LED irradiation device, hydrogel photocrosslinking method, and drug efficacy evaluation method using same

Abstract

The present invention relates to an LED irradiation device, a hydrogel photocrosslinking method, and a drug efficacy evaluation method using same. The LED irradiation device according to the present invention comprises: a plate including a plurality of wells; and a light generator installed at the bottom of the plate, wherein the light generator may include a plurality of LED light sources that irradiate light in the direction of the plate.

Description

LED irradiation device, hydrogel photocrosslinking method, and drug efficacy evaluation method using the same

[0001] The present invention relates to an LED irradiation device, a hydrogel photocrosslinking method, and a drug efficacy evaluation method using the same.

[0002] Hydrogel-based 3D cell culture is widely used to model the microphysiological microenvironment of natural tissues. Hydrogels provide complex biological environments by manipulating their physicochemical and mechanical properties using various materials and cross-linking methods. Among the various properties of hydrogels, mechanical properties are closely related to cell-extracellular matrix (ECM) interactions and mechanical signaling, and thus play a crucial role in various cellular functions, including cell proliferation. Therefore, controlling the mechanical properties of hydrogels is crucial for tissue physiology studies. Furthermore, developing a platform that can produce multiple samples by manipulating the mechanical properties of hydrogels is essential for effective drug testing.

[0003] Controlling the mechanical properties of hydrogels hinges on material selection and cross-linking methods. Among various methods, photocross-linking, which utilizes light energy to form covalent bonds linking polymer chains without heat or other harsh chemical reactions, offers significant advantages in controlling the mechanical properties of hydrogels. Compared to other methods, photocross-linking can be performed rapidly, typically within seconds to minutes, and allows for a wide range of modifications to the mechanical properties of hydrogels, making it a widely used method for fabricating 3D cell culture scaffolds. Despite these advantages, photocross-linking suffers from ultraviolet (UV) toxicity, which can be harmful to cells. Research into photocross-linking using visible light has improved biocompatibility, leading to potential applications in the development of in vitro biomodels and tissue regeneration. However, limitations persist, including the limited availability of light sources, resulting in batch variations due to differences in sample position and light exposure. Furthermore, when fabricating multiple hydrogels using a single light source, precise mechanical property control and significant property variation are difficult, and compatibility with high-throughput analytical equipment remains challenging. Therefore, to address these challenges, it is essential to develop a platform that utilizes minimally toxic materials and can consistently produce a variety of samples. The development of such a platform is a critical prerequisite for drug development and evaluation, which require consistent and numerous samples. Furthermore, the physical properties of hydrogels, the 3D cell culture scaffolds that serve as the cell environment in drug development, can influence cell response to drugs and signal transduction. Therefore, it is necessary to secure a library with diverse hydrogel properties for use in drug screening and efficacy evaluation.

[0004] The background technology described above is something that the inventor possessed or acquired in the process of deriving the disclosure of the present application, and cannot necessarily be said to be a publicly known technology disclosed to the general public prior to the present application.

[0005] In order to solve the above-described problem, the present invention provides an LED irradiation device including a plurality of LED light sources and a photocrosslinking method.

[0006] The present invention aims to provide an LED irradiation device, a photocrosslinking method, and a drug efficacy evaluation method using the same, which enable high-throughput analysis by controlling various mechanical properties of a hydrogel.

[0007] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the relevant technical field from the description below.

[0008] An LED irradiation device according to the present invention comprises: a plate including a plurality of wells; and a light generator installed at the bottom of the plate; wherein the light generator may include a plurality of LED light sources that irradiate light in the direction of the plate.

[0009] According to one embodiment, the LED light source is formed on a circuit board, and the circuit board may be of a microstrip type.

[0010] According to one embodiment, the well may individually correspond to the LED light source, and the center of the LED light source may be arranged in a straight line with the center of the well.

[0011] According to one embodiment, the plurality of LED light sources may be arranged in a plurality of columns or rows, and the plurality of columns or rows may be electrically controlled independently.

[0012] In one embodiment, the electrical manipulation may be to control one or more of the intensity, wavelength, and irradiation time of the light.

[0013] In one embodiment, the plate comprises from 6 to 96 wells, the wells having a size of from 1 mm to 50 mm.

[0014] In one embodiment, the distance between the bottom of the plate and the LED light source may be from 2 mm to 20 mm.

[0015] According to one embodiment, the LED light source may have an emission angle of 80° to 140° and a size of 0.5 mm to 20 mm.

[0016] In one embodiment, the light may have an intensity of 500 lux to 2500 lux and a wavelength of 350 nm to 600 nm.

[0017] In one embodiment, a hydrogel may be positioned in the well, and the hydrogel may be photocrosslinked by irradiating the light.

[0018] In one embodiment, the hydrogel may comprise at least one selected from the group consisting of collagen, gelatin, chitosan, fibrin, agar, starch, alginate, hyaluronic acid, dextran, elastin, carrageenan, polyethylene glycol, and decellularized tissue-derived extracellular matrix.

[0019] In one embodiment, the hydrogel may have one or more mechanical properties, and the hydrogel library may be formed simultaneously on a single plate.

[0020] In one embodiment, it may be compatible with high-throughput analysis equipment.

[0021] The hydrogel photocrosslinking method according to the present invention comprises the steps of: positioning hydrogels in each of a plurality of wells; and irradiating each of the plurality of wells with an LED.

[0022] According to one embodiment, the step of irradiating the LED may be using an LED irradiation device according to the present invention.

[0023] According to one embodiment, the step of irradiating the LED may be such that the light intensity is 500 lux to 2500 lux, the wavelength is 350 nm to 600 nm, and the light irradiation time is 5 seconds to 20 minutes.

[0024] In one embodiment, the mechanical properties of the photocrosslinked hydrogel may be from 0.1 kPa to 100 kPa, and the difference in properties between the photocrosslinked hydrogels may be within ±10%.

[0025] In one embodiment, the hydrogel may comprise cells.

[0026] A drug efficacy evaluation method according to the present invention comprises the steps of: photocrosslinking a hydrogel containing cells; adding a cell culture medium and culturing the cells; treating a drug; and analyzing; wherein the photocrosslinking step may be photocrosslinking using an LED irradiation device according to the present invention or photocrosslinking using a hydrogel photocrosslinking method according to the present invention.

[0027] In one embodiment, the photocrosslinking step may be forming a hydrogel library having one or more mechanical properties, and the analyzing step may be analyzing drug efficacy using the library.

[0028] The present invention provides an LED irradiation device including a plurality of LED light sources to control various mechanical properties of hydrogels and enable high-throughput analysis.

[0029] Specifically, the LED irradiation device according to the present invention can independently, accurately, and quickly control multiple LEDs, making it suitable for precise pharmaceutical testing using uniform samples. In particular, it can be used as a device for evaluating and screening drug efficacy and for examining cellular responses to drugs, as it can secure a library of hydrogels with different physical properties from a single plate.

[0030] Figure 1 is a schematic diagram showing an LED irradiation device according to the present invention.

[0031] Figure 2 is a schematic diagram showing a side view of a part of an LED irradiation device according to the present invention.

[0032] Figure 3 is a schematic diagram showing a drug efficacy evaluation method according to the present invention.

[0033] Figure 4 is a schematic diagram showing the internal structure and hydrogel photocrosslinking of an LED irradiation device according to an embodiment of the present invention.

[0034] Fig. 5 is a circuit diagram of an LED irradiation device according to an embodiment of the present invention.

[0035] Fig. 6 is a photograph showing light irradiation using an LED irradiation device according to an embodiment of the present invention.

