KR102780671B1 - A preparation method of extracellular vesicles with cellular spheroid and extracellular vesicles made thereby - Google Patents

Abstract

The present invention relates to a method for forming a core-shell structure in-situ by dispensing a droplet containing a thermosensitive hydrogel, an ionic solution, and cells into an ionically cross-linked hydrogel, thereby producing a cell aggregate inside the structure, and to a cell aggregate produced by this method. The method has the advantage of excellent reproducibility, thereby enabling quality control and rapid mass production.

Description

Method for preparing cell aggregates or extracellular vesicles and extracellular vesicles prepared by the method {A PREPARATION METHOD OF EXTRACELLULAR VESICLES WITH CELLULAR SPHEROID AND EXTRACELLULAR VESICLES MADE THEREBY}

The present invention relates to a method for producing extracellular vesicles by dispensing a thermosensitive hydrogel, an ionic solution, and a droplet containing cells into an ionically cross-linked hydrogel to form a core-shell structure in-situ , producing cell aggregates inside the structure, and then culturing the cell aggregates in 3D, and to extracellular vesicles produced by the method.

[Project ID Number] S3197954

[Ministry Name] Ministry of SMEs and Startups

[Research Management Specialist Agency] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Small and Medium Enterprise Technology Development Support Project

[Research Project Name] Development of High-Efficiency Extracellular Vesicle Production and Extracellular Vesicle Therapeutic Technology

[Contribution rate] 50/100

[Organization] Spebio Co., Ltd.

[Research Period] 2021.10.01. ~ 2023.09.30.

[Project ID Number] 2021-IT-RD-0042-01

[Ministry Name] Ministry of Science and ICT

[Research Management Specialist Agency] Research and Development Special Zone Promotion Foundation

[Research Project Name] Technology Transfer Commercialization (Gangso History)

[Research Project Name] Technology for producing extracellular vesicles using 3D bioprinting-based cell spheroids

[Contribution rate] 50/100

[Organization] Spebio Co., Ltd.

[Research Period] 2021.07.01. ~ 2023.06.30

Most of the cellular structures that make up the human and animal bodies are organized in three-dimensional configurations. This three-dimensional structure of cells results in complex interactions between cells that are difficult to replicate in two-dimensional monolayer cell cultures.

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

Cellular spheroids are an example of a typical three-dimensional cell culture model and are applied in basic research and clinical pharmacology. These cellular spheroids refer to aggregated cell clusters with a diameter of about several hundred micrometers.

As a method for forming existing cell aggregates, there are dynamic culture methods such as the hanging drop method or rotary stirring, but the culture of cell aggregates using these methods can take up to about 4 days, and the success rate is only about 50%. In addition, there is a problem that cell loss occurs during the exchange process because it is difficult to exchange the cell culture medium.

Among existing methods for producing other cell aggregates, there exists a method for producing cell aggregates by injecting cells into microwells to which cells cannot attach and then inducing aggregation of the injected cells.

For these methods, a complex and lengthy microfabrication process involving photolithography and soft lithography is required to produce microwells.

The method for manufacturing cell aggregates using these microwells also still has the problem that it is difficult to control the size of the cell aggregates and that the process of recovering or harvesting the manufactured 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 provides a method for continuously manufacturing a cell aggregate by discharging a first composition including a thermosensitive hydrogel and cells dissolved in an ionic solution into a second hydrogel, causing the second hydrogel to react with the ionic solution along the outer surface of the first composition discharged within the second hydrogel to form a cross-linked hydrogel membrane (shell), and then causing cells to precipitate and aggregate inside the cross-linked hydrogel membrane, and a cell aggregate manufactured by this method. In addition, the present invention provides a method for obtaining extracellular vesicles by three-dimensionally culturing the cell aggregate manufactured in this way.

In addition, in the dispensing step of discharging a composition drop containing a hydrogel and cells into another hydrogel, the volume of the composition drop discharged at one time can be reduced by using a separate external force generating device such as an electric field or magnetic field rather than relying solely on gravity. This increases the number of drops that can be discharged using the same amount of the hydrogel and cell-containing composition, thereby improving the process efficiency for continuously manufacturing cell aggregates.

