JP7430895B2 - Method for producing a hydrogel structure, and method for culturing and analyzing biological samples using the hydrogel structure - Google Patents

Description

The present invention relates to a hydrogel structure that can be applied as a scaffold for three-dimensional cell culture, and to a method for culturing and analyzing biological samples using the hydrogel structure.

In recent years, since induced pluripotent stem cells (iPS cells) have been established, their application to regenerative medicine is expected due to the development of differentiation techniques into various stem cells and the construction of mass culture systems. In particular, in the application of induced pluripotent stem cells to cell transplantation, research is progressing using stem cells cultured in dispersed culture such as culture dishes and cells that have been induced to differentiate. However, since cells under these conditions differ from the in-vivo environment, there are problems with differences in quantitative performance and decreased functionality due to immune rejection during cell transplantation. In other words, in multicellular organisms such as humans, most of the cells that make up the living body, excluding blood cells and humoral immune cells, are adherent cells, and these adherent cells adhere to each other or adhere to the extracellular matrix. A three-dimensional structure is formed by adhering to the constituent molecules. Therefore, for applications in regenerative medicine, three-dimensional cell culture technology is considered important in order to exhibit functions closer to those of living tissue environments.

Generally, in order to three-dimensionally culture cells, a method is adopted in which cells are cultured while attached to or encapsulated in a predetermined scaffold structure. Various materials have been studied as scaffold structures, such as collagen (Non-Patent Documents 1 and 2), hyaluronic acid (Non-Patent Document 3), alginic acid (Non-Patent Document 4), and chitosan (Non-Patent Document 5). , polyethylene glycol (PEG), a synthetic polymer (Non-Patent Document 6), etc. are known, among which methods that utilize collagen, which is the main constituent molecule of the human extracellular matrix, have been studied for a relatively long time. It has become a general-purpose method in recent years (for example, it is also applied as an extracellular matrix product that enables more stable cell culture by applying it to culture dishes, etc.) (Non-patent Document 7: (See product information website).

On the other hand, flow cytometry technology is known, which enables high-speed analysis of cell characteristics at the single cell level and separation and collection of specific cell populations. Flow cytometry technology is widely used in basic research in the life science field, drug discovery fields such as drug evaluation, and regenerative medicine fields such as cell transplantation. However, cells that are the target of flow cytometry technology are generally disaggregated into single cell levels by enzyme treatment, etc., and their state differs from that found in living tissues. It was not suitable for analysis. In this regard, if we can develop a scaffold material that is suitable for three-dimensional culture of cells and can be applied to various analytical techniques, it will become possible to perform high-throughput analysis of higher-order cell functions, which will be useful for research in the life science field. is expected to accelerate.

On the other hand, regarding scaffold structures using collagen, for example, Patent Document 1 discloses that cells are suspended in a collagen solution containing medium components, and then the cells are suspended in an organic solvent that is nontoxic to the cells and immiscible with water. Disclosed is an invention of a method for producing cell-embedded collagen gel beads, which is characterized by dispersing a suspension and gelling it. Then, various cells such as hamster pancreatic Langer's islet-derived insulin-producing cell line, rat normal kidney cell line, Swiss 3T3 cells (fibroblast-like cells), and hamster lung-derived V79 cells were embedded in the collagen gel beads in a specified medium. It is described that it can be cultured.

Ming-Thau Sheu et al. “Characterization of collagen gel solutions and collagen matrices for cell culture” Biomaterials, 2001 : 22(13) pp1713-1719. A. Matsuhashi et al. “Fabrication of fibrillized collagen microspheres with the microstructure reassembling an extracellular matrix” Soft Matter, 2015 : vol.11, pp2844-2851. Shaoquan Bian et al. "The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cell culture, Colloids and Surfaces B", Biointerfaces, 2016 : vol.140(1), pp 392-402. Li X et al. “Culture of neural stem cells in calcium alginate beads”, Biotechnol Prog., 2006 : 22(6), pp1683-9. Zhewei Chen et al. "Phase-separated chitosan-fibrin microbeads for cell delivery", J Microencapsul., 2011 : 28(5), pp344-352. Cer E et al. “Polyethylene glycol-based cationically hydrocharged gel beads as a new microcarrier for cell culture” J Biomed Mater Res B Appl Biomater. 2007 : 80(2):406-14. Product information website of Thermo Fisher Scientific Life Science Technologies Japan Co., Ltd. (“Gibco Extracellular Matrix Products”, https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/jp/catalogs/dl -GIB049-B1511IH.pdf)

Japanese Unexamined Patent Publication No. 64-71487

However, when a collagen solution is dispersed in an organic solvent as described in Patent Document 1, a relatively firm gel can be prepared, but in order to keep the cells alive, it is necessary to wash them with ethanol etc. There were problems such as damage to cells. In particular, nervous system cells are susceptible to damage, and although it is possible to wash them thoroughly after gel formation and attach them to the gel surface and culture them, it is difficult to encapsulate the cells inside the gel during gel formation and then culture them. Therefore, it has been difficult to culture a neural network-like structure inside the gel.

Therefore, an object of the present invention is to provide a hydrogel structure using collagen, which can be prepared under biocompatible and mild conditions as a scaffold structure for three-dimensional culture of cells, and which can be used for various analyses. It is an object of the present invention to provide a method for producing a hydrogel structure, which yields a structure applicable to various technologies. Another object of the present invention is to provide a method for culturing and analyzing a biological sample using the hydrogel structure.

In order to achieve the above purpose, the present inventors conducted extensive research and found that when heparin, a glycosaminoglycan that constitutes the extracellular matrix, acts on collagen, a hydrogel structure that meets the above purpose is formed. The present inventors have discovered that it is possible to do this, and have completed the present invention.

That is, in a first aspect of the present invention, a hydrogel material-containing solution containing collagen is added to a sugar chain-containing solution containing sugar chains belonging to glycosaminoglycans. The present invention provides a method for producing a hydrogel structure, which is characterized by forming a hydrogel structure having an interface with a medium comprising:

In the method for producing a hydrogel structure, the sugar chain belonging to the glycosaminoglycans is preferably heparin.

Furthermore, in the method for producing a hydrogel structure, the collagen is preferably acid-soluble collagen and/or enzyme-solubilized collagen.

Further, in the method for producing a hydrogel structure, it is preferable that the pH of the hydrogel material-containing solution is adjusted to pH 6 or higher, and the pH of the sugar chain-containing solution is adjusted to pH 7 or lower.

