Biocompatible structures for connecting and culturing biomaterials

Abstract

The present invention relates to a method for producing a biocompatible structure for the connection and culture of biological materials, a method for culturing aggregates of biological materials, and the use of a biocompatible structure for the connection and culture of biological materials.

Description

[Technical Field]

[0001] The present invention relates to a method for producing biocompatible structures for connecting and culturing biomaterials, a method for culturing aggregates of biomaterials, and the use of biocompatible structures for connecting and culturing biomaterials. [Background technology]

[0002] Traditionally, living cells have been cultured in vitro, i.e., in a culture dish. Such cultures are mainly carried out by the following three methods: 1. Culture on a plastic or glass surface, usually coated with a gel, protein, or extracellular matrix peptide, or on an uncoated plastic or glass surface (culture on an uncoated plastic surface is called "adherent cell culture"), 2. Culture on a semipermeable membrane ("Transwell"), and 3. Three-dimensional culture (e.g., suspension culture).

[0003] The first two methods described above allow for the cultivation of cells or cell mixtures derived from (primary) tissues or stem cells, and some degree of cell maturation and functionality can be obtained. However, such cells have little physiological function and it is almost impossible to reproduce complex intercellular and inter-tissue interactions.

[0004] Suspension culture of cell aggregates, such as spheroids and organoids, has the advantage of allowing the formation of complex structures and interactions from cells (usually self-organizing cells). For example, spheroids can be produced by autoaggregation. In suspension culture of cell aggregates, single or mixed cells cultured with adhesion can be aggregated in a container to autonomously form spherical masses or differentiate into complex organoids resembling organs. One example is retinal organoids, which form a structured, photosensitive retina. Intestinal organoids also exhibit intestinal transport function and can assist in vivo intestinal villi. Other examples include brain organoids and kidney organoids.

[0005] However, most of these organoids and spheroids remain as individual organ / tissue systems and cannot cooperate to form complex organ systems. Furthermore, organoids and spheroids generally cannot form vascular or ductal structures that extend or invade other areas of the body from the formed organ during normal development.

[0006] The formation of complex organ systems, vascular structures, or conduit structures, as described above, can be achieved to some extent by utilizing so-called assembloids. An assembloid is formed by fusing several organoids to create a morphologically functional unit. One example is the cortico-spinal-muscle assembloid, which consists of a brain region (cortex), a spinal cord spheroid (spinal cord), and muscle cells. See Anderson et al., Generation of Functional Human 3D Cortico-Motor-Assembloids. Cell, Volume 183, 2020, pages 1913-1929, e1-10.

[0007] However, like organoids, assembleroids are difficult to give directionality to, and most fuse randomly with each other. Also, organoids and assembleroids are often of uneven size, making it difficult to align them correctly in space. One organoid or assembleroid often "absorbs" another, becoming larger than the others. Furthermore, the possibility of obtaining organoids or assembleroids that retain their structure is extremely limited. Organoids and assembleroids can only be connected "in series," and cannot be connected in any other direction. Moreover, there is no simple method for attaching individual cells, substances, or functional structures, such as immune cells, to fused organoids or assembleroids.

[0008] Under these circumstances, the present invention aims to provide a method that can avoid, or at least reduce, the drawbacks of culture methods known in the prior art. [Overview of the Initiative] [Means for solving the problem]

[0009] The fundamental objective of this invention is to achieve a method for producing biocompatible structures ("linkerspheres") for connecting and culturing biomaterials, and this method is 1) A step of providing a nonpolar, non-aqueous, biocompatible liquid, preferably mineral oil; 2) A step of introducing a solution of a biocompatible matrix material into the biocompatible liquid to obtain an emulsion; 3) A step of incubating the emulsion; and 4) A step of forming a three-dimensional structure from the biocompatible matrix material by incubation of the emulsion to obtain a linker sphere. Includes. [Modes for carrying out the invention]

[0010] According to the present invention, "biocompatibility" means the property of being non-toxic to biological substances. In relation to the linker sphere and matrix material according to the present invention, "biocompatibility" means that the matrix material is particularly suitable as a substrate for culturing and growing biological substances.