This is a method for manufacturing a cell aggregate according to one embodiment of the invention, comprising: a step of preparing a first composition including a hydrogel and cells; a dispensing step of discharging a drop of the first composition into a second hydrogel; a step of forming a core-shell structure by allowing the second hydrogel to react with the ionic solution along an outer surface of the first composition drop discharged into the second hydrogel to form a cross-linked hydrogel shell; a step of changing a temperature so that a phase of a thermosensitive hydrogel within the cross-linked hydrogel shell changes from a gel phase to a liquid phase; And the inside of the cross-linked hydrogel film changes into a liquid phase, and the cells are precipitated and aggregated to form a cell aggregate; and in the dispensing step, an external force is applied around the nozzle where the first composition drop is formed, thereby discharging a drop having a reduced volume compared to the drop volume discharged by gravity alone.

It is preferable that the above first composition includes a thermosensitive hydrogel and cells dissolved in an ionic solution.

The above external force can be selected from a force generated by an electric or magnetic field, a force caused by an airflow, a force caused by vibration applied to a nozzle, and a force resulting from depressurization.

The thermosensitive hydrogel preferably comprises at least one selected from the group consisting of gelatin, Pluronic F127 or Poly(Nisoprolylacrylamide), the second hydrogel preferably comprises at least one selected from the group consisting of alginate and chitosan, and the ion solution may comprise at least one ion selected from the group consisting of calcium and potassium.

In the above dispensing step, the size of the first composition to be discharged can be controlled by a change in the discharge conditions or the size of the external force, and it is preferable that the discharge conditions are the discharge time or the discharge volume.

Another embodiment of the invention may include a cell aggregate obtained by this method, a method for culturing this cell aggregate to obtain extracellular vesicles, and extracellular vesicles obtained by this method.

This is another embodiment of the invention, comprising: a step of preparing a first composition including a hydrogel and cells; a dispensing step of discharging a drop of the first composition into a second hydrogel; a step of forming a core-shell structure by allowing the second hydrogel to react with the ionic solution along an outer surface of the first composition drop discharged into the second hydrogel to form a cross-linked hydrogel shell; a step of changing a temperature so that a phase of a thermosensitive hydrogel in the cross-linked hydrogel shell changes from a gel phase to a liquid phase; a step of forming a cell aggregate in which the inside of the cross-linked hydrogel shell changes into a liquid phase and the cells are precipitated and aggregated; And a step of culturing the cell aggregate to obtain extracellular vesicles; and in the dispensing step, an external force is applied around a nozzle where a first composition drop is formed, thereby discharging a drop having a reduced volume compared to a drop volume discharged by gravity alone. A method for producing extracellular vesicles using cell aggregates is provided.

In addition, in another embodiment, a step of preparing a first composition including a hydrogel and cells; a dispensing step of discharging a drop of the first composition into a second hydrogel; a step of forming a core-shell structure by allowing the second hydrogel to react with the ionic solution along an outer surface of the first composition drop discharged into the second hydrogel to form a crosslinked hydrogel shell; a step of changing a temperature so that a phase of a thermosensitive hydrogel in the crosslinked hydrogel shell changes from a gel phase to a liquid phase; a step of forming a cell aggregate in which the inside of the crosslinked hydrogel shell changes to a liquid phase and the cells are precipitated and aggregated; a step of freezing and storing the cell aggregate obtained in the cell aggregate forming step; a step of thawing the frozen-stored cell aggregate; And a step of culturing the thawed cell aggregate to obtain extracellular vesicles; and in the dispensing step, an external force is applied around the nozzle where the first composition drop is formed, thereby discharging a drop having a reduced volume compared to the drop volume discharged by gravity alone. A method for producing extracellular vesicles using cell aggregates may be included.

The method for producing cell aggregates according to the present invention has the advantage of excellent reproducibility, enabling quality control and rapid mass production, compared to existing methods for producing cell aggregates that are difficult to mass-produce due to problems such as high operator-to-operator variability, labor-intensiveness, time-consumingness, and difficulty in quality control.

In addition, since cells aggregate in the empty space within the cross-linked hydrogel membrane (shell), the size of the obtained cell aggregate can be controlled by controlling the amount of the composition discharged, and cell contamination can be effectively prevented, and there is an effect of obtaining a stable cell aggregate without damage.

Through the method for producing cell aggregates according to the present invention, a high yield, uniform and stable cell aggregates can be produced in large quantities, and there is a point where extracellular vesicles can be effectively obtained by culturing the cell aggregates produced in this way in three dimensions.

