KR20220036395A - Method for preparing collagen filled highly porous microsphere for delivery and formation of cell spheroid - Google Patents

Abstract

The present invention relates to a method for manufacturing porous microspheres filled with collagen for delivery and formation of cell spheroid, which protects cell spheroid from physical damage. According to the present invention, the method comprises the following steps: preparing a first polymer solution including a biodegradable polymer and a pore inducer; preparing a second polymer solution containing polyvinyl alcohol (PVA); forming microdroplets while supplying the first polymer solution in a dispersed phase and supplying the second polymer solution in a continuous phase in a microfluidic channel; forming porous particles by removing the pore inducer while freeze-drying the microdroplets; and impregnating collagen and cells into the porous particles.

Description

Manufacturing method of highly porous biodegradable microparticles filled with collagen for cell spheroid formation and delivery

The present invention relates to a method for manufacturing high porous biodegradable microparticles filled with collagen for cell spheroid formation and delivery, and collagen-filled high porosity biodegradable for cell spheroid formation and delivery by the manufacturing method Provides micro-particles.

In general, in cell culture, single-layer cells spread in the two-dimensional direction are cultured, so the two-dimensional cell culture has a limitation in that it does not properly simulate the cellular environmental conditions in our body.

A three-dimensional cell culture technology has been developed, and the three-dimensional cell culture technology collectively refers to a cell culture model that allows cells to grow in all dimensions or interact with the surrounding environment by artificially creating an environment similar to a living body in vitro.

Unlike the two-dimensional cell culture model, the three-dimensional cell culture model with excellent bioreactivity is made of a combination of cells, extracellular matrix, cell culture vessel, and cell growth factors, so that the cell culture model varies according to various combinations.

The 3D cell culture model is an animal that evaluates the effects of regenerative medicine, hybrid artificial organs, bio-useful material production, investigation and exploration of biological tissue or organ functions, screening of new drugs, and endocrine disrupting substances. It has potential to be used in industries in each field, such as an experimental alternative method and a cell chip having a sensor function.

Recently, culturing of spheroids, which is a three-dimensional cell tissue having a function equivalent to that of a living body, is attracting attention. In order to induce cell aggregation to mimic cancer, and to induce normal secretion of insulin for the treatment of diabetes, a method of transplanting aggregated cells is used in transplanting pancreatic cells, and stem cell research As various methods have been attempted for the three-dimensional culture of embryonic stem cells as they mature and applied to the study of various differentiation mechanisms, mass production technology and commercialization of spheroids are required.

Therefore, the production technology of 3D spheroids and cell culture technology using 3D spheroids that can not only guarantee better and more reliable precision but also minimize physical damage during injection into the body and provide a stable tissue regeneration function. development is needed

The present invention, in order to solve the above-mentioned problems, while having a uniform particle distribution, it is possible to form a plurality of spheroids on the particles, which is advantageous for tissue regeneration, and when injecting the spheroids into the body, To provide a method for preparing collagen-filled porous particles for spheroid formation and delivery, which can minimize possible physical damage.

The present invention is to provide a collagen-filled porous particle for spheroid formation and delivery, prepared by the method for producing the collagen-filled porous particle according to the present invention.

According to an embodiment of the present invention, preparing a first polymer solution comprising a biodegradable polymer and a pore derivative; Preparing a second polymer solution containing PVA (polyviniyalcohol); forming microdroplets while flowing the first polymer solution from the microfluidic channel to the dispersed phase and the second polymer solution as a continuous phase; forming porous particles by removing the pore derivative while freeze-drying the microdroplets; and impregnating collagen and cells in the porous particles; It relates to a method for producing collagen-filled porous particles, including, for spheroid formation and delivery.

According to an embodiment of the present invention, the biodegradable polymer is, poly-(β-caprolactone) (poly(β-carprolactone), PCL), poly(lactic-co-glycolic acid) (poly(lactic-coglycolic acid) ), PLGA), polyglycolic acid (poly(glycolic acid), PGA) and polylactic acid (PLA), wherein the pore derivative contains at least one selected from the group consisting of, camphene. And it may include one or more selected from the group consisting of gelatin.