[0011] According to the present invention, "biomolecules" include living cells, aggregates of living cells, organs, and parts thereof.

[0012] According to the present invention, the nonpolar, non-water-miscible, biocompatible liquid provided in step (1) is a liquid that behaves like mineral oil and is 100% immiscible with water, thereby forming the emulsion of the present invention and being non-toxic to biological substances.

[0013] The liquid provided in step (1) is preferably "mineral oil" (CAS number: 8042-47-5; EC number: 232-455-8), that is, preferably a highly viscous bioreagent with quality and purity suitable for use in the field of molecular biology, for example, by layering it on top of an aqueous solution and performing density gradient centrifugation (quality level: 200). Suitable mineral oils for the present invention include mineral oil from Sigma-Aldrich (M5904), mineral oil from Carl Roth (#8904), and mineral oil from Merck (#107160; #113898). The mineral oil according to the present invention is also called "paraffin oil" or "petrolatum oil". The mineral oil is preferably provided in a container, and more preferably in a micro-reaction vessel of appropriate size, for example, a container with a capacity of 2 ml, 1.5 ml, 0.5 ml, 0.2 ml (e.g., an "Eppendorf" tube).

[0014] According to the present invention, the "matrix material" is a mixture of molecules that exist in liquid form in step 2 and can be transformed into a tissue-like solid form by the action of physical or chemical phenomena. The solid matrix material provides a biocompatible structure, preferably a network-like structure, on which biomolecules can be cultured. Examples of matrix materials suitable for the present invention include hydrogels, basement membrane-like matrices (e.g., Matrigel; Matrigel preferably contains a high amount of laminin), and other biocompatible gel-like substances.

[0015] In step (2), the introduction of the biocompatible matrix material solution into the mineral oil is carried out using a suitable dispensing device (e.g., a pipette). Since this aqueous matrix material is fluid, it is preferable to introduce it into the mineral oil in the form of droplets to form an emulsion.

[0016] The size of the linker sphere according to the present invention can be easily controlled by changing the volume of the matrix material. The larger the volume of the matrix material, the larger the linker sphere, and the smaller the volume of the matrix material, the smaller the linker sphere.

[0017] According to the present invention, in step (3), incubation is performed over a period of time during which the formation of linker spheres becomes possible, preferably for at least 10 seconds to a maximum of 60 minutes.

[0018] The exact incubation time is determined by those skilled in the art and depends on the type of gelation process. For example, Matrigel is incubated at a high temperature. Other hydrogels require a "crosslinking agent" or similar binding chemical. Thus, incubation can be from a few seconds to a few minutes (up to 1 hour).

[0019] In step (4) of the method of the present invention, due to the interfacial effect between the aqueous biocompatible matrix material and the surrounding mineral oil, the aqueous biocompatible matrix material in the emulsion is presumed to be spherical, and thus is named "linker spheres".

[0020] In this way, the object underlying the present invention can be fully achieved.

[0021] The inventors have found that by using the method of the present invention, a biocompatible structure in the form of linker spheres can be produced, and this linker sphere is a useful tool for tissue engineering that can at least partially or completely overcome the drawbacks of the prior art.

[0022] According to the present invention, the linker sphere obtained by the method of the present invention is suitable for culturing biological substances inside it and / or on its surface, and is configured to enable such culturing. Further, since the linker sphere functions as a crosslink or a connection path between different types of biological substances or between their aggregates, it is suitable for connecting biological substances and is configured to enable such connection ("linker" = connection, "sphere" = sphere). Also, since the linker sphere can culture biological substances, a biological substance or a part thereof can grow through the inside of the linker sphere or can grow on the surface of the linker sphere. Therefore, a connection can be constructed between the first biological substance or its aggregate and the second biological substance or its aggregate, and further between another biological substance or its aggregate via the linker sphere.

[0023] Furthermore, the linker sphere provides a physiological environment for biological substances that enables the formation of blood vessel pathways and duct pathways that extend or penetrate from one organ to another biological region during the development process of an organism. The linker sphere obtained according to the present invention can reproduce complex cell-cell interactions, tissue-tissue interactions, and organ-organ interactions. Therefore, the linker sphere is particularly suitable for studying complex biological structures (such as organs and organ systems).