In addition, in the dispensing step, by using an external force generating device such as a separate electric field, magnetic field, etc. instead of relying solely on gravity, the volume of the composition drop discharged at one time can be reduced, thereby increasing the number of drops that can be discharged using the same amount of hydrogel and cells containing the composition, and there is an advantage in that the efficiency of continuous production of cell aggregates can be maximized.

In addition, the cell aggregates and extracellular vesicles manufactured according to one embodiment of the present invention can be effectively utilized in various bio-industries, such as drug test chips for new drug development and production of various artificial tissue mimics for tissue regeneration.

Figure 1 schematically illustrates a process for manufacturing a cell aggregate according to one embodiment of the present invention.
Figure 2 is a result of observing the change in size of a hollow sphere-shaped bead in which a crosslinked hydrogel membrane (shell) is formed according to the discharge volume during a manufacturing process according to one embodiment of the present invention.
Figure 3 shows the results of sequentially observing the process of obtaining actual cell aggregates using a manufacturing method according to one embodiment of the present invention.
Figure 4 is a diagram schematically illustrating a process for manufacturing extracellular vesicles according to another embodiment of the present invention.
Figure 5 shows photographs of each step (S1, S2, S3) that was actually experimented with according to the method presented in Figure 4.
Figure 6 summarizes the results of conventional 2D cell culture and 3D cell aggregate culture according to the present invention, comparing the production amount of extracellular vesicles (EVs) obtained per volume of culture solution, and Figure 7 compares the production amount of extracellular vesicles (EVs) obtained per cell.
Figure 8 is a schematic comparison of the natural falling of a drop (a) and forced falling by an external force (b) in the dispensing step.
Fig. 9 is a photograph of a device according to a specific example for forced dropping of a drop by external force, and Fig. 10 (a) and (b) are photographs showing the influence of an external force applied to a drop before (a) and after (b) applying an electric field, respectively.
Figure 11 shows the experimental results comparing the total volume of dispensed drops depending on the presence or absence of external force.

Before explaining in detail the preferred embodiments of the present invention below, it should be noted that the terms or words used in the claims of this specification should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts consistent with the technical idea of the present invention.

Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather includes other components, unless otherwise specifically stated.

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

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A method for manufacturing a cell aggregate according to one embodiment of the present invention comprises the steps of: preparing a first composition including a thermosensitive hydrogel dissolved in an ionic solution and cells, as shown in FIG. 1; a dispensing step of discharging the first composition into a second hydrogel; a step of forming a cross-linked hydrogel shell along an outer surface of the first composition discharging within the second hydrogel by reacting the second hydrogel with the ionic solution; and a cell aggregate forming step in which cells are precipitated and aggregated within the cross-linked hydrogel shell.

The above ionic solution is not particularly limited as long as it is an ionic solution in which the heat-sensitive hydrogel can be easily dissolved and can crosslink the second hydrogel, and is preferably an aqueous calcium chloride solution containing at least one selected from the group consisting of calcium and potassium.

The first composition above is a composition in which living cells are uniformly mixed in an ionic solution in which a thermosensitive hydrogel is dissolved, and may contain living cells of about 10 ³ to 10 ⁸ , and the ion concentration contained in the ionic solution may be contained in a range of about 10 to 700 mM. At this time, if the ion concentration is too low, a hydrogel film formed in a subsequent process may not be formed, and if it is too high, the thickness of the formed shell may become too thick, resulting in insufficient space for cell aggregates to form inside, or a problem in which the shell thickness is formed unevenly may occur.

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 ion solution. If the composition exceeds the above range, it may be difficult to discharge in the subsequent dispensing step, or the discharged shape may not be uniform.

In the dispensing step, the first composition having these composition components and composition ranges is dispensed into a reservoir in which the second hydrogel is stored via a pipette, a dispenser, or a 3D printer. In the dispensing step, it is preferable that the first composition is uniformly dispensed in a constant amount, because this can affect the uniformity of cell aggregates formed in a subsequent step and the production rate of extracellular vesicles produced from these cell aggregates.

When the first composition discharged into the storage tank in which the second hydrogel is stored comes into contact with the second hydrogel, the ion solution contained in the first composition and the second hydrogel react in-situ to crosslink, thereby forming a hydrogel film (shell) on the outer periphery of the discharged first composition.

The hydrogel film formed along the outer surface of the first composition that is discharged above is formed by the second hydrogel undergoing an instantaneous cross-linking reaction by the ionic solution, so that a hollow structure is formed inside in which living cells, a heat-sensitive hydrogel, and an ionic solution that did not participate in the formation of the hydrogel film (shell) exist.

