KR20240041567A - Membrane thermally bonded structure and method for manufacturing same - Google Patents

Abstract

The present invention relates to a structure in which a membrane is thermally bonded and a method for manufacturing the same. More specifically, by thermally bonding the porous nanomembrane to the conjugate to improve the bonding strength between the membrane and the conjugate, other obstacles that may arise during analysis of experiments and research (e.g., bond separation) are prevented in advance. Alternatively, it relates to a structure with a heat-bonded membrane that prevents receptors (e.g., cultured cells) accommodated in the conjugate from leaking out of the conjugate during experiments and research, and a method of manufacturing the same.

Description

Membrane heat bonded structure and method of manufacturing the same {MEMBRANE THERMALLY BONDED STRUCTURE AND METHOD FOR MANUFACTURING SAME}

The present invention relates to a structure in which a membrane is thermally bonded and a method for manufacturing the same. More specifically, by thermally bonding the porous nanomembrane to the conjugate to improve the bonding strength between the membrane and the conjugate, other obstacles that may arise during analysis of experiments and research (e.g., bond separation) are prevented in advance. Alternatively, it relates to a structure with a heat-bonded membrane that prevents receptors (e.g., cultured cells) accommodated in the conjugate from leaking out of the conjugate during experiments and research, and a method of manufacturing the same.

A porous nanomembrane is a membrane formed by intertwining multiple polymer fibers, and is a membrane tens to hundreds of nanometers thick with numerous penetration pores that selectively allow specific substances to pass through.

Porous nanomembranes are characterized by being inexpensive, simple to process, transparent, and easy to absorb/desorb, allowing them to be used both inside and outside the body. In addition, it is known to have higher application value because the size of the permeation pore and the thickness of the membrane can be freely adjusted by adjusting the type and concentration of the solvent.

Porous nanomembranes are mainly used in cell culture or cell research and analysis. In this case, the porous nanomembrane serves as a cell culture surface in cell culture or as a cell membrane in cell research and analysis.

In order for this porous nanomembrane to fully perform its role, the porous nanomembrane must be attached or bonded to a container containing cells. Conventional nanomembrane bonding technology only involves applying adhesive to the cell container or using adhesive tape (double-sided tape). ), causing many problems in cell culture and cell research. For example, the method of applying adhesive may have a negative effect on the experimental environment, such as the inherent substances of the adhesive slowing down cell culture, and the method of applying adhesive tape may be easily peeled off over time, or even if attached, there may be gaps in the adhesive surface. Many problems occurred, such as cells leaking out.

The present invention was developed with an eye on this problem, and not only can solve the technical problems discussed above, but also provide additional technical elements that cannot be easily devised by those skilled in the art. It has been done.

Korean Patent Publication 10-2022-0116708A (2022.08.23)

The purpose of the present invention is to provide a structure that can further improve the bonding strength between the membrane and the bonded body compared to the conventional adhesive application method or adhesive tape attachment method, and to ensure that the bonding surface remains strong even after bonding, and a method of manufacturing the same. Do it as

In addition, the present invention applies heat to the bonding surface in order to thermally bond the membrane to the bonded body, by applying a specific temperature at which heat is not conducted to the non-bonded side of the membrane during bonding with the membrane and no thermal deformation occurs, Provides a structure in which the optimal membrane is thermally bonded.

The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present invention proposes a structure in which a membrane is thermally bonded to solve the above problems. In this case, the structure includes a membrane made of nanofiber material; and a bonded body that is a bonding object of the membrane, wherein the membrane is formed by contacting a joint of the bonded body to which heat of a specific temperature is applied, and is thermally deformed by contact and bonded to the bonded body. ; And it may include a surface other than the bonding surface, a transparent surface having a porous structure.

Additionally, in the structure to which the membrane is thermally bonded, the specific temperature may be within the range of [melting point (Tm)] to [melting point (Tm) +10°C] of the membrane.

In addition, in the structure in which the membrane is thermally bonded, the bonding surface is a surface in which the nanofiber structure is deformed and the permeability function is reduced compared to before deformation, and the passage of microcells or fluids may be significantly restricted. .

In addition, in the structure in which the membrane is thermally bonded, the structure may be characterized by having a value of the undamaged transmission surface compared to the damaged transmission surface at different ratios depending on the approaching speed of the membrane and the joint.

On the other hand, a method of manufacturing a structure in which a membrane is thermally bonded according to another embodiment of the present invention includes the steps of applying heat at a specific temperature to the joint of the bonded body; And a step of thermally bonding the joint of the bonded body and the membrane by approaching them, wherein the membrane after the thermal bonding step includes a bonding surface and a permeable surface, and the bonding surface is heated at a specific temperature. It is formed by contacting the joint of the assembly to which heat is applied, and is a surface that is thermally transformed by contact and joined to the assembly, and the transparent surface is a surface other than the joint surface, and has a porous structure. can do.

