Hemispherical microporous membrane and method for manufacturing same

Abstract

The present invention relates to a hemispherical microporous membrane, a method for manufacturing same, and a use thereof as an in vitro model for culturing spheroids or organoids. Since an in vitro substrate is not used, excellent viability and uniform control of the size, shape, and other characteristics of spheroids or organoids cultured inside a hemispherical porous PDMS membrane are possible, and microscale spheroids or organoids can be manufactured precisely and rapidly.

Description

Hemispherical microporous membrane and method for manufacturing the same

[0001] The present invention relates to a hemispherical microporous membrane, a method for producing the same, and use thereof in a spheroid or organoid disease model.

[0002] Physiologically similar 3D tumor models are being utilized in various in vitro studies, including tissue response to drugs. In particular, there is ongoing interest and active research and development in 3D tumor models, focusing on the microenvironment and signaling regulators closely related to maintenance and differentiation. Techniques for culturing in vitro tumor models that can be cultured in 3D, physiologically similar to organs, are also being continuously researched and developed.

[0003] However, existing methods for fabricating 3D in vitro tumor models utilize an in vitro matrix, making it difficult to develop models tailored to each tumor progression stage. Furthermore, 3D in vitro tumor models containing an in vitro matrix exhibit heterogeneity in viability, size, and shape, limiting their ability to simulate biomechanical stresses experienced in vivo. Furthermore, the densely enclosing extracellular matrix is ​​not completely biodegradable, and the remaining matrix hinders the penetration of test drugs, making drug screening studies challenging.

[0004] Therefore, research on an in vitro tumor platform that can overcome the problems of existing in vitro tumor models using extracellular matrices and perform accurate drug testing is needed.

[0005] Meanwhile, methods for manufacturing biocompatible polydimethylsiloxane (PDMS) porous membranes widely used in bio-research include thermal curing, salt casting, gas foaming, phase separation, and freeze drying. These existing porous membrane manufacturing methods, as described above, involve multiple process steps, making them complex, time-consuming, and expensive. Furthermore, these technologies have the disadvantage of not being able to precisely control pore size, porosity, and air permeability because the pore shape is determined by the type of salt crystals mixed. Therefore, there is a need for a manufacturing technology for hemispherically patterned porous PDMS membranes that is simple to manufacture, minimizes manufacturing costs and time, and allows for free control of the pore characteristics and mechanical properties that determine the characteristics of the porous membrane.

[0006] Accordingly, the inventors of the present invention completed the present invention by manufacturing a hemispherical porous PDMS membrane and confirming its use in a spheroid or organoid disease model.

[0007] The purpose of the present invention is to provide a hemispherical microporous membrane, a method for producing the same, and a use thereof for a spheroid or organoid disease model.

[0008] In order to achieve the above object, the present invention provides a method for manufacturing a hemispherical microporous membrane, comprising: a first step of silanizing a substrate; a second step of spin-coating a mixture of polydimethyl siloxane (PDMS) and a crosslinking agent onto the substrate; a third step of placing a polydimethyl siloxane (PDMS) mask patterned in a hole shape on the coating layer to induce a capillary phenomenon; and a fourth step of pressurizing and depressurizing vapor to form hemispherical micropores.

[0009] In addition, the present invention provides a hemispherical microporous membrane manufactured by the manufacturing method of the present invention.

[0010] Additionally, the present invention provides an in vitro model of a spheroid or organoid comprising a hemispherical microporous membrane according to the present invention.

[0011] In addition, the present invention provides a microfluidic chip including a hemispherical microporous membrane according to the present invention.

[0012] In addition, the present invention provides a drug screening method using a spheroid or organoid in vitro model according to the present invention.

[0013] The hemispherical porous PDMS membrane of the present invention is made by spin-coating a thin layer of PDMS, a flexible elastic material, and then imparting porosity to it, thereby achieving permeability. The multi-array porous structure facilitates the circulation of cell culture medium and simultaneously provides elastic stimulation through fluid-structure interaction (FSI). Because it facilitates the supply of nutrients and CO2 gas exchange necessary for culture, it can accelerate spheroid growth even without an extracellular matrix.

[0014] The inherently hydrophobic surface of PDMS material is modified to become hydrophilic, lowering the surface energy of the hemispherical porous PDMS membrane. This effectively increases culture medium circulation and facilitates CO2 gas exchange.

