KR20150125853A - Method of fabricating scaffold for non-autologous cell encapsulation - Google Patents

Abstract

The present invention relates to a scaffold production method for transplanting non-autologous cells. According to an embodiment of the present invention, a flat heat-dissipation device integrally comprises: a lower case; a microfiber structure formed on an inner surface of the lower case; and a supporting projection attached from each other to face the lower case and partially protruding toward the microfiber structure to support the same, and the flat heat-dissipation device further comprises an upper case. In the microfiber structure, a liquid flow is formed as a coolant liquid invades therein. Moreover, a gas flow is formed by vaporized coolant gas at a space between the upper and lower cases.

Description

[0001] METHOD OF FABRICATING SCAFFOLD FOR NON-AUTOLOGOUS CELL ENCAPSULATION [0002]

The present invention relates to a scaffold manufacturing method, and more particularly, to a scaffold manufacturing method for a tachy cell implantation.

Currently, millions and hundreds of millions of patients worldwide suffer from diseases such as Parkinson's Disease and diabetes, which are caused by the failure of normal hormone secretion, and most of them deal with symptoms by continuously injecting drugs . However, it is a reality that it is difficult to cure these diseases with the current drug administration method which is close to the side of the symptom suppression rather than the root cause of the disease and the secretory system itself.

However, as drug delivery system technology has been developed in recent years, a new approach is being investigated. This is a method of continuously releasing proteins and drugs in the body rather than administering drugs externally. This can be seen as a significant benefit because it does not require continuous administration of medications and can reduce patient suffering and reduce social costs incurred during outpatient treatment. However, drug delivery systems developed for this purpose are limited in their application due to their limited duration or weak mechanical strength.

Based on the technical background as described above, the present invention combines a drug delivery system technique and a scaffold production technique used for tissue engineering to treat diseases requiring drug administration on a regular and long-term basis, And to produce scaffolds capable of sustained drug release over a long period of time.

In addition, the present invention intends to produce a scaffold that does not suffer from external force even if it is implanted in most parts of the body by combining the drug delivery system and the free shape fabrication technique described above.

According to another aspect of the present invention, there is provided a method for manufacturing a scaffold for a different cell embryo, comprising the steps of: preparing a frame structure using an organic-inorganic resin; injecting the gel solution into the frame structure together with a culture solution containing cells; And gelling the gel solution containing the cells in the mold structure.

The step of fabricating the mold structure may include forming the organic-inorganic resin layer by using a three-dimensional printing technique for forming a three-dimensional shape by extruding or injecting the material.

The step of injecting the gel solution into the mold structure together with the culture solution containing the cells may include injecting a gel solution mixed with the culture solution containing the cells between the gaps of the mold structure, Injecting the gel solution after injecting the gel solution into the mold structure, injecting the gel solution, and injecting the culture solution containing the cells.

The step of gelating the gel solution containing the cells in the mold structure may include gelation by injecting a curing agent into the gel solution, and the curing agent may include an ion-binding solution.

The ion-binding solution may include any one of a calcium chloride ion solution, a calcium sulfate ion solution, and a calcium carbonate ion solution.

The step of gelling the gel solution containing the cells in the mold structure may include a step of inserting a photo initiator, a monomer or a polymer into the gel solution, Or a photocurable gel is used, and in the state that the gel solution containing the cells is injected between the gaps of the mold structure, the photocurable gel or the photocurable gel is reacted And gelling the gel solution by irradiating light.

The cell-binding substance may include an Arginyl-glycyl-aspartic acid (RGD) family.

In the case where the organic-inorganic resin is a photo-curable organic-inorganic resin, the step of fabricating the frame structure comprises: laminating a two-dimensional layer using a projection-based microstereolithography equipment to produce the frame structure of a three- .

The method may further include forming the concave and convex portions on the surface of the frame structure by placing the frame structure into ethanol, IPA (isopropanol) or acetone containing fine particles, and using a vortex generator. Alternatively, the method may further comprise the step of forming irregularities on the surface of the mold structure using a sandblaster, or coating the surface of the mold structure with any one of polyadpamine, RGD, and mussel adhesive proteins.

