KR102246224B1 - A preparation method of cellular spheroid and cellular spheroid made thereby - Google Patents

Abstract

The present invention relates to a method for preparing a cellular spheroid in a structure by dispensing a liquid drop including heat-sensitive hydrogel, an ionic solution and cells in ion-crosslinking hydrogel to form a core-shell structure in-situ, and the cellular spheroid prepared by the method. Due to excellent repeatability, quality control and rapid mass production are possible.

Description

Method for producing a cell aggregate, and a cell aggregate produced by this method {A PREPARATION METHOD OF CELLULAR SPHEROID AND CELLULAR SPHEROID MADE THEREBY}

The present invention relates to a method for preparing a cell aggregate inside the structure by dispensing a droplet including a heat-sensitive hydrogel, an ionic solution, and a cell into an ion-crosslinked hydrogel to form a core-shell structure in-situ. .

Most of the cellular structures that make up the human and animal body are organized in a three-dimensional form. The three-dimensional structure of these cells results in interactions between complex cells that are difficult for two-dimensional monolayer cell cultures to mimic.

Cells behave more naturally when cultured in a three-dimensional environment, but formation of three-dimensional cell cultures is mostly difficult and expensive. Existing in vitro cell culture can only provide a typical two-dimensional cell culture model.

Cellular spheroid is an example of a typical three-dimensional cell culture model, and has been applied to basic research and clinical pharmacology. These cell aggregates refer to clusters of aggregated cells having a diameter of about several hundred micrometers.

As the conventional method of forming cell aggregates, dynamic culture methods such as hanging drop method or rotary agitation exist, but cultivation of cell aggregates according to these methods may take up to about 4 days, and the success rate is also It stays at about 50%, and it is difficult to exchange the cell culture solution, so there is a problem that cell loss occurs during the exchange process.

FIG. 1 exemplarily shows several methods for preparing conventional cell aggregates, FIG. 1(a) is a scaffold dish capable of preparing cell aggregates, and FIG. 1(b) shows cells in vitro. It schematically shows the process of agglutination, and FIG. 1(c) schematically shows the process of preparing a cell aggregate by a hanging drop method.

As another conventional method for producing cell aggregates, there is a method of injecting cells into microwells to which cells cannot be attached, and then inducing aggregation of the injected cells to prepare cell aggregates.

In the case of this method, in order to fabricate a microwell, a complicated and long micromachining process is required that accompanies photolithography and soft-lithography.

In the method for producing cell aggregates using such microwells, there is still a problem in that it is difficult to control the size of the cell aggregates, and the process of recovering or harvesting the produced cell aggregates is difficult.

Publication Patent No. 2018-0032597

In order to effectively solve the problems of the prior art discussed above, the present invention discharges a first composition comprising a heat-sensitive hydrogel and cells dissolved in an ionic solution as a second hydrogel, and discharges the first composition from the second hydrogel. After the second hydrogel reacts with the ionic solution along the outer circumferential surface of the first composition to form a crosslinked hydrogel membrane (shell), cells are precipitated and aggregated inside the crosslinked hydrogel membrane, thereby forming cell aggregates. It provides a method for continuously preparing and cell aggregates prepared by this method. In addition, it is to further provide a method of obtaining exosomes from the cell aggregates that are continuously produced and recovered.

A method of preparing a cell aggregate according to an embodiment of the present invention includes: preparing a first composition comprising a heat-sensitive hydrogel and cells dissolved in an ionic solution; A dispensing step of discharging the first composition as a second hydrogel; Forming a crosslinked hydrogel membrane (shell) by reacting a second hydrogel with the ionic solution along the outer circumferential surface of the first composition discharged from the second hydrogel; And forming a cell aggregate in which cells are precipitated and aggregated in the crosslinked hydrogel membrane.

The heat-sensitive hydrogel preferably includes at least one or more selected from the group consisting of gelatin, Pluronic F127, or Poly(N-isoprolylacrylamide), and the second hydrogel is selected from the group consisting of alginate and chitosan It may contain any one or more, and the ionic solution is more preferably containing any one or more selected from the group consisting of calcium and potassium.

In the step of forming the cell aggregate, the temperature may be changed so that the phase of the heat-sensitive hydrogel in the crosslinked hydrogel membrane (shell) is changed, and in the dispensing step, the size of the first composition to be discharged is, It is preferable to control through a change in the discharge condition, and the discharge condition may be a discharge time or a discharge volume.