[0015] Flexible, thin, porous PDMS membranes can control their mechanical properties depending on the mixing ratio of the PDMS solution. Even with the same fluid flow, the intensity of the mechanical stimulus generated within the porous structure can be easily varied, thereby facilitating uniform mixing of cultured cells. By imparting biomechanical movement to spheroid cultures, they can simulate the in vivo microenvironment.

[0016] Additionally, when cultured cells are injected at different ratios onto a patterned hemispherical porous PDMS membrane, the cells are gathered at the center of the hemispherical structure by gravity and can be rapidly grown into spheroid or organoid models at each stage of disease progression.

[0017] Because no in vitro substrate is used, the superior viability and uniform control of the size, shape, etc. of spheroids or organoids cultured inside the hemispherical porous PDMS membrane are possible, and sophisticated micro-spheroids or organoids can be produced in a short period of time.

[0018] Figure 1 is a schematic diagram showing the manufacturing process of a hemispherical porous PDMS membrane.

[0019] Figure 2 is a schematic diagram showing a hole pattern mask of a hemispherical porous PDMS membrane and cells located therein.

[0020] Figure 3 is a schematic diagram showing the biomechanical stimulation of a hemispherical porous PDMS membrane by fluid flow.

[0021] Figure 4 is a schematic diagram showing the formation of a spheroid model at each stage of disease progression by controlling the ratio of cultured cells.

[0022] Figure 5 shows an image of a hemispherical porous PDMS membrane and its MicroCT image.

[0023] Figure 6 shows a MicroCT image of a hemispherical porous PDMS membrane. The lower membrane is not represented in the image due to resolution issues.

[0024] Figure 7 shows an SEM image of a hemispherical porous PDMS membrane.

[0025] Figure 8 shows an SEM image of a hemispherical porous PDMS membrane.

[0026] Figure 9 shows a microscope image of a hemispherical porous PDMS membrane.

[0027] Figure 10 shows a monitoring image of breast cancer cells cultured on a hemispherical porous PDMS membrane.

[0028] Hereinafter, the present invention will be described in more detail. However, the present invention may be implemented in many different forms, and the present invention is not limited to the embodiments described herein, but is defined solely by the claims set forth below.

[0029] Additionally, the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to limit the invention. Throughout the specification of the present invention, the term "including" a component does not exclude other components, but rather implies the inclusion of other components, unless specifically stated otherwise.

[0030] The present invention relates to a hemispherical microporous membrane and a method for manufacturing the same.

[0031] The above hemispherical microporous membrane can be manufactured according to the process illustrated in FIG. 1. For example, the method can be manufactured by, but is not limited to, a first step of silanizing a substrate; a second step of spin-coating a mixture of polydimethyl siloxane (PDMS) and a crosslinking agent onto the substrate; a third step of placing a polydimethyl siloxane (PDMS) mask patterned in a hole shape on the coating layer to induce a capillary phenomenon; and a fourth step of pressurizing and depressurizing vapor to form hemispherical micropores.

[0032] Since the substrate of the above hemispherical microporous membrane can be controlled to have either hydrophilicity or hydrophobicity, any hydrophilic or hydrophobic substrate commonly used in the field can be used without limitation. For example, the substrate may be selected from the group consisting of silicon wafers, glass, and quartz, but is not limited thereto. The substrate may be cleaned with acetone and isopropanol, and then the surface may be silanized by coating a silanizing solution, but is not limited thereto. The porous membrane produced by the silanization may be easily separated from the substrate, but is not limited thereto.

[0033] The mixture of the above polydimethyl siloxane (PDMS) and the crosslinking agent may be mixed at a weight ratio of 5 to 20:1. For example, the mixture of polydimethyl siloxane (PDMS) and the crosslinking agent may be mixed at a weight ratio of 10:1, but is not limited thereto.

[0034] The third and fourth steps may be performed after spin-coating polydimethyl siloxane (PDMS) on the above substrate and before the polydimethyl siloxane (PDMS) is cured, but are not limited thereto. The uncured polydimethyl siloxane (PDMS) solution within the coating layer may be formed in a hemispherical shape on the inner wall surface of the pattern of the polydimethyl siloxane (PDMS) mask by capillary action, but are not limited thereto.

[0035] In the fourth step, micropores may be formed in a hemispherical polydimethylsiloxane (PDMS), but are not limited thereto.

[0036] The above hemispherical microporous membrane can be manufactured using, but is not limited to, high-pressure saturated steam technology (HPSS). The high-pressure saturated steam technology may be manufactured by increasing the pressure by supplying steam to pressurize the internal bubbles of the hemispherical polydimethyl siloxane (PDMS), and then reducing the pressure and temperature by discharging the steam to connect the internal bubbles to form pores, but is not limited thereto. The high-pressure saturated steam technology may be performed by, for example, an internal bubble rising step, a cross-linking step, an internal bubble pressurization step, and an internal bubble connection step, but is not limited thereto.