Further, in the method for producing a hydrogel structure, the hydrogel structure has a granular shape, a major axis of 20 μm or more, and/or a circularity of 50% or more. It is preferable.

Further, in the method for producing a hydrogel structure, the hydrogel material-containing solution contains a living biological sample, and the hydrogel structure containing the living biological sample is obtained as the hydrogel structure. is preferred.

Furthermore, in the above method for producing a hydrogel structure, the biological sample is preferably a cell sample derived from one or more types selected from the group consisting of unicellular organisms and multicellular organisms.

On the other hand, in a second aspect of the present invention, at least a living biological sample is attached to or included in the hydrogel structure obtained by the method for producing a hydrogel structure, and The present invention provides a method for culturing a biological sample, which is characterized by culturing together with a medium.

In addition, in a third aspect of the present invention, the hydrogel structure obtained by the above-mentioned method for producing a hydrogel structure is cultured together with a medium suitable for the biological sample. A culture method is provided.

Furthermore, in a fourth aspect, the present invention is characterized in that a biological sample cultured within the hydrogel structure obtained by the method for culturing a biological sample according to the second or third aspect is subjected to analysis. The present invention provides a method for analyzing biological samples.

In the biological sample analysis method described above, it is preferable that the analysis is performed by means of analyzing the entire hydrogel structure.

On the other hand, in its fifth aspect, the present invention provides the use of sugar chains belonging to glycosaminoglycans for preparing a hydrogel structure.

In the above use, the sugar chain belonging to the glycosaminoglycans is preferably heparin.

According to the present invention, a hydrogel structure using collagen can be prepared as a scaffold structure for three-dimensional culture of cells under mild biocompatible conditions without using an organic solvent. It is possible to provide a method for producing a hydrogel structure, which can yield a structure that can be applied to various analytical techniques. Furthermore, a method for culturing and analyzing a biological sample using the hydrogel structure can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic explanatory diagram showing an embodiment of a method for manufacturing a hydrogel structure according to the present invention. 2(a) is an example of a phase contrast microscope image, and FIG. 2(b) is an example of a hydrogel structure (gel 2 is a chart showing the outer circumferential length of each particle of particles (particles) in the form of a histogram. 3(a) is an example of a confocal microscope image, and FIG. 3(b) is an example of a hydrogel structure (gel 2 is a chart showing the roundness of each particle of particles (particles) as a histogram. 4(a) is an example of a phase contrast microscope image, and FIG. 4(b) is an example of a hydrogel structure ( 2 is a chart showing the roundness of each particle of gel particles in the form of a histogram. 5 is a chart showing the results of CD spectrum measurement of a glucose solution, a collagen solution, or a mixed solution of collagen and glucose in Test Example 3, and FIG. 5(a) shows the results when the solution was prepared under the condition of pH 3. , FIG. 5(b) shows the results when the solution was prepared under the condition of pH 5, and FIG. 5(c) shows the result when the solution was prepared under the condition of pH 7. 6(a) is a chart showing the results of CD spectrum measurement of a collagen solution, a heparin solution, or a mixed solution of collagen and heparin in Test Example 3, and FIG. 6(a) shows the results when the solution was prepared under the condition of pH 3. , FIG. 6(b) shows the results when the solution was prepared under the condition of pH 5, and FIG. 6(c) shows the result when the solution was prepared under the condition of pH 7. This is an example of an image obtained by 3D observation of the Neuro ball prepared using primary cultured rat cerebral cortex cells in Test Example 4, and Fig. 7(a) is a confocal microscope image of the sample on the third day of culture. This is an example of an image, and FIG. 7(b) is an example of a confocal microscope image of a sample on the 10th day of culture. This is an example of a confocal microscope image when 3D observing the Neuro ball produced using human iPS cell-derived neural progenitor cells in Test Example 5, and the confocal microscope image of the sample on the 10th day of culture. This is an example. Image showing the results of Ca ²⁺ imaging measurement on the Neuro ball on the 10th day of culture in order to investigate the functionality of the Neuro ball produced using primary cultured rat cerebral cortex cells in Test Example 6. FIG. 9(a) is an example of a phase contrast microscope image taken when performing Ca ²⁺ imaging measurement and a fluorescence microscope image in the same field of view, and FIG. 9(b) is an example of a fluorescence microscope image taken when performing Ca 2+ imaging measurement. These are examples of fluorescence microscope images at 9.43 seconds from the start, 19.47 seconds from the start, and 28.6 seconds from the start when observing seven bright spots with strong fluorescence intensity for 2 minutes. 9(c) is a chart showing the results of examining changes in fluorescence intensity over time for each of the above-mentioned bright spots. In Test Example 6, in order to investigate the functionality of Neuro balls prepared using primary cultured rat cerebral cortex cells, the Neuro balls on the 10th day of culture were injected with bicuculline, an antagonist of GABA _A receptors. ) are images and charts showing the results of Ca ²⁺ imaging measurement after dropping a 10 μM solution of 10 μM solution. Figure 10(a) is a phase contrast microscope image taken during Ca ²⁺ imaging measurement and the same field of view. Fig. 10(b) is an example of a fluorescence microscope image at 48.4 seconds from the start and 48.4 seconds from the start when seven bright spots with strong fluorescence intensity were arbitrarily observed for 2.5 minutes. This is an example of a fluorescence microscope image at 55.1 seconds and 61.8 seconds from the start, and FIG. 10(c) is a chart showing the results of examining changes in fluorescence intensity over time for each of the bright spots. In Test Example 6, in order to investigate the functionality of the Neuro ball produced using primary cultured rat cerebral cortex cells, a prescribed drug was dropped onto the Neuro ball on the 23rd day of culture and action potential measurement was performed. 11(a) is an image showing the Neuro ball mounted on a planar microelectrode array, and FIG. 11(b) is an image showing the results when no drug was administered. ("before" in the figure), when bicuculline (Bicucullne), an antagonist of GABA _A receptor, is instilled ("Bicucullne 10 μM" in the figure), CNQX, an antagonist of AMPA type receptors, is added together with bicuculline (Bicucullne). This is a chart showing the results of observing the firing frequency due to Ca ²⁺ oscillation for 30 seconds for each case of dripping (“CNQX 30 μM” in the figure), and FIG. 11(c) shows the results of the firing frequency obtained. It is a graph showing relative values when no drug is administered as 100%. These are images showing the results of creating a brain tumor model using Cell ball in Test Example 7, and FIG. 12(a) shows samples on the 2nd, 5th, 18th, and 60th days of culture. FIG. 12(b) is an example of a confocal microscopic image of a Cell ball for a sample on the fifth day of culture. This is an image showing the results of constructing a cancer microenvironment model using a Cell ball in Test Example 8, and shows the results of a single culture of human glioma cells, a single culture of astrocytes, and a co-culture of human glioma cells and astrocytes. These are examples of phase contrast microscopic images of each Cell ball for samples on the 2nd day, 8th day, and 14th day of culture, respectively. This is an image showing the results of constructing a cancer microenvironment model using a Cell ball in Test Example 8, and shows the results of a single culture of human glioma cells, a single culture of astrocytes, and a co-culture of human glioma cells and astrocytes. This is an example of an image obtained by HE staining a section of a Cell ball for each sample on the 16th day of culture. This is a chart showing the results of evaluating the cancer microenvironment model using Cell balls in Test Example 8, and FIG. Figure 14(b) shows the results of measuring the brightness of each cell ball for each number of days of culture, showing the average value and standard deviation. The following graph is shown below.