According to the present invention as described above, the bonding force between the membrane and the bonded body is improved, and the bonding surface is maintained firmly even after bonding. Furthermore, damage to the membrane is minimized, and the structure with the membrane bonded is strengthened. This has the effect of allowing the structure to play its role in cell culture or cell research.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

1 is a diagram showing a membrane bonded to various conjugates.
Figure 2 is a diagram showing an example of use of a cell culture vessel that is a membrane and a conjugate.
Figure 3 shows the process of thermally bonding the membrane to the bonded body.
Figure 4(a) is a diagram showing the nanofiber structure of the bonded surface, the transparent surface, and the interface between the bonded surface and the transparent surface of the membrane, and Figure 4(b) is a diagram showing the nanofiber structure of the undamaged surface, the damaged surface, and the bonded surface. This is a drawing showing the formation.
Figure 5 is a diagram comparing the conventional membrane-attached body attachment method and the membrane-conjugated body thermal bonding method according to the present invention through drawings or captured images.
Figure 6 is a diagram showing [Experimental Example 1] of the present invention.
Figure 7 is a diagram showing [Experimental Example 2] of the present invention.
Figure 8 is a diagram showing [Experimental Example 3] of the present invention.
Figure 9 is a diagram specifically showing a method of manufacturing a thermally bonded membrane structure according to the present invention.
Figure 10 is a diagram showing how a user forms a bonding surface and a transmission surface of a desired shape.
Figure 11 shows a structure with a heat-bonded membrane impregnated with a hydrogel composition.
Figure 12 shows a blood brain barrier (BBB) model using Brain Microvascular Endothelial Cells (BMECs) derived from induced pluripotent stem cells (iPSC).

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms, and the present embodiments are merely intended to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with meanings that can be commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless clearly specifically defined. The terms used in this specification are for describing embodiments and are not intended to limit the invention. As used herein, singular forms also include plural forms, unless specifically stated otherwise in the context.

Terms such as “first” and “second” are used to distinguish one component from another component, and the scope of rights should not be limited by these terms. For example, a first component may be named a second component, and similarly, the second component may also be named a first component.

As used herein, “comprises” and/or “comprising” refers to a referenced component, step, operation and/or element that includes one or more other components, steps, operations and/or elements. Does not exclude presence or addition.

The present invention relates to a structure in which a nanomembrane is thermally bonded and a method for manufacturing the same. The present invention relates to a structure in which a nanomembrane is thermally bonded and a method for manufacturing the same. By thermally bonding a porous nanomembrane (10) to a bonded object (20), the bonding force between the membrane (10) and the bonded object (20) (or By maximizing adhesion, it is intended to solve various problems (e.g. joint separation, leakage, etc.) that were revealed in the method of using existing adhesives or adhesive tapes.

As briefly mentioned in the background previously, a porous nanomembrane refers to a membrane formed by entangling multiple polymer fibers, and refers to a membrane with a thickness of tens of nm to tens of um having a plurality of penetration pores that selectively transmit specific substances.

Figure 1 is a diagram showing the membrane 10 bonded to various bonded bodies 20. Referring to FIG. 1, the membrane 10 may be bonded to a cell culture vessel 20a or a microfluidic chip 20b, which is the conjugate 20.

In this detailed description, the conjugate 20 will be mainly described based on the cell culture vessel 20a among the various conjugates 20, but the conjugate 20 is not limited thereto and is used in cell culture, cell research, and In the analysis, if the membrane 10 is a structure with a surface that can be bonded, it can be included within the scope of the bonded body 20. Or, furthermore, even if it is not necessarily related to cell culture, research, or analysis, any type of structure can be included as a type of conjugate 20 as long as the membrane 10 includes a bondable surface.

Figure 2 shows an example of use of a cell culture vessel that is a membrane and conjugate. Figure 2 is a diagram to aid understanding of how the structure of the membrane and conjugate to be discussed in this detailed description can be utilized.

Referring to FIG. 2, a membrane 100 is attached and bonded to one side of the cell culture container 200, and the cell culture container 200 to which the membrane 100 is bonded is inserted into the opening 310 of the plate 300. You can.

In this way, when the membrane is bonded to the cell culture vessel, the permeable pores of the membrane allow oxygen and nutrients contained in the fluid to be more smoothly supplied to the cells in the cell culture vessel, thereby further improving cell proliferation and differentiation. It can be induced to promote it. Additionally, the membrane at this time can also serve as a cell culture surface on which cells are cultured.

In addition, since the membrane is made of a plurality of polymer nanofibers, it can have a structure similar to the basement membrane in vivo, and thus can be used to create an environment similar to the blood flow environment in vivo.

In this way, the structure formed by the membrane and the conjugate can be used to create a variety of environments in cell culture and analysis, and the purpose of the present invention is to make it possible to easily create such a highly useful structure without defects.

Figure 3 shows the step of thermally bonding the membrane 10 to the bonded body 20.

Step (A) in FIG. 3 is a step of partially applying heat to the joint portion 21 of the joined body 20 in order to thermally bond the membrane 10 to the joined body 20. Here, the joint portion 21 refers to the part of the structure of the bonded body 20 that directly touches the membrane 10. In FIG. 3, the spout of the cylindrical bonded body 20 corresponds to the bonded portion 21. You can. The joined body 20 may be basically made of a heat-resistant material. In some cases, even if the entire structure of the joined body 20 is not heat-resistant, at least the joint portion 21 may be made of a heat-resistant material.

Meanwhile, the bonding surface 11 of the membrane 10 refers to the surface in contact with the bonding portion 21 of the bonded body 20, and the bonding surface 11 and the transmission surface 12, that is, the membrane 10, are connected to each other. Among the surfaces, a boundary may exist between the transparent surfaces 12 that play a role in transmitting fluid. Also, for reference, the bonding surface 11 has the characteristic that the passage of cells or fluid is significantly restricted after thermal bonding, and is a concept that includes an area or surface where there is no space between the nanofiber structures and the bonded body by crushing them. It can be understood as Significantly restricted passage of cells (microcells) or fluid means that passage of cells or fluid at the bonding surface 11 is limited to less than 5% compared to the permeable surface 12. However, the most desirable state is the bonding surface 11 in which cells or fluid cannot pass through, that is, 0%. On the other hand, if the temperature of the joint 21 of the bonded body 20 is lower than the melting point of the membrane, the bonding itself will not occur and the bonding surface 11 will not be created, and the temperature of the joint 21 will be lower than the melting point of the membrane. It will be understood that the bonding surface 11 will be created only when bonded to the membrane in a higher heated state.