[0036] Figure 7 shows the luminosity of an LED irradiation device according to an embodiment of the present invention.

[0037] Figure 8 shows the results of analyzing the temperature change of the bottom of the well plate of the LED irradiation device according to an embodiment of the present invention according to the light exposure time.

[0038] Figure 9 shows the results showing the Young's modulus of a photocrosslinked hydrogel according to the light irradiation time according to an embodiment of the present invention.

[0039] Figure 10 shows the results showing the swelling ratio of a photocrosslinked hydrogel according to the light irradiation time according to an embodiment of the present invention.

[0040] Figure 11 is an SEM image showing the pore size of a photocrosslinked hydrogel according to the light irradiation time according to an embodiment of the present invention.

[0041] Figure 12 shows the results showing the change in dry weight over time of a photocrosslinked hydrogel according to an embodiment of the present invention.

[0042] Figure 13 is a photograph showing the change in time of a photocrosslinked hydrogel according to an embodiment of the present invention.

[0043] Figure 14 is an image showing cell survival according to light irradiation time in a photocrosslinked hydrogel according to an embodiment of the present invention.

[0044] Figure 15 is a graph showing the quantitative results of cell survival and relative cell activity according to the light irradiation time in a photocrosslinked hydrogel according to an embodiment of the present invention.

[0045] Figure 16 shows the results of gene expression of osteoblast markers according to light irradiation time in a photocrosslinked hydrogel according to an embodiment of the present invention.

[0046] Figure 17 is a schematic diagram of the morphological changes of mesenchymal stem cells according to the stiffness of a photocrosslinked hydrogel according to an embodiment of the present invention and an F-actin immunostaining image.

[0047] Figure 18 is a result showing the difference in relative gene expression of biomarkers when a drug is treated in a hydrogel having various stiffnesses by crosslinking according to an embodiment of the present invention.

[0048] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be modified in various ways, and the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included within the scope of the patent application.

[0049] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0050] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0051] In addition, when describing with reference to the attached drawings, the same components will be given the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing an embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted. In addition, when describing a component of an embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only to distinguish the component from other components, and the nature, order, or sequence of the component is not limited by the terms. When a component is described as being "connected," "coupled," or "connected" to another component, it should be understood that the component may be directly connected or connected to the other component, but another component may also be "connected," "coupled," or "connected" between each component.

[0052] Components included in one embodiment and components with common functions will be described using the same names in other embodiments. Unless otherwise stated, the descriptions given in one embodiment can be applied to other embodiments, and detailed descriptions will be omitted to the extent of overlap.

[0053]

[0054] An LED irradiation device according to the present invention comprises: a plate including a plurality of wells; and a light generator installed at the bottom of the plate; wherein the light generator may include a plurality of LED light sources that irradiate light in the direction of the plate.

[0055] Fig. 1 is a schematic diagram illustrating an LED irradiation device according to the present invention. Referring to Fig. 1, the LED irradiation device includes a plate (102) including a plurality of wells (101) and a light generator (103) installed at the bottom of the plate, and may include a plurality of LED light sources (104) in the light generator (103). The LED irradiation device according to the present invention can uniformly irradiate a large number of samples simultaneously by including a plurality of wells (101) and a plurality of light sources (104).

[0056] Specifically, the light generator may further include a dial, a circuit board, a power supply, a resistor, a heat sink, a timer remote control, an aluminum enclosure case, and the like.

[0057] The above dial can control the intensity, wavelength, irradiation time, etc. of the light of the LED light source, and can be controlled by individual lines according to the dial.

[0058] The above power supply device may use a power supply dedicated to the light source to suppress the flicker phenomenon.

[0059] The above resistors may preferably be 6-way resistors and may be used to independently control the intensity, wavelength, irradiation time, etc. of light from multiple LED light sources.

[0060] The above heat sink is intended to suppress heat emitted from the circuit board and can help dissipate heat by making surface contact with the surface of the circuit board.

[0061] The above aluminum enclosure case can ultimately dissipate heat energy accumulated inside the case and can have a body grounding purpose to stabilize power and signal noise.

[0062] The above timer remote control may utilize radio frequency signals, as long as there is no problem in operating the relay inside the case. The timer remote control can remotely control the light output of the LED light source of the light generator.

[0063]

[0064] According to one embodiment, the LED light source is formed on a circuit board, and the circuit board may be of a microstrip type.

[0065] The circuit board is included in the light generator, and an LED light source can be formed on the circuit board. The circuit board is configured in a microstrip type, which allows for a large surface copper area, thereby having the advantage of controlling temperature increases by dissipating heat energy emitted from the LED light source to the surface.

[0066]

[0067] According to one embodiment, the well may individually correspond to the LED light source, and the center of the LED light source may be arranged in a straight line with the center of the well.

[0068] The above well and the LED light source correspond to one LED light source per well, and the center of each well and the center of each LED light source can be arranged in a straight line. When the wells and LED light sources correspond individually and their centers are aligned, uniform light can be irradiated to each well. In particular, by aligning the centers of the wells and the LED light sources, the distance, light intensity, and light emission angle can be effectively optimized.

[0069]

[0070] According to one embodiment, the plurality of LED light sources may be arranged in a plurality of columns or rows, and the plurality of columns or rows may be electrically controlled independently.

[0071] The above LED light sources may be arranged in multiple columns or rows, and may be arranged to match the arrangement of wells within the plate.

[0072] The above multiple columns or rows can be electrically manipulated independently for each column or row, so that different electrical manipulations can be applied to each column or row. This may be done by a resistor included in the light generator.

[0073]

[0074] In one embodiment, the electrical manipulation may be to control one or more of the intensity, wavelength, and irradiation time of the light.

[0075] That is, multiple columns or rows can control the intensity, wavelength, and irradiation time of light equally or differently for each column or row.

[0076]

[0077] In one embodiment, the plate comprises from 6 to 96 wells, the wells having a size of from 1 mm to 50 mm.

[0078] The above plate may preferably comprise 6, 12, 24, 48 or 96 wells.

[0079] The well is not limited in shape as long as it can contain a sample, but may be a square cylinder, a cylinder, a rhombus cylinder, etc., and preferably a cylinder. The size of the well refers to the longest length passing through the center of the bottom of the well, and preferably may be a diameter. The size of the well may preferably be 1 mm to 40 mm; 10 mm to 40 mm; 10 mm to 30 mm; or 20 mm to 30 mm.

[0080] If the size of the well is less than the above range, the amount of sample required for drug evaluation may be too small, which may cause difficulties in analysis. If the size of the well is greater than the above range, too much sample may be required, which may result in high costs.

[0081]

[0082] In one embodiment, the distance between the bottom of the plate and the LED light source may be from 2 mm to 20 mm.

[0083] Fig. 2 is a schematic diagram illustrating a side view of a portion of an LED irradiation device according to the present invention. Fig. 2 illustrates a side view crossing the center of one well and one LED light source, wherein the bottom of the plate may be the bottom (201) of a well within the plate, and the distance from this to the LED light source (202) may be optimized to irradiate uniform light.

[0084] The distance may preferably be 2 mm to 18 mm; 4 mm to 18 mm; 4 mm to 16 mm; 6 mm to 16 mm; 6 mm to 14 mm; 8 mm to 14 mm; or 8 mm to 12 mm.

[0085] If the distance between the plate bottom and the LED light source is less than the above range, there may be a problem in uniformly irradiating the entire sample or a problem in which the heat of the LED is directly transferred to the sample, and if it is more than the above range, there may be a problem in which the efficiency of light transfer to the sample is reduced.