There are no particular restrictions on the origin of the collagen used in the present invention. For example, the collagen used in the present invention is extracted from collagen-containing tissues (skin, bones, etc.) of mammals, fish and shellfish, etc. using various extraction methods, and the purity is determined as necessary. It is possible to use a material that has been subjected to a treatment such as salting out in order to increase the viscosity. Depending on the extraction method, for example, acid-soluble collagen obtained by extraction with dilute acid, enzyme-solubilized collagen obtained by solubilizing with enzyme, alkali-solubilized collagen obtained by solubilizing with alkali, etc. Can be mentioned. Among these, acid-soluble collagen and enzyme-solubilized collagen are preferably used from the viewpoint of solubility in water and aqueous buffer solutions. There are various types of collagen depending on their physiological functions in the body, but type I collagen, which produces the elasticity and strength of the skin, has previously been shown to be useful as a scaffold material for cell culture. It is a type of collagen that can be suitably used in the present invention. Furthermore, there are products that are commercially available for use in cell culture, such as culture plate coats, and such commercially available products may also be used. Note that, for the purpose of reducing antigenicity, etc., the terminal telopeptide of the collagen molecule used may be removed by enzymatic treatment or the like. That is, it may be atelolated collagen. One type of collagen may be used alone, or two or more types may be used in combination.

The sugar chains belonging to the glycosaminoglycans used in the present invention are not particularly limited, but include, for example, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, and the like. Glycosaminoglycans are unbranched glycans in which one of the repeating disaccharide units is always an amino sugar. More specifically, one of the disaccharide units is an amino sugar consisting of galactosamine or glucosamine, and the other is a uronic acid consisting of glucuronic acid or iduronic acid, or galactose. Because a carboxyl group exists in uronic acid, a sulfate group is introduced into the hydroxyl group of a sugar, or an acetyl group is introduced into the amino group of an amino sugar, they are generally strongly negatively charged. . In biological tissues, they are known as sugar chains that exist on cell surfaces and extracellular matrices, and are also called mucopolysaccharides. In the present invention, those derived from such biological tissue may be used, and those obtained by arbitrarily modifying the amount or ratio of introduced sulfate groups, acetyl groups, or other functional groups may be used. One type of sugar chain belonging to glycosaminoglycans may be used alone, or two or more types may be used in combination. Moreover, it is particularly preferable to use heparin.

In the present invention, a hydrogel material-containing solution containing the above-mentioned collagen is added to a sugar chain-containing solution containing sugar chains belonging to the above-mentioned glycosaminoglycans, and the solution is mixed with a medium consisting of the sugar chain-containing solution. A hydrogel structure having an interface therebetween is formed.

The concentration of collagen in the hydrogel material-containing solution is preferably 0.1 to 10 mg/mL, more preferably 0.5 to 3 mg/mL. Further, the pH of the hydrogel material-containing solution is preferably pH 6 or higher, more preferably pH 6 to 10, and even more preferably pH 6.5 to 8. Furthermore, the concentration of sugar chains belonging to glycosaminoglycans in the sugar chain-containing solution is preferably 0.01 to 100 mg/mL, more preferably 1 to 10 mg/mL. Further, the pH of the sugar chain-containing solution is preferably pH 7 or less, more preferably pH 3 or more and pH 7 or less, and even more preferably pH 4 or more and pH 6 or less.

Here, to explain the present invention more specifically with reference to FIG. 1, in the embodiment shown in FIG. After collecting the sample, the tip of the pipette is immersed in the sugar chain-containing solution 2, and it is slowly released by normal operation of the pipette. As a result, the hydrogel material-containing solution 3 forms an interface with the medium made of the heparin-containing solution 2, and gels approximately in its shape, yielding a hydrogel structure 4. After forming the hydrogel structure 4, from the state using the sugar chain-containing solution 2 as a medium (state indicated by reference numeral 10 in the figure), to the state using the sugar chain-containing solution 2 as a medium, and from the state using an appropriate buffer solution, medium, etc. for storage and preservation, etc. The medium may be replaced with a storage solution 5 (state indicated by reference numeral 20 in the figure).

In addition, when adding the hydrogel material-containing solution to the sugar chain-containing solution, the "addition" may be spouting into the solution, as in the embodiment shown in FIG. 1, or dropping from outside the solution. It may be layered on the surface of the solution, or it may be dispersed into the solution with stirring. In other words, the added hydrogel material-containing solution may be separated from the medium consisting of the sugar chain-containing solution. It is sufficient to form an interface, and the mode is not limited. However, in order to obtain a spherical hydrogel structure with higher roundness, it is more preferable to use a mode in which the hydrogel structure is ejected into a solution as shown in FIG.

In addition, although FIG. 1 shows a form in which the hydrogel material-containing solution 3 is ejected from the tip of a normal micropipettor, the hydrogel material-containing solution 3 is ejected from a device such as a nozzle having a predetermined diameter and shape. By spouting out, a hydrogel structure of a desired size and shape can be formed. For example, it is possible to prepare one having a diameter of about 1 cm. Further, the hydrogel structure 4 may optionally contain a biological sample (indicated by reference numeral 6 in the figure) such as cells.