And washing the mold structure.

The step of washing the mold structure may be performed by exposing the mold structure to ultraviolet rays in a state of being immersed in IPA (isopropanol), ethanol or water after putting the mold structure into acetone or ethanol.

The cleaning of the mold structure may include using a vortex generator or an ultrasonic washer in which the mold structure is placed in ethanol, IPA (isopropanol) or acetone.

The step of injecting the gel solution together with the cell-containing culture solution into the mold structure is carried out by mixing 2% (w / v) of the alginate gel solution with the cell culture solution at a ratio of 1: 1 w / v) concentration of cells.

The gel solution may be a gel solution having Arginyl-glycyl-aspartic acid (RGD) attached thereto.

According to the above-described method for manufacturing a scaffold for differentiated cell transplantation, it is possible to produce a scaffold that does not suffer from external force even if it is implanted in most parts of the body.

FIG. 1 is a conceptual diagram for explaining the principle of a scaffold for other cell transplantation.
FIG. 2 is a view for explaining a free shape fabrication technique in a method of manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention. Referring to FIG.
FIG. 3 is a block diagram of a projection-based microstereolithography equipment in a method of manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.
FIG. 4 is a conceptual diagram of a three-dimensional printer in which a material is extruded or injected in a method of manufacturing a scaffold for tagging cells according to an embodiment of the present invention.
FIG. 5 is a plan view, (b) side view, and (c) perspective view showing a structure of a frame structure manufactured by the method of manufacturing a scaffold for a different cell implant according to an embodiment of the present invention.
FIG. 6 is an image showing a frame structure manufactured by projection-based microstereolithography in the method of manufacturing a scaffold for tagging cells according to an embodiment of the present invention.
FIG. 7 is a schematic view showing a process of binding a framework and a gel in a method of manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.
FIG. 8 is an image showing cell behavior in a scaffold when anchorage-dependent cells are injected into a scaffold fabricated by a method for manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.
FIG. 9 is a graph showing the concentration profile of dopamine released from a scaffold when dopamine-releasing cells are injected into a scaffold fabricated by a method for manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and the same or similar components are denoted by the same reference numerals throughout the specification.

A common drug delivery system (DDS) is designed to release the required drug in the required amount in a specific system and then release it at a specific concentration in the body. However, these systems do not release any more drugs if they consume a certain amount of the drug. To overcome this point, the idea is to utilize living cells as a kind of "drug factory". For example, a system that puts dopamine-releasing cells into the body and steadily releases the dopamine secreted by the cells in need. These systems can release drugs steadily as long as the cells do not die, but there is a limit to the limited supply of cells. Thus, the use of xenogeneic cells is actively considered. In order to avoid the post-transplantation immune response, xenogeneic cells can be protected from immune rejection by encapsulation in a material such as hydrogel.

However, since the hydrogel is inherently weak in mechanical strength, there is a fear that the hydrogel may be damaged by movement in the body. According to the embodiment of the present invention, a hybrid scaffold can be manufactured by fusing a frame structure and a hydrogel to maintain a high mechanical strength. Hydrogels can be used for cell-based drug delivery systems (DDS) by incorporating drug-secreting cells.

FIG. 1 is a conceptual diagram for explaining the principle of a scaffold for other cell transplantation.

Referring to FIG. 1, the hybrid scaffold includes a framework and a hydrogel. The frame structure is a skeletal structure that not only maintains high strength but also serves as a site where adhesion-dependent cells can be attached, thereby maintaining the original characteristics of the cells. The hydrogel containing cells in the empty space of the frame structure functions to protect the transplanted cells from the host immune rejection reaction as previously known. Since the mechanical characteristics of the frame structure reflect the mechanical characteristics of the hybrid scaffold, the material and structure of the frame structure can be varied depending on the purpose of the hybrid scaffold. The material must not only be a biocompatible material, but also have no biodegradable properties if necessary. From a structural point of view, if a thick structure is constructed to maintain high strength and rigidity, the ratio of void space is reduced to reduce the amount of cells that can be implanted, and oxygen and nutrient delivery efficiency into the structure There is a possibility of falling.