According to an embodiment of the present invention, preparing the polymer solution includes: preparing a solution of a biodegradable polymer; preparing a pore derivative solution; and mixing the biodegradable polymer solution and the pore derivative solution; Including, the mixing ratio (weight ratio) of the biodegradable polymer solution to the pore derivative solution may be 75: 25 to 25: 75.

According to an embodiment of the present invention, the porous particles may have a particle size of 100 μm to 500 μm and a pore size of 1 μm to 100 μm.

According to an embodiment of the present invention, the step of impregnating collagen and cells in the porous particles may include: modifying the surface of the porous particles; the surface-modified porous particles; and mixing a first collagen solution containing cells; centrifugation at a temperature of 5° C. or less; mixing the porous particles and a second collagen solution after the centrifugation; and dispersing the porous particles in a solution and curing the collagen impregnated in the porous particles.

According to an embodiment of the present invention, the step of curing the collagen may be curing at a temperature of 35 °C to 40 °C.

According to an embodiment of the present invention, the step of curing the collagen may be to fill the collagen in 50% to 100% by volume in the pores.

According to an embodiment of the present invention, the solution in the step of curing the collagen is a cell culture solution, and after the step of curing the collagen, culturing the cells; It may further include.

According to an embodiment of the present invention, the cells may be liver cells.

According to an embodiment of the present invention, the step of surface-modifying the porous particles may include immersing the porous particles in an alcohol solution having 1 to 3 carbon atoms; and washing the porous particles with deionized water and PBS solution; may include.

According to one embodiment of the present invention, it relates to a collagen-filled porous particle, prepared by the method according to the present invention, for hepatic spheroid formation and delivery.

The present invention uses a microfluidic system to prepare porous particles having a uniform size, and by using them to produce cell spheroids, a plurality of spheroids are formed in one particle, which is advantageous for tissue regeneration and in vivo. When spheroids are injected, it is possible to protect cell spheroids from physical damage. In addition, the present invention can provide a cell spheroid that can be utilized as an in vivo regenerative engineering and cell therapeutic agent.

FIG. 1 exemplarily shows a droplet formation process by a microfluidic system in the method for producing collagen-filled porous particles according to the present invention, according to an embodiment of the present invention.
FIG. 2 exemplarily shows a process for forming porous particles in the method for producing collagen-filled porous particles according to the present invention, according to an embodiment of the present invention.
FIG. 3 exemplarily shows the collagen impregnation process, cell culture process and utilization in the method for producing collagen-filled porous particles according to the present invention, according to an embodiment of the present invention.
4 is an image showing the results of cell collagen hardening and cell culture of the spheroids prepared in Examples according to an embodiment of the present invention.
5 is an image showing the cytotoxicity (Live/Dead cell analysis) analysis results of the spheroids prepared in Examples according to an embodiment of the present invention.
6 is an image showing the analysis result of the number of cells located in the spheroids prepared in Examples 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. In describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the terms used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or a custom in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Like reference numerals in each figure indicate like elements.

Throughout the specification, when a member is said to be located "on" another member, this includes not only a case in which a member is in contact with another member but also a case in which another member exists between the two members.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components.

The present invention relates to a method for manufacturing collagen-filled porous particles for spheroid formation and delivery according to the present invention, and according to an embodiment of the present invention, the manufacturing method is uniform using a microfluidic system It is possible to provide porous particles having a three-dimensional structure and biodegradability that can reduce physical damage during in vivo injection while having particle distribution. In addition, it is possible to provide porous particles, which can be prepared as excellent cell spheroids due to good cell culture.

According to an embodiment of the present invention, the manufacturing method comprises the steps of preparing a first polymer solution; preparing a second polymer solution; forming microdroplets; forming porous particles; and impregnating the collagen and cells.