For reference, in this detailed description, the structure is formed by thermally bonding the membrane 10 to the bonded body 20, that is, the bonding surface 11 of the membrane 10 is thermally bonded to the bonding portion 21 of the bonded body 20. The structure is referred to as 'structure (1) in which the membrane (10) is thermally bonded'.

Continuing the explanation of FIG. 3 again, in step (B) after step (A) of FIG. 3, the membrane 10 is bonded by approaching and contacting the joint portion 21 of the heat-applied body 20. . This step is performed by allowing the joint portion 21 of the bonded body 20 to approach the membrane 10 while the membrane 10 is fixed so that it cannot move, or by fixing the bonded body 20 so that it cannot move. It can be implemented by bringing the membrane 10 closer to the joint 21 or by allowing the membrane 10 and the joint 21 to approach each other.

Meanwhile, in step (B), the membrane 10 may be approached and joined to the joint 21 while maintaining a flat state, but is not necessarily limited thereto. In other words, the membrane 10 can be approached and joined to the joint 21 while maintaining a curved surface having a predetermined curvature value rather than being flat. The predetermined curvature value may be determined according to the intention of the person designing the structure. For example, (i) when it is desired to implement a nanomembrane filter in a sagging state, thereby cultivating cells within the curved surface, a relatively large curvature is required. The value can be determined, or (ii) a relatively small curvature when it is desired to prevent the durability of the transmission surface 12 from being reduced due to slight shrinkage of the fiber structure when the fiber structure is hardened by contact and bonding with the joint 21. You can set the value. In other words, even if shrinkage of the fiber structure occurs due to bonding, the membrane 10 with a pre-curvature value was used in anticipation of this, and as a result, a flat transparent surface 12 will be obtained after bonding.

For reference, the above embodiments (i) and (ii) refer to additional embodiments, and the bonding process according to the present invention is basically premised on an embodiment in which a flat membrane without curvature is bonded to the bonded body. Let us remind ourselves once again that this is the case.

On the other hand, in step (B), a process of controlling the approach speed between the membrane 10 and the joint 21 may also be performed. Bonding is achieved by a change in the fiber structure from the point where the joint 21 and the membrane 10 come into contact, or in some cases, before the contact occurs. At this time, the membrane 10 and the joint 21 approach. By adjusting the speed from 0.1mm/s to 100mm/s, it is possible to manufacture the structure desired by the designer. The magnitude of this joining speed may vary depending on the area of the joining part 21, the thickness of the membrane 10, or the physical properties of the joining part 21.

In some cases, even when adjustment of the transmission surface 12 is necessary, the area of the transmission surface 12 according to the needs of the designer (experimenter) can be realized by controlling the approach speed. As will be explained in more detail in the description of FIG. 4(b), the transparent surface 12 can be further divided into an undamaged surface 12a and a damaged surface 12b. The undamaged surface 12a refers to the transparent surface 12. Among the inner areas, it means an area that is not damaged by heat, and the damage surface 12b, on the other hand, means an area that is damaged by heat. At this time, it should be noted that the damaged surface 12b and the previously described joint surface 11 refer to different surfaces, and the joint surface 11 is a surface that is assumed to be in contact with the joint 21. Let me remind you once again. On the other hand, when the transmission surface 12 can be divided into an undamaged surface 12a and a damaged surface 12b, the area of the surface capable of penetration (transmission surface) on the transmission surface 12 can be adjusted according to the approaching speed. You can. For example, when it is desired to minimize the damaged surface 12b of the transparent surface 12, the access speed of at least one of the membrane 10 and the joint 21 is relatively small, that is, the access time is increased to achieve bonding. can do. At this time, the temperature of the joint 21 may be relatively lower compared to the case where the damaged surface 12b is widened, which will be described later. On the other hand, when it is desired to further widen the damaged surface 12b of the transparent surface 12, the approaching speed is increased so that the heated joint 21 contacts the surface of the membrane 10 at a high temperature as much as possible to prevent damage. The surface area can be made larger. At this time, the temperature of the joint 21 may be relatively higher than in the previous case because sufficient jointing must be achieved within a short period of time.

Meanwhile, finally, in step (C) after step (B) of FIG. 3, the nanofiber structure of the bonding surface 11 in contact with the bonding portion 21 is thermally deformed and hardened to form a gap between the bonding portion 21 and the bonding surface 11. This is the stage where a bond is formed. In the drawing of step (C) of FIG. 3, the joint portion 21 and the joint surface 11 are not shown separately, which means that the joint portion 21 and the joint surface 11 are completely joined and cannot be distinguished from each other. This is to emphasize that structural modification has occurred, especially the structural modification of the nanofibers that make up the membrane.

Figure 4(a) is a diagram showing the nanofiber structure of the bonding surface 11, the transmission surface 12, or the interface 13 between the bonding surface 11 and the transmission surface 12 of the membrane 10. .