[0024] In one embodiment of the method of the present invention, the biocompatible matrix material is a basement membrane-like matrix.

[0025] According to the present invention, the "basement membrane-like matrix" is a complex mixture of biomolecules used as a growth substrate, that is, a complex mixture of biomolecules used as a matrix or cell substrate in three-dimensional cell culture and three-dimensional tissue engineering. The basement membrane-like matrix is ​​a secretion purified from the mouse sarcoma cell line Engelbreth-Holm-Swarm (EHS cells), and its composition is similar to the extracellular matrix of the basement membrane of animal cells. The basement membrane-like matrix contains laminin, entactin, collagen, heparan sulfate proteoglycan, etc. This approach has the advantage of employing a particularly suitable biocompatible matrix material according to the present invention. The basement membrane-like matrix forms a gel-like structure, or hydrogel, with a network formed by the polymerization of proteins contained in its composition at approximately 37°C, but becomes liquid at lower temperatures, such as 4°C. Compared to polylysine-coated cell culture matrices, the basement membrane-like matrix can form a complex and physiological three-dimensional cell network structure. For an overview of basement membrane-like matrices, see, for example, Hughes et al: “A complex protein mixture required for optimal growth of cell culture” in Proteomics, Volume 10, Issue 9, 2010, ISSN 1615-9861, pp. 1886-1890.

[0026] Suitable basement membrane-like matrices for the present invention include Matrigel (Corning Life Sciences), BME, and EHS matrix.

[0027] In one embodiment of the present invention, in step (2) of the method of the present invention, a solution of a growth factor-reduced (GFR) basement membrane-like matrix is ​​introduced.

[0028] This approach has the advantage of using a matrix with reduced growth factors (e.g., EGF) that can affect the cells constituting the aggregate. For example, neurons are preferably cultured on such a matrix to reduce such effects. A suitable growth factor-reduced basement membrane-like matrix according to the present invention is Corning® Matrigel® Growth Factor-Reduced (GFR) Basement Membrane Matrix, LDEV-Free (product number: 354230, Corning Life Sciences).

[0029] In one embodiment of the present invention, step (3) of the method of the present invention is performed in which the emulsion is treated to solidify the biocompatible matrix material, and this operation is preferably performed by heating the emulsion, preferably at about 37°C, and more preferably for about 15 to 30 minutes.

[0030] This approach has the advantage of converting linker spheres into a manageable solid form suitable for culture purposes by crosslinking the proteins contained in the biocompatible matrix material or its composition. Coagulation or crosslinking forms a solid hydrogel or gel-like structure, which enables the culture and growth of biomolecules within it. Heat treatment can be easily performed by placing the reaction vessel containing the emulsion in a water bath.

[0031] In a further embodiment of the method of the present invention, the solution of the biocompatible matrix material includes a cell culture medium.

[0032] This method creates the physiological conditions necessary for linker spheres to be used for culturing biomaterials immediately after their creation. Any culture medium can be suitably used in this invention, and the specific culture medium is selected by an expert depending on the biomaterial being cultured or the biomaterial to be connected to the linker sphere. For example, suitable cell culture media for culturing astrocytes are N2 medium and "B27-based retinal differentiation medium" (BRDM medium).

[0033] According to a further embodiment of the present invention, the solution of the biocompatible matrix material preferably contains biomaterials and living cells.

[0034] According to this method, the target biomaterial or living cells are already incorporated into the linker sphere during its manufacture. Such linker spheres containing biomaterial can be used as independent culture units or for connecting units or aggregates of biomaterial.

[0035] In one embodiment of the present invention, the solution of the biocompatible matrix material contains a dye.

[0036] This method has the advantage of improving the visibility of the linker spheres, thereby facilitating handling and making them easier to use in subsequent applications. Suitable dyes are biocompatible dyes, which are dissolved, for example, in a buffer.

[0037] In one embodiment of the present invention, in step (2) of the method of the present invention, the solution of the biocompatible matrix material is preferably introduced into the mineral oil as a fluid droplet using a pipette tip.