[0037] The internal bubble rising step involves placing a substrate coated with a mixture of polydimethylsiloxane (PDMS) and a crosslinking agent into a chamber of a high-pressure saturated steam system, and increasing the internal temperature of the chamber to raise the internal bubbles of the mixture. In other words, the internal bubbles trapped within the thermally crosslinked polymer are raised. At this time, the chamber, which has an internal temperature of room temperature (20±5℃), can be heated to increase the internal temperature to 95 to 105℃ for 10 to 20 minutes. For example, it can be increased to 100℃ for 15 minutes. If the internal temperature of the chamber is increased below 95℃ in this step, steam generation may be small, and crosslinking may occur slowly. If it exceeds 105℃, sufficient steam is generated, but crosslinking may occur too quickly, leaving insufficient time for pore formation. Furthermore, if the internal temperature of the chamber is increased for less than 10 minutes, pore formation is not sufficient. If it exceeds 20 minutes, pore formation is sufficient, but may be inefficient.

[0038] The cross-linking step can be performed by increasing the internal chamber temperature slightly beyond the increased internal chamber temperature, so that the internal bubbles of the mixture are automatically aligned according to size and cross-linking can proceed in a portion of the mixture. In this step, the internal chamber temperature can be increased from 95 to 105°C to 115 to 125°C for 1 to 5 minutes. For example, it can be increased to 120°C for 3 minutes. Through this process, large bubbles are arranged on top and small bubbles are arranged on the bottom, so that a membrane having a gradient structure with different bubble sizes in the cross-section can be manufactured (Figs. 7 to 9b).

[0039] The internal bubble pressurization step can pressurize the internal bubbles of the mixed solution by supplying steam until the water vapor content inside the chamber becomes saturated, thereby increasing the internal pressure of the chamber. This can create a network structure in which the bubbles are interconnected. In this step, the internal pressure of the chamber can be increased to 0.10 to 0.14 MPa by supplying steam at 115 to 125°C for 15 to 25 minutes. It is preferable to increase the internal pressure of the chamber to 0.12 MPa at 120°C for 20 minutes.

[0040] The internal bubble connection step can be performed by discharging steam to the outside of the chamber, thereby lowering the internal pressure and temperature of the chamber, thereby allowing the internal bubbles of the mixture to connect with each other. This occurs when the pressure caused by the steam is released, causing the internal bubbles of the mixture to burst and connect with each other. This allows for the production of a porous membrane through which air can freely pass. To achieve this, the internal pressure of the chamber can be lowered to 0 MPa and the internal temperature to room temperature by discharging steam for 35 to 45 minutes, preferably for 40 minutes. If the steam discharge time exceeds 45 minutes, the internal bubbles may not burst properly, resulting in unconnected areas.

[0041] Furthermore, the present invention relates to an in vitro model of a spheroid or organoid comprising the hemispherical microporous membrane of the present invention (Fig. 2).

[0042] The above spheroid or organoid in vitro model may include cultured cells. For example, it may include single cultured cells or co-cultured cells, but is not limited thereto. The co-cultured cells may include normal cells and diseased cells. For example, it may include normal cells and cancer cells, but is not limited thereto. By adjusting the ratio of the cultured cells, a spheroid model according to the progression stage of the disease can be formed. For example, a steroid or organoid model according to the progression stage of liver cancer can be formed by adjusting the ratio of hepatocytes and hepatocellular carcinoma, but is not limited thereto (Fig. 4). In addition, effective drugs can be screened by treating the spheroid or organoid model according to the progression stage of the disease, but is not limited thereto (Fig. 4).

[0043] The circulation of the culture medium is facilitated by the fluid flow of the culture medium inside the hemispherical microporous membrane, and biomechanical stimulation can be provided, so that the microenvironment in vivo can be simulated, but is not limited thereto (Fig. 3).

[0044] The above hemispherical microporous membrane can be cut into any desired shape for use. For example, it can be divided into various shapes such as square, rectangular, and circular, but is not limited thereto.

[0045] The present invention also relates to a microfluidic chip comprising a hemispherical microporous membrane.

[0046] In addition, the present invention relates to a drug screening method using the spheroid or organoid in vitro model of the present invention.