As the thermosensitive hydrogel, at least one selected from the group consisting of gelatin, Pluronic F127 or Poly(N-isoprolylacrylamide) can be used, and as the second hydrogel, at least one selected from the group consisting of alginate and chitosan can be used.

The phase of this thermosensitive hydrogel can be changed through temperature change in the cell aggregate formation step described below. For example, by increasing the temperature of the storage tank in which the second hydrogel is stored to about 30 degrees or more, the gel phase of the thermosensitive hydrogel existing inside the hydrogel membrane (shell) formed in the hollow sphere shape formed in-situ can be changed to a liquid phase.

It is preferable that the above-mentioned heat-sensitive hydrogel includes at least one selected from the group consisting of gelatin, Pluronic F127 or Poly(N-isoprolylacrylamide), the second hydrogel may include at least one selected from the group consisting of alginate and chitosan, and it is more preferable that the above-mentioned ionic solution includes at least one selected from the group consisting of calcium and potassium.

By changing the phase of the thermosensitive hydrogel present inside the hydrogel hollow membrane (shell) in this way, the cells present together with the thermosensitive hydrogel inside the hydrogel hollow membrane (shell) naturally settle to the bottom of the hydrogel hollow membrane (shell) by gravity.

These settled cells go through the cell aggregate formation step, and by changing the temperature so that the phase of the thermosensitive hydrogel within the cross-linked hydrogel membrane (shell) changes, the cells naturally form cell aggregates within the hollow hydrogel membrane (shell) due to cell sedimentation.

Therefore, in the above dispensing step, the size of the first composition to be discharged is preferably controlled by changing the discharge conditions, and the size of the hydrogel hollow membrane (shell), which is the space forming the cell aggregate, is determined by the discharge time or the discharge volume, etc.

When the cell aggregates are formed in this way within the hydrogel hollow membrane (shell), a recovery step of decomposing the cross-linked hydrogel membrane (shell) to recover the cell aggregates can be further performed. The process of decomposing the cross-linked hydrogel membrane (shell) can be performed through various known methods, for example, a method that does not affect the cell aggregates, such as decomposition using enzymes, heat, electric fields, and magnetic fields, can be appropriately mixed.

In another embodiment of the present invention, a three-dimensional cell aggregate manufactured through these processes can be mentioned. In the case of culturing a three-dimensional cell aggregate according to the present invention, the three-dimensional culture has characteristics most similar to actual cell aggregates compared to cell aggregates obtained through conventional two-dimensional culture, and has the advantage of being able to more easily obtain a large amount of extracellular vesicles without applying a separate external stimulus or condition.

Therefore, another embodiment of the present invention includes a method of obtaining extracellular vesicles by culturing a cell aggregate obtained by the method for producing such a cell aggregate, and extracellular vesicles obtained in this manner.

The above extracellular vesicles can be obtained by culturing the cell aggregates obtained after the cell aggregate formation process described above. Although the extracellular vesicles can be obtained by going through a culturing step after forming the cell aggregates, it is also possible to obtain the extracellular vesicles by freezing and storing the obtained cell aggregates, and then thawing the frozen cell aggregates when the extracellular vesicles are needed, and culturing the thawed cell aggregates.

That is, after forming cell aggregates, it is possible to produce extracellular vesicles in a continuous process, but it is also possible to produce the required amount of extracellular vesicles by culturing the cell aggregates at the desired time by storing the cell aggregates for a certain period of time through a cryopreservation process after forming the cell aggregates and then thawing and culturing the cell aggregates at the required time.

Meanwhile, the first composition, in which living cells are uniformly mixed in the ionic solution in which the thermosensitive hydrogel is dissolved, is discharged in the form of drops into the second hydrogel through a dispensing step (see FIGS. 1, 4, and 8). At this time, the dispensed drops can be performed in a natural falling manner due to gravity, as shown in FIG. 8(a). In this case, the discharged drops provide a space in which the included living cells can aggregate (see FIG. 4).

However, the size of the cell aggregates actually formed (diameter D2) is significantly smaller than the size of the drops (diameter D1). This is because, although the size of the cell aggregates formed within the drops is not greatly affected by the volume of the drops discharged, a certain level (approximately 5 to 6 ㎕) of drop size is required for them to fall naturally by gravity during the dispensing step.