[0038] This method allows for particularly efficient production of the linker spheres of the present invention. In the production of linker spheres, first, a pipette containing a solution of biocompatible matrix material is held just above the oil level. When using a micropipette, it is preferable to press it to the first stop. This operation forms a droplet at the tip of the pipette. Next, by immersing the pipette tip in the oil, the droplet separates from the tip, sinks into the oil, and settles at the bottom of the reaction vessel.

[0039] In a further developed version of the present invention, after step (4), step 5) is performed to isolate the linker spheres from the emulsion.

[0040] This method has the advantage that linker spheres can be separated from the emulsion and prepared for further use.

[0041] In one embodiment of the method of the present invention, after step (5), step 6) washing the isolated linker spheres is performed, which is preferably done using an aqueous solution and more preferably using a cell culture medium.

[0042] This method has the advantage of separating any remaining mineral oil from the linker spheres. This washing process can be repeated to completely remove any excess mineral oil from the linker spheres so that the residual oil does not adversely affect subsequent cultivation.

[0043] In another embodiment of the present invention, an additional step is performed after step (4) and before step (5), in which an aqueous solution, preferably an aqueous buffer, is introduced into the 4') emulsion to form an aqueous phase, and the linker spheres are moved into this aqueous phase.

[0044] This method produces a two-phase system consisting of an oil phase and an aqueous phase. The formed aqueous linker spheres can be easily isolated and purified by moving them from the oil phase to the aqueous phase. Generally, any commercially available biocompatible buffer, such as PBS buffer, can be suitably used. The specific buffer is selected by an expert depending on the specific biomaterial being cultured or connected to.

[0045] In another embodiment of the present invention, the isolated linker spheres, which may have been washed, are transferred to a culture vessel, preferably a culture dish.

[0046] This method allows linker spheres to be placed in an environment where they can be directly used for culturing and connecting biomaterials. For example, commercially available culture dishes such as Petri dishes are suitable, and the type of culture dish is selected according to the specific application of the linker spheres.

[0047] In one embodiment of the method of the present invention, isolated linker spheres, which may be washed and moved, are cultured preferably at about 37°C, more preferably under about 20 vol% O2, and even more preferably under about 5 vol% CO2, for at least about 12 hours.

[0048] This method has the advantage of being able to utilize parameters that are particularly suitable for culturing linker spheres.

[0049] Another aspect of the present invention is a method for culturing and / or connecting aggregates of biomaterials, 1) A step of bringing aggregates of biomaterials into contact with biocompatible structures ("linkerspheres") for linking and culturing biomaterials in a suitable culture medium to obtain a composite of the aggregates of biomaterials and the linkerspheres, and 2) A step of culturing the complex of the biomolecular aggregate and the linker sphere. Includes, The linker sphere is obtained by the manufacturing method of the present invention. Regarding the method.

[0050] The features, characteristics, developments, and advantages of the manufacturing method of the present invention also apply to the culture method of the present invention.

[0051] According to the present invention, the "culturing" of the complex of biomolecular aggregates and linker spheres is carried out under standard cell culture conditions, for example, in an incubator at approximately 37°C in nutrient medium, and under 5% CO2 where applicable.

[0052] By using the linker spheres of the present invention, aggregates of biomaterials can be connected or fused at a predetermined distance determined by the linker spheres. In one embodiment of the present invention, two or more linker spheres can be placed between aggregates, and the number of linker spheres placed between aggregates may be any number. Since a certain distance is ensured by the linker spheres, the aggregates to be fused can be reliably spatially separated, making it difficult for the aggregates to intertwine and grow together, thus preventing one aggregate from growing more than another. Furthermore, by utilizing the linker spheres, multiple aggregates can be connected to a single aggregate, for example, a four-leaf clover-shaped structure can be obtained.

[0053] By connecting or fusing multiple aggregates via multiple linker spheres, theoretically infinitely large and complex arrangements can be formed in a manner similar to building blocks ("Lego systems").

[0054] Furthermore, linker spheres can be used to create pathway systems that connect aggregates of biomaterials, for example, perfusible blood vessels, nerve pathways, or extracellular matrix (ECM) structures. These can also be combined. Such pathway systems can be provided as additional spheroid structures (e.g., vascular organoids) or embedded in linker spheres during the manufacturing process. Moreover, it is easily possible to fuse multiple linker spheres containing different types of cells.