[0086]

[0087] According to one embodiment, the LED light source may have an emission angle of 80° to 140° and a size of 0.5 mm to 20 mm.

[0088] Referring to FIG. 2, the light irradiation angle (203), which indicates the degree to which light spreads when light irradiated from an LED light source (202) shines on the bottom (201) of a well within a plate, can be controlled.

[0089] The above-mentioned luminescence angle may preferably be 90° to 140°; 90° to 130°; 100° to 130°; 100° to 120°.

[0090] If the emission angle is below the above range, there may be a limitation in the light's uniform distribution throughout the sample, and if it exceeds the above range, there may be a problem in that the light escapes outside the well and interferes with the surrounding sample. The emission angle within the above range may be a range that allows the light to be evenly irradiated without escaping outside the well, taking into account the distance between the plate bottom and the LED when the center positions of the well and the LED are aligned. This can minimize interference of the light from other LEDs in each well.

[0091] The size of the LED light source may preferably be 0.5 mm to 15 mm; 1 mm to 15 mm; 1 mm to 10 mm; or 5 mm to 10 mm.

[0092] If the size of the LED light source is less than the above range, there may be limitations in LED manufacturing or output, and if it exceeds the above range, there may be a problem of requiring unnecessary high costs.

[0093] The size of the above well and the size of the LED light source may be 4:1 to 2:1.

[0094]

[0095] In one embodiment, the light may have an intensity of 500 lux to 2500 lux and a wavelength of 350 nm to 600 nm.

[0096] The light intensity is measured at a distance of 1 cm from the light source, and may preferably be 500 lux to 2000 lux; 500 lux to 1500 lux; 500 lux to 1000 lux; 1000 lux to 2500 lux; 1000 lux to 2000 lux; 1000 lux to 1500 lux; 1500 lux to 2500 lux; 1500 lux to 2000 lux; or 2000 lux to 2500 lux.

[0097] If the light intensity is below the above range, there may be a problem of insufficient hydrogel crosslinking when used for photocrosslinking, and if it exceeds the above range, there may be a problem of negatively affecting the survival of cells within the hydrogel.

[0098] The wavelength of the light may be a visible light wavelength that is not biologically toxic. Preferably, the wavelength may be 350 nm to 550 nm; 350 nm to 500 nm; 350 nm to 450 nm; 350 nm to 400 nm; 400 nm to 600 nm; 400 nm to 550 nm; 400 nm to 500 nm; 400 nm to 550 nm; 450 nm to 600 nm; 450 nm to 550 nm; 450 nm to 500 nm; 500 nm to 600 nm; 500 nm to 550 nm; or 550 nm to 600 nm.

[0099]

[0100] In one embodiment, a hydrogel may be positioned in the well, and the hydrogel may be photocrosslinked by irradiating the light.

[0101] Referring to FIG. 2, a hydrogel (204) may be positioned at the bottom (201) of a well within a plate. The hydrogel (204) may be photocrosslinked by light irradiated by an LED light source (202).

[0102] The LED irradiation device according to the present invention can irradiate light for photocrosslinking of hydrogels. Photocrosslinking can solidify hydrogels in a short period of time and control the mechanical properties of the solidified hydrogels. Photocrosslinked hydrogels can provide a complex biological environment through their physicochemical and mechanical properties, and the mechanical properties can play an important role in cellular functions, including proliferation through mechanotransduction related to cell-extracellular matrix (ECM) interactions. Therefore, controlling the mechanical properties of hydrogels is important for tissue physiology research, and it is necessary to produce multiple samples by manipulating the mechanical properties of hydrogels. The present invention can simultaneously and uniformly photocrosslink hydrogels by positioning hydrogels in each of multiple wells and irradiating them with multiple LEDs. Therefore, the photocrosslinked hydrogels can produce three-dimensional tissue replicas that reproduce the mechanical properties of various tissues in the body.

[0103] The hydrogel may be positioned at the center of the well. This enables uniform light irradiation from the LED light source, and as a result, the hydrogel may be uniformly photocrosslinked.

[0104] In the LED irradiation device according to the present invention, the alignment of the center of the well and the center of the LED light source, the distance to the light source, the light emission angle, the light intensity, etc. are optimally formed, thereby enabling effective control of the mechanical properties of the hydrogel without deviation, and high-throughput analysis may be possible.

[0105]

[0106] In one embodiment, the hydrogel may comprise at least one selected from the group consisting of collagen, gelatin, chitosan, fibrin, agar, starch, alginate, hyaluronic acid, dextran, elastin, carrageenan, polyethylene glycol, and decellularized tissue-derived extracellular matrix.

[0107] The above hydrogel can be used without significant limitations, but preferably, it may be a composite hydrogel composed of one or more hydrogel components.

[0108]

[0109] The above hydrogel may include a photocatalyst. The photocatalyst may initiate photocrosslinking in response to light when irradiated with light, and may preferably be eosin Y.

[0110]

[0111] In one embodiment, the hydrogel may have one or more mechanical properties, and the hydrogel library may be formed simultaneously on a single plate.

[0112] The above hydrogel may have mechanical properties by photocrosslinking. Since it can exhibit various mechanical properties depending on light conditions, in the LED irradiation device according to the present invention, a plurality of hydrogels in a plurality of wells are independently controlled by a plurality of columns or rows of LED light sources, and hydrogels in the same column or row may have the same mechanical properties, but hydrogels in different columns or rows may have more than one mechanical property depending on different light conditions.

[0113] Accordingly, a hydrogel library comprising multiple hydrogels having various mechanical properties by photocrosslinking can be formed, which can be formed simultaneously within a single plate.

[0114] This enables the mass production of uniform 3D cell tissue mimic samples with various mechanical properties as a diverse hydrogel library is formed, and can be used as a device for drug efficacy and evaluation and screening according to mechanical properties, and for confirming cell responses to drugs.

[0115]

[0116] In one embodiment, it may be compatible with high-throughput analysis equipment.

[0117] As the LED irradiation device according to the present invention enables the production of a large number of samples that are uniformly processed, continuous mass analysis can be performed.

[0118]

[0119] The hydrogel photocrosslinking method according to the present invention comprises the steps of: positioning hydrogels in each of a plurality of wells; and irradiating each of the plurality of wells with an LED.

[0120] According to one embodiment, the step of irradiating the LED may be using an LED irradiation device according to the present invention.

[0121] Photocrosslinked hydrogels can provide a complex biological environment through their physicochemical and mechanical properties. These mechanical properties are closely related to mechanical signaling associated with cell-extracellular matrix (ECM) interactions, and thus play a crucial role in various cellular functions, including cell proliferation. Therefore, controlling the mechanical properties of hydrogels is crucial for tissue physiology research. The multiple wells of the hydrogel photocrosslinking method according to the present invention may be formed on a single plate. Each of the multiple wells may be illuminated by multiple LEDs. Accordingly, a single hydrogel may be positioned in a single well and illuminated by a single LED.

[0122] Preferably, the hydrogel may be photocrosslinked using an LED irradiation device according to the present invention. The LED irradiation device of the present invention can uniformly and consistently irradiate light from each LED to each well under optimized conditions such as distance, light intensity, and light emission angle, thereby reducing light interference to other wells, thereby simultaneously providing uniform photocrosslinked hydrogels.

[0123]

[0124] According to one embodiment, the step of irradiating the LED may be such that the light intensity is 500 lux to 2500 lux, the wavelength is 350 nm to 600 nm, and the light irradiation time is 5 seconds to 20 minutes.

