KR102160587B1 - Composition of liquid bath for dipping hydrogel droplet - Google Patents

Abstract

The present invention provides a liquid bath composition for dipping hydrogel droplets, including a surfactant, a thickener and, if necessary, a curing agent. The liquid bath composition for dipping hydrogel droplets of the present invention successfully implements a three-dimensional spherical spheroid that is difficult to implement on a flat substrate, and can obtain the spheroid with a uniform size, thereby being usefully used as a technique capable of obtaining the three-dimensional spherical spheroid with a uniform size.

Description

Composition of liquid bath for dipping hydrogel droplet}

The present invention relates to a liquid bath composition for dipping hydrogel droplets, and more particularly, to a liquid bath composition for dipping hydrogel droplets comprising a surfactant, a thickener and, if necessary, a curing agent.

Recently, organs and tissue transplants are increasing worldwide to regenerate or replace damaged organs or tissues in the human body. However, the shortage of donors for organ transplantation is increasing, and therefore, research is being conducted to develop artificial tissues and organs to solve this problem. As an example, in order to develop artificial tissues and organs, a method of injecting cells suitable for the tissues and organs to be produced into a three-dimensional support, and providing an appropriate culture environment for the injected cells to produce the final artificial tissues and organs Is being used. In addition, 3D bioprinting technology that outputs bio-ink with cells is drawing attention.

3D bioprinting is a technology that prints living cells with bio-ink to create a three-dimensional artificial structure or support that can be implanted in the human body. Using this 3D bioprinting technology, organoids and organ-on chips are used. -a-chip), tissue and organ analogues for animal testing, and other studies that can help cure diseases are being actively conducted.

In recent years, by using advanced 3D cell culture methods such as tumor spheroids, stem cell organoids, and tissue engineering through 3D bioprinting, the actual in vivo cellular responses have been implemented with more detailed modeling. In this process, 3D bioprinting systems of various structures have been attempted, and with this, detailed problems arising in the cell culture process have emerged.

In other words, spheroids made by the conventional agglomeration method have a problem that uniformity is low, and the hanging-drop method can produce uniform spheroids, but the difficulty of operation is high and errors are likely to occur depending on the researcher performing it. have.

Korean Patent Publication No. 10-2018-0117179 (October 26, 2018)

While the inventors of the present invention are researching a technology that can rapidly produce a uniform spheroid, a liquid bath is used to create a spheroid, which is a three-dimensional spherical cell structure that is difficult to implement on a flat substrate. By implementing it, it was found that it was possible to reproducibly implement a three-dimensional spherical structure by putting the bath composition in an existing container used by users rather than a special container.

Accordingly, an object of the present invention is to provide a liquid bath composition for dipping hydrogel droplets comprising a surfactant, a thickener, and, if necessary, a curing agent, which is a composition capable of implementing the spheroid.

According to an aspect of the present invention, a liquid bath composition for dipping hydrogel droplets comprising a surfactant and a thickener is provided.

In one embodiment, the surfactant may be one selected from the group consisting of polyoxyethylene-polyoxypropylene copolymer, sorbitan ester, polyoxyethylene sorbitan, polyoxyethylene ether, and mixtures thereof.

In one embodiment, the surfactant may be included in an amount of 1 to 10% by weight based on the total weight of the composition.

In one embodiment, the thickener is 1 selected from the group consisting of gellan gum, carrageenan gum, locust bean gum, xanthan gum, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl starch phosphate, poloxamer, and mixtures thereof. It can be a bell.

In one embodiment, the thickener may be included in 0.001 to 10% by weight based on the total weight of the composition.

In one embodiment, the liquid bath composition for dipping droplets may additionally include a curing agent.

In one embodiment, the curing agent is transglutaminase, genipin, glutaraldehyde, diisocyanates, carbodiimides, calcium chloride, thrombin, And it may be one selected from the group consisting of a mixture thereof.

In one embodiment, the curing agent may be included in 1 to 10% by weight based on the total weight of the composition.

In one embodiment, the liquid bath composition for dipping hydrogel droplets of the present invention may include poloxamer (polyoxyethylene-polyoxypropylene copolymer), gellan gum, and transglutaminase.

In one embodiment, the liquid bath composition for dipping hydrogel droplets of the present invention comprises 1 to 10% by weight of poloxamer (polyoxyethylene-polyoxypropylene copolymer), 0.001 to 10% by weight of gellan gum, and trans It may contain 1 to 10% by weight of glutaminase.