The scaffold for the transgene cell transplantation according to an embodiment of the present invention can provide an environment in which the other cell can survive by inhibiting the immune rejection even if the transgene that can cause the immune rejection is transplanted into the body. To this end, oxygen, nutrients, and waste materials must be able to interact with the outside to enable cell survival, but it is necessary to inhibit contact with cells through the permeation of antibodies and the like. Therefore, a scaffold can be manufactured by combining a gel for protecting cells from an antibody or the like and a frame structure for preventing the gel from breaking due to external force due to weak strength.

Hereinafter, a method for preparing a scaffold for other cell transplantation having such a function and structure will be described.

First, a frame structure is manufactured using an organic-inorganic resin.

The step of fabricating the mold structure may include forming a three-dimensional mold structure by stacking two-dimensional layers using a projection-based microstereolithography equipment, or performing three-dimensional printing Inorganic resin layer may be laminated by using the above-mentioned technique. When the projection-based microstereolithography technology equipment is used, the organic-inorganic resin may be a photo-curable organic-inorganic resin.

Next, the gel solution is injected into the mold structure together with the culture medium containing the cells.

The step of injecting the gel solution into the mold structure together with the culture solution containing the cells may include injecting a gel solution mixed with the culture solution containing the cells between the gaps of the mold structure, Spraying the gel solution after spraying on the mold structure, spraying the gel solution, and spraying the culture solution containing the cells.

Next, the gel solution containing the cells is gelled in the mold structure.

The step of gelling the gel solution containing the cells in the mold structure may be performed by injecting a curing agent into the gel solution, and the curing agent may be an ion-binding solution. As the ion-binding solution, any one of a calcium chloride ion solution, a calcium sulfate ion solution, and a calcium carbonate ion solution may be selected and used.

In another embodiment, the step of gelling the gel solution containing the cells in the mold structure may be performed by inserting a photo initiator, a monomer or a polymer into the gel solution, Attaching a substance capable of cell binding to the gel material of the template structure or using a photo-curable gel, and in the state where the gel solution containing the cells is injected between the gaps of the template structure, the photoinitiator or the photo- The gel solution can be gelated by irradiating light having a wavelength equal to or less than a wavelength band. The cell-binding substance may include an Arginyl-glycyl-aspartic acid (RGD) family.

On the other hand, it is possible to form concave and convex portions on the surface of the frame structure so that cells are easily attached to the frame structure.

That is, the above-mentioned frame structure is put into ethanol, IPA (isopropanol) or acetone containing fine particles and the surface of the frame structure is formed by using a vortex generator, or by using a sandblaster, Or by coating the surface of the frame structure with a material selected from the group consisting of polydodamine, RGD, and mussel adhesive proteins.

The gel containing cells for the crossing cell transplantation can not exclude the possibility of gel breakage due to external force because of its low mechanical strength. However, this can be solved by installing the frame structure on the outside of the gel so that the gel is not directly exposed to external force. For the purpose of protecting the gel, the frame structure should be designed to have sufficient mechanical strength and have a space to fill the gel inside. In addition, in order to prevent the patient from feeling a foreign body when inserted into the body, And can be made as small as several millimeters. In this embodiment, a frame structure can be manufactured using a free-form production equipment or a three-dimensional printer, which will be described in detail with reference to the drawings.

FIG. 2 is a view for explaining a free shape fabrication technique in a method of manufacturing a scaffold for a different cell implant according to an embodiment of the present invention, and FIG. 3 is a diagram illustrating a structure of a projection-based microstereolithmetic technology equipment .

A device called projection based Micro-stereolithography (pMSTL) is a three-dimensional printing apparatus, and a free shape manufacturing technique is a method of laminating a two-dimensional layer of a three-dimensional shape as shown in FIG. Among the three-dimensional printing methods, stereolithography is a method of making a three-dimensional shape by curing a resin by selectively irradiating light to a photopolymer.