According to an embodiment of the present invention, preparing the first polymer solution includes preparing a first polymer solution including a solvent, a biodegradable first polymer (or, referred to as a biodegradable polymer), and a pore derivative and the first polymer solution may be in a state in which the first polymer and the pore derivative are dissolved in a solvent.

In one embodiment of the present invention, the pore derivative includes a monoterpene-based compound, gelatin, or two. For example, the monoterpene-based compound includes camphene, CAMPANE, CAMPHANE, delta. -3-carene (δ-3-carene), P-cymene (p-cymene), limonene, myrcene, cis-ocimene, alpha-phellandrene (α) -phellandrene), beta-phellandrene, alpha-pinene, beta-pinene, sabinene, alpha-terpinene, gamma -Terpinene (γ-terpinene), terpinene (terpinolene), and alpha- may include at least one selected from the group consisting of thujene (α-thujene), preferably camphene (camphene).

In one embodiment of the present invention, the first polymer, that is, the biodegradable polymer is poly-(β-caprolactone) (poly(β-carprolactone), PCL), poly(lactic acid-co-glycolic acid) (poly(lactic acid) -coglycolic acid), PLGA), polyglycolic acid (poly(glycolic acid), PGA), polylactic acid (poly(lactic acid), PLA), polyanhydrides (poly(anhydrides)), polyorthoesters , polyethylene glycol (poly (ethyleneglycol)), polyurethane (polyurethane), polyacrylic acid (polyacrylic acid), poly-N-isopropyl acrylamide (poly (N-isopropyl acrylamide), CAMPHANE and poly (ethylene oxide)-poly ( Propylene oxide)-poly (ethylene oxide) copolymer (poly (ethyleneoxide)-poly (propyleneoxide)-poly (ethyleneoxide) copolymer) may include at least one selected from the group consisting of.

In one embodiment of the present invention, the first polymer and the pore derivative may be mixed after each dissolved and/or dispersed in a solvent. That is, the step of preparing the first polymer solution, preparing a solution of the biodegradable polymer; preparing a pore derivative solution; and mixing the biodegradable polymer solution and the pore derivative solution. The solvent of each solution may be the same or different.

For example, the first polymer may be included in 5 wt% to 10 wt% of the biodegradable polymer solution, and the pore derivative may be included in an amount of 40 wt% to 80 wt% in the pore derivative solution.

For example, in the step of mixing the biodegradable polymer solution and the pore derivative solution, the mixing ratio (weight ratio) of the biodegradable polymer solution to the pore derivative solution may be 75: 25 to 25: 75, the ratio range When included in it, a low pore ratio can be formed, and a decrease in the strength of the particles can be prevented due to an increase in the pore ratio. That is, as the mixing ratio of the pore derivative increases, the strength may decrease due to the increase in the internal pore ratio, thereby making it vulnerable to strength.

In one embodiment of the present invention, the first polymer, 1% to 10% by weight of the first polymer solution; 5% to 10% by weight; or 3 wt% to 5 wt%, wherein the pore derivative is 40 wt% to 99 wt% of the first polymer solution; 50% to 95% by weight; Or 80 wt% to 90 wt% may be included. When included within the above range, it is possible to provide porous particles with increased pore size and pore volume. For example, when the concentration of the pore derivative is 80% or more, it may be advantageous for the production of porous particles with large pores.

As an example of the present invention, the solvent may be included in an amount of 5% to 50% by weight of the remaining amount or the first polymer solution. The solvent is water and/or an organic solvent, and the organic solvent is dichloromethane, dimethyl carbonate, chloroform, an alcohol having 1 to 5 carbon atoms, hexane, heptane, pentane, octane, acetone, ethyl acetate, methylene chloride, carbon tetrachloride, Benzene, o-dichlorobenzene, toluene, xylene, pentane, mesitylene, cyclohexane, diethyl ether, tetrachloroethylene acetonitrile, dimethyl sulfoxide, dimethylformamide, trichloroethylene and oleic acid It may contain one or more selected from the group consisting of oleic acid, and is preferably a non-polar solvent to form a dispersed phase, for example, dichloromethane, dimethyl carbonate, chloroform, cyclohexane, carbon tetrachloride, ethyl acetate, toluene, pentane, hexane, heptane, octane, oleic acid and linoleic acid.