According to the present invention, the liquid bath composition for dipping hydrogel droplets containing a surfactant, a thickener and, if necessary, a curing agent receives the droplets discharged from the nozzle of the hydrogel discharge device and is separated from the nozzle. By keeping the shape and minimizing the impact and allowing it to float inside the bath composition, it is possible to produce spheroids faster and more uniformly than the coagulation method or the hanging drop method, which is a technology that used a bioreactor or low attachment plate in the spheroid manufacturing technology. In addition, it can be applied to high-throughput screening (HTS) analysis as it can be used as an automated method regardless of the skill maturity of the researcher, and it can successfully implement a three-dimensional spherical spheroid that is difficult to implement on a flat substrate. It turns out that there is.

Therefore, the liquid bath composition for dipping hydrogel droplets comprising a surfactant, a thickener and, if necessary, a curing agent of the present invention can obtain a three-dimensional spherical spheroid in a uniform size in the field of spheroid production such as cell culture It can be usefully used as a technique.

1 is a schematic diagram summarizing a method for manufacturing spheroids.
2 is a spheroid photograph of A549 (lung cancer cell line) cultured in a culture plate.
3 is a spheroid photograph of HT29 (colorectal cancer cell line) cultured in a culture plate.
Figure 4 is a photograph of A549 [lung cancer cell line] spheroid observed with a fluorescence microscope [top left: Bright Field image, top right: Merged fluorescence image, bottom left: Calein AM (Green Fluorescence) image, live cells, bottom right: Ethidium homodimer-1 (Red Fluorescence) image, dead cells].
5 is a photograph of observing DU145 (prostate cancer cell line) spheroid (top left: Bright Field image, top right: Merged fluorescence image, bottom left: Calein AM (Green Fluorescence) image, live cells, bottom right: Ethidium homodimer-1 (Red Fluorescence) image, dead cells].
6 is a photograph of observation of HT29 (colorectal cancer cell line) spheroid [top left: Bright Field image, top right: Merged fluorescence image, bottom left: Calein AM (Green Fluorescence) image, live cells, bottom right: Ethidium homodimer-1 (Red Fluorescence) image, dead cells].
7 is a photograph of observation of HCT116 (colorectal cancer cell line) spheroid [top left: Bright Field image, top right: Merged fluorescence image, bottom left: Calein AM (Green Fluorescence) image, live cells, bottom right: Ethidium homodimer-1 (Red Fluorescence) image, dead cells].
8 is a photograph of the MCF-7 (breast cancer cell line) spheroid observed (top left: Bright Field image, top right: Merged fluorescence image, bottom left: Calein AM (Green Fluorescence) image, live cells, bottom right: Ethidium homodimer-1 ( Red Fluorescence) image, dead cells].
9 is a photograph of various sizes of A549 spheroids manufactured with a 3D bioprinter.
10 is a photo of the first day of the organoid produced by the 3D bioprinter (inset is a photo of the organoid formed by the conventional technology).
11 is a photograph of the 7th day of organoids produced with a 3D bioprinter.
Figure 12 is a graph showing the size (Size of organoids) and standard deviation (Size uniformity) distribution of the aggregation-based organoid (Aggregation-based organoid) and the organoid (Bioprintin-based organoid) produced by a bioprinter.

The term “droplet” as used herein refers to any binding volume of a hydrogel material (which may be a liquid or gel). The volume of water droplets is generally less than 1.0 mL, but may be larger than this for viscous substances, and the single binding volume of the medium is referred to as the droplet.

As used herein, "spheroid" refers to a three-dimensional structure formed such that cells are aggregated and generally have a circular or elliptical cross section.

The present invention provides a liquid bath composition for dipping hydrogel droplets comprising a surfactant and a thickener.

The liquid bath composition is obtained by using a liquid bath to receive droplets discharged from the nozzle of the hydrogel discharge device in order to obtain a three-dimensional spherical cell structure that is difficult to implement on a flat substrate as a spheroid close to the spherical shape. It is a liquid bath composition that embodies the spheroid, which is a spherical cell structure.