The pMSTL apparatus 100 used to fabricate a frame structure according to an embodiment of the present invention is an apparatus using the stereolithography technology. The apparatus 100 includes an ultraviolet (UV) lamp 110, Is transmitted to the photocurable resin selectively by using a Digital Micro Mirror (DMD) 120 to form a two-dimensional shape, and the process is repeated to produce a three-dimensional structure. Therefore, compared with the method of drawing a line to form a two-dimensional shape and then forming a three-dimensional shape by stacking the two-dimensional shape, the production speed is generally high. Also, when a plurality of structure layers are projected onto the window 130 by using the characteristics of the pMSTL to construct a structure by forming a two-dimensional shape at a time, a large amount of structures can be manufactured at a time. By using these characteristics, it is possible to further reduce the manufacturing time when a large number of structures are manufactured as compared with the general photo-shaping method.

FIG. 4 is a conceptual diagram of a three-dimensional printer in which a material is extruded or injected in a method of manufacturing a scaffold for tagging cells according to an embodiment of the present invention.

Referring to FIG. 4, there is a method of extruding or injecting a material as another embodiment of a method of manufacturing a frame structure, and in particular, a multi head deposition system (MHDS) 200 can be used. The equipment named as MHDS has several heads with material inserted, and each head can operate independently. Through this, the material for forming the mold is inserted into the head No. 1, the culture liquid containing the cells is inserted into the head No. 2, and the gel solution is inserted into the head No. 3, and when the two-dimensional layer is laminated, A desired material can be extruded into a three-dimensional shape. This method does not require a separate washing process unlike the case of using pMSTL.

The ultimate goal of tissue engineering is to replace human tissues that are deficient and destroyed by producing human tissue similar to real ones. For this purpose, materials applied to scaffolds must satisfy both biocompatibility and biodegradability. However, since the scaffold in the embodiment of the present invention aims at surviving the other cell as long as possible, the scaffold should not be broken, so it is appropriate to use a material having biocompatibility but low or no biodegradability.

5 is a plan view (a), a side view (b), and a perspective view (c) of a frame structure according to an embodiment of the present invention.

The size of the frame structure is 1.5 mm x 1.5 mm x 1.5 mm cube shape. It is intended to make the size smaller so that the patient does not feel foreign body as much as possible after the transplantation, and at the same time, It can be inserted into the body through a tube. Meanwhile, since the frame structure is small in size and can maintain the strength to protect the gel while filling the gel therein, the frame structure may have a shape as shown in FIG.

The material of the frame structure of the present embodiment may be various materials, but it is biocompatible and advantageous in that it has little or no biodegradability. For example, a photo-curable resin called Ormocomp (Ormocomp®, Micro-resist technology GmbH, Berlin, Germany) can be used, which is biocompatible and biodegradable. Therefore, in this embodiment, a three-dimensional Ormocomp frame structure can be manufactured using pMSTL.

When the Ormocomp mold structure designed as above is manufactured using pMSTL, the non-hardened Ormocomp surrounds the hardened Ormocomp mold structure, and a cleaning process is required to remove it.

In order to prevent the destruction of the structure during the cleaning process, the intensity of the structure can be improved by increasing the illumination time. At this time, in order to prevent the difference between the desired shape and the actual formed shape from occurring due to overcuring, pMSTL is used to form a desired shape by locally irradiating only the region where overcorrection occurs, The structure may be cleaned by using a suitable solvent other than the exclusive cleaning agent, and post curing may be applied using a UV gun to improve the strength.

As the cleaning method of the frame structure, a cleaning solvent such as acetone, ethanol, IPA or the like is used. In this case, the cleaning speed can be increased by generating strong turbulence using a vortex generator or an ultrasonic cleaner.

As another example, by increasing the surface roughness of the mold structure, the adhesion of the cells can be improved. To this end, fine particles are added to the solvent during the cleaning process using a solvent such as IPA, ethanol, or acetone, and a vortex generator or an ultrasonic washing machine It is possible to improve the adhesion of cells by providing irregularities on the surface of the order of several nm or 탆.