According to an embodiment of the present invention, the preparing of the second polymer solution comprises preparing a second polymer solution that can be applied as a non-dispersed phase (ie, continuous phase) in a microfluidic system, wherein the second polymer is It is a dilute concentration of 10% by weight or less in the second polymer solution, which can help to form uniform microdroplets by providing flow from the microfluidic system to the non-dispersed phase (ie, continuous phase). For example, the solvent, including water, an organic solvent, or both, room temperature to 100 ℃; Alternatively, a second polymer solution in which the second polymer is sufficiently dissolved may be prepared by heating at a temperature of 50 to 90°C.

As an example of the present invention, the second polymer is a polymer having solubility in water, and may be polyvinyl alcohol. The solvent may be selected from the solvents mentioned in the first polymer solution, and is preferably a hydrophilic solvent and water to act as a continuous phase.

According to an embodiment of the present invention, the forming of the microdroplets is a step of forming microdroplets in the microfluidic channel, that is, referring to FIG. 1 , the first polymer solution as a dispersed phase in the microfluidic channel. and forming microdroplets while flowing the second polymer solution in a continuous phase. When using such a microfluidic system, particles having a uniform size can be formed, and microporous particles can be formed through the drying process mentioned in the next step.

According to an embodiment of the present invention, the step of forming the porous particles is a step of removing the pore derivative from the microdroplets to form the porous particles. For example, referring to FIG. 2 , in the first polymer After the solvent is removed from the micro-droplets in which the pore derivative is distributed, the pore derivative is removed through freeze-drying to form porous particles. For example, the solvent is added to the microdroplets in the second polymer solution for 1 hour or more; more than 2 hours; Alternatively, after stirring for 2 to 20 hours, it can be removed by washing, for example, washing with deionized water. The freeze drying is carried out at a pressure of 30 Pa to 60 Pa, -30 °C to -40 °C or 25 °C to 50 °C temperature for 40 hours to 80 hours; or 45 to 75 hours.

As an example of the present invention, the porous particles may have a particle size of 100 μm to 500 μm and a particle size of 1 μm to 100 μm; 10 μm to 100 μm; Alternatively, it may have a pore size of 50 μm to 90 μm.

According to an embodiment of the present invention, the step of impregnating the collagen and cells is a step of impregnating the collagen and cells in the pores of the porous particles, for example, the step of impregnating the collagen and cells in the porous particles , surface-modified porous particles can be applied. More specifically, referring to Figure 3, the step of modifying the surface of the porous particles; mixing the surface-modified porous particles with a first collagen solution containing cells; centrifugation at low temperature; mixing the porous particles and a second collagen solution after the centrifugation; and curing the collagen impregnated in the porous particles; may include

In one embodiment of the present invention, the step of modifying the surface of the porous particle is to use a hydrophilic organic solvent for the porous particle, for example, the porous particle

Soaking in an alcohol solution having 1 to 3 carbon atoms; and washing the porous particles with deionized water and a buffer solution. The buffer may be, but is not limited to, TEA buffer, Tris buffer, phosphate buffer (Phosphate-buffered saline, PBS), pyrophosphate buffer, HEPES buffer, POPSO buffer, and the like.

According to an embodiment of the present invention, the step of centrifuging at a low temperature, a temperature of 5 ℃ or less; Alternatively, centrifugation at 2 ° C. to 1 ° C. to precipitate collagen and porous particles impregnated with cells, and removing the supernatant to remove excess collagen (and / or cells).

According to an embodiment of the present invention, the mixing of the porous particles and the second collagen solution includes the porous particles obtained after the centrifugation step and collagen in the same or different amounts as the first collagen solution. It is a step of mixing the second collagen solution containing By removing the excess collagen, it is possible to prevent the formation of collagen lumps other than collagen filled in the microparticles.