The surfactant included in the liquid bath composition in the present invention may be one selected from the group consisting of polyoxyethylene-polyoxypropylene copolymer, sorbitan ester, polyoxyethylene sorbitan, polyoxyethylene ether, and mixtures thereof. have. Specifically, the surfactant is a polyoxyethylene-polyoxypropylene copolymer, for example, poloxamer (Poloxamer), Pluronic ^{® F-127 (Pluronic ® F} -127)], sorbitan esters [e.g. , and the like span ^TM (span ^TM)], polyoxyethylene sorbitan [e.g., tween ^TM (tween ^TM)], polyoxyethylene ethers [for example, Breeze ^TM (Brij ^TM)].

In one embodiment, the surfactant may be included in an amount of 1 to 10% by weight, preferably 2 to 8% by weight, and more preferably 4 to 6% by weight, based on the total weight of the composition.

The thickener contained in the composition of the present invention maintains a spherical spheroid shape at the time when the droplet discharged from the nozzle of the hydrogel discharge device is separated from the nozzle by adjusting the viscosity of the bath composition to an appropriate range, and minimizing impact. It plays a role in allowing the bath composition to float inside.

Examples of suitable thickeners that can be used in the composition of the present invention include gellan gum, carrageenan gum, locust bean gum, xanthan gum, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl starch phosphate, poloxamer, and mixtures thereof. It may be one selected from the group.

In one embodiment, the thickener may be included in 0.001 to 10% by weight, preferably 0.005 to 5% by weight, more preferably 0.01 to 1% by weight, based on the total weight of the composition.

In one embodiment, the liquid bath composition for dipping droplets may additionally include a curing agent. At this time, the curing agent serves to cure the hydrogel droplets discharged from the nozzle of the hydrogel discharge device.

The curing agent may be used without limitation as long as it is a material capable of eventually curing the hydrogel droplet by curing the material contained in the hydrogel droplet. For example, transglutaminase (gelatin curing agent), genipin ), gelatin curing agents such as glutaraldehyde, diisocyanates, and carbodiimides; Alginate curing agents such as calcium chloride; Fibrinogen curing agents such as thrombin; And it may be one selected from the group consisting of a mixture thereof.

In one embodiment, the curing agent may be included in 1 to 10% by weight, preferably 2 to 5% by weight based on the total weight of the composition.

In one embodiment, the liquid bath composition for dipping hydrogel droplets of the present invention may include a poloxamer (polyoxyethylene-polyoxypropylene copolymer), gellan gum and transglutaminase as a preferred composition, and the most preferred As a composition, poloxamer (polyoxyethylene-polyoxypropylene copolymer) 1 to 10% by weight, gellan gum 0.001 to 10% by weight, and 1 to 10% by weight of transglutaminase may be included based on the total weight of the composition. .

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Spheroid production

(1) Overview

The following method was performed to implement a three-dimensional spherical cell structure that is difficult to implement on a planar substrate using a liquid bath. The container used does not require a special container, but users can use it by putting the bath into the container that they have been using, and this technology can be applied if it is a device that can put a drop of bio-ink in the water surface of the bath.

(2) Technical content

The composition of the liquid bath is shown in Table 1 below, and the composition of the bioink is shown in Table 2 below.

material Mixing ratio role Pluronic F-127 5% Thickener/Surfactant Gellan gum 0.03-0.33% Thickener Transglutaminase 3% Hardener (gelatin)

material Mixing ratio* role gelatin 6-7.5% Extracellular matrix (ECM) Gellan gum 0.45-0.67% Thickener

Pluronic F-127 was used for mixing a solution dissolved in DPBS (Dulbecco's Phosphate Buffered Saline) at 15%, and a solution dissolved in DPBS (Dulbecco's Phosphate Buffered Saline) at 10% transglutaminase was used for mixing. .

Gellan gum was used by mixing a solution dissolved in 1% in tertiary distilled water so as to have the above mixing ratio.

Transglutaminase used as a curing agent is included in the composition of a liquid bath to cure the gelatinous component of the bio-ink, and can be removed from the composition or replaced with another curing material depending on the characteristics of the bio-ink to be output into the liquid bath.

The liquid bath composition can be adjusted by varying the ratio of the thickener according to the viscosity of the bio-ink to be used. The combination of a liquid bath and bioink is important for the formation of a sphere.

2. How to make spheroids

(1) Overview

It is difficult to uniformly fabricate a three-dimensional spherical cell structure on a flat substrate, and this was to be solved with a liquid bath and a bio-ink discharging device. By this method, it is possible to manufacture a three-dimensional spherical cell structure having reproducibility, and the size of the sphere can be adjusted according to the diameter of the nozzle, and a viscous material that is difficult to use a micropipette can be uniformly discharged by using bioprinting. Made it possible.