[0125] The intensity of the light may preferably be 500 lux to 2000 lux; 500 lux to 1500 lux; 500 lux to 1000 lux; 1000 lux to 2500 lux; 1000 lux to 2000 lux; 1000 lux to 1500 lux; 1500 lux to 2500 lux; 1500 lux to 2000 lux; or 2000 lux to 2500 lux.

[0126] If the light intensity is below the above range, there may be a problem of insufficient hydrogel cross-linking, and if it exceeds the above range, there may be a problem of negatively affecting the survival of cells within the hydrogel.

[0127] The wavelength of the light may be a visible light wavelength that is not biologically toxic. Preferably, the wavelength may be 350 nm to 550 nm; 350 nm to 500 nm; 350 nm to 450 nm; 350 nm to 400 nm; 400 nm to 600 nm; 400 nm to 550 nm; 400 nm to 500 nm; 400 nm to 550 nm; 450 nm to 600 nm; 450 nm to 550 nm; 450 nm to 500 nm; 500 nm to 600 nm; 500 nm to 550 nm; or 550 nm to 600 nm.

[0128] The above light irradiation time may preferably be 5 seconds to 15 minutes; 30 seconds to 15 minutes; 30 seconds to 10 minutes; 1 minute to 10 minutes; or 1 minute to 5 minutes.

[0129] If the light intensity is below the above range, there may be a problem of insufficient hydrogel cross-linking, and if it exceeds the above range, there may be a problem of negatively affecting the survival of cells within the hydrogel.

[0130]

[0131] After the step of irradiating the LED, the well temperature may increase by less than 5°C compared to before the irradiation.

[0132] When irradiating with an LED, there may be a problem of heat generation and an increase in the temperature of the bottom of the well, but the hydrogel photocrosslinking method according to the present invention solves the temperature increase problem by using the LED irradiation device according to the present invention, so that photocrosslinking can be performed stably.

[0133]

[0134] In one embodiment, the mechanical properties of the photocrosslinked hydrogel may be from 0.1 kPa to 100 kPa, and the difference in properties between the photocrosslinked hydrogels may be within ±10%.

[0135] Hydrogels can have mechanical properties through photocrosslinking. These mechanical properties can indicate rigidity, and by possessing properties within the above range, they can be used to simulate some of the rigidity of human organ tissues.

[0136] Since various mechanical properties can be exhibited depending on the light conditions, it is possible to have the same mechanical properties while being uniformly photocrosslinked under the same light conditions using multiple LEDs. The difference in the properties of each hydrogel photocrosslinked under the same conditions can be within ±10% of the average properties, and it can be uniformly photocrosslinked. This solves the problem of controlling inhomogeneous tissue properties that arises from using a single light source for multiple samples or not considering sample size, and it can be used for precise pharmaceutical testing using homogeneous samples because it can be controlled accurately and quickly.

[0137]

[0138] In one embodiment, the hydrogel may comprise cells.

[0139] The above cells may be positioned within the hydrogel in a capsule form, and cell growth (proliferation, differentiation, etc.) may occur depending on the mechanical properties of the hydrogel photocrosslinking.

[0140] The above cells are not particularly limited, and may be any cells, especially animal-derived cells of organ tissues. Preferably, the cells may include at least one selected from the group consisting of blast cells, hepatocytes, fibroblasts, myoblasts, adult stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, and umbilical cord blood stem cells.

[0141] The cells may be present in an amount of 0.01 wt% to 10 wt% of the hydrogel.

[0142] Cell differentiation can be affected by the mechanical properties of hydrogels, i.e., stiffness, and can be effectively used to evaluate drugs that exhibit various effects according to changes in cell differentiation.

[0143]

[0144] A drug efficacy evaluation method according to the present invention comprises the steps of: photocrosslinking a hydrogel containing cells; adding a cell culture medium and culturing the cells; treating a drug; and analyzing; wherein the photocrosslinking step may be photocrosslinking using an LED irradiation device according to the present invention or photocrosslinking using a hydrogel photocrosslinking method according to the present invention.

[0145] Figure 3 is a schematic diagram illustrating a drug efficacy assessment method according to the present invention. For example, a method may be used to photocrosslink a hydrogel containing cells using an LED irradiation device that illuminates multiple LEDs across multiple wells, followed by cell culture and drug treatment. Drug efficacy can then be assessed through high-speed analysis.

[0146] Controlling the mechanical properties of cell scaffolds, such as hydrogels, is crucial because the stiffness of the in vivo cellular microenvironment is linked to cell-ECM interactions, further influencing accurate cellular responses during drug evaluation. Therefore, the differentiation of drug-responsive cells can be controlled by the stiffness of photocrosslinked hydrogels, thereby enabling evaluation of drug delivery efficiency by reproducing the mechanical properties of various tissues in the body.

[0147] The above photocrosslinking step may be photocrosslinking using an LED irradiation device according to the present invention or a hydrogel photocrosslinking method according to the present invention, and thus, a large number of samples can be uniformly photocrosslinked simultaneously, enabling analysis of a large number of samples.

[0148] The above culturing step may be culturing for 1 to 15 days after adding the cell culture medium.

[0149] The step of treating the above drug may be treating the drug for 1 to 10 days.

[0150] The above-described analysis step may be for analyzing the effectiveness of a drug. According to the present invention, high-throughput analysis is possible using a hydrogel containing a large quantity of uniformly photocrosslinked cells, enabling effective evaluation of drug efficacy.

[0151]

[0152] In one embodiment, the photocrosslinking step may be forming a hydrogel library having one or more mechanical properties, and the analyzing step may be analyzing drug efficacy using the library.

[0153] The above photocrosslinking step is to irradiate the hydrogel with light to perform photocrosslinking, and the light may have an intensity of 500 lux to 2500 lux, a wavelength of 350 nm to 600 nm, and an irradiation time of 5 seconds to 20 minutes. When light is irradiated within the above range and photocrosslinking proceeds, the survival of cells within the hydrogel may not be significantly affected.

[0154] The hydrogel may have mechanical properties due to photocrosslinking. Accordingly, since multiple columns or rows can be independently controlled by the LED irradiation device according to the present invention, a library of hydrogels having one or more mechanical properties can be simultaneously created on a single plate, thereby forming a hydrogel library comprising multiple hydrogels having various mechanical properties due to photocrosslinking.

[0155] The hydrogel library enables the production of uniform 3D cell-tissue mimic samples with diverse mechanical properties, similar to those of the body. These hydrogels can be used to assess drug efficacy and screening based on mechanical properties, as well as to determine cellular responses to drugs. In particular, since drug efficacy can vary depending on the properties of surrounding tissues, the hydrogel library can be used to replicate the mechanical properties of various tissues in the body, enabling the evaluation of drug delivery efficiency to reach target tissues.

[0156]

[0157] Hereinafter, the present invention will be described in more detail by way of examples and comparative examples.