As another example, the surface of the frame structure can be coated with a material having good adhesion, such as polyadopamine, RGD, and mussel adhesive protein having good mutual adhesion with cells.

Fig. 6 shows the structure of the Ormocomp frame structure produced through such a process. It can be seen that the Ormocomp frame structure maintains its shape in spite of the low curing area.

The scaffold for transplanting a cell according to an embodiment of the present invention needs to inhibit the immune rejection upon transplantation into the body. For this purpose, the scaffold can use a gel as mentioned above. The size of the micropores in the gel is due to the oxygen and nutrients required for cell survival, the waste products and proteins produced from the cells, but not the antibodies. Since the scaffold for the different cell transplantation needs to survive the tag cells for a long time in the body, alginate with little or no biodegradability can be used.

There are many ways to make the gel solution into gel. Among them, ion crosslinking method in which a gel is made by adding calcium ion or the like, or a shared crosslinking method in which a shared crosslinking agent is added can be used. In addition, there is a cell cross-linking method. The cell cross-linking method improves the strength of the alginate gel by attaching Arginyl-glycyl-aspartic acid (RGD) to the alginate and binding the RGD and the cell receptor (Recptor) Even if the gel is applied to the gel, the receptor of the RGD and the cell binds again and returns to the gel state. However, since the strength of the cell crosslinking method itself is known to be weak, it can be used together with ion crosslinking.

Examples of the calcium ion used include calcium chloride, calcium sulfate and calcium carbonate. Of these, calcium chloride has a high gelation speed while calcium sulfate and calcium carbonate have a slow gelation rate. If the gelation speed is fast, the gelation reaction may start unevenly at each position. However, since the size of the gel produced in this embodiment is as small as several millimeters, there is an Ormocomp structure that protects the gel from external force Therefore, the intensity deviation may not be a big problem.

Alginate (A0682, Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 4% (w / v) was used in this example since the degradation rate is slower and the size of the fine pore is smaller as the concentration of the gel in the gel solution is higher. (Cell / alginate) solution containing cells at a concentration of 2% (w / v) made by mixing a 1: 1 solution of the cell / RGD alginate ) Solution was used as the alginate gel.

It is very difficult to put the cells and alginate into the Ormocomp mold structure in the gel state. Therefore, in this embodiment, the Ormocomp mold structure is prepared in advance and a cell / (No RGD / RGD) alginate solution is inserted After that, the calcium ion solution can be administered to make the gel inside the Ormocomp frame structure.

FIG. 7 is a schematic view showing a process of binding a framework structure to an alginate gel in a method for manufacturing a scaffold for tagged cell transplantation according to an embodiment of the present invention.

Referring to FIG. 7, the Ormocomp mold structure 50 is put in ethanol, UV sterilized for a day, and stored in a PBS solution at 37 ° C for one day. Thereafter, the PBS solution filled inside the structure is removed by using a vacuum chamber. This is because if the PBS solution remains inside the Ormocomp framework, the alginate gel does not penetrate into the Ormocomp framework due to the surface tension between the PBS and the Ormocomp framework. . If the inside of the Ormocomp frame structure is empty, insert the cell / alginate solution into the Ormocomp frame structure, add calcium ion solution, and gel over 20 minutes. After this process, the cell / alginate gel is formed inside the Ormocomp frame structure. And the combination of the cell, alginate gel, and Ormocomp structure made this way becomes a scaffold for other cell transplantation.

FIG. 8 is an image showing cell behavior in a scaffold when anchorage-dependent cells are injected into a scaffold fabricated by a method for manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.

Referring to FIG. 8, when the actual cells are injected into the scaffold, the cells can survive for about two months without significant problems. In particular, since cells are adhesion-dependent cells, .

FIG. 9 is a graph showing the concentration profile of dopamine released from a scaffold when dopamine-releasing cells are injected into a scaffold fabricated by a method for manufacturing a scaffold for a tagged cell implant according to an embodiment of the present invention.

