX used 3x10 ⁵ cells, 10 pmol of aptamer, and 1.44 ml of buffer. Compared to the materials used in the experiment, Chip SELEX required 16 times fewer cells, 22 times less platamer, and 21 times less buffer than the conventional method.

Conventional methods require a flask or plate, which results in a very large amount of cells, an embryo, and a buffer. Also, the cells have to be scratched off the plate, damaging the cells and causing them to burst. However, in the Chip SELEX technology according to the present invention, the elution buffer is used in the elution step, and the tympanum can be obtained without the by-products in dead cells without affecting the cells.

As a result, the Chip SELEX according to the present invention is economical, effective and can be performed faster. Because the chip has fewer volumes, you can cultivate a variety of cells without contamination. It may be preferred to cultivate hard-to-obtain cells or small amounts of cells.

Also, the Chip SELEX according to the present invention can be used for multiplex selection. Since the chip contains six chambers, it is possible to culture other types of cell lines without contamination. Since the chamber of the chip is small in volume, the amount of cells required is small. Approximately 10 ⁵ cells per chamber are sufficient.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Novel Process of Preparing Cell Targeting Aptamers <130> P14-B303 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Aptamer <400> 1 acgctcggat gccactacag gcatccccat gtgaacaatc gcattgtgat tgttacggtt 60 tccgcctcat ggacgtgctg gtgac 85 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 acgctcggat gccactacag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gtcaccagca cgtccatgag 20

Claims (7)

A method for preparing an abdominal cell binding to a cell comprising the steps of:
Culturing the cells in a chip comprising a single or a plurality of chambers;
Binding the cell-specific platemer to the cell;
Washing the cells with the aptamer bound thereto; And
And eluting the platemer bound to the cell with an elution buffer.
The method according to claim 1,
The chamber aptamer method comprising the 1X10 ⁵ ~ 9X10 ⁵ cells.
The method according to claim 1,
Lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt;
The method according to claim 1,
The elution buffer is aptamer method characterized in that it comprises at least one selected from the group consisting of MgCl _2, NaCl, NaOH and HCl.
The method according to claim 1,
The elution buffer is aptamer method, characterized in that MgCl _2.
6. The method of claim 5,
Wherein the elution buffer has a concentration of 5mM-5M.
The method according to claim 1,
Wherein the elution buffer is treated for 10 to 30 minutes.

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