Therefore, if the drops are discharged only in a natural falling manner in the dispensing step, a drop volume that is too large compared to the size of the cell aggregates actually formed is bound to be formed, and therefore the number of drops that can be obtained through a composition in which living cells are uniformly mixed in an ionic solution in which a certain amount of thermosensitive hydrogel is dissolved is bound to be limited, and thus the amount of cell aggregates obtained is also limited.

That is, in another embodiment of the invention, an external force generating device capable of generating an external force by forming an electric field or a magnetic field around the discharge port of the dispensing unit where a drop is formed, as shown in FIG. 8(b), may be added.

By additionally providing an external force generating device around the discharge port of the dispensing unit in this way, even if the size of the drop formed at the discharge port is smaller than the volume that can be naturally dropped due to gravity, forced falling is possible due to an external force applied by an additional electric or magnetic field, so that the size of the discharged drop can be reduced.

This can increase the number of drops that can be obtained through a composition in which living cells are uniformly mixed in an ionic solution in which a certain amount of thermosensitive hydrogel is dissolved, and due to the increased number of drops, the number of cell aggregates formed and obtained within the drops increases, so that the effect of increased productivity can be expected.

In the dispensing step, the external force generating device formed around the discharge port of the dispensing unit may be, but is not limited to, an electric field or magnetic field forming device, and any device capable of applying an additional external force to a drop formed at the discharge port of the dispensing unit may be used without limitation. For example, any device capable of applying an additional external force to the drop by forming an air current, applying vibration to the discharge nozzle, or forming a reduced pressure condition such as a vacuum may be used.

Hereinafter, specific actions and effects of the present invention will be explained through specific examples of the present invention. However, these are presented as preferred examples of the present invention, and the scope of the rights of the present invention is not limited by the examples.

[Manufacturing Example 1: Manufacturing 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 to ensure that the cells were uniformly distributed within the culture medium.

Gelatin powder (cold water fish, or porcine skin) was dissolved in distilled water to prepare 1 ml of a 10 wt% gelatin solution, and CaCl ₂ (Calcium chloride, Molecular Weight: 111.0, Sigma Aldrich) was additionally mixed into the gelatin solution to a concentration of 500 mM, thereby preparing a bioink (first mixture) containing 5 wt% gelatin and cells.

[Manufacturing Example 2]

A 5% alginate solution was prepared and filled into an open-top container, the container was placed under a volumetric precision dispenser, and the bioink (first mixture) prepared in Manufacturing Example 1 was transferred to a syringe for a volumetric precision dispenser, and any residual bubbles inside the syringe and the volumetric precision dispenser were removed.

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

Calcium ions contained in the bioink thus discharged are ion-cured in-situ upon contact with the alginate solution filling the container, and the first mixture, bioink, forms hollow sphere-shaped beads.

At this time, the size of the hollow sphere-shaped beads created was confirmed by changing the volume of the bioink discharged, and the results are summarized in Table 1 and Figure 3 below.

Bioink discharge volume [nl] 100 200 700 Ejection time [sec] 1 Nozzle size [㎛] 200 Hollow spherical bead diameter
[mm] 1 1.4 2 Fig. 3 (a) (b) (c)

As confirmed in the results of Table 1 and Fig. 3 above, it was found that the diameter of hollow spherical beads generated in-situ was effectively controlled by controlling the discharge volume of bioink (first mixture). This discharge process was performed at room temperature, and it is also possible to add a Peltier cooling system with a temperature control function along the outer surface of the dispenser syringe if necessary.

[Manufacturing Example 3]

200 nl of bioink was dispensed using the same method as in Manufacturing Examples 1 and 2, and fluorescent staining was performed to observe the cell aggregation process within the hollow spherical beads.

Cell tracking dye is a non-fluorescent dye, but it is a hydrophobic substance that fluoresces when it enters a living cell. It has the advantage of easily penetrating living cells and the fluorescent substance remains even after cell division, allowing detection in proliferating cells.

In this manufacturing 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, and tracking dye green working solution was added and reacted at approximately 37°C for approximately 30 minutes. After washing three times with HHBS, the cells were pelleted using a centrifuge, and 1 ml DMEM was added to create a cell suspension. After mixing with hydrogel and going through the same process as Manufacturing Examples 1 and 2, the fluorescently stained cells were confirmed at a wavelength of 490/525 nm, and the results are also illustrated by process in Fig. 4.