[0055] Linker spheres can contain any ECM structure and therefore can mimic biological / pathway structures.

[0056] Even if the aggregates are of different sizes, they can be easily fused together via the linker spheres of the present invention simply by connecting each of these aggregates to the linker spheres of the present invention, and then a pathway system can be arranged between these aggregates.

[0057] In one embodiment of the culture method of the present invention, the aggregate of biomaterial is cut before contacting the linker sphere, preferably with micro-scissors, and more preferably the aggregate of biomaterial is brought into contact with the linker sphere at its cut surface.

[0058] This means allows the shape and size of the aggregate to be adapted to the linker sphere and / or the desired connection. The cut surface forms a particularly good contact area, enabling reliable and good connection with the biomaterial aggregate.

[0059] In one embodiment of the culture method of the present invention, the culture is carried out at approximately 37°C, preferably under approximately 20% O2, and more preferably under approximately 5% CO2.

[0060] This method has the advantage of providing those skilled in the art with culture conditions that have been proven particularly effective according to their expertise.

[0061] In another embodiment of the culture method of the present invention, the aggregate of biomaterial is an organoid and / or spheroid and / or assemblyroid.

[0062] This method has the advantage of reducing, and even avoiding, known drawbacks in prior art relating to organoids and assembleroids. By placing linker spheres between aggregates of biomaterials, different types of tissues do not fuse directly, and such indirect fusion is not normally observed in living organisms. Furthermore, by inserting linker spheres, the spatial orientation of the aggregates can be precisely and arbitrarily determined. On the other hand, direct fusion usually results in random fusion without directionality, especially when aggregates of various sizes are used. Moreover, the method of the present invention has succeeded for the first time in oriented multiple aggregates "in series".

[0063] In one embodiment of the culture method of the present invention, the aggregate of biomaterial is selected from the group consisting of retinal organoids, vascular organoids, brain organoids, and neurospheroids.

[0064] This means that the culture method of the present invention can be applied to biological aggregates that exist in complex environments in nature, and the present invention makes it possible for the first time to reproduce biological aggregates under such complex environments. "Retinal organoids" are three-dimensional structures of retinal cells differentiated from pluripotent stem cells, possessing lamination, intercellular interactions, and cell-type specific diversity that mimics the human embryonic retina. Furthermore, retinal organoids can also form photosensitive photoreceptors with special structures (internal and external segments) of retinal cells. "Vascular organoids" are three-dimensional structures that can be formed from pluripotent stem cells and contain vascular cells such as endothelial cells and pericytes. Vascular organoids self-organize a three-dimensional capillary network surrounded by a basement membrane. "Brain organoids" are organoids formed from pluripotent stem cells and can reflect the organization and cellular diversity of specific brain regions. For example, thalamic organoids contain neurons found in the thalamus. Furthermore, according to the present invention, a "neurospheroid" is a spherical aggregate of nerve cells (neurons, glial cells, and their progenitor cells) that can be produced from pluripotent stem cells. This aggregate may include organized regions (e.g., neural rosettes or cortical-like regions) and unorganized regions.

[0065] In one embodiment of the culture method of the present invention, the aggregate of biomaterial contains astrocytes, and preferably contains astrocytes derived from embryonic pluripotent stem cells (iPSCs).

[0066] This method has the advantage of being able to utilize cells that are particularly important for tissue engineering, and especially cells of great interest in neuroscience research and drug testing. The stem cells are preferably derived from humans or animals.

[0067] In one embodiment of the present invention, in the culture method of the present invention, after step (2), a step (3) is performed in which steps (1) and (2) are repeated.

[0068] This method allows for the creation of tissue structures without length limitations and enables the connection of any number of linker spheres and biomolecular aggregates. Therefore, according to the present invention, steps (2) and (3) can be repeated at least once, at least twice, at least three times, at least four times, ... at least ten times, at least twenty times, at least 100 times, and so on.

[0069] Another aspect of the present invention relates to the use of linker spheres obtained by the manufacturing method of the present invention for linking and culturing biomaterials.