After the step of forming the cell aggregate, a recovery step of recovering the cell aggregate by decomposing the crosslinked hydrogel membrane (shell); may be further included.

In another embodiment of the present invention, a cell aggregate prepared by this method may be mentioned, and a method of obtaining an exosome from such a cell aggregate may also be included.

The method for producing a cell aggregate according to the present invention has high reproducibility compared to the conventional method for producing a cell aggregate, which is difficult to mass-produce due to the problem of high variability per worker, labor intensive, and time-consuming, and difficulty in quality control. As it is excellent, there is an advantage that enables quality control and rapid mass production.

In addition, as cells are aggregated in empty spaces in the crosslinked hydrogel membrane (shell), by controlling the amount of the composition to be discharged, not only the size of the obtained cell aggregate can be controlled, but also contamination of the cells can be effectively prevented. , There is an effect of obtaining a stable cell aggregate without damage.

Through the method for producing a cell aggregate according to the present invention, uniform and stable cell aggregates can be produced in large quantities with high yield, and there is a peak that can effectively obtain exosomes from the cell aggregates thus prepared.,

In addition, the cell aggregate prepared according to an embodiment of the present invention has an effect that can be used in various bio industries, such as a drug test chip for new drug development and the production of various artificial tissue mimetics for tissue regeneration.

1 schematically shows a method for preparing a conventional cell aggregate.
2 schematically shows a process of preparing a cell aggregate according to an embodiment of the present invention.
3 is a result of observing a change in the size of a hollow sphere-shaped bead in which a crosslinked hydrogel film is formed according to a discharge volume in the manufacturing process according to an embodiment of the present invention.
4 is a result of sequential observation of a process of obtaining an actual cell aggregate by a manufacturing method according to an embodiment of the present invention.

Before describing in detail through a preferred embodiment of the present invention, terms or words used in the claims of the present specification should not be construed as being limited to a conventional or dictionary meaning, but a meaning consistent with the technical idea of the present invention. And should be interpreted as a concept.

Throughout the present specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

Hereinafter, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art can implement various modified forms of the contents described in the present specification within the scope of the present invention.

A method of preparing a cell aggregate according to an embodiment of the present invention includes: preparing a first composition comprising a heat-sensitive hydrogel and cells dissolved in an ionic solution; A dispensing step of discharging the first composition as a second hydrogel; Forming a crosslinked hydrogel membrane (shell) by reacting a second hydrogel with the ionic solution along the outer circumferential surface of the first composition discharged from the second hydrogel; And forming a cell aggregate in which cells are precipitated and aggregated in the crosslinked hydrogel membrane (see FIG. 2).

The ionic solution is not particularly limited as long as it is an ionic solution capable of easily dissolving the heat-sensitive hydrogel and crosslinking the second hydrogel, and preferably containing at least one selected from the group consisting of calcium and potassium, and calcium chloride. An aqueous solution can be used.

The first composition is a homogeneous mixture of living cells in an ionic solution in which a heat-sensitive hydrogel is dissolved, ^{and may contain about 10 3} to 10 ⁸ living cells, and the ion concentration contained in the ionic solution is about 10 to It may be included in the range of 700 mM, but if the ion concentration is too low, the hydrogel film formed in the subsequent process may not be formed, and if the ion concentration is too high, the thickness of the formed shell becomes too thick to form cell aggregates inside. There may be a problem in that the space becomes insufficient or the shell thickness is formed unevenly.

The heat-sensitive hydrogel can be dissolved in a range of about 1 to 10 parts by weight based on 100 parts by weight of the ionic solution, and if it is out of the above composition range, it is difficult to discharge in the subsequent dispensing step, or the discharged shape is uniform. I can't.

In the dispensing step, the first composition having such a composition component and composition range is discharged to a storage tank in which the second hydrogel is stored through a pipette, a dispenser, or a 3D printer (see process A in FIG. 2). In this dispensing step, it is preferable that the first composition is uniformly discharged in a certain amount, since this may affect the uniformity of the cell aggregates formed in the subsequent step and the production rate of exosomes produced from such cell aggregates. to be.

When the first composition discharged to the storage tank in which the second hydrogel is stored is in contact with the second hydrogel, the ionic solution contained in the first composition and the second hydrogel react in-situ to crosslink, thereby 1 A hydrogel film is formed on the outer periphery of the composition (see process B in FIG. 2).