(2) Technical content

The manufacturing method is summarized and illustrated in FIG. 1, and the manufacturing procedure is as follows.

① The output bath was prepared by putting it in a container to be printed, and the bio-ink (hydrogel) mixed with cells was put into the printing barrel and mounted on the printer.

② The ink was discharged in the size of the spheroid to be produced and formed into droplets (Beading step). At this time, the size of the droplet can be adjusted according to the discharge conditions such as nozzle diameter, discharge time, and pressure, and should be optimized according to the specifications of the used discharge device.

③ The nozzle with ink was lowered to the surface of the output bath so that ink droplets entered the output bath (Dipping Gelation step). The formed bioink droplets were separated from the nozzle due to a change in surface tension due to dipping. If the tip of the nozzle goes inside the water surface more than the size of the ink droplet, the sphere is damaged, so it is necessary to optimize the position of the nozzle according to the manufacturing size. This step is the principle of separating the bioink droplets by changing the surface tension due to dipping. In this case, the combination of the liquid bath and the bioink is important in order to be separated while maintaining the shape of the droplets created outside the bath.

④ The output nozzle was moved to the origin and the droplet was separated from the nozzle.

⑤ If necessary, it was cured at 37°C for 30 minutes. At this time, the curing conditions may vary depending on the characteristics of the bioink to be used.

3. Spheroid production

Cancer cell spheroids were prepared as follows with a 3D bioprinter.

① Made in the pellet of the cells to the output Lloyd (10 ⁸ cells) was a bio-ink, preheated at 37 ℃ (0.67% gellan gum + 6% gelatin in DPBS) was suspended in 1 mL (suspension) loaded into the output barrel of 3D bio printer I did. A liquid bath (Gellan Gum 0.1%, Pluronic F-127 5%, Transglutaminase 3% in DPBS) was added to a 24-well plate at 400 μl/well.

② A 30G (blunt tip) was used for the nozzle, and the pressure was output to a 24-well plate filled with a liquid bath at 20 kPa.

③ It was cured in an incubator (37° C.) for 30 minutes.

④ The liquid bath was removed, washed with 1 mL of DPBS, and then the spheroids were cultured in a low attachment well plate.

-Culture medium: RPMI + 10% FBS + 1% antibiotic antimycotic solution, 1 mL/well

-Culture conditions: 37 ℃, 5% CO ₂

Pictures of the culture plate containing spheroids are shown in FIGS. 2 (A549, lung cancer cell line) and FIG. 3 (HT29, colon cancer cell line).

4. Live/Dead assay of cancer cell spheroids made with 3D bioprinter

① A pellet (10 ⁸ cells) of cells to be output spheroids was suspended in 1 mL of bio-ink (0.67% gellan gum + 6% gelatin in DPBS) preheated at 37° C. and loaded into the output barrel of a 3D bioprinter. A 24-well plate was prepared by filling a liquid bath with 400 μl/well.

② A 30G (blunt tip) was used for the nozzle, and the pressure was output to a 24-well plate filled with a liquid bath at 20 kPa.

③ It was cured at 37°C for 30 minutes.

④ The liquid bath was removed, washed with 1 mL of DPBS, and then spheroids were cultured in a low-adhesion well plate. A culture solution (RPMI + 10% FBS + 1% antibiotic antimycotic solution) was used at 1 mL/well and cultured for 24 hours (37° C., 5% CO ₂ ).

⑤ The culture solution was removed, washed twice with 1 mL/well of DPBS, and treated with a fluorescence reagent (Fluorescence reagent: DPBS solution with Ethidium homodimer-1 concentration 4 μM, Calcein AM concentration 1 μM).

⑥ After reaction at room temperature for 30 minutes, it was observed with a fluorescence microscope (Ex/Em: Calcein=494/517 nm, Ethidium homodimer-1 528/617 nm).

Figure 4 shows a photograph of A549 (lung cancer cell line) spheroid.

Figure 5 shows a picture of DU145 (prostate cancer cell line) spheroid.

Figure 6 shows a picture of HT29 (colorectal cancer cell line) spheroid.

Fig. 7 shows a picture of observing HCT116 (colorectal cancer cell line) spheroid.