[0070] The features, characteristics, developments, and advantages of the manufacturing method and culture method of the present invention also apply to the use of the present invention.

[0071] The features described above and those described below are not limited to the specific combinations shown, but can also be used in other combinations or individually without departing from the scope of the present invention.

[0072] The present invention will be described in more detail below with reference to examples. The features described in the examples are not only applicable in relation to the following specific examples, but are also considered to belong to the present invention in their individual forms. [Brief explanation of the drawing]

[0073] Refer to the attached drawings for the following details. [Figure 1] This is a schematic diagram illustrating the manufacturing process of the linker sphere of the present invention. [Figure 2] (a) shows a schematic diagram of the astrolinker, and (b) to (e) show microscopic images. [Figure 3] A schematic diagram (upper panel) illustrating the connection process between a whole or halved organoid and a linker sphere, and a microscopic image of the ligation product (lower panel) are shown. [Figure 4](a) Microscopic images of the ligation process between retinal organoids and linker spheres, (b) suspension culture of the ligation product, (c) extension of axons from retinal organoids into the interior of linker spheres, (d) ligation product obtained by single ligation, and (e) ligation product obtained by double ligation. [Figure 5-6] Microscopic images of the dual connection between neurospheres, retinal organoids, and linker spheres are shown. [Figure 7] Microscopic images of the dual connection between neurospheres, retinal organoids, and astrolinkers are shown. [Figure 8] A schematic diagram of the composite optic nerve model using the linker sphere of the present invention is shown. [Figure 9] This is a schematic diagram illustrating the assistance of linker spheres for angiogenesis according to the present invention. [Figure 10] This is a schematic diagram illustrating the concept of multiple connections. [Examples]

[0074] 1. Manufacturing of Linker Spheres overview Figure 1 shows a schematic diagram illustrating the manufacturing process of linker spheres. In the first step, shown on the far left, mineral oil is placed in a reaction vessel. A liquid biocompatible matrix material, which may be mixed with living cells and / or other biomaterials such as culture media, is pipetted into the vessel.

[0075] In the next step, a droplet of the biocompatible matrix material solution is introduced into the mineral oil. To perform this dropwise dispensing, the biocompatible matrix material at the tip of the pipette is brought into contact with the surface of the mineral oil. Next, this single drop is released from the pipette, allowing it to settle at the bottom of the reaction vessel filled with mineral oil.

[0076] In the next step, the reaction vessel containing the formed emulsion is heat-treated. This heat treatment is performed, for example, by placing the reaction vessel in a water bath and incubating it at 37°C for 30 minutes. The proteins contained in the biocompatible matrix material are cross-linked, causing the biocompatible matrix material to solidify and form a gel-like structure, or hydrogel. This is called a "linker sphere."

[0077] In the final step shown in Figure 1, the linker spheres are washed and then transferred to the culture medium.

[0078] detail Linker spheres containing cells The following describes the technical details regarding the production of cell-containing linker spheres, using astrocytes, known as "Astrolinker," as an example.

[0079] The necessary materials are as follows: • Growth factor-reducing Matrigel (Corlight Life Sciences) • Mineral oil (Sigma-Aldrich) • N2 medium (DMEM / F12 medium supplemented with Glutamax, 2% hormone mix, 1% non-essential amino acids (NEAA), and 1% antibiotic-antifungal agent (Anti-Anti); all manufactured by Thermo Fisher Scientific) • BRDM medium (DMEM / F12 (3:1) medium supplemented with Glutamax, 2% vitamin A-free B27, 1% amino acids, and 1% NEAA; all manufactured by Thermo Fisher Scientific) ·ASC ++ Culture medium (N2 medium + 10 ng / ml epidermal growth factor (EGF) + 10 ng / ml fibroblast growth factor 2 (FGF2)) • BRDM FBST (DMEM / F12 (3:1) medium supplemented with Glutamax, 10% FBS, 2% B27, 1% amino acids, 1% NEAA (all from Thermo Fisher Scientific), and 100 μM taurine) • Magnesium / calcium-free PBS (PBS) -- (Thermo Fisher Scientific) • TrypLE (Thermo Fisher Scientific) 1.5ml reaction tube 15ml conical tube • Heating block for tubes • Non-adhesive 24-well plate or non-adhesive 48-well plate • A 1000 μl pipette tip with the tip cut (with scissors) 37℃ water bath • A bucket filled with ice • Micro scissors (FST Co.) • Non-tissue culture treated V-type 96-well plate (Sarstedt)

[0080] Linker spheres containing astrocytes are prepared using the following method.