Figure 4(a) (A) shows the nanofiber structure with the membrane 10 fixed for bonding, or the transparent surface 12 after bonding but without deformation of the fiber structure. Referring to (A) of Figure 4(a), it can be seen that the transmission surface 12 is formed in a structure in which a plurality of polymer fibers are entangled with each other, forming a plurality of penetration holes that selectively transmit specific substances. . Since the transmission surface 12 with these characteristics is a surface practically used in experiments, it will generally have a relatively large area ratio compared to the bonding surface 11. At this time, the diameter of the nanofiber may be on the order of tens of nanometers to several micrometers, the number of micropores per unit volume may vary depending on the thickness, and the thickness of the membrane 10 made of such nanofibers is several hundred nanometers. It may be on the order of meters to tens of micrometers.

Referring to (B) of FIG. 4(a), after thermal bonding of the membrane 10 and the bonded body 20, the interface 13 may be formed between the transparent surface 12 and the bonding surface 11. can confirm. Here, the boundary surface 13, as the term suggests, is a surface that separates the boundary between the transmission surface 12 and the bonding surface 11. In some cases, the boundary surface 13 is a fluid (or fine fluid) transmitted by the transmission surface 12. It can also play a role in preventing substances) from leaking or running off.

For reference, the nanofiber structure of the interface 13 is partially crushed due to thermal deformation, but the remaining part is transparent, so not only does it play the role of a barricade described above, but it also partially connects the transparent surface 12 and the bonding surface 11. can perform its role.

Referring to (C) of Figure 4(a), after thermal bonding of the membrane 10 and the bonded body 20, the nanofiber structure of the bonded surface 11 is crushed and hardened again by thermal deformation, resulting in permeability. It is transformed into a state without pores, and the bonding surface 11 of this modified structure prevents cells or fluid contained in the bonded body 20 from leaking to the outside of the boundary surface 13. The bonding surface 11 may consist only of areas where no clearly distinguishable nanofiber structure exists when compared to the transparent surface 12, or even if it exists, it is 200% larger than the diameter of the nanofibers on the transparent surface. It can be characterized by the presence of nanofibers. In addition, according to the characteristics of the bonding surface 11 as above, the interface 13 between the transparent surface 12 and the bonding surface 11 may show characteristics in the process of changing the diameter of the nanofiber by more than 200%. .

In this way, the role of preventing mobile bodies such as cells accommodated in the conjugate 20 from escaping outside the boundary 13 has an important influence on measuring the results of cell culture and cell research/analysis. Specifically, the main purpose of cell culture is to proliferate cells, but if cells escape from the conjugate 20, not only will this purpose not be achieved properly, but it will also cause problems of obtaining poor experimental results. However, the conventional technology for bonding the membrane 10 to the bonded object 20 utilized a method of applying an adhesive liquid to the bonded object 20 or attaching (taping) an adhesive, and this technology (20) did not solve the problem of cell detachment. Accordingly, to emphasize once again, the present invention seeks to solve the above problem by thermally bonding the membrane 10 and the bonded body 20.

Figure 4(b) shows a plane of the bonded membrane 10, in which the bonding surface 11, the transmission surface 12, and their boundary surfaces 13 are shown, and further the transmission surface ( It can be seen that in 12), the undamaged surface 12a and the damaged surface 12b are shown again. The transmission surface 12 does not necessarily include the damaged surface 12b, and depending on the bonding method, it may be composed of only the undamaged surface 12a without damaging the membrane.

For reference, the undamaged surface 12a and the damaged surface 12b can be defined based on the following criteria.

For example, the damaged surface 12b may be defined as a surface with a light transmittance of 70% or more, and the non-damaged surface 12a may be defined as the remaining area. In the case of the damaged surface 12b, light or fluid transmittance increases and the size of micropores increases, and the rigidity of the nanofiber membrane increases, which may have a negative effect on cell culture. In contrast, the damaged surface 12b can be defined as an area where the light transmittance is 90% or more, and the non-damaged surface 12a can be defined as other areas. In this way, the damaged surface 12b can be divided and defined based on an area having a relatively higher transmittance compared to the non-damaged surface 12a and a point where the transmittance critically varies.

As another example, the undamaged surface 12a and the damaged surface 12b of the membrane may be distinguished by roughness. Generally, in areas where nanofibers are damaged, the layered structure of nanofiber strands disappears and the surface becomes smooth. Considering these characteristics, when observing from a side cross section, the ridges (nanofiber structure) visible when the nanofibers are crushed. The area where the difference in thickness between the highest point of the nanofiber structure) and the trough (the lowest point of the nanofiber structure) is several tens of nm or less can be defined as the damaged surface 12b. Additionally, the non-damaged surface 12a may be defined as an area other than the damaged surface 12b. For reference, in the case of the undamaged surface 12a, the nanofiber strands will be maintained without changing their state, and at this time, the thickness difference between the crest and valley of the nanofiber as seen from the side cross section may be hundreds of nm to several um.

As another example, the undamaged side 12a and the damaged side 12b of the membrane may be defined based on the ratio of nanofiber diameters. Specifically, the damaged surface (12b) can be defined as an area that has increased by more than tens to hundreds of% compared to the diameter of a normal nanofiber, and the undamaged surface (12a) can be defined as an area that is not the damaged surface (12b). You can. For reference, the normal diameter range of nanofibers may mean the range within which the nanofiber diameters observed within any given membrane can be included.

As another example, the undamaged surface 12a may be defined as a surface where nanofibers having a diameter of several tens of nanometers to several micrometers are observed, and the damaged surface 12b may be defined as the remaining area.

As another example, the damaged surface 12b may be defined as an area where the thickness of the membrane is reduced by more than 30% compared to the non-damaged surface 12a.