The hydrogel membrane formed along the outer circumferential surface of the discharged first composition is formed by performing an instantaneous crosslinking reaction of the second hydrogel by the ionic solution, so that the inside is a living cell, a heat-sensitive hydrogel, and a hydrogel membrane (shell A hollow structure in which an ionic solution that did not participate in the formation of) is formed.

The heat-sensitive hydrogel may be at least any one or more selected from the group consisting of gelatin, Pluronic F127, or Poly(N-isoprolylacrylamide), and the second hydrogel may be any selected from the group consisting of alginate and chitosan. More than one may be used.

Such a heat-sensitive hydrogel may change its phase through a temperature change in the step of forming a cell aggregate to be described later, for example, by raising the temperature of the storage tank in which the second hydrogel is stored to about 30 degrees or more, It is possible to change the gel phase of the heat-sensitive hydrogel present inside the hydrogel membrane (shell) formed in the form of a hollow sphere formed in-situ into a liquid phase.

The heat-sensitive hydrogel preferably includes at least one or more selected from the group consisting of gelatin, Pluronic F127, or Poly(N-isoprolylacrylamide), and the second hydrogel is selected from the group consisting of alginate and chitosan It may contain any one or more, and the ionic solution is more preferably containing any one or more selected from the group consisting of calcium and potassium.

By changing the phase of the heat-sensitive hydrogel that exists inside the hydrogel hollow membrane (shell), the cells that exist together with the heat-sensitive hydrogel inside the hydrogel hollow membrane (shell) are naturally softened by gravity. It settles down the inside of the hollow membrane (see process C in FIG. 2).

The cells thus sedimented go through the step of forming a cell aggregate.By changing the temperature so that the phase of the heat-sensitive hydrogel in the crosslinked hydrogel membrane (shell) is changed, the inside of the hydrogel hollow membrane (shell) According to the precipitation of the cells, cells naturally form cell aggregates with each other (see process D in FIG. 2).

Therefore, in the dispensing step, the size of the first composition to be discharged is preferably controlled through a change in discharge conditions, and through a discharge time or discharge volume, a hydrogel hollow membrane (shell This is because the size of) is determined.

When the cell aggregate is formed in the hydrogel hollow membrane (shell), a recovery step of decomposing the crosslinked hydrogel membrane (shell) to recover the cell aggregate; may be further performed. The process of decomposing the crosslinked hydrogel membrane (shell) can be carried out through various known methods, for example, decomposition using enzymes, heat, electric field, magnetic field, etc. It can be done by mixing.

As another embodiment of the present invention, there may be mentioned a three-dimensional cell aggregate prepared through these processes. The three-dimensional cell aggregate according to the present invention is the most similar to the actual cell aggregate compared to the cell aggregate obtained through the conventional two-dimensional culture. It has a characteristic, and there is an advantage that a large amount of exosomes can be obtained more easily even without applying a separate external stimulus or condition.

Accordingly, another embodiment of the present invention includes a method of obtaining an exosome from a cell aggregate obtained by the method of producing such a cell aggregate, and an exosome obtained in this manner.

Hereinafter, specific actions and effects of the present invention will be described through specific embodiments of the present invention. However, this has been presented as a preferred example of the present invention, and the scope of the present invention is not limited according to the embodiment.

[Production Example 1: Preparation of the first mixture]

6 x 10 ⁵ cells (ex. MIN-6 cells) were prepared through cell culture, and the cells were suspended in 1 ml of culture media, so that the cells were uniformly distributed in the culture medium.

After dissolving gelatin powder (cold water fish, or porcine skin) in distilled water to prepare 1 ml of a 10 wt% gelatin solution, CaCl ₂ (Calcium chloride, Molecular Weight: 111.0, Sigma Aldrich) was added to the gelatin solution to a concentration of 500 mM. By mixing, a bio-ink (first mixture) containing 5 wt% gelatin and cells was prepared.

[Production Example 2]

After preparing a 5% Alginate solution and filling it in a container with an open top, the container was placed under a volumetric precision dispenser, and the bioink (first mixture) prepared in Preparation Example 1 was transferred to a syringe for a volumetric precision dispenser. After containing, residual bubbles inside the syringe and the volumetric precision dispenser were removed.

The dispenser with an inner diameter of 200 μm is discharged under the conditions of a preset discharge volume (range of 100 nl to 700 nl) and discharge time (1 second per shot). At this time, position adjustment was performed so that the bioink (first mixture) discharges discharged through the drive control of the x-y-z stage interlocked with the dispenser do not overlap each other and maintain a certain distance.