[0158] However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

[0159]

[0160] Example

[0161] LED investigation device

[0162] FIG. 4 is a schematic diagram showing the internal structure of an LED irradiation device and hydrogel photocrosslinking according to an embodiment of the present invention. The photocrosslinking system consists of a microstrip circuit board, a 520 nm LED (INNOCEM, South Korea), a 220 V power supply (MEANWELL, Taiwan), 6-way resistors, a timer remote control, and an aluminum enclosure case. The wavelength band of the LED is 515 nm to 525 nm, the light emitting area is 5 mm × 5 mm, and the irradiation angle is 120°. 24 LEDs are completely fixed to the circuit board and positioned at the center of each well of a 24-well plate. FIG. 5 is a circuit diagram of an LED irradiation device according to an embodiment of the present invention. Referring to FIG. 5, a 6-way resistor is connected to each of the six lines to adjust the light intensity of four LEDs corresponding to one vertical line of the well plate in six steps. A radio frequency-based wireless switch that receives a remote signal from the timer controller to control the irradiation time was integrated into the electronic circuit. The system's electronic circuit was completed by connecting the power supply for the light source to this circuit. The completed circuit was connected to an aluminum enclosure designed to maintain a 1 cm distance between the well plate and the LED. Referring to Figure 4, by controlling the light irradiation time, multiple cell-containing hydrogels with diverse mechanical properties can be simultaneously generated.

[0163]

[0164] Analysis of photothermal characteristics of LEDs

[0165] The luminosity and spectrum of the LED were measured in a darkroom at room temperature using a spectrum measuring device (UPRtek, Taiwan). The distance between the measuring device and the LED was 1 cm, which is the same distance as the distance between the LED and the bottom of the well plate in the LED irradiation device. The temperature change of the well plate due to LED heating was measured at room temperature using a thermal imaging camera (BOSCH, Germany). The temperature change was monitored at four points on the bottom of the well plate at 1-minute intervals for 10 minutes. The absorption wavelength spectrum of EY used in the hydrogel construction was measured using a spectrophotometer (SpectraMax M4, Molecular Devices, USA) to confirm that it overlapped with the actual emission wavelength spectrum of the LED used in the system. A quartz cuvette (Sigma-Aldrich, USA) with a 1.0 cm path length was filled with a 0.01 mM EY (Sigma-Aldrich) solution, and the spectrum was recorded in the range of 450 nm to 650 nm.

[0166]

[0167] Hydrogel preparation

[0168] GelMA hydrogel was prepared. To prepare the hydrogel, 25% (w / v) GelMA with a degree of substitution of 90% (Rokit Healthcare, Korea), 1 mM EY (Sigma-Aldrich) in Dulbecco's phosphate-buffered saline (DPBS, Gibco, USA), 4% (w / v) triethanolamine (TEA; Sigma-Aldrich) in DPBS, and 4% (w / v) 1-vinyl-2-pyrrolidinone (VP; Sigma-Aldrich) in DPBS were prepared. To produce a final 10% GelMA hydrogel solution of 300 μL, 120 μL of GelMA solution, 30 μL of EY solution, 15 μL of TEA solution, 15 μL of VP solution, and 120 μL of DPBS were mixed. The final concentrations in the hydrogel were 10% (w / v) GelMA, 0.1 mM EY, 0.2% TEA, and 0.2% VP. For photocrosslinking of GelMA hydrogel, 10 μL of hydrogel solution was dropped into the center of each well of a 24-well plate and irradiated with an LED irradiation device at six intensities for 0.5, 1, 2, 4, or 8 min.

[0169]

[0170] Scanning electron microscope imaging

[0171] The pore size and morphology of GelMA hydrogels were investigated using a scanning electron microscope (SEM; JEOL, Tokyo, Japan). GelMA hydrogel structures were fabricated in polydimethylsiloxane (PDMS; Dow Corning, USA) molds (height 1.5 mm, diameter 4 mm) and photocrosslinked for 0.5, 1, 2, 4, or 8 min. After photocrosslinking, the GelMA hydrogels were washed twice with distilled water and then freeze-dried to produce freeze-dried GelMA hydrogel samples, which were stored at -80 °C before measurement. The GelMA hydrogel samples were fixed to carbon tape and coated with gold using a sputter coater (Bal-Tec, USA) at 60 mA for 60 s. SEM images were taken at 5.0 kV, and the pore area (S0) of GelMA hydrogels in the SEM images was measured using ImageJ software (NIH, MD, USA), and the pore size was calculated considering the circle using Equation 1 below.

[0172] [Formula 1]

[0173]

[0174] Young's modulus measurement

[0175] The Young's modulus of GelMA hydrogels was measured using a universal testing machine (Shimadzu, Japan). GelMA hydrogels were prepared in PDMS molds (height 1.5 mm, diameter 4 mm), photocrosslinked for 0.5, 1, 2, 4, and 8 min, and then kept overnight at 37°C in DPBS solution before measurement. Considering that the contact area between the hydrogel and the testing machine increases with applied pressure, only the slope of the initial linear cross-section was calculated according to the equation Young's modulus = (force / area) / (Δheight / height). To analyze the slope, the samples were compressed to 10% strain.

[0176]

[0177] Swelling test

[0178] Dry weight of GelMA hydrogel was measured and W D was recorded. Then, GelMA hydrogel was immersed in DPBS at 37°C for 24 h until equilibrium swelling occurred. After incubation, excess DPBS was gently removed using filter paper and W S The weight was measured, and the swelling ratio was calculated using the following equation 2.

[0179] [Formula 2]

[0180]

[0181] cell culture

[0182] Human bone marrow-derived mesenchymal stem cells (hMSCs, Lonza, Switzerland) were maintained in T-75 flasks containing hMSC growth medium BulletKit™ (PT-3001, Lonza) at 37°C in a 5% (v / v) CO2 incubator. When the cells reached 90% confluence, they were detached using 0.25% (w / v) trypsin-EDTA (Gibco). The medium was changed every 2 days. For osteogenic differentiation of hMSCs, cells were cultured in hMSC osteogenic differentiation medium BulletKit™ (PT-3002; Lonza). For 3D hMSC culture, cells were detached using 0.25% (w / v) trypsin-EDTA, mixed, and approximately 1 × 10 in the final GelMA hydrogel solution. 6 Cell mL -1 The final cell concentration was reached.

[0183]

[0184] Hydrogel degradation evaluation

[0185] The degradation properties of GelMA hydrogels were measured in the presence and absence of hMSCs (1 x 10 6 Cell mL -1GelMA hydrogels (10 μl) with or without hMSCs were dropped and photocrosslinked in a 24-well plate for 0.5, 1, 2, 4, or 8 min. After osteogenic differentiation of cells for 0, 1, 4, or 7 days, samples were collected, lyophilized, and individually weighed.

[0186]

[0187] Cell viability assessment

[0188] To measure the viability of 3D cultured hMSCs, hMSCs (1 x 10 6 Cell mL -1 ) was dropped into the center of a well of a 24-well plate and photocrosslinked for 1, 2, 4, and 8 min. After photocrosslinking, the cells were cultured in osteogenic differentiation medium for 0, 3, or 7 days. The viability of hMSCs in GelMA hydrogels was assessed using the CellTiter-Glo 3D Cell Viability Assay Kit (Promega, USA). Briefly, equal volumes of reagent and cell culture medium were added to each well, and the cells were lysed by shaking at room temperature for 20 min. After incubation, luminescence was measured using a microplate reader (Tecan, Switzerland).

[0189]

[0190] real-time polymerase chain reaction (qRT-PCR)

[0191] Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to analyze the mRNA levels of runt-related transcription factor 2 (RUNX2), distal-less homeobox 5 (DLX5), bone morphogenetic protein 6 (BMP6), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), and c-Myc in hMSCs. Total mRNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Germany). Total RNA (200 ng of hMSC total RNA) was reverse transcribed using ReverTra Ace qPCR RT Master Mix with a gDNA removal kit (Toyobo, Japan). GAPDH was used as an internal control. PCR was performed using miScript SYBR Green PCR.