When looking at the membrane 10 after being joined in this way, the planar structure is as shown in FIG. 4(b). In general, when it is considered that correct bonding has been achieved between the membrane 10 and the bonded body 20, the damaged surface 12b is It will be defined to mean less than 1% of the total transmission surface 12.

Figure 5 is a diagram comparing the conventional method of adhering the membrane 10 to the bonded body 20 and the thermal bonding method of the membrane 10 to the bonded body 20 according to the present invention through drawings or captured images.

Figures 5(A) and 5(B) show cell leakage when tested using a conventional adhesive tape (Figure 5(A)) and cell leakage when the thermal bonding method according to the present invention is used. This is to compare the appearance ((B) in Figure 5).

Comparing Figure 5 (A) and Figure 5 (B), it can be seen that some of the cells in the conventional adhesive tape method invaded the interface and leaked, and the cells in the heat bonding method according to the present invention It can be seen that it exists only inside the permeable surface 12 within the interface 13. For reference, in the conventional adhesive tape method, cells that invade the interface and break away cannot properly receive nutrients or oxygen, so the corresponding cells The desired experimental data cannot be obtained, and overall cell culture may not be performed properly.

Figures 5(C) and 5(D) show cell outflow in the conventional adhesive tape method (Figure 5(C)) and cell outflow in the thermal bonding method according to the present invention (Figure 5). (D)) is an illustrative drawing.

As shown in (C) of FIG. 5, cells in the conventional taping method remain in the gap between the adhesive surface 11a even if the adherend 20 and the membrane 10 are attached through adhesive tape. It could be a leak.

On the other hand, referring to (D) of FIG. 5, the cells in the thermal bonding method according to the present invention do not leak out due to bonding to the bonded body 20 by thermal deformation of the nanofibers of the membrane 10, By allowing movement only within the transmission surface 12, high-quality experimental data and excellent cell culture effects can be achieved.

In this way, it can be seen that the nanofiber structure of the membrane 10, which is thermally modified by thermal bonding, can prevent cell escape and is an important factor in obtaining good results in cell culture and cell research/analysis.

On the other hand, in order to form the nanofiber structure of the membrane 10, the joint 21 must be heated to a 'specific temperature'. To define the range of this specific temperature, it is [melting point of the membrane] to [melting point of the membrane. + 10°C] can be defined as a specific range of preferred temperatures.

As an example, when heat bonding a nanofiber membrane 10 made of PCL (polycaprolactone), the 'range of specific temperature' may be 62°C to 68°C, and if any one value is set as a specific temperature, it is preferably 65°C. It can be.

The basis that the above temperature can form the optimal membrane 10 nanofiber structure for thermal bonding can be found through [Experimental Example 1], [Experimental Example 2], and [Experimental Example 3] of FIGS. 6 to 8. I want to see it.

For reference, the temperature of the joint 21 at the moment the membrane contacts it is an important factor in determining the state of the joint surface, and this temperature is [melting point of the membrane] ~ [melting point of the membrane + 10°C. ] (For convenience, this temperature range is referred to as the contact temperature range), as described above, that the bonding surface, transmission surface, and interface desired by the user (experimenter) can be formed. However, at this time, the joint 21 may be heated to a temperature higher than the contact temperature range (for convenience, this is referred to as a preset temperature) before contacting the membrane, which lasts until the joint 21 contacts the membrane. This takes into account the situation in which the joint 21 is naturally cooled during the time taken. The preset temperature may be determined by considering the time or cooling rate from when the junction 21 is heated until it contacts the membrane, and may preferably be 20°C higher than the contact temperature range.

Figure 6 is a diagram showing [Experimental Example 1] of the present invention.

[Experiment Example 1]

Figure 6 (A) is a graph showing how the temperature of the bonding surface 11 and the transmission surface 12 of the membrane 10 changes depending on the temperature applied to the bonding portion 21 of the bonded body 20, This graph shows the temperature at which the nanofiber structure deforms, ' (hereinafter referred to as melting point) '60°C is indicated as a reference line. [Experimental Example 1] of the present invention attempts to analyze data using this melting point, and derives the temperature at which this melting point forms the optimal nanofiber structure of the membrane 10 for thermal bonding.

For reference, the reason why the temperatures of the bonding surface 11 and the transparent surface 12 are different while thermal bonding is in progress is that the bonding surface 11 is in direct contact with the bonding portion 21 of the heat-heated member 20. Therefore, while it has a relatively high temperature compared to the transparent surface 12, the transparent surface 12 is adjacent to the joint 21, but is not in direct contact with it, and therefore has a relatively low temperature compared to the joint surface 11. Understand that you have shame.

When referring to Figure 6 (A), the temperature applied to the joined body 20, more precisely, the joined portion 21 ( ) is 55℃, 60℃, 65℃, 70℃, 75℃. Monitoring the temperature of the joint surface (11) and the transmission surface (12), when heated to 55℃, 60℃, the joint surface (11) and the temperatures of the transmission surface 12 are both It was found that it did not reach . Conversely, when heated to 70°C or 75°C, the temperatures of both the joint surface 11 and the transmission surface 12 are It was found that significant deformation occurred in the nanofiber structure. On the other hand, when the joint 21 is heated to 65°C, the temperature of the joint surface 11 is While exceeding , the temperature of the transmission surface 12 is It was found that the nanofiber structure could be maintained in the most ideal state by not exceeding .

Meanwhile, Figure 6 (B) shows a photograph actually taken of the nanofiber structure of the joint surface 11 and the transmission surface 12 according to the heating temperature.