As for the temperature conditions, for the hydrogel material-containing solution, it is preferably carried out in the range of 0°C to room temperature, and more preferably in the range of 0 to 10°C. If the temperature is too high, the collagen-containing hydrogel material-containing solution itself may become kelized. Furthermore, when a living biological sample is included, the activity of the sample may be adversely affected. Further, the temperature of the sugar chain-containing solution may be in the range of 0 to 40°C, and for example, it is typically room temperature, which is more convenient for manufacturing operations.

The hydrogel structure obtained according to the present invention preferably has a granular shape and a major axis of 20 μm or more. If it is too small, it tends to be difficult to uniformly disperse cells and the like when they are encapsulated therein. Further, the circularity is preferably 50% or more, more preferably 80% or more. If the shape of the hydrogel structure is within this range, cells can be grown more uniformly when encapsulating cells and performing three-dimensional culture.

The hydrogel structure obtained by the present invention can be suitably used as a scaffold material for three-dimensional culture of living biological samples, especially cells. In other words, by attaching living cells to or encapsulating them in a hydrogel structure and culturing them with a medium appropriate for the cells, the cells grow while being three-dimensionally organized using the hydrogel structure as a scaffold. As a result, a cell network is formed that mimics the biological tissue environment. This makes it possible to analyze such cell networks in conditions closer to the living tissue environment. It is also conceivable that it could be used in pathological elucidation, drug discovery, regenerative medicine, and general basic scientific research on cell networks. In this case, there are no particular restrictions on the type of cells, and cells derived from unicellular organisms such as Escherichia coli, yeast, fungi, diatoms, and flagellates may be used, as well as cells from higher animals such as mammals, and vegetables. Cells derived from multicellular organisms such as cells of higher plants such as , fruit trees and trees may be used, or two or more different types of cells may be used.

In order to attach or encapsulate at least a living biological sample in the hydrogel structure obtained by the present invention, after preparing the hydrogel structure, desired cells are suspended in a solution such as an appropriate medium, and the cells are Although it can also be achieved by immersing the hydrogel structure in a suspension, adding such a cell suspension to the hydrogel structure, dropping it, coating it, etc., the method according to the present invention The hydrogel structure is formed without using organic solvents or extreme temperature conditions. Therefore, for example, when producing a hydrogel structure as described above, living cells may be encapsulated in the hydrogel material-containing solution together with a suitable culture medium if necessary. According to this embodiment, cells are encapsulated in a living state inside the resulting hydrogel structure. By transferring this to a well-known culture device or the like and culturing it with an appropriate medium, three-dimensional cell culture can be easily performed.

According to the present invention, as shown in the Examples below, a neural network-like model, which has been difficult to prepare in the past as a biological tissue environment model, can be obtained with a very simple operation. Furthermore, a cancer microenvironment model composed of cancer cells and their surrounding cells can be obtained with very simple operations. Furthermore, since the gel particles after forming such a model are samples that can be directly subjected to immunochemical measurements, electrophysiological measurements, or flow cytometry analysis, there is a limited scope for subsequent analysis methods. This makes it easy to analyze the formation process of biological tissue networks.

One example is the application of this technology to techniques that allow secretions released from cell networks to remain within hydrogel structures. For example, when a substance such as amyloid β is cultured in a normal culture dish, it is washed away when the medium is replaced, making it impossible to reproduce the deposition of amyloid β that occurs in living organisms. Since the hydrogel structure obtained by the present invention allows substances such as amyloid β to remain, it is possible to create a better pathological model.

Another example is application to a technology in which the number, type, and compound of cells are controlled and encapsulated within a hydrogel structure. This allows control of cell density, unlike spheroids, which are made up only of cells. Spheroids made up only of cells cannot allow light to reach inside, but if the hydrogel structure obtained by the present invention is used, light can reach inside, making it possible to detect internal cell information.

Furthermore, as another example, the structure can be labeled by incorporating fluorescent beads or the like, and by changing the type of fluorescent beads, multiple types of properties can be analyzed at once. This enables high-throughput analysis using flow cytometry or multi-well plates.

Furthermore, as described above, there is no restriction on the type of cells contained within the hydrogel structure. Therefore, after encapsulating cells for medical treatment such as iPS cells and adult stem cells and fixing them in a hydrogel structure by culturing under predetermined conditions, the cells obtained in this way can be used with the structure or further. It is also possible to develop it into regenerative medical technology, such as separating it and administering it into the body.

The present invention will be described in more detail below with reference to Examples, but these Examples do not limit the scope of the present invention in any way.

<Test Example 1> (Effect of pH of heparin-containing solution)
A hydrogel structure was prepared by ejecting a solution containing a hydrogel material with a micropipette in a heparin-containing solution according to the embodiment illustrated in FIG.

Specifically, a heparin-containing solution was prepared by dissolving heparin sodium (derived from porcine intestinal tract, Tokyo Kasei Kogyo Co., Ltd.) to a final concentration of 1 mg/mL in PBS. At that time, the pH was adjusted to pH 3, pH 5, or pH 7 with 1M HCl. On the other hand, as a hydrogel material-containing solution, a collagen gel precursor solution (product name "Collagen I, Rat Tail", containing 3 mg/mL of type I collagen derived from rat tail, thermoplastic Fisher Scientific Life Science Technologies Japan Co., Ltd.) was used, 10x PBS and 1M NaOH were added to this, and the mixture was further diluted with neuro basal medium so that the collagen concentration was 15 mg/mL and the pH was 7.0. Prepared.

To squirt the hydrogel material-containing solution into the heparin-containing solution, collect 1 μL of the hydrogel material-containing solution into a 10 μL tip using a micropipette (Eppendorf), and then This was done by immersing the tip of the heparin-containing solution in approximately 1 mL of a 24-well plate and slowly releasing it using the same normal operation with a micropipette. As a result, the hydrogel material-containing solution formed an interface with the medium consisting of the heparin-containing solution, and gelled approximately in its shape, yielding a hydrogel structure. As for the temperature conditions, all the hydrogel material-containing solutions containing collagen were operated while being cooled on ice, while the heparin-containing solutions were heated to 37°C. Under these conditions, gelation and precipitation were observed immediately after ejection. Thereafter, the heparin-containing solution was removed by suctioning with a pipette, the medium was replaced with neuro basal medium, and the medium was subjected to the following microscopic observation.

(Observation using a phase contrast microscope)
An image was taken with a phase contrast microscope (T-2000, Nikon Corporation) (objective magnification 10x), and by image analysis, the outer circumference length of the particles of the imaged hydrogel structure was measured. The roundness was determined according to (1).