[0081] Human iPSC-derived astrocytes were differentiated according to the method of Krencik et al. (2011) (Directed differentiation of functional astroglial subtypes from human pluripotent stem cells, Nat. Protoc. 6(11): 1710-7, doi:10.1038 / nprot.2011.405). In each experiment, astrocytes were thawed and divided into 24-well or 48-well plates. ++ Cultivate in culture medium. Six to seven days before starting this experiment, ASC ++ Treat astrocytes in culture medium with 1 ng / ml CNTF, changing the medium every other day. Transfer at least one well that reaches confluence to PBS. --Wash very carefully and detach the astrocytes from the wells by adding TrypLE and incubating at 37°C for 2 minutes. After confirming that the cells are single cells, stop the reaction by adding twice the volume of N2 medium as the TrypLE-containing medium. Transfer the cells to a 15 ml conical tube and centrifuge at 1500 × g for 2 minutes. Discard the supernatant, resuspend the cells in an appropriate volume of N2 medium, dilute 1:1 with trypan blue, and count the number of cells (in approximately 500 μl to 1000 μl) on a Neubauer hemocytometer to identify dead cells. Transfer the required number of cells to a 1.5 ml Eppendorf tube. 10,000 cells are needed to prepare one astrolinker, each 2.5 μl in size.

[0082] Next, the harvested cells are centrifuged at 800×g for 2 minutes to re-pelletize them. The supernatant is discarded as completely and carefully as possible. The cell pellet is then resuspended in cold BRDM medium to obtain a cell suspension of 1.25 μl per astrolinker (for example, to obtain 10 astrolinkers from 100,000 cells, use 10 μl of medium). The resulting cell suspension is then stored on ice. Growth factor-reduced Matrigel (thawed overnight in the refrigerator) is added to the chilled cell suspension in a 1:1 ratio, and the resulting solution is gently and thoroughly mixed, taking care not to introduce air bubbles. Matrigel can be stained with ink or other dyes to make the astrolinkers more visible in later steps. In this experiment, PBS was used. -- A 5% ink diluted to 1:1000 was used.

[0083] Before starting the next step, add approximately 50 μl of mineral oil to a 1.5 ml reaction tube (prepare one tube per astrolinker) and store at room temperature until ready to use.

[0084] Next, transfer 2.5 μl of the mixture of astrocytes and Matrigel into the reaction tube containing mineral oil using a thin-walled 10 μl pipette tip (preferably cooled in advance). To create an astro-linker, first, hold the pipette with 2.5 μl of the mixture of astrocytes and Matrigel aspirated into the tip just above the oil surface and press to the first stop. By this operation, a droplet is formed at the tip of the pipette. Next, immerse the pipette tip and the droplet at its tip in the oil liquid, so that the droplet detaches from the tip and sinks into the oil liquid. Discard the remaining liquid in the pipette. Place the tube containing the astro-linker on the heating block (37 °C) as quickly as possible and rapidly solidify it. This operation is necessary to prevent the cells from dispersing unevenly within the droplet. Next, incubate the tube at 37 °C for 15 - 30 minutes.

[0085] After incubation, add approximately 200 μl of pre-warmed PBS -- (37 °C). This operation is performed to transfer the formed linker sphere from the oil phase to the aqueous phase. Transfer the linker sphere and PBS -- (and as little oil liquid as possible) into a Petri dish (e.g., 6 cm) containing pre-warmed PBS -- at 37 °C. At this time, use a 1000 μl pipette tip with a cut tip to avoid damaging the linker sphere. In this way, the linker sphere is washed and the oil liquid is removed. This washing step can be repeated to remove the residue of the oil liquid. Transfer the washed astro-linker into a non-tissue culture-treated 48-well plate containing 250 μl of ASC ++ medium pre-warmed to 37 °C. Again, use a pipette tip with a cut tip. The astro-linker is cultured at least overnight at 37 °C under 20% O2 and 5% CO2, and the medium is exchanged (half of the medium is exchanged) every 2 - 3 days until use or fixation.