[0192]

[0193] Cell morphology analysis

[0194] hMSCs differentiated on GelMA hydrogels for 7 days were fixed with 4% (v / v) paraformaldehyde (Electron Microscopy Sciences, USA) for 20 min and treated with DPBS containing 0.1% (w / v) Triton X-100 (Sigma-Aldrich) and 5% bovine serum albumin (Santa Cruz Biotechnology, USA) for 30 min at room temperature. Cells were immunostained for F-actin using Alexa Fluor 594-conjugated phalloidin (1:400, Invitrogen, USA). Fluorescence images were obtained using a confocal microscope (LSM710; Carl Zeiss, Germany). To perform principal component analysis (PCA), five principal components were selected based on cell shape features (polarity, number of protrusions, circularity, robustness, and major axis length). PCA was performed using the 'prcomp' function in the 'stat' package in R (v3.1.1).

[0195]

[0196] PTH drug evaluation

[0197] hMSC(1 x 10 6 Cell mL -1 ) was placed in a 24-well plate and photocrosslinked for 1, 2, or 4 min. After culturing hMSCs in GelMA hydrogels for 3 or 7 days, the cells were treated with 10, 50, 100, and 200 ng mL -1 The cells were treated with parathyroid hormone (PTH; Tocris, UK) for 1 or 3 days. After PTH treatment, the proliferation of hMSCs was assessed using the CellTiter-Glo 3D Cell Viability Assay kit (Promega), and the mRNA levels of c-Myc, ALP, and OCN were examined by qRT-PCR to evaluate the osteogenic differentiation of hMSCs. The group not treated with PTH served as the control group.

[0198]

[0199] Statistical analysis

[0200] All experiments were performed at least three times, and all numerical data are expressed as mean ± standard deviation (SD). Statistical significance was determined using a two-tailed Student's t-test. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0201]

[0202] Experimental example

[0203] Well plate-based LED irradiation device

[0204] A photocrosslinking system was fabricated using a multi-well plate-based LED irradiation device integrated with LEDs. Photocrosslinkable GelMA hydrogels were used to control the stiffness of the hydrogels using the LED irradiation device. Light intensity was adjusted in six steps using a power dial, while exposure time per line was controlled using a timer remote control. Figure 6 is a photograph illustrating light irradiation using an LED irradiation device according to an embodiment of the present invention. To minimize cytotoxicity, a 520 nm LED was used instead of the UV light typically used in the photocrosslinking process. Eosin Y (EY), the photoinitiator in the GelMA hydrogel mixture, reacts with 520 nm visible light to induce crosslinking of the GelMA hydrogel. The mechanical properties of the GelMA hydrogels can be optimally tuned over a wide range by controlling light intensity or exposure time. Using a well-plate-format LED irradiation device for high-throughput analysis, multiple samples with diverse mechanical properties can be simultaneously acquired within a single well plate.

[0205]

[0206] Optical and thermal validation of the photocrosslinking system

[0207] To evaluate the performance and uniformity of the LED irradiation device for photocrosslinking hydrogels, the emission spectrum, intensity, uniformity, and heat generation of the light emitted from the LED were evaluated. First, we investigated whether the emission wavelength spectrum of the LED used in the system was suitable for the absorption reaction of EY. EY generates radical species triggered by LED illumination, which are transferred to the methacrylamide and methacrylate groups of GelMA, leading to covalent bonding of the remaining groups and subsequent crosslinking. However, the absorption spectrum of EY is known to vary slightly depending on concentration and temperature, and the emission spectrum of the LED is also known to be affected by the applied voltage. Measurements of the absorption spectrum of EY and the emission spectrum of the LED confirmed that both exhibited a peak at 520 nm. This agreement between the two spectra suggests that the LED in the system can induce radical generation by EY, leading to crosslinking of GelMA hydrogels.

[0208] Figure 7 illustrates the luminosity of an LED irradiation device according to an embodiment of the present invention. The figure illustrates the method and location for measuring luminosity, and referring to the graph above, it can be confirmed that the luminosity of the LED is gradually adjusted in six stages ranging from 500 to 2000 lx. Furthermore, it can be observed that a consistent level of luminosity is maintained for each of the six lines (4 LEDs per line) of the well plate at a luminosity of 2000 lx (graph below).

[0209] Figure 8 shows the results of analyzing the temperature change at the bottom of a well plate of an LED irradiation device according to an embodiment of the present invention as a function of light exposure time. Since high temperatures can weaken the mechanical properties of GelMA hydrogel and be harmful to cells, the amount of heat generated by the LED during photocrosslinking was investigated. During 10 minutes of irradiation at maximum light intensity, the temperature at each point of the well plate increased by less than 3°C, indicating that GelMA hydrogel photocrosslinking can be stable.

[0210] In summary, it was confirmed that the light intensity of each well of the well plate could be consistently and robustly adjusted, and thermal disturbances during the photocrosslinking process were negligible. This suggests that the mechanical properties of the hydrogel can be widely and appropriately controlled by adjusting the light exposure time, and that cell damage due to temperature changes may not be significant. Using the LED irradiation device according to the present invention, uniform samples with diverse mechanical properties can be generated by controlling the light intensity and exposure time.

[0211]

[0212] Mechanical properties of photocrosslinked hydrogels

[0213] The mechanical properties of GelMA are controlled by various factors, such as the degree of substitution, composition, photoinitiator concentration, and the amount of light used for crosslinking. Using an LED irradiation device according to the present invention, changes in the mechanical properties of GelMA hydrogels were evaluated by controlling the light exposure time and intensity. First, the Young's modulus of the hydrogel was investigated according to light intensity while the irradiation time was fixed at 2, 4, and 8 minutes. During 2 minutes of light exposure, the Young's modulus of the hydrogel gradually increased within the range of 1–6 kPa as the light intensity increased. However, the range of this change was not large. A rapid change in Young's modulus was observed between 1411 and 1731 lx at 4 and 8 minutes of light exposure, and it stabilized above this intensity. Figure 9 shows the results of the Young's modulus of photocrosslinked hydrogels according to the light irradiation time according to an example of the present invention. Referring to Fig. 9, it can be seen that the Young's modulus of the hydrogel can be controlled over a much wider range of 1 to 30 kPa by changing the light irradiation time while fixing the light output level at the maximum (about 2000 lx). Next, the swelling ratio, which is correlated with the physical properties of the hydrogel such as the degree of crosslinking, viscoelastic properties, and pore size, was measured. Fig. 10 shows the results showing the swelling ratio of the photocrosslinked hydrogel according to the light irradiation time according to an embodiment of the present invention. Referring to Fig. 10, the swelling rate of the hydrogel decreased overall as the irradiation time increased. When the hydrogel was photocrosslinked for 0.5 minutes, it swelled about 20 times compared to the dry state. However, when photocrosslinked for 8 minutes, the hydrogel swelled about 6 times compared to the dry state. Fig. 11 is an SEM image showing the pore size of the photocrosslinked hydrogel according to the light irradiation time according to an embodiment of the present invention. Referring to Figure 11, the pore size of GelMA hydrogel was investigated according to the light irradiation time through microstructural analysis using SEM imaging. As the irradiation time increased, the pore size of GelMA hydrogel decreased and the microstructural distribution became denser.Additionally, a degradation test was conducted to determine whether the photocrosslinked GelMA hydrogels were stable as long-term cell supports. Figure 12 shows the results of the dry weight change over time of photocrosslinked hydrogels according to an example of the present invention. We confirmed that there was no significant degradation of GelMA hydrogels without hMSCs. When cells were included, the GelMA hydrogels showed a slight degradation up to day 1, but were mostly stable for 8 days. The rapid decrease in dry weight on day 1 may be due to the removal of uncrosslinked GelMA strands during washing. In contrast, the GelMA hydrogels showed rapid degradation even at the very early stage of culture, when the irradiation time was very short. Figure 13 shows photographs of photocrosslinked hydrogels according to an example of the present invention over time. Referring to Figure 13, in particular, all hydrogels irradiated for 0.5 minutes degraded after 4 days, indicating that the 0.5 minute irradiation time was not sufficient for the 3D culture scaffold.