Circularity = 4 x π x area ÷ (circumference) ² ...(1)

(Observation using a confocal microscope)
According to a conventional method, particles of the hydrogel structure were fluorescently stained using collagen type I antibody C2456 (Sigma-Aldrich), and Z-axis cross-sectional images were taken using a confocal microscope (TCS SP8, Leica).

FIG. 2A shows an example of an image taken using a phase contrast microscope about the obtained hydrogel structure (gel particles). Further, FIG. 2B shows a histogram of the outer circumferential length of each particle of the obtained hydrogel structure (gel particles). Moreover, FIG. 3A shows an example of a confocal microscope image of the obtained hydrogel structure (gel particles). Further, FIG. 3B shows a histogram of the circularity of each particle of the obtained hydrogel structure (gel particles).

As a result, especially when the pH of the heparin-containing solution was adjusted to pH 3 or pH 5, as seen in the phase contrast microscopy image shown in Figure 2A, the obtained hydrogel structure (gel particles) While the planar projection shape was approximately circular, when the pH of the heparin-containing solution was adjusted to pH 7, the obtained hydrogel structure (gel particles) had an elliptical or circular planar projection shape. It was observed that the shape collapsed without any problems. Accordingly, the results of measuring the circumference of each particle also showed a tendency for the circumference to increase as the pH value increased (FIG. 2B). FIG. 3A shows an example of a three-dimensional fluorescence image obtained using a confocal microscope to investigate the structure of gel particles. As a result of measuring the circularity of each particle of the obtained hydrogel structure (gel particles) from the Z-axis cross-sectional image, it was confirmed that the circularity tended to increase as the pH value decreased (Fig. 3B).

From the above, in order to obtain a hydrogel structure that is closer to a spherical shape, the pH value of the heparin-containing solution must be below a predetermined value, for example, at least a value less than 7 according to this test example. It became clear that there was a need.

<Test Example 2> (Effect of heparin concentration in heparin-containing solution)
In the preparation of the hydrogel structure shown in Test Example 1, the final concentration of heparin sodium when preparing the heparin-containing solution was changed to 10 mg/mL, 1 mg/mL, or 0.1 mg/mL. A hydrogel structure was prepared in the same manner as in Example 1. Note that the pH of the hydrogel material-containing solution was adjusted to pH 3.5. The obtained hydrogel structure was observed with a phase contrast microscope in the same manner as in Test Example 1, and the roundness was determined.

FIG. 4A shows an example of the phase contrast microscope image. Further, FIG. 4B shows a histogram of the roundness of each particle of the obtained hydrogel structure (gel particles).

As a result, in particular, from the roundness measurement results shown in FIG. 4B, the hydrogel structures prepared under the conditions of a final concentration of heparin sodium of 10 mg/mL or 1 mg/mL when preparing a heparin-containing solution, More than 90% had a roundness value of 0.95 or more, whereas when the final concentration of heparin sodium when preparing a heparin-containing solution was 0.1 mg/mL, the roundness value The proportion of hydrogel structures with 0.95 or more was 16%.

From the above, in order to obtain a more spherical shape of the hydrogel structure, the heparin concentration of the heparin-containing solution should be at least a predetermined concentration, for example, at least 0.1 mg/mL, according to this test example. It became clear that the concentration needed to exceed the

<Test Example 3> (Structural analysis of collagen by CD spectrum measurement)
In order to understand the mechanism of how hydrogel structures such as those shown in Test Examples 1 and 2 are formed in a heparin-containing solution, structural analysis of collagen was performed using CD spectroscopy. Heparin is a polymeric polysaccharide made by polymerizing β-D-glucuronic acid or α-L-iduronic acid and D-glucosamine, and its basic composition is a structure containing D-glucose whose hydroxyl group is modified with a sulfate group, etc. It can be said that there is. Therefore, in this test example, a solution was prepared in which collagen was mixed with heparin and D-glucose, and CD spectra were measured. Specifically, a test sample was prepared as follows, its CD spectrum was measured, and the results were evaluated.

(Preparation of test sample)
(1) Glucose solution: D-glucose was dissolved in purified water to a concentration of 0.20 mg/mL, and the pH was adjusted to pH 3, pH 5, or pH 7 with 1M HCl.

(2) Collagen solution: The collagen gel precursor solution (product name "Collagen I, Rat Tail") used in Test Examples 1 and 2 was dissolved in purified water so that the collagen concentration was 0.30 mg/mL, and at that time, The pH was adjusted to pH 3, pH 5, or pH 7 with 1M HCl.

(3) Mixed solution of collagen and glucose: Collagen gel precursor solution (product name "Collagen I, Rat Tail") used in Test Examples 1 and 2 and D- Glucose was dissolved in purified water, and the pH was adjusted to pH 3, pH 5, or pH 7 with 1M HCl.

(4) Heparin solution: The heparin sodium used in Test Examples 1 and 2 (derived from pig intestine, Tokyo Kasei Kogyo Co., Ltd.) was dissolved in PBS to a final concentration of 0.20 mg/mL, and at that time, dissolved in 1M HCl. The pH was adjusted to pH 3, pH 5, or pH 7.

(5) Collagen and heparin mixed solution: Collagen gel precursor solution (product name "Collagen I, Rat Tail") used in Test Examples 1 and 2 and heparin sodium so that the concentration is the same as the above collagen solution and heparin solution. (derived from pig intestinal tract, Tokyo Kasei Kogyo Co., Ltd.) was dissolved in PBS, and the pH was adjusted to pH 3, pH 5, or pH 7 with 1M HCl.

(CD spectrum measurement)
Measurement was performed using a circular dichroism dispersion meter (J-720WI-L, JASCO Corporation) in a quartz cuvette with an optical path length of 1 mm at 5°C.

FIG. 5 shows the results of CD spectra of a glucose solution, a collagen solution, and a mixed solution thereof under each condition of pH 3, pH 5, or pH 7.

As shown in FIG. 5, the spectral data of the CD spectrum characteristically measured for collagen molecules found in collagen solutions hardly changed even when mixed with D-glucose.

FIG. 6 shows the results of CD spectra of a heparin solution, a collagen solution, and a mixed solution thereof under each condition of pH 3, pH 5, or pH 7.