According to an embodiment of the present invention, the curing of the collagen impregnated in the porous particles comprises dispersing the porous particles in a solution to harden the impregnated collagen, wherein the solution is a cell culture solution, and 35° C. to 40° C. Curing the collagen in a temperature incubator. The cell culture medium may be applied by appropriately selecting a cell culture medium known in the technical field of the present invention according to the culture target, and is not specifically mentioned herein.

As an example of the present invention, less than 100% by volume in the pores through the curing process; 90% by volume or less; Or 20% by volume to 70% by volume of collagen may be filled. The hardened collagen may act as an extracellular matrix for cell culture and support.

According to an embodiment of the present invention, the method may further include culturing cells after curing the collagen. This is done in a cell culture solution, and a cell culture solution known in the technical field of the present invention can be applied, and it is not specifically mentioned herein. The cell culture medium may be appropriately selected depending on the type of cell, for example, 37 ° C., 5% CO ₂ Cultured in an incubator that provides a growth environment, it is possible to form cell spheroids.

As an example of the present invention, the cultured cells may be appropriately selected depending on the use of the porous particles of the present invention, and the cultured cells can be applied to cells known in the art. For example, cells isolated from tissue, muscle, skin, organ, bone, bone marrow, cartilage, blood, etc., more specifically, epithelial cells, fibroblasts, osteoblasts, chondrocytes, cardiomyocytes, hepatocytes, human-derived cord blood cells , mesenchymal stem cells, endothelial progenitor cells, embryonic stem cells, myoblasts, cardiac stem cells, and the like. In addition, the mesenchymal stem cells are not limited thereto, but may be isolated from bone marrow, muscle, fat, umbilical cord blood, amniotic membrane, or amniotic fluid. The vascular endothelial progenitor cells are not limited thereto, but may be isolated from blood, umbilical cord blood, embryos, or bone marrow.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited to the following examples.

Example

Method for producing porous micro-particles

After dissolving 5% polycaprolactone (PCL) and 80% camphene porogen in each vial in chloroform, the two solutions were mixed at 6:4 (wt.%/wt.%). A 2% polyvinyl alcohol solution was prepared by heating at 90° C. for at least 8 hours using deionized water.

A 5% polycaprolactone solution as a dispersed phase and a 2% polyvinyl alcohol solution as a continuous phase were put into the microfluidic system and flowed. The prepared microdroplets were placed in a beaker with 2% polyvinyl alcohol solution and stirred at 300 RPM. After 5 hours of washing with deionized water, the pore-conducting material was removed through freeze-drying. where % is wt.%.

Liver spheroid culture method using collagen-filled porous particles

For surface modification and washing of the porous particles, they were immersed in 70% alcohol for about 1 hour, and then the ethanol was washed with deionized water and PBS solution. 2% collagen and hepatocytes were mixed, a vial with porous particles was put, and pipetting was thoroughly mixed with .

As shown in FIG. 3 , the particles were down using a centrifuge at 3000 RPM for 5 minutes and excess collagen was removed as much as possible. Using a stirrer, collagen and porous particles were mixed again. The collagen-filled porous particles were added to the cell culture solution, and the collagen was cured by putting it in an incubator at 37° C. for at least 1 hour.

The images of porous particles, cell seeding, and cell seeding after 1 day, after collagen curing, and after 5 days after collagen curing are shown in FIG. 4 . In FIG. 4 , after cells containing collagen were included in the cells and cured at 37° C. for one hour, cells not contained in collagen were removed, and then microparticles were observed from 1 to 5 days after removing the escaped cells. It can be confirmed that the cells are located inside the collagen through an optical microscope.

5 shows cell viability was confirmed through live dead analysis in porous microparticles. It can be seen that the cells gradually increase to form cell spheroids inside the porous particles.

6, DAPI analysis was performed to stain cell nuclei in order to clearly identify the number of cells located in the porous microparticles, and it can be confirmed that a large number of cells are located inside the pores of the particles in the form of spheroids as time passes. .