[0214] These results confirm that the mechanical properties of GelMA hydrogels can be readily controlled by light exposure time, indicating that light exposure time influences the crosslinking of GelMA hydrogels. The relatively low stiffness of culture supports mimicking the extracellular matrix (ECM) of conventional hydrogels has been a recurring issue, but in the present invention, GelMA hydrogels irradiated with light for more than 4 minutes exhibited a significantly higher elastic modulus (range 0.5–30 kPa) compared to the elastic modulus of conventional hydrogels composed of collagen or Matrigels (range 0.1–2 kPa). This characteristic, combined with the ability to generate high-throughput samples using a well-plate-based approach, indicates the potential to construct diverse microenvironments with diverse mechanical properties.

[0215]

[0216] Effect of light irradiation time on cell viability within hydrogels

[0217] The biggest drawback of photocrosslinking is its potential cytotoxicity. Many other photocrosslinking methods involving UV light can directly affect cellular DNA, making them unsuitable for creating cell-containing hydrogels. The use of a 520 nm visible light LED and EY can significantly overcome this issue. However, prolonged exposure to the high concentration of radicals generated during the crosslinking process can lead to cell death. Therefore, the effect of light irradiation time on cell viability was investigated. Figure 14 is an image showing cell viability according to light irradiation time in a photocrosslinked hydrogel according to an embodiment of the present invention. The scale bar is 100 μm. In live and dead staining of hMSCs, minimal dead cells were observed at 1-4 minutes of light irradiation. However, at 8 minutes, cell death significantly increased, even on day 0, indicating that this was due to damage caused by radicals during light irradiation. Figure 15 is a graph showing the quantitative results of cell viability and relative cell activity according to light irradiation time in a photocrosslinked hydrogel according to an embodiment of the present invention. Quantitative analysis revealed that cell viability significantly decreased with photoexposure times longer than 4 minutes, with the majority of cells in the 8-minute group dying compared to the 1-minute group. Over time, the number of cells with similar proliferation rates significantly recovered in the 1-, 2-, and 4-minute groups. Consequently, most cells remained viable and maintained a constant level of cell activity for photoexposure times shorter than 4 minutes. The viability time for these cells may vary depending on the hydrogel composition, light intensity and type, and the concentration of the photoinitiator.

[0218] Excessively low cell viability in 3D scaffolds increases errors and leads to inaccurate experimental results. Generally, increasing the stiffness of GelMA hydrogels leads to decreased cell viability. Therefore, to obtain successful cell-laden hydrogel samples, a specific range of light exposure times is necessary when controlling the mechanical properties of the hydrogels through photocrosslinking. Despite significant cell death at 8 minutes of exposure, no significant changes in mechanical properties were observed between hydrogels exposed to light for 4 and 8 minutes. These results can provide important clues for optimizing experimental conditions to study cell behavior in response to changes in mechanical properties.

[0219]

[0220] Effect of hydrogel stiffness on osteogenic differentiation of hMSCs

[0221] Bone is an organ with unique mechanical properties of the ECM. hMSCs play a crucial role in bone, differentiating into osteoblasts responsible for bone formation. Essentially, bone stiffness is a critical factor in hMSC differentiation into osteoblasts and the overall process of bone formation. It is generally known that hMSCs grown in a high-stiffness environment undergo accelerated osteogenic differentiation. In the present invention, we investigated the osteogenic differentiation of hMSCs under various light exposure times ranging from 1 to 4 minutes, and found that the cells exhibited minimal cytotoxicity and maintained cellular activity. Figure 16 shows the results of gene expression of osteoblast markers according to light exposure time in a photocrosslinked hydrogel according to an embodiment of the present invention. Analysis was performed on days 3 and 7 after differentiation of hMSCs within the hydrogel. Referring to Figure 16, prolonged light exposure, which is expected to increase stiffness, significantly enhanced the expression of early osteoblast markers, including Runx2, DLX5, and BMP6, in hMSCs on day 3. Among late osteoblast markers, OPN expression slightly increased, whereas OCN showed no significant change. Furthermore, the expression of ALP, a late osteoblast marker, decreased in high-stiffness hydrogels. At day 7, the expression of markers associated with MSC osteogenic lineage commitment, particularly Runx2 and DLX5, decreased or further decreased with increasing hydrogel stiffness. BMP6 expression was slightly upregulated with increasing hydrogel stiffness. However, the degree of expression enhancement was reduced compared to day 3. In contrast, the gene expression levels of osteoblast markers ALP, OCN, and OPN increased. These results confirmed that increased hydrogel stiffness due to prolonged light exposure enhanced the osteogenic differentiation of hMSCs. This can be understood as a result of mechanical interactions between the hydrogel and cells. Consequently, increased hydrogel stiffness affects the mechanotransduction of hMSCs, accelerating osteogenic differentiation.

[0222] In addition, we investigated the osteogenic differentiation of hMSCs encapsulated in hydrogels while controlling the hydrogel stiffness. As the hydrogel stiffness increased, the shape of hMSCs changed, and as osteogenic differentiation occurred, the MSC morphology could gradually change into a dendritic shape. Figure 17 is a schematic diagram of the morphological changes of mesenchymal stem cells according to the stiffness of photocrosslinked hydrogels according to an embodiment of the present invention, and an F-actin immunostaining image. The F-actin immunostaining image was obtained by sequentially irradiating with light for 1, 2, and 4 minutes. On day 7, the cells were uniaxially elongated in the low-stiffness GelMA hydrogel (1-minute group), while they exhibited a dendritic shape in the relatively stiff hydrogel (2-minute and 4-minute groups). Changes in the polarity, number of protrusions, circularity, and firmness of hMSCs when encapsulated in GelMA hydrogels were investigated. We observed a decrease in cell polarity and rigidity and an increase in the circularity and number of protrusions of hMSCs encapsulated within GelMA hydrogels with longer photoirradiation time, indicating that cells exhibited stronger osteogenic differentiation in hydrogels with longer photoirradiation time.

[0223] Using the LED irradiation device of the present invention, we successfully controlled the stiffness of GelMA hydrogels, and confirmed that higher hydrogel stiffness led to more effective induction of osteogenic differentiation of hMSCs. This suggests that high-throughput cell-laden hydrogels with diverse mechanical properties facilitate a comprehensive understanding of cell-ECM interactions. Furthermore, image analysis based on the correlation between cell morphology and bone differentiation can be combined with deep learning technology, which is considered a novel drug evaluation approach in the future.