As shown in FIG. 6, the spectral data of the CD spectrum characteristically measured for collagen molecules found in collagen solutions changed significantly when mixed with heparin. More specifically, in the vicinity of 200 nm to 210 nm, the optical rotation shifted to the positive side in the figure, while the peak in the vicinity of 210 nm to 240 nm tended to fall to the negative side in the figure, and the tendency of this change was observed at pH 3, The rate of change was highest in the order of pH 5 and pH 7.

From the above, it was suggested that collagen gelation is promoted in a heparin-specific manner by causing some kind of interaction between heparin and collagen. It was also revealed that structural changes occur more easily under acidic conditions.

<Test Example 4> (Preparation of Cell ball: Neuro ball using primary cultured rat cerebral cortex cells)
In order to investigate whether the hydrogel structure shown in Test Examples 1 and 2 is suitable as a scaffold structure for three-dimensional culture of cells, primary cultured rat cerebral cortex cells collected from a rat fetus (E18) were used. We attempted to create a Cell ball using this method. Here, a cell ball is a cell containing a desired cell, which is obtained by attaching or encapsulating a living cell to a hydrogel structure and culturing it with a medium appropriate for the cell. It is a hydrogel structure.

Specifically, the hydrogel structure was prepared in the same manner as Test Examples 1 and 2, except that the final concentration of heparin sodium in the heparin-containing solution was 1.0 mg/mL, and the pH was 3.5. In addition, a cell suspension suspended in neuro basal medium was added to the collagen-containing hydrogel material-containing solution to a cell concentration of 1.3 x 10 ⁶ cells/mL. , suspended (collagen concentration 15 mg/mL, pH 7.0). Using this hydrogel material-containing solution, a hydrogel structure was prepared in the same manner as in Test Examples 1 and 2 except for the above.

After replacing the medium of the hydrogel structure containing living cells prepared as described above from the heparin-containing solution with neuro basal medium, the structure was transferred to a conventional 12-well plate incubator. Culture was performed under conditions of 5% CO ₂ and 37° C., and immunochemical staining was performed on samples on the 3rd and 10th days of culture. Specifically, immunochemical staining was performed as follows.

(immunochemical staining)
After culturing, the medium was replaced with a 4% paraformaldehyde solution and fixed at 4°C for 10 minutes. Next, it was fixed using methanol (-20°C) for 10 minutes, and further fixed using 0.2% Triton-X (dissolved in PBS) for 5 minutes. Thereafter, in order to reduce non-specific staining, the cells were reacted for 10 minutes with Preblock buffer (0.05% Triton-X, 5% fatal bovine serum in PBS), and stained with Preblock buffer containing a specific primary antibody. The primary antibodies used were mouse anti-MAP2 (M4403, Sigma-Aldrich) and mouse anti-β-tubulin III (T8578, Sigma-Aldrich) as specific antibodies for MAP2 and β-Tubulin III, which are known as neuronal markers, respectively. ), and rabbit anti-synaptophysin (MAB329, MILLIPORE) was used as a specific antibody against synaptophysin, which is known as a presynaptic marker. Each of these primary antibody reagents for labeling neurons and presynapses was added to Preblock buffer at a ratio of 1:1000 and used as the primary antibody solution, and the sample was immersed in the primary antibody solution at 4°C. and incubated overnight. After that, the sample was washed twice for 5 minutes at room temperature with Preblock buffer, and the secondary antibodies were Alexa Fluor 546 (anti-rabbit IgG, Molecular Probes) and Alexa Fluor 488 (anti-mouse IgG, Molecular Probes). In order to identify the nucleus, Hoechst 33258 was added to the Preblock buffer at a ratio of 1:1000, and the sample was immersed in the secondary antibody solution and incubated for 1 hour. Thereafter, the samples were washed with Preblock buffer twice for 5 minutes at room temperature, and then twice with PBS every 10 minutes. This sample was subjected to fluorescence observation using a confocal microscope (TCS SP8, Leica) equipped with a cooled CCD camera (Luca, Andor).

FIG. 7A shows an example of an image obtained by 3D observation using a confocal microscope for a sample on the third day of culture. Further, FIG. 7B shows an example of an image obtained by 3D observation using a confocal microscope for a sample on the 10th day of culture. As shown in these figures, the results of 3D observation using a confocal microscope, which includes fluorescence observation of neuronal markers MAP2 or β-Tubulin III and presynaptic marker Synaptophysin, indicate that on the third day of culture, The extension of neuron processes within the gel particles was confirmed (FIG. 7A), and on the 10th day of culture, the formation of synapses on the neurites extended within the gel particles was confirmed (FIG. 7B).

From the above, by using the hydrogel structure of the present invention, a cell ball that mimics the biological tissue environment, that is, a neuron network-like structure was formed in a three-dimensional microspace. It has become clear that it is possible to construct a neuro ball. Furthermore, this Neuro ball could be further cultured for a long period of about one month.

<Test Example 5> (Preparation of Cell ball: Neuro ball using human iPS cell-derived neural progenitor cells)
In order to investigate whether the hydrogel structures shown in Test Examples 1 and 2 can be applied to human cells, human iPS cell-derived neural progenitor cells (XCell Science) were used to form a Cell ball. I tried to make it. To prepare a hydrogel structure containing living cells, human iPS cell-derived neural progenitor cells are added to a solution containing the hydrogel material to a cell concentration of 3.0 x 10 ⁵ cells/mL. The same procedure as in Test Example 4 was conducted except that the suspension was suspended (collagen concentration: 15 mg/mL, pH 7.0). In addition, the culture medium, culture conditions, immunochemical staining after culture, microscopic observation, etc. were conducted in the same manner as in Test Example 4.

FIG. 8 shows an example of an image obtained when a sample on the 10th day of culture was observed in 3D using a confocal microscope. As shown in this figure, from the results of 3D observation using a confocal microscope accompanied by fluorescence observation for β-Tubulin III, a neuronal marker, human iPS cell-derived neural progenitor cells were found within gel particles on the 10th day of culture. differentiated into nerve cells, and neurite outgrowth was confirmed.

From the above, not only the rat fetus-derived cells shown in Test Example 4 but also the human iPS cell-derived cells can be used to create a cell ball that imitates the living tissue environment, that is, in a three-dimensional microspace. It has now become clear that it is possible to construct a Neuro ball with a neuron network-like structure. Furthermore, this Neuro ball could be further cultured for a long period of about one month.