The present invention provides a method for producing collagen-filled biodegradable porous particles for culturing and delivering liver spheroids, and after dispersing and mixing a biodegradable polymer and a pore-conductor in an organic solvent, a polymer through a microfluidic system The solution was flowed into the dispersed phase and the PVA solution was flowed into the continuous phase to prepare particles of uniform size, and then porous particles were prepared by removing the pore-covalent conductor through freeze-drying. After surface modification of the prepared porous particles, the collagen solution in which the cells were mixed was filled and remained, thereby preparing biodegradable porous particles filled with collagen. Cell spheroids were manufactured using the collagen-filled porous particles, and the cell spheroids can form a plurality of spheroids in one particle, so it is advantageous for tissue regeneration, and when injecting spheroids into the body It is possible to protect the cell spheroids from physical damage that may occur during injection.

As described above, although the embodiments have been described with reference to the limited embodiments and drawings, various modifications and variations are possible from the above description by those skilled in the art. For example, even if the described techniques are performed in an order different from the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or substituted by other components or equivalents Appropriate results can be achieved. Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (11)

Preparing a first polymer solution comprising a biodegradable polymer and a pore derivative;
Preparing a second polymer solution containing PVA (polyviniyalcohol);
forming microdroplets while flowing the first polymer solution from the microfluidic channel to the dispersed phase and the second polymer solution as a continuous phase;
forming porous particles by removing the pore derivative while freeze-drying the microdroplets; and
impregnating the porous particles with collagen and cells;
containing,
Method for preparing collagen-filled porous particles for spheroid formation and delivery.
According to claim 1,
The biodegradable polymer is, poly-(β-caprolactone) (poly(β-carprolactone), PCL), poly(lactic-co-glycolic acid) (poly(lactic-coglycolic acid), PLGA), polyglycolic acid ( containing at least one selected from the group consisting of poly(glycolic acid), PGA) and polylactic acid (PLA),
The pore derivative will include at least one selected from the group consisting of camphene and gelatin,
A method for preparing collagen-filled porous particles.
According to claim 1,
The step of preparing the polymer solution,
preparing a solution of a biodegradable polymer;
preparing a pore derivative solution; and
mixing the biodegradable polymer solution and the pore derivative solution;
including,
The mixing ratio (weight ratio) of the biodegradable polymer solution to the pore derivative solution is 75: 25 to 25: 75,
A method for producing collagen-filled porous particles.
According to claim 1,
The porous particles will have a particle size of 100 μm to 500 μm and a pore size of 1 μm to 100 μm,
A method for preparing collagen-filled porous particles.
According to claim 1,
The step of impregnating collagen and cells in the porous particles,
modifying the surface of the porous particle;
the surface-modified porous particles; and mixing a first collagen solution containing cells;
centrifugation at a temperature of 5° C. or less;
mixing the porous particles and the second collagen solution after the centrifugation; and
dispersing the porous particles in a solution and curing the collagen impregnated in the porous particles;
which includes,
A method for preparing collagen-filled porous particles.
6. The method of claim 5,
The step of curing the collagen is to cure at a temperature of 35 ℃ to 40 ℃,
A method for preparing collagen-filled porous particles.
6. The method of claim 5,
The step of curing the collagen is to fill the collagen in 50% to 100% by volume in the pores,
A method for preparing collagen-filled porous particles.
6. The method of claim 5,
The solution in the step of curing the collagen is a cell culture solution,
After curing the collagen, culturing the cells;
which further comprises
A method for preparing collagen-filled porous particles.
According to claim 1,
The cell is a liver cell,
A method for preparing collagen-filled porous particles.
6. The method of claim 5,
The step of surface-modifying the porous particles,
immersing the porous particles in an alcohol solution having 1 to 3 carbon atoms; and
washing the porous particles with deionized water and PBS solution;
which includes,
A method for preparing collagen-filled porous particles.
It is prepared by the method of claim 1,
Collagen-filled porous particles for liver spheroid formation and delivery.

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