Referring to (B) of FIG. 6, 'the temperature applied to the bonded body 20 ( )' is 55°C, the bonding surface 11 does not melt properly and only a portion of the nanofiber structure is deformed, and at this temperature, thermal bonding between the membrane 10 and the bonded body 20 is not properly achieved, and some bonding occurs. However, it was found that the adhesion between the membrane 10 and the conjugate 20 was weak, so they could be easily separated and problems with cell leakage may occur.

'Temperature applied to the bonded body 20 ( )' is 65°C, the nanofiber structure of the bonding surface 11 is deformed by heat and has bonding strength with the bonded body 20, and at this time, the nanofiber structure of the transparent surface 12 is not thermally deformed. It was found that the fiber structure was optimal for thermal bonding.

Meanwhile, the 'temperature applied to the bonded body 20 ( )' is 75°C, not only is the nanofiber structure of the bonding surface 11 deformed by heat, but also a part of the transmission surface 12 adjacent to the bonding surface 12 is thermally deformed, thereby reducing the transmission of the transmission surface 12. It was found that some functions may be lost. Through the above [Experimental Example 1], it was found that the 'specific temperature' that forms the nanofiber structure of the membrane 10 in thermal bonding is 65 ℃, and when thermal bonding is performed within 75 ℃, normal thermal bonding occurs. It was confirmed that the structure (1) was obtained as a result, and through this, it was confirmed that the temperature required for thermal bonding was within the range of the melting point of the membrane (10) or 10°C greater than the melting point of the membrane (10). .

Figure 7 is a diagram showing [Experimental Example 2] of the present invention.

[Experiment Example 2]

[Experimental Example 2] of the present invention is a diagram showing the heat conduction phenomenon through color during the thermal bonding process between the membrane 10 and the bonded body 20.

7 (A) to (C) respectively show the 'temperature applied to the bonded body 20 ( )' shows the heat conduction phenomenon between the membrane 10 and the bonded body 20 when 'is 55°C, 65°C, and 75°C. In addition, Figures 7 (A) to (C) indicate the temperature of the corresponding location through color, and the temperature of the corresponding color is indicated on the right (e.g., dark red = 80 ℃, dark blue = 24 ℃)

First, (A) in FIG. 7 shows the 'temperature applied to the bonded body 20 ( )' is 55℃. In this case, the joint surface 11 has not yet reached the temperature corresponding to the melting point (60℃: yellow), so it can be inferred that thermal deformation has not been properly achieved. .

(B) in Figure 7 shows the 'temperature applied to the bonded body 20 ( )' is 65°C. In this case, the bonding surface 11 reaches the melting point range and undergoes thermal deformation, and at the same time, a bonding phenomenon can occur between the membrane 10 and the bonded body 20. can be seen. Meanwhile, it can be seen that the temperature conducted to the transparent surface 12 by the heated heat is within the range below the melting point (yellow), so the transparent surface 12 will not undergo thermal deformation.

(C) in Figure 7 shows the 'temperature applied to the bonded body 20 ( )' is 75°C. In this case, the joint surface 11 will reach a range exceeding the melting point and undergo thermal deformation, and due to the heat conduction phenomenon, the adjacent transparent surface 12 will also have a temperature above the melting point. It can be seen that the conduction occurred and thermal deformation occurred.

Through the above [Experimental Example 2], at 65°C, which is the 'specific temperature' of the present invention, thermal deformation occurs only at the joint surface 10, and heat conduction phenomenon is also conducted at a temperature below the melting point (60°C: yellow). It was found that the transparent surface 12 can form an optimal nanofiber structure of the membrane 10 that is not thermally deformed.

Figure 8 is a diagram showing [Experimental Example 3] of the present invention.

[Experiment Example 3]

[Experiment Example 3] of the present invention is an experiment that once again supports that the 'specific temperature' of 65°C, that is, the same temperature as the melting point of the membrane, is the optimal temperature. 55°C, 65°C, and 75°C at the joint 21. The actual product when the membrane 10 is actually heat bonded after being heated to a temperature of ℃ is compared, and the range in which the transparent surface 12 is not damaged when heated to the temperature listed above is graphed.

First, Figure 8 (A) shows a case where heat of 55°C is applied to the joint portion 21 of the bonded body 20 during thermal bonding, and the structure at this time has a relatively bright color on the bonded surface 11. The characteristic shown was that the fiber structure was not easily deformed.

Figure 8 (B) shows a case where heat of 65°C is applied to the joint portion 21 of the bonded body 20 during thermal bonding. Referring to Figure 8 (B), the structure at this time is the bonding surface. (11) showed a relatively dark color compared to (A), that is, there was a deformation of the fiber structure. In addition, the boundary between the bonding surface 11 and the transmission surface 12 was revealed quite clearly, and in particular, the transmission surface 12 area appeared to maintain a bright color because the fiber structure was not deformed.

Figure 8 (C) shows a case where heat of 75°C is applied to the joint portion 21 of the bonded body 20 during thermal bonding, and a portion of the transparent surface 12 to be placed on the existing bonded body 20 is shown. Heat above the melting point was conducted and thermal deformation was confirmed. In other words, it can be seen that the area that can be used as the transmission surface 12, that is, the ‘proportion of the undamaged transmission surface (Y-axis in (D) of FIG. 8)’ has decreased.

Meanwhile, Figure 8 (D) is a graph showing the ‘proportion of undamaged transmission surface (ratio of normal transmission surface 12)’ of the transmission surface 12 according to the applied temperature.