Figure 8 shows a picture of the MCF-7 (breast cancer cell line) spheroid.

5. Analysis of size distribution of A549 spheroids made with 3D bioprinter

<Test method>

① A pellet of cells (10 ⁸ cells) to be output spheroids was suspended in 1 mL of bioink (0.67% gellan gum + 6% gelatin in DPBS) preheated at 37° C. and loaded into the output barrel of a 3D bioprinter. A 24-well plate was prepared by filling a liquid bath with 400 μl/well.

② A 30G (blunt tip) was used for the nozzle, and the pressure was output to a 24-well plate filled with a liquid bath at 20 kPa.

③ It was cured at 37°C for 30 minutes.

④ The liquid bath was removed, washed with 1 mL of DPBS, and observed under a microscope to obtain an image of each spheroid. The obtained image is shown in FIG. 9.

⑤ After measuring the diameter of the spheroid using image J software, the average value and the standard deviation were calculated. As a result, the average diameter was measured to be 645.78 μm and the standard deviation was 49.46 μm.

6. Organoid culture and size uniformity analysis made with 3D bioprinter

① Human embryonic stem cells (H9) pellets (10 ⁸ cells) are suspended in 1 mL of bioink (0.67% gellan gum + 6% gelatin in DPBS) preheated at 37°C to print out a 3D bioprinter. Loaded into the barrel. A 24-well plate was prepared by filling a liquid bath with 400 μl/well.

② A 30G (blunt tip) was used for the nozzle, and the pressure was output to a 24-well plate filled with a liquid bath at 20 kPa.

③ It was cured at 37°C for 30 minutes.

④ The liquid bath was removed, washed twice with 1 mL/well of DPBS, and then the spheroid was incubated in the culture solution.

-Culture medium: Essential8 (Gibco)

-Culture conditions: CERO (benchtop incubator & bioreactor), 37℃, 5% CO ₂

FIG. 10 shows a photo of the first day of the organoid produced by the 3D bioprinter (the insertion diagram is a photo of the organoid formed by the conventional technology), and FIG. 11 shows the photo of the day 7 of the organoid produced by the 3D bioprinter. Indicated.

In addition, Fig. 12 shows a graph showing the size of organoids and standard deviation (Size uniformity) distribution of an aggregation-based organoid and a bioprintin-based organoid. .

As shown in FIGS. 10, 11, and 12, the organoid size is more uniform in the case of a bioprintin-based organoid, compared to the conventional aggregation-based organoid. Can be seen.

Claims (10)

In the liquid bath composition for dipping hydrogel droplets comprising a surfactant and a thickener, the surfactant is a polyoxyethylene-polyoxypropylene copolymer, characterized in that the liquid bath composition for dipping hydrogel droplets. delete The liquid bath composition for dipping hydrogel droplets according to claim 1, wherein the surfactant is contained in an amount of 1 to 10% by weight based on the total weight of the composition. The method of claim 1, wherein the thickening agent is selected from the group consisting of gellan gum, carrageenan gum, locust bean gum, xanthan gum, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl starch phosphate, poloxamer, and mixtures thereof. Liquid bath composition for dipping hydrogel droplets, characterized in that the species. The liquid bath composition for dipping hydrogel droplets according to claim 1, wherein the thickener is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. The liquid bath composition for dipping a hydrogel droplet according to claim 1, wherein the liquid bath composition for dipping a droplet further comprises a curing agent. The method of claim 6, wherein the curing agent is transglutaminase, genipin, glutaraldehyde, diisocyanates, carbodiimides, calcium chloride, thrombin. , And a liquid bath composition for dipping hydrogel droplets, characterized in that one selected from the group consisting of a mixture thereof. The liquid bath composition for dipping hydrogel droplets according to claim 6, wherein the curing agent is contained in an amount of 1 to 10% by weight based on the total weight of the composition. The hydrogel droplet dipping according to claim 6, wherein the surfactant is a poloxamer (polyoxyethylene-polyoxypropylene copolymer), the thickener is gellan gum, and the curing agent is transglutaminase. Liquid bath composition. The method of claim 9, comprising 1 to 10% by weight of poloxamer (polyoxyethylene-polyoxypropylene copolymer), 0.001 to 10% by weight of gellan gum, and 1 to 10% by weight of transglutaminase based on the total weight of the composition. Liquid bath composition for dipping hydrogel droplets, characterized in that.

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