As confirmed in (a) to (c) of Fig. 2, hollow sphere-shaped beads were formed in the alginate solution by discharging bioink using a volumetric precision dispenser, and cell aggregates of living cells were confirmed within the hollow sphere-shaped beads.

[Manufacturing Example 4]

Another embodiment of the present invention will now be described with respect to a method for producing extracellular vesicles using cell aggregates. As schematically illustrated in Fig. 4, gelatin powder in the form of 5% (bovine skin, type B, Sigma Aldrich) was dissolved in 3-distilled water at a ratio of 5% at 70°C, and then calcium chloride (CaCl ₂ ) at a concentration of 10 to 30 mM was added to produce Solution A.

Solution B was prepared by dissolving sodium alginate (Sigma Aldrich) powder in powder form in distilled water at a concentration of 0.5 to 2% at room temperature.

At this time, hMSCs (Human mesenchymal stromal cells) cultured using α-MEM (FBS 10%, Antibiotic-Antimycotic 1%) were mixed in Solution A at a concentration of 1–10×10 ⁵ cells/ml at room temperature.

After mixing 2 ml of Solution A prepared in this way and hMSCs (3×10 ⁵ cells/ml) using a pipette at room temperature, the material was supplied to the syringe of the precision injection module.

Solution B, prepared in a previously prepared conical tube, was dispensed under the conditions of 50 kPa pneumatic pressure of the precision injection module, approximately 6 ㎕ of droplet discharge, and 0.05 s of discharge time (S1).

A droplet of Solution A mixed with cells forms a bead with a shell through ionic cross-linking when it comes into contact with Solution B (S2).

After separating Solution B and Bead, they are transferred to a cell culture vessel, the cell culture medium is injected, and when cultured in an incubator environment (37℃) for 12 hours, the cells will aggregate inside (S3).

The results of the actual observation of the processes of S1 to S3 are presented in the photograph in Fig. 5.

[Manufacturing Example 5]

In order to confirm the efficiency of extracellular vesicle production using a 3D culture method using cell aggregates according to an embodiment of the present invention, extracellular vesicles (EVs) were produced and compared with those produced using a conventional two-dimensional (2D) cell culture method.

hMSCs (3×10 ⁵ cells/ml) were cultured in a cell culture dish using α-MEM (Exosome-depleted FBS 10%, Antibiotic-Antimycotic 1%), and after 2 days of culture, EVs were extracted using an isolation kit product and quantitatively analyzed.

The cell aggregates obtained through Manufacturing Example 4 were cultured in the same manner using α-MEM (Exosome-depleted FBS 10%, Antibiotic-Antimycotic 1%), and after 2 days of culture, EVs were extracted using an isolation kit product and quantitatively analyzed.

As confirmed in the results of FIGS. 6 and 7, the EV production per culture volume (ml) (FIG. 6) and EV production per cell (FIG. 7) were compared, and it was confirmed that the production of EVs was higher in the 3D culture using the cell aggregate according to the present invention than in the conventional 2D cell culture. It can be seen that the 3D cell culture technology using the cell aggregate manufactured according to the present invention is an effective method for producing extracellular vesicles (EVs).

[Manufacturing Example 6]

In the same manner as in Manufacturing Example 4, a Solution A droplet containing cells was discharged, and at this time, an electrode capable of forming an electric field was positioned on the side of the nozzle, which is the discharge port (see Fig. 9).

When the volume of the discharged drop was about 6㎕, the drop fell naturally due to gravity from the tip of the nozzle, but when it was about 3㎕, the drop was only stuck at the tip of the nozzle and was not discharged (see Fig. 10(a)). At this time, when a voltage of 13,000 V was applied to the electrode located on the side of the nozzle to form an electric field, the drop was forcibly dropped as if being dragged in the direction of the electrode from the tip of the nozzle, as shown in Fig. 10(b).

This means that if the volume (or size) of the drop is too small, the applied gravity is not sufficient and the drop is not discharged in a form that is stuck at the tip of the nozzle. However, if an external force such as an electric field is additionally applied, the drop stuck at the tip of the nozzle is forced to fall (or forcibly discharged).

Figure 11 summarizes the results of a comparative experiment comparing the volume (discharge amount) of drops that can be discharged by the natural falling method by gravity with the volume (discharge amount) of drops that can be discharged by the forced falling method when an electric field is additionally used as an external force. As can be seen in the results of Figure 11, when an electric field is additionally used as an external force, the volume of drops that can be discharged is about 1/3 of the volume of drops that can be discharged by the natural falling method by gravity. This means that when an electric field is additionally used as an external force, about 3 times more drops can be discharged (based on the same bio-ink composition), suggesting a significant improvement in the productivity of cell aggregates.