[0224]

[0225] Differential responses of hMSCs to PTH treatment in hydrogels of different stiffness

[0226] The LED irradiation device of the present invention offers the advantage of performing various experiments in a consistent and standardized plate-based manner. This approach provides a valuable foundation for high-throughput drug testing. In this study, we investigated whether the LED irradiation device can be used as a platform to evaluate cellular drug responses according to the various mechanical properties of the hydrogel, a cellular microenvironment. Among many osteogenic drugs, parathyroid hormone (PTH) is a representative drug approved for the treatment of osteoporosis. Rather than targeting a specific signaling pathway, PTH exerts distinct effects on all bone cell types throughout the bone formation process, inducing synergistic effects. PTH can stimulate hMSC proliferation, enhance bone differentiation from preosteoblasts to osteoblasts, and enhance bone mineral secretion from osteoblasts. The effects of PTH on the various maturations of hMSCs in various physical microenvironments are not well understood, and understanding the precise effects of PTH on the complex in vivo bone formation process is crucial. According to the present invention, photocrosslinking using an LED irradiation device allowed manipulation of the mechanical properties of GelMA hydrogels and control of the degree of osteogenic differentiation of hMSCs. Therefore, by controlling the level of hMSC differentiation based on the mechanical properties of the hydrogel, we aimed to confirm that the expected effects of PTH may vary depending on the degree of cell maturation.

[0227] First, the overall experimental process and the PTH concentration in the cells were optimized. The experiment was conducted by photocrosslinking MSC-containing GelMA hydrogels, followed by osteogenic differentiation for 3 or 7 days, and then treating the cells with PTH for 1 day (100 ng mL). -1 A significant increase in cell proliferation was observed at the above PTH concentrations, and in subsequent experiments, the PTH concentration was 100 ng mL -1was determined. Figure 18 shows the results showing the difference in relative gene expression of biomarkers when drugs were treated in hydrogels with various stiffnesses that were photocrosslinked according to an embodiment of the present invention. Referring to Figure 18, the results of PTH treatment after 3 and 7 days of osteogenic differentiation of hMSCs in photocrosslinked gels are shown, respectively. When treated with PTH, the expression level of the proliferation marker c-Myc was enhanced in hMSCs cultured in low-stiffness hydrogels by treatment with PTH. In contrast, when treated with PTH, the expression of the osteogenic marker Runx2 and the mineral synthesis-related marker OCN was enhanced in hMSCs cultured in stiffer hydrogels. A similar pattern was observed at 7 days of differentiation, except for c-Myc. After 3 days of osteogenic differentiation followed by 3 days of PTH treatment, PTH did not affect the expression of c-Myc and Runx2 in hMSCs, but the expression of OCN was observed to increase.

[0228] Consequently, by adjusting the stiffness of the GelMA hydrogel, we were able to modulate the degree of MSC bone differentiation, inducing variations in the degree of differentiation and ultimately leading to PTH-induced proliferation or osteogenic fate. PTH primarily binds to the PTH type 1 receptor on various bone cell types, enhancing the cyclic AMP-dependent signaling cascade. This signaling cascade is generally known to positively influence both proliferation and osteogenic differentiation. Numerous studies have shown that PTH treatment primarily enhances proliferation in osteoblast precursor cells and differentiation in late osteoblasts. Furthermore, when the osteogenic differentiation period or PTH treatment duration was extended, proliferation was not induced and the expression of osteogenic differentiation markers increased in cases where osteoblast differentiation had already occurred in hMSCs. These results suggest that optimizing specific culture and drug treatment timelines, related to culture duration and drug treatment duration, is essential for efficiently evaluating drug effects based on the degree of specific cell differentiation.

[0229]

[0230] The present invention developed a photocrosslinking system using an LED irradiation device capable of simultaneously generating multiple samples in a multi-well plate, allowing the mechanical stiffness of hydrogels to be tuned over a wide range (0.5-30 kPa). By simply controlling the irradiation time, we observed various levels of osteogenic differentiation of hMSCs in response to various levels of hydrogel stiffness without cytotoxicity. Furthermore, we demonstrated that PTH can induce differential responses to osteogenic differentiation under these different gel properties. This demonstrates that this system, capable of easily and rapidly regulating various mechanical properties at high throughput, can serve as a research platform for various tissues or cells sensitive to changes in ECM mechanical properties. This provides a more comprehensive view of the physical properties of the cell-ECM, enabling the evaluation of many other newly developed osteogenic drugs from a more multidimensional perspective. Furthermore, this system holds promise for exploring the relationship between drug penetration and tissue stiffness. Ultimately, the LED irradiation device of the present invention offers significant potential for advancing research on in vivo physiological processes and various drug evaluations facilitated by the generation of multicellular cell-containing hydrogels with a wide range of mechanical properties.

[0231]

[0232] Although the embodiments have been described above, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0233] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0234]

Claims

1. A plate comprising a plurality of wells; and A light generator installed at the bottom of the above plate; The above light generator includes a plurality of LED light sources that irradiate light in the direction of the plate. LED investigation device.

2. In paragraph 1, The above LED light source is formed on a circuit board, The above circuit board is of the microstrip type. LED investigation device.

3. In paragraph 1, The above well corresponds individually to the LED light source, The center of the LED light source is arranged in a straight line with the center of the well. LED investigation device.

4. In paragraph 1, The above plurality of LED light sources are arranged in multiple columns or rows, The above plurality of columns or rows are independently controlled for electrical operation, LED investigation device.

5. In paragraph 4, The above electrical manipulation is to control one or more of the intensity, wavelength, and irradiation time of light. LED investigation device.

6. In paragraph 1, The above plate contains 6 to 96 wells, The above well has a size of 1 mm to 50 mm, LED investigation device.

7. In paragraph 1, The distance between the bottom of the plate and the LED light source is 2 mm to 20 mm. LED investigation device.

8. In paragraph 1, The above LED light source has an emission angle of 80° to 140°, The size is 0.5 mm to 20 mm, LED investigation device.

9. In paragraph 1, The above light has an intensity of 500 lux to 2500 lux, having a wavelength of 350 nm to 600 nm, LED investigation device.

10. In paragraph 1, The hydrogel is positioned in the above well, The hydrogel is photocrosslinked by irradiating the above light. LED investigation device.

11. In paragraph 10, The hydrogel comprises at least one selected from the group consisting of collagen, gelatin, chitosan, fibrin, agar, starch, alginate, hyaluronic acid, dextran, elastin, carrageenan, polyethylene glycol, and decellularized tissue-derived extracellular matrix. LED investigation device.

12. In paragraph 10, The above hydrogel has one or more mechanical properties, The hydrogel library is formed simultaneously on a single plate. LED investigation device.

13. In paragraph 1, Compatible with high-throughput analysis equipment, LED investigation device.

14. A step of positioning hydrogels in each of a plurality of wells; and A step of irradiating each of the plurality of wells with an LED; Hydrogel photocrosslinking method.

15. In paragraph 14, The step of investigating the LED is to use the LED investigation device of the first clause. Hydrogel photocrosslinking method.

16. In paragraph 14, The step of examining the above LED is such that the light intensity is 500 lux to 2500 lux, The wavelength is 350 nm to 600 nm, The light exposure time is from 5 seconds to 20 minutes, Hydrogel photocrosslinking method.

17. In paragraph 14, The mechanical properties of the above-mentioned photocrosslinked hydrogel are 0.1 kPa to 100 kPa, The difference in physical properties between the above-mentioned photocrosslinked hydrogels is within ±10%. Hydrogel photocrosslinking method.

18. In paragraph 14, The above hydrogel contains cells, Hydrogel photocrosslinking method.

19. A step of photocrosslinking a hydrogel containing cells; A step of adding cell culture medium and culturing cells; Step of processing the drug; and comprising the steps of: The above photocrosslinking step is photocrosslinking using the LED irradiation device of claim 1 or photocrosslinking using the hydrogel photocrosslinking method of claim 14. Drug efficacy evaluation method.

20. In paragraph 19, The above photocrosslinking step forms a hydrogel library having one or more mechanical properties, The above analyzing step is to analyze drug efficacy using the library. Drug efficacy evaluation method.

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