[0047] The present invention will be described in more detail below through examples. However, the following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0048] <Example>

[0049] Example 1. Fabrication of a hemispherical microporous PDMS membrane

[0050] A PDMS solution containing 3 g of silicone elastomer base and 0.3 g of curing agent (Sylgard 184, Dow Corning Corp., Midland, MI, USA) was spin-coated on a silicon wafer at 5500 rpm for 30 s without degassing. Then, a PDMS mask patterned with pre-made hole shapes was placed on the PDMS solution before it was cured to induce capillary action. Then, it was placed in the chamber of a solidification saturated vapor system, and the temperature inside the chamber was increased to 100°C for 15 min, and then increased from 100°C to 120°C for 3 min. Steam was supplied at 120°C for 20 min to increase the pressure inside the chamber to 0.12 MPa, and then the steam was exhausted for 40 min to lower the pressure inside the chamber to 0 MPa and the temperature inside the chamber to room temperature, thereby fabricating a porous membrane.

[0051] As a result, as shown in Figures 5 to 9b, pores were formed in a hemispherical shape inside the porous PDMS membrane, with large bubbles arranged above and small bubbles arranged below. It was confirmed that the cross-section was formed in a gradient structure with different bubble sizes.

[0052] Example 2.

[0053] MCF-7 (breast cancer cells) cells were cultured in a cell incubator within the porous membrane manufactured in Example 1 and monitored over time.

[0054] As a result, as shown in Fig. 10, the cells clump together to form spheroids about 10 hours after cell injection into the hole, and the spheroids are maintained even on the 4th day.

[0055] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0056] The structural and functional properties of this invention make it a valuable tool in life sciences and medicine. Rapid functional cell organization through 3D cell culture enables more precise drug efficacy testing, disease modeling, regenerative medicine therapeutics, and new drug development platforms. It can also serve as a fundamental technology in diverse fields, including regenerative medicine and tissue engineering.

Claims

1. The first step is to silanize the substrate; A second step of spin coating a mixture of polydimethyl siloxane (PDMS) and a crosslinking agent onto the substrate; A third step of inducing a capillary phenomenon by placing a polydimethyl siloxane (PDMS) mask patterned in a hole shape on the coating layer; and Step 4: Pressurizing and depressurizing the steam to form hemispherical micropores; A method for manufacturing a hemispherical microporous membrane including:

2. In paragraph 1, A manufacturing method wherein the third and fourth steps are performed after the second step and before the polydimethyl siloxane (PDMS) is cured.

3. In paragraph 1, A manufacturing method wherein, in the third step, the polydimethyl siloxane (PDMS) solution of the coating layer is formed in a hemispherical shape on the inner wall surface of the pattern of the polydimethyl siloxane (PDMS) mask by a capillary phenomenon.

4. In paragraph 3, A manufacturing method wherein, in the fourth step, micropores are formed in the hemispherical polydimethyl siloxane (PDMS).

5. In paragraph 3, A manufacturing method wherein, in the fourth step, the pressure is increased by supplying steam so that the internal bubbles of the hemispherical polydimethyl siloxane (PDMS) are pressurized, and the pressure and temperature are reduced by discharging steam so that the internal bubbles are connected to each other.

6. In paragraph 1, A manufacturing method wherein the above substrate is hydrophilic or hydrophobic.

7. In paragraph 1, A manufacturing method, wherein the substrate is selected from the group consisting of silicon wafer, glass and quartz.

8. A hemispherical microporous membrane manufactured by the manufacturing method of any one of claims 1 to 7.

9. An in vitro model of a spheroid or organoid comprising a hemispherical microporous membrane according to Article 8.

10. In paragraph 9, A spheroid or organoid in vitro model comprising cultured cells.

11. In paragraph 10, A spheroid or organoid in vitro model, wherein the cultured cells are selected from the group consisting of normal cells, cancer cells, and combinations thereof.

12. In paragraph 11, A spheroid or organoid in vitro model that forms a spheroid model according to the stage of disease progression by controlling the ratio of normal cells and cancer cells.

13. In paragraph 9, A spheroid or organoid in vitro model that facilitates circulation of a culture medium and provides biomechanical stimulation by the fluid flow of the culture medium inside the hemispherical microporous membrane.

14. In paragraph 9, The above hemispherical microporous membrane can be cut into a desired shape and used as a spheroid or organoid in vitro model according to the intended use.

15. A microfluidic chip comprising a hemispherical microporous membrane according to claim 7.

16. A drug screening method using a spheroid or organoid in vitro model according to Article 8.

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