The present invention is not limited to the specific embodiments and descriptions described above, and anyone with ordinary skill in the art to which the present invention pertains may make various modifications without departing from the gist of the present invention as claimed in the claims, and such modifications fall within the protection scope 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 (12)

A step of preparing a first composition comprising a thermosensitive hydrogel and cells dissolved in an ionic solution;
A dispensing step of discharging a drop of the first composition into a second hydrogel;
A step of forming a core-shell structure by forming a cross-linked hydrogel film (shell) along the outer surface of the first composition drop discharged into the second hydrogel by reacting the second hydrogel with the ionic solution;
A step of changing the temperature so that the phase of the thermosensitive hydrogel within the cross-linked hydrogel membrane (shell) changes from a gel phase to a liquid phase; and
A step of forming a cell aggregate in which the interior of the cross-linked hydrogel membrane changes into a liquid phase, and the cells precipitate and aggregate;
A method for producing a cell aggregate, characterized in that, in the dispensing step, an external force is applied around a nozzle where a first composition drop is formed, thereby discharging a drop having a reduced volume compared to a drop volume discharged by gravity alone. delete A method for producing a cell aggregate, characterized in that in claim 1, the external force is selected from a force generated by an electric field or a magnetic field, a force caused by an air current, a force caused by vibration applied to a nozzle, and a force caused by depressurization. A method for producing a cell aggregate, characterized in that in claim 1, the thermosensitive hydrogel comprises at least one selected from the group consisting of gelatin, Pluronic F127, and Poly(Nisoprolylacrylamide). A method for producing a cell aggregate, characterized in that in claim 1, the second hydrogel comprises at least one selected from the group consisting of alginate and chitosan. A method for producing a cell aggregate in claim 1, wherein the ion solution contains at least one ion selected from the group consisting of calcium and potassium. A method for producing a cell aggregate, characterized in that in the first paragraph, in the dispensing step, the size of the first composition to be discharged is controlled by a change in the discharge conditions or the size of the external force. A method for producing a cell aggregate, characterized in that in claim 7, the discharge condition is a discharge time or a discharge volume. delete A step of preparing a first composition comprising a thermosensitive hydrogel and cells dissolved in an ionic solution;
A dispensing step of discharging a drop of the first composition into a second hydrogel;
A step of forming a core-shell structure by forming a cross-linked hydrogel film (shell) along the outer surface of the first composition drop discharged into the second hydrogel by reacting the second hydrogel with the ionic solution;
A step of changing the temperature so that the phase of the thermosensitive hydrogel within the cross-linked hydrogel shell changes from a gel phase to a liquid phase;
A step of forming a cell aggregate in which the inside of the cross-linked hydrogel membrane changes into a liquid phase, and the cells precipitate and aggregate; and
A step of culturing the above cell aggregates to obtain extracellular vesicles; comprising;
A method for producing extracellular vesicles using cell aggregates, characterized in that, in the dispensing step, an external force is applied around a nozzle where a first composition drop is formed, thereby discharging a drop having a reduced volume compared to a drop volume discharged by gravity alone. A step of preparing a first composition comprising a thermosensitive hydrogel and cells dissolved in an ionic solution;
A dispensing step of discharging a drop of the first composition into a second hydrogel;
A step of forming a core-shell structure by forming a cross-linked hydrogel film (shell) along the outer surface of the first composition drop discharged into the second hydrogel by reacting the second hydrogel with the ionic solution;
A step of changing the temperature so that the phase of the thermosensitive hydrogel within the cross-linked hydrogel shell changes from a gel phase to a liquid phase;
A cell aggregate formation step in which the interior of the cross-linked hydrogel membrane changes into a liquid phase, and the cells precipitate and aggregate;
A step of freezing and storing the cell aggregates obtained in the above cell aggregate formation step;
A step of thawing the frozen cell aggregates; and
A step of culturing thawed cell aggregates to obtain extracellular vesicles; comprising;
A method for producing extracellular vesicles using cell aggregates, characterized in that, in the dispensing step, an external force is applied around a nozzle where a first composition drop is formed, thereby discharging a drop having a reduced volume compared to a drop volume discharged by gravity alone.
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