Referring to (D) of FIG. 8, when a temperature exceeding 65°C is applied, heat above the melting point is conducted to the transmission surface 12, and the surface that is not damaged by thermal deformation gradually decreases, and the 'undamaged transmission surface' The 'ratio' showed a decrease, and as the temperature increased, the undamaged surface gradually decreased, and at 85℃, it was confirmed that the undamaged transparent surface dropped to the 10% range.

Through [Experimental Example 3], we were able to once again support the experimental results of [Experimental Example 2]. In thermal bonding, when heat higher than the melting point of the membrane is applied to the joint, heat is also applied to the transmission surface (12). It was confirmed that the proportion of the undamaged surface, that is, the non-damaged surface 12a, among the transmission surfaces 12 gradually decreased.

Figure 9 is a diagram showing a method of manufacturing the structure (1) thermally bonded to the membrane (10) of the present invention.

Referring to FIG. 9, the method of manufacturing the structure 1 according to the present invention may first include a step (S101) of applying heat at a specific temperature to the joint portion 21 of the bonded body 20. Here, the joint portion 21 of the bonded body 20 refers to a surface that corresponds to and is joined to the bonding surface 11 of the membrane 10 when the membrane 10 is bonded to the bonded body 20. In addition, the specific temperature referred to here is when the bonding surface 11 reaches the melting point, which is the temperature at which the membrane 10 begins to undergo thermal deformation, and the melting point occurs on the transparent surface 12 adjacent to the bonding surface 11 during the thermal bonding process. It refers to the temperature below which heat can be conducted.

After step S101, a step (S102) of bonding both components may be included by allowing the joined portion 21 of the heated member 20 to approach the membrane 10. At this time, joining efficiency can be improved by applying a predetermined pressure (pressing pressure) in the direction being joined.

Although not shown in FIG. 9, after step S102, a step of allowing the bonded state to rest for a predetermined period of time may be added to provide time for the bonded surface 11 to undergo thermal deformation and harden again.

Figure 10 is a diagram showing how a user forms a bonding surface 11 and a transparent surface 12 of a desired shape.

The user can form the bonding surface 11 and the transmission surface 12 of the desired shape, and after heat bonding, the transmission surface 12 of a specific shape (triangle, square, or star shape) or In order to form the joint surface 11, the user forms the desired shape on one surface of the heating element 30 (e.g., a sculpture), and applies heat to one surface of the heating element 30 containing the formed shape. By stamping the surface of the membrane (10) as if stamping it, the transmission surface (12) or bonding surface (11) of the desired shape can be obtained.

Meanwhile, the structure (1) created through the process described above is subsequently further impregnated into a hydrogel, allowing the permeable surface (12) of the structure (1) to have a function similar to the characteristics of the extracellular matrix in the actual body. You can do it. Porous membranes made of general plastic materials have a limitation that their form and physical properties are very different from those of the extracellular matrix, so they largely lack similarity in form or function to cells in the body during cell culture. To overcome this, the extracellular matrix and its Hydrogels with more similar shapes and properties can be used. On a structure that has gone through the hydrogel impregnation stage (hereinafter abbreviated as a hydrogel structure), cells grow with a physical/chemical structure more similar to that of the body and feel stimulation, and as a result, the above hydrogel structure is more similar to the body. It can be a means by which the form and function of cells can be expressed.

Figure 11 shows the structure (1) to which the membrane is thermally bonded is impregnated with the hydrogel composition (50). As can be seen in the figure, the transmission surface of the structure (1) with the hydrogel composition (50) placed thereon. (12) can be sufficiently immersed in the hydrogel composition (50) to impregnate the membrane area of the permeable surface (12). At this time, the transmission surface 12 may have a mechanical strength of several tens of KPa to several GPa depending on the characteristics of the nanofiber structure. Thanks to these characteristics, a hydrogel with weak mechanical strength is impregnated into the nanofiber, resulting in a hydrogel. The culture environment can have excellent mechanical strength.

Meanwhile, nanofibers mentioned in this detailed description include silk fibroin, collagen, gelatin, cellulose, alginate, polycaprolactone, polyurethane, and polyvinylidene fluoride (PVDF). , polystyrene, polycarbonate, polyethyleneterephthalate (PET), polytetrafluoroethylene (PTFE), polylactic acid, polyvinyl alcohol (PLA Polyvinil alcohol, PVA) , Polymethacrylate (PMMA), polyethylene glycol (Polyethylene oxide, PEO), nylon (nylon-4,6), polyglycolic acid (PGA), and polylactic-co-glycolide (Poly( It may include at least one polymer nanofiber selected from the group consisting of (lactic-co-glycolic acid), PLGA), and preferably at least one polymer nanofiber selected from bio-derived materials such as silk fibroin, collagen, and gelatin. It may include fibers, and more preferably may be polymer nanofibers of silk fibroin. When using silk fibroin, there may be an advantage in that it has a strong bond with hydrogel.

Meanwhile, the hydrogel includes one or more thin film components selected from the group consisting of collagen, gelatin, agarose, extracellular matrix (ECM)-derived materials, Matrigel, and Geltrex. It can be done, preferably collagen. When cells are cultured on a hydrogel containing collagen, which accounts for a large portion of the extracellular matrix components, there is an advantage in that the survival rate, substrate adhesion, and functionality of the cells are improved. For example, the hydrogel composition may include 1 to 20% by weight of the thin film component; And it may contain 80 to 99% by weight of PBS or cell culture medium. More preferably, it may contain 3 to 30% by weight of the thin film component and 70 to 97% by weight of PBS or cell culture medium. At this time, in addition to the thin film components, water, PBS, cell culture medium, or acid/base solution may be further included. Specifically, the additional ingredients may be included so that the pH of the hydrogel composition is 5 to 9, and when collagen is included in the hydrogel thin film, it can have a three-dimensional structure when the pH is at the level of 7, forming three-dimensional cells. A hydrogel thin film with a structure suitable for culture can be provided.