<Test Example 6> (Functional evaluation of Neuro ball)
Functional evaluation was performed on the Cell ball Neuro ball obtained in Test Example 4, which was prepared using primary cultured rat cerebral cortex cells. That is, in order to investigate the functionality related to the neural network, Ca ²⁺ imaging measurement was performed on the sample on the 10th day of culture, and action potential measurement was performed on the sample on the 23rd day of culture. Specifically, measurements were performed as follows and the results were evaluated.

(Ca ²⁺ imaging measurement)
Ca ²⁺ imaging measurement of Neuro ball was performed using a calcium fluorescent indicator (Oregon Green ^TM 488 BAPTA-1, Thermo Fisher Scientific Logo). Observation of intracellular calcium was performed using an inverted fluorescence microscope (BX50, Olympus Corporation) equipped with a cooled CCD camera. The composition of artificial cerebrospinal fluid (aCSF) was NaCl 127mM, NaHCO ₃ 26mM, KCI 1.5mM, KH ₂ PO ₄ 1.24mM, MgSO ₄ 1.4mM, CaCl ₂ 2.4mM, and Glucose 10mM. A solution containing a calcium indicator at a concentration of 4 μM in artificial cerebrospinal fluid was loaded by standing in a 5% CO ₂ incubator at 37° C. for 60 minutes. Thereafter, in order to enhance spontaneous combustion, the solution was replaced with a solution that did not contain MgSO ₄ and had a calcium ion concentration of 1.2 mM CaCl ₂ . Fluorescence images were taken (30 frames/sec) of a Neuro ball set on the sample stage of an inverted fluorescence microscope under the same 37°C environment as in vivo, and the images were analyzed.

FIG. 9A shows a phase contrast microscope image taken when performing Ca ²⁺ imaging measurement and a fluorescence microscope image in the same field of view. Here, in the fluorescence microscope image of FIG. 9A, seven bright spots with high fluorescence intensity are shown as observation targets for fluorescence oscillation. Further, FIG. 9B shows fluorescence microscope images at 9.43 seconds from the start, 19.47 seconds from the start, and 28.6 seconds from the start when observing the bright spot for 2 minutes. Further, FIG. 9C shows the results of examining the change in fluorescence intensity over time for each of the above-mentioned bright spots.

As a result, as shown in FIG. 9, Ca ²⁺ oscillation was observed from one cell out of seven randomly selected cells in the field of view under the microscope.

On the other hand, in order to confirm whether the inhibitory synapses within the Neuro ball are functioning, a 10 μM solution of bicuculline, a _GABAA receptor antagonist, was added to the Neuro ball on the 10th day of culture. When we performed the same observation as above, we observed Ca ²⁺ oscillation from all 7 cells out of 7 randomly selected cells in the field of view under the microscope (Fig. 10A to Fig. 10A). 10C: Fluorescence microscopy images at 48.4 seconds from start, 55.1 seconds from start, and 61.8 seconds from start when observed over 2.5 minutes).

(action potential measurement)
As shown in FIG. 11A, Neuro balls on the 23rd day of culture were mounted on a planar microelectrode array, and action potentials were measured. In order to investigate whether excitatory-inhibitory synapses are functioning, drug responses were detected using synapse-related drugs. Specifically, a 10 μM solution of Bicuculline, which is an antagonist of the GABA _A receptor described above, was dropped into a Neuro ball on the 23rd day of culture, and then action potentials were measured. A group was established. Furthermore, after the addition of Bicuculline, a 30 μM solution of CNQX (6-Cyano-7-nitroquinoxaline-2,3-dione), which is an antagonist of AMPA type receptors, was added. A test group was established in which action 0 potential was measured.

Figure 11B shows the cases in which no drug was administered (“before” in the figure), the case in which bicuculline (Bicucullne), a GABA _A receptor antagonist, was instilled (“Bicucullne 10 μM” in the figure), and the cases in which bicuculline (Bicucullne) and The results are shown in which the firing frequency due to Ca ²⁺ oscillation was observed for 30 seconds when CNQX, an antagonist of AMPA type receptors, was dropped (“CNQX 30 μM” in the figure). Further, FIG. 11C shows a chart in which the obtained firing frequency results are graphed as relative values when the time of no drug administration is set as 100%.

As a result, as shown in FIGS. 11B and 11C, administration of bicuculline increased the firing frequency, and administration of CNQX suppressed the increase in firing frequency. Therefore, it was considered that functional inhibitory synapses and excitatory synapses were formed.

Based on the above, the Neuro ball produced using the hydrogel structure of the present invention is a method that can construct a neural network that expresses functions such as inhibitory synapses and excitatory synapses, It was shown that this is a sample preparation method that can detect changes.

<Test Example 7> (Preparation of brain tumor model using Cell ball)
In order to investigate whether the hydrogel structures shown in Test Examples 1 and 2 are suitable as scaffold structures for the production of brain tumor models, we used human glioma cells (from the RIKEN BioResource Center), which are neuroglial cells. An attempt was made to create a Cell ball using the following method. To prepare a hydrogel structure containing living cells, human glioma cells are added to a hydrogel material-containing solution to a cell concentration of 3.0 x 10 ⁵ cells/mL, and suspended. The test was carried out in the same manner as in Test Example 4, except that (collagen concentration was 15 mg/mL, pH 7.0). In addition, immunochemical staining and microscopic observation after culturing were performed in the same manner as in Test Example 4. However, the culture conditions were DMEM medium conditions. In addition, for immunochemical staining after culturing, a specific antibody against β-actin, ab8227 (abcam), was used at a dilution of 1:1000.

FIG. 12A shows an example of an image obtained when each cell ball obtained was observed using a phase contrast microscope for samples on the 2nd, 5th, 18th, and 60th days of culture. . Moreover, FIG. 12B shows an example of an image obtained when the obtained Cell ball was observed in 3D using a confocal microscope for a sample on the 5th day of culture.

As a result, as shown in FIG. 12A, on the 5th day of culture, it was confirmed that the gel particles were reduced in size due to cell proliferation within the hydrogel structure (gel particles). On the 18th day of culture, cells were frequently observed coming out of the hydrogel structure (gel particles), and enlargement of the gel particles was confirmed. On the 60th day of culture, cells were observed to spread on the petri dish and proliferate. In addition, as shown in Figure 12B, when we performed 3D observation using a confocal microscope of each cell ball obtained for the sample on the 5th day of culture, we found that cell nuclei were locally observed as indicated by the arrows. It was confirmed that the tumor was growing and had a morphology characteristic of a brain tumor.