The calcium ions contained in the discharged bio-ink are ion-cured in-situ as the alginate solution filling the container contacts, and the bio-ink, the first mixture, forms beads in the shape of a hollow sphere.

At this time, the size of the hollow sphere-shaped beads generated while changing the volume of the bio-ink discharged was confirmed, and the results are summarized in Table 1 and FIG. 3 below.

Bio ink discharge volume [nl] 100 200 700 Discharge time [sec] One Nozzle size [㎛] 200 Bead diameter in the form of a hollow sphere
[What is the unit?] One 1.4 2 Figure 3 (a) (b) (c)

As can be seen from the results of Table 1 and FIG. 3, by controlling the discharge volume of the bio-ink (first mixture), it was found that the diameter of the hollow sphere-shaped beads generated in-situ is effectively controlled.

This discharge process was performed at room temperature, and it is possible to add a Peltier cooling system having a temperature control function along the outer circumferential surface of the dispenser syringe, if necessary.

[Production Example 3]

Bio-ink 200nl was discharged in the same manner as in Preparation Examples 1 and 2, and fluorescence staining was performed to observe the aggregation process of cells in the hollow sphere-shaped beads.

Cell tracking dye is a non-fluorescent dye, but it is a hydrophobic substance that emits fluorescence when it enters a living cell. It can be easily transmitted to living cells, and a fluorescent substance remains after cell division, so that it can be confirmed even in proliferating cells.

In this preparation example, MIN-6 cells were fluorescently stained using a cell tracking dye kit. 5 x 10 ⁶ live cells were prepared, washed with 1x PBS, followed by adding a tracking dye green working solution, and reacted at about 37°C for about 30 minutes. After washing three times with HHBS, the cells were pelleted using a centrifuge, and then 1ml DMEM was added to make a cell suspension, and then mixed with a hydrogel to perform the same procedures of Preparation Examples 1 and 2, Cells stained with fluorescence at a wavelength of 490/525 nm were confirmed, and the results are also shown in FIG. 4 for each process.

As can be seen from the results of FIG. 4, by discharging bio-ink using a volumetric precision dispenser, a hollow sphere-shaped bead was formed in the alginate solution, and alive in a hollow sphere-shaped bead. Cell aggregates of cells could be identified.

The present invention is not limited to the specific embodiments and description described above, and any person with ordinary knowledge in the technical field to which the present invention pertains without departing from the gist of the present invention claimed in the claims can implement various modifications. And, such modifications are within the scope of protection of the present invention.

10: nozzle
100: heat sensitive hydrogel
200: ion
300: cell
350: first composition
400: second hydrogel
500: hydrogel membrane
600: precipitated cells
700: cell aggregate

Claims (10)

In the method of producing a cell aggregate,
Preparing a first composition comprising a heat-sensitive hydrogel and cells dissolved in an ionic solution;
A dispensing step of discharging the first composition drops into a second hydrogel;
Forming a crosslinked hydrogel membrane (shell) by reacting a second hydrogel with the ionic solution along the outer circumferential surface of the first composition drop discharged into the second hydrogel to form a core-shell structure;
Changing the temperature so that the phase of the heat-sensitive hydrogel in the crosslinked hydrogel membrane is changed from a gel phase to a liquid phase; And
A method for producing a cell aggregate comprising a; step of forming a cell aggregate in which the inside of the crosslinked hydrogel membrane is changed to a liquid state, and the cells are precipitated and aggregated. The method of claim 1,
Heat-sensitive hydrogel, characterized in that it comprises at least any one or more selected from the group consisting of gelatin, Pluronic F127 or Poly (N-isoprolylacrylamide), a method for producing a cell aggregate. The method of claim 1,
The second hydrogel, characterized in that it comprises any one or more selected from the group consisting of alginate and chitosan, a method for producing a cell aggregate. The method of claim 1,
The ionic solution, characterized in that it contains any one or more ions selected from the group consisting of calcium and potassium, a method for producing a cell aggregate. delete The method of claim 1,
In the dispensing step, the size of the first composition to be discharged is controlled through a change in discharge conditions. The method of claim 6,
The discharge condition is a method of producing a cell aggregate, characterized in that the discharge time or the discharge volume. The method of claim 1,
After the cell aggregate formation step, a recovery step of recovering the cell aggregate by decomposing the crosslinked hydrogel membrane (shell); a method for producing a cell aggregate further comprising. The cell aggregate produced by the method according to any one of claims 1 to 4 and 6 to 8. A method for obtaining exosomes from the cell aggregate according to claim 9.

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