FIG. 9 shows that when dopamine-releasing cells are injected into the scaffold, the dopamine produced by the survival of the cells is released well into the scaffold. Especially, in the case of scaffolds, dopamine secretion was higher than other cells injected without frame structure, even though other variables such as cell number were the same. It can be seen that the frame structure helps to improve the cell function more than the basic hydrogel-based DDS by providing not only the basic function of increasing the mechanical strength but also the site where cells can grow and attach.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, And it goes without saying that the invention belongs to the scope of the invention.

Claims (17)

Fabricating a frame structure using an organic-inorganic resin;
Injecting the gel solution into the mold structure together with the culture solution containing the cells;
Gelling the gel solution containing the cells in the mold structure;
≪ / RTI > wherein said scaffold is a scaffold. The method according to claim 1,
Wherein the step of fabricating the mold structure comprises:
Inorganic resin layer by using a three-dimensional printing technique for forming a three-dimensional shape by extruding or injecting a material. The method according to claim 1,
The step of injecting the gel solution into the mold structure together with the culture medium containing the cells may include:
Injecting a gel solution mixed with a culture solution containing the cells between the gaps of the frame structure, spraying the culture solution containing the cells onto the frame structure, spraying the gel solution, spraying the gel solution And optionally injecting a culture medium containing the cells. The method according to claim 1,
The step of gelling the gel solution containing the cells in the mold structure comprises:
And injecting a curing agent into the gel solution to cause gelation. 5. The method of claim 4,
Wherein the curing agent comprises an ion-binding solution. 6. The method of claim 5,
Wherein the ion-binding solution comprises any one of a calcium chloride ion solution, a calcium sulfate ion solution, and a calcium carbonate ion solution. The method according to claim 1,
The step of gelling the gel solution containing the cells in the mold structure comprises:
A photo initiator, a monomer or a polymer is inserted into the gel solution, a substance capable of cell binding is attached to the gel material of the gel solution, a photo-curable gel is used,
Irradiating light of a wavelength shorter than a wavelength range at which the photoinitiator or the photo-curable gel is reacted in a state where the gel solution containing the cells is injected between gaps of the mold structure to gellify the gel solution, A method for manufacturing a scaffold for implantation. 8. The method of claim 7,
Wherein the cell binding-capable material comprises Arginyl-glycyl-aspartic acid (RGD) -based material. The method according to claim 1,
Wherein the organic-inorganic resin is a photo-curable organic-inorganic resin,
Wherein the step of fabricating the mold structure comprises:
A method for fabricating a scaffold for other cell implantation in which a two-dimensional layer is laminated using a projection-based microstereolithography equipment to produce the frame structure of a three-dimensional shape. The method according to claim 1,
The step of forming a concave / convex pattern on the surface of the frame structure using a vortex generator by adding the frame structure to ethanol, IPA (isopropanol) or acetone containing fine particles, Gt; The method according to claim 1,
Forming a concavo-convex pattern on the surface of the frame structure using a sandblaster. The method according to claim 1,
Wherein the surface of the frame structure is coated with a material selected from the group consisting of polydodamine, RGD, and mussel adhesive proteins. The method according to claim 1,
Further comprising the step of washing the mold structure. 14. The method of claim 13,
Wherein the cleaning of the mold structure comprises:
Wherein the frame structure is immersed in any one of IPA (isopropanol), ethanol, or water, followed by exposure to ultraviolet light to cure the scaffold. 14. The method of claim 13,
Wherein the cleaning of the mold structure comprises:
Wherein the frame structure is placed in ethanol, IPA (isopropanol) or acetone, and a vortex generator or an ultrasonic washing machine is used. The method according to claim 1,
The step of injecting the gel solution into the mold structure together with the culture medium containing the cells may include:
And providing an alginate gel solution containing cells at a concentration of 2% (w / v) made by mixing the alginate gel solution at a concentration of 4% (w / v) with the culture solution containing the cells at a ratio of 1: A method for manufacturing a scaffold for cell implantation. 17. The method of claim 16,
Wherein the gel solution is a gel solution with Arginyl-glycyl-aspartic acid (RGD) attached thereto.

Images