From the above, it is clear that by using the hydrogel structure of the present invention, it is possible to construct a brain tumor model in a cell ball that mimics the biological tissue environment, that is, in a three-dimensional microspace. Ta.

<Test Example 8> (Construction of cancer microenvironment model using Cell ball and evaluation of cancer growth ability by constituent cell types)
In recent years, cancer research has focused not only on research on cancer cells themselves, but also on research on the cancer microenvironment, including surrounding cells. If it is possible to reproduce the cancer microenvironment, including surrounding cells, in vitro, it is expected to be applied to, for example, understanding the mechanism of cancer microenvironment formation and evaluating the development of anticancer drugs. Therefore, in this test example, human glioma cells, which are neuroglial cells, were the target, and human glioma cells and astrocytes, which are peripheral cells of the brain, were cultured alone in the hydrogel structure of the present invention, and they were cultured. The proliferation ability of glioma cells was evaluated in the case of co-culture.

A hydrogel structure containing living cells was prepared in the same manner as in Test Example 4. However, in the hydrogel material-containing solution (collagen concentration: 15 mg/mL, pH 7.0), (1) in the case of single culture of human glioma cells, the cell concentration is ^3.0 (2) In the case of monoculture of astrocytes, the cell concentration is 3.0 × 10 ⁵ cells/mL; (3) In the case of co-culture of human glioma cells and astrocytes, Each cell was added and suspended to the same cell concentration as above, and then used.

After replacing the medium of the hydrogel structure containing the living cells prepared above with a DMEM medium from the heparin-containing solution, the hydrogel structure was transferred to a conventional 12-well plate incubator and cultured in this manner. was carried out under the conditions of 5% CO ₂ and 37°C, and the samples obtained on the 2nd, 4th, 6th, 8th, 10th, 12th, and 14th days of culture were A section of each cell ball was prepared and stained with HE (Hematoxy-Eosin). Furthermore, the length of the outer periphery of each of the obtained Cell balls was measured by observation using a phase contrast microscope as in Test Examples 1 and 2.

Figure 13A shows samples taken on the 2nd, 8th, and 14th days of culture for single culture of human glioma cells, single culture of astrocytes, and co-culture of human glioma cells and astrocytes. An example of an image obtained when a ball is observed using a phase contrast microscope is shown. In addition, FIG. 13B shows the cell balls obtained for samples on the 16th day of culture for each of human glioma cell single culture, astrocyte single culture, and co-culture of human glioma cells and astrocytes. An example of an image obtained when a section was stained with HE is shown.

As a result, as shown in Figure 13A, no tumor formation was observed in astrocytes alone culture, whereas in human glioma cells alone culture and co-culture of human glioma cells and astrocytes, the number of culture days Cell proliferation within the hydrogel structure (gel particles) was observed depending on the conditions, and the formation of black cell clusters resembling brain tumors was observed. Furthermore, as shown in Figure 13B, the results of HE staining showed that tumor formation was similarly not observed in astrocytes alone cultured, whereas tumor formation was observed in specific locations in human glioma cells alone cultured. The situation was confirmed. Furthermore, in co-culture of human glioma cells and astrocytes, the tumor was observed to expand and infiltrate into the hydrogel structure (gel particles).

On the other hand, in Figure 14A, in order to evaluate the difference in cancer growth ability between single culture of human glioma cells and co-culture of human glioma cells and astrocytes, each cell ball was A graph showing the results of measuring the length of the outer circumference in terms of average value and standard deviation is shown. Further, FIG. 14B shows a graph showing the results of measuring the brightness of each Cell ball for each number of days of culture in terms of average values and standard deviations. Note that "**" shown in the figure indicates that the statistically significant difference determined by the T-test is less than 1% in risk.

As shown in Figure 14A, the length of the circumference of each cell ball is significant depending on the number of days of culture in both single culture of human glioma cells and co-culture of human glioma cells and astrocytes. and was the lowest in co-culture. This was thought to be because the collagen fibers were stretched due to cell proliferation within the hydrogel structure (gel particles), reducing the circumference of the particles. Furthermore, as shown in FIG. 14B, when the average value of the brightness of each cell ball was measured for each culture day, the brightness decreased the most in co-culture of human glioma cells and astrocytes. This decrease in brightness was thought to be due to an increase in cell clusters such as brain tumors due to cell proliferation.

Based on the above, by using the hydrogel structure according to the present invention, a cell ball that imitates the living tissue environment can be created, that is, a cell ball that includes cancer cells and surrounding cells in a three-dimensional microspace. It has become clear that it is possible to construct a cancer microenvironment model that

Claims (9)

A hydrogel material-containing solution containing collagen is added to a sugar chain-containing solution containing sugar chains belonging to glycosaminoglycans to form a hydrogel structure having an interface between the solution and the medium made of the sugar chain-containing solution. A method for producing a hydrogel structure, characterized in that the sugar chain belonging to the glycosaminoglycans is heparin, and the pH of the sugar chain-containing solution is adjusted to pH 7 or less. A method for producing a hydrogel structure. The method for producing a hydrogel structure according to claim 1, wherein the collagen is acid-soluble collagen and/or enzyme-solubilized collagen. The method for producing a hydrogel structure according to claim 1 or 2, wherein the pH of the hydrogel material-containing solution is adjusted to pH 6 or higher. The hydrogel structure has a granular shape, a major axis of 20 μm or more, and/or a circularity of 50% or more, according to any one of claims 1 to 3. A method for producing a hydrogel structure. The method according to any one of claims 1 to 4, wherein the hydrogel material-containing solution contains a living biological sample, and the hydrogel structure is obtained as the hydrogel structure containing the living biological sample. A method for producing a hydrogel structure. 6. The method for producing a hydrogel structure according to claim 5, wherein the biological sample is a cell sample derived from one or more types selected from the group consisting of unicellular organisms and multicellular organisms. At least a living biological sample is attached to or encapsulated in the hydrogel structure obtained by the method for producing a hydrogel structure according to any one of claims 1 to 4, and a culture medium corresponding to the biological sample is prepared. 1. A method for culturing a biological sample, comprising culturing together with a biological sample. A method for culturing a biological sample, comprising culturing the hydrogel structure obtained by the method for producing a hydrogel structure according to claim 5 or 6 together with a medium suitable for the biological sample. A method for analyzing a biological sample , characterized in that the biological sample cultured in the hydrogel structure obtained by the method for culturing a biological sample according to claim 7 or 8 is subjected to analysis together with the hydrogel structure .