In addition, the hydrogel structure according to the present invention may have a three-dimensional structure by impregnating the transparent surface 12 inside the hydrogel. Therefore, cells can be cultured in the three-dimensional structure of the permeable surface, and as described above, the hydrogel structure has superior transparency compared to conventional cell culture membranes, making it easier to observe cells during cell culture.

In addition, cells that can be cultured in the hydrogel structure according to the present invention may be fibroblasts, vascular endothelial cells, smooth muscle cells, nerve cells, cartilage cells, bone cells, skin cells, Schwann cells, or stem cells, but are not limited thereto. no.

On the other hand, after the structure (1) is immersed in the hydrogel composition and the transparent surface (12) is impregnated, an additional drying step may be added. Drying time may be 10 minutes or more, for example, 30 minutes or more and 6 hours or less at 20 to 50°C. If the time is less than 30 minutes, the hydrogel is not sufficiently dried, which may cause a problem in which the membrane shape of the permeable surface 12 is not formed. However, the drying time is not limited to this, and the drying time may be adjusted depending on the drying method. The drying may be performed, for example, by natural drying, oven drying, hot air drying, etc., but is not limited thereto. Preferably this is carried out by natural drying.

On the other hand, using the hydrogel structure according to the present invention, it is possible to create a cerebrovascular cell barrier model with a form and function similar to that of cerebrovascular tissue in the body. As briefly mentioned earlier, the hydrogel structure can create an environment similar to the cell production environment in the actual human body, and this can also be applied to the culture of cerebrovascular cells. That is, the hydrogel structure according to the present invention can provide rigidity and ease of observation suitable for culturing cerebrovascular cells, and the organelle structure, arrangement, and shape of cerebrovascular cells cultured on the hydrogel structure are similar to those of cerebrovascular cells in the body. It becomes possible to construct an in vitro blood-brain cell barrier model that is induced similarly to cells and contains physiologically similar levels of barrier function and metabolic capacity. As a result, in fields where in vitro cerebrovascular models are used, such as brain disease drug evaluation and brain physiology research, phenomena similar to drug absorption and metabolism in actual cerebrovascular vessels in the body can be simulated, observed, and analyzed in vitro. There is an effect.

Figure 12 shows a blood brain barrier (BBB) model using Brain Microvascular Endothelial Cells (BMECs) derived from induced pluripotent stem cells (iPSC). Referring to the drawing, the entire culture system can be configured with a hydrogel structure at the top and a culture plate at the bottom, where cerebrovascular cells differentiated from induced pluripotent stem cells (iPS) are combined with the hydrogel bound to the top. It can be confirmed that a blood-brain barrier model that mimics the cerebrovascular structure in the human body can be formed by culturing on the transparent surface 12 of the structure and culturing Astrocytes/Pericytes on the lower culture plate.

Examples of the structure (1) in which the above membrane (10) is thermally bonded, a method of manufacturing the same, a hydrogel structure in which this structure is impregnated into a hydrogel, and a method of forming a blood-brain barrier model using the same are provided. I looked.

The present invention is not limited to the specific embodiments and application examples described above, and various modifications can be made by those skilled in the art without departing from the gist of the invention as claimed in the claims. Of course, these modified implementations should not be understood separately from the technical idea or outlook of the present invention.

1: Structure 20b: Microfluidic chip
10: membrane 21: junction
11: joint surface 30: heating body
12: Transmissive surface 100: Membrane
13: interface 200: cell culture vessel
20: conjugate 300: plate
20a: cell culture vessel 310: opening

Claims (5)

In a structure in which a membrane is thermally bonded,
Membrane made of nanofiber material; and
A conjugate to which the membrane is bonded;
Including,
The membrane is,
A bonding surface formed by contacting a joint of the bonded object to which heat of a specific temperature is applied, and thermally deformed by contact and bonded to the bonded object; and
A surface other than the bonding surface, a transparent surface having a porous structure;
Including,
A structure with a thermally bonded membrane.
According to paragraph 1,
The specific temperature is,
Characterized in that it is within the range of melting point (Tm) to melting point (Tm) +10°C of the membrane,
A structure with a thermally bonded membrane.
According to paragraph 2,
The joint surface is,
A surface in which the nanofiber structure is modified and the permeability function is reduced compared to before modification, and the passage of fine cells or fluid is significantly restricted.
A structure with a thermally bonded membrane.
According to paragraph 3,
The structure is characterized in that it has a value of the undamaged transmission surface compared to the damaged transmission surface at different ratios depending on the approaching speed of the membrane and the joint,
A structure with a thermally bonded membrane.
In the method of manufacturing a structure with a thermally bonded membrane,
Applying heat at a specific temperature to the joint of the bonded body; and
A step of thermally bonding the bonded portion of the bonded body and the membrane by approaching them;
Characterized by including,
The membrane after the thermal bonding step includes a bonding surface and a transparent surface,
The bonding surface is formed by contacting the joint of the bonded object to which heat of a specific temperature is applied, and is a surface that is thermally deformed by contact and bonded to the bonded object,
The transparent surface is a surface other than the bonding surface, and is characterized in that it has a porous structure,
A method of manufacturing a structure with a thermally bonded membrane.

Images