[0086] Figure 2a shows a schematic diagram of an astrolinker. Figure 2b shows an astrolinker (linker sphere carrying astrocytes) after 1 day of culture. Figure 2c shows an astrolinker carrying astrocytes pre-transfected with a lentiviral construct (Lenti-GFAP-GFP) that expresses green fluorescent protein (GFP) under the control of the glial fibrillary acidic protein (GFAP) promoter. Figures 2d and 2e show a partial three-dimensional reconstruction of an astrolinker carrying astrocytes expressing GFP under the control of the GFAP promoter, as shown in Figure 2c. Figure 2d shows a fluorescence image stained with magenta, and Figure 2e shows an image with height indicated in pseudocolor.

[0087] Linker spheres that do not support cells Cell-free linker spheres are prepared using the same protocol as described above.

[0088] Instead of astrocytes, only culture medium (e.g., BRDM medium) is added to the GFR-Matrigel. All subsequent steps are the same. Cell-free linker spheres can be cultured in a medium or buffer preheated to 37°C, and the type of medium or buffer is not important.

[0089] 2. Creating a dual connection Human iPSC-derived retinal organoids (ROs) differentiated according to previously published protocols (Zhong et al. 2014, Achberger et al. 2019) are selected 40 to 80 days after differentiation induction.

[0090] On day 1 (the day after the creation of the linker spheres), before connecting the retinal organoids to the linker spheres, cut the retinal organoids in half with micro-scissors and transfer them to a non-tissue culture-treated V-bottom 96-well plate. Place one halved retinal organoid into each well. Next, add 1000 μl of astrolinker or cell-free linker sphere to each well containing the retinal organoids using a pipette tip. Using a fine needle or small pipette tip, place the retinal organoids and linker spheres under a microscope. Position the retinal organoids so that the cut surface is in direct contact with the astrolinker / linker sphere. Then, very carefully place the plate in an incubator (37°C, 20% O2, 5% CO2), taking care not to disturb the arrangement of the retinal organoids.

[0091] A triple connection can also be created the following day or at a later date.

[0092] 3. Construction of a triple connection The following day, thalamic organoids (TOs) differentiated according to a previously published protocol (Xiang 2019) are selected 40-80 days after differentiation induction. One thalamic organoid is added to each of the V-bottom 96-well plates containing the double connections (astrolinker / linker sphere + retinal organoid). Here, a triple connection is created by directly positioning the thalamic organoid on the opposite side of the astrolinker / linker sphere from the side to which the retinal organoid is attached, using a fine needle or small pipette tip. This step is crucial for connecting cells via astrolinkers rather than directly. Carefully, without moving the organoids, the plates are left to stand overnight at 37°C in a 20% O2 and 5% CO2 incubator.

[0093] The triple-connection culture can be performed at a later date.

[0094] Figure 3 shows a schematic diagram of the connection process. The scale bar shown in the two microscope images at the bottom of the figure corresponds to a distance of 1000 μm.

[0095] 4. Triple-connected culture The following day, prepare a 48-well plate for non-tissue culture treatment using 250 μl of BRDM medium preheated to 37°C. Transfer the triple-connected organoids to this 48-well plate (using a 1000 μl pipette tip with the tip cut) and incubate at 37°C under 20% O2 and 5% CO2. Observe astrolinkers under a microscope to observe axonal projections between retinal and thalamic organoids. For this purpose, GFP can be stably transduced into the retinal organoids (for example, using a lentiviral vector). Change the medium for the double-connected or triple-connected organoids every 2-3 days using warmed BRDM medium (250 μl per well, half the volume of medium replaced).

[0096] Figure 4b shows microscopic images illustrating the results of the connection process between linker spheres and retinal organoids. Figure 4b shows the linker sphere-retinal organoid complex in suspension culture. Figure 4c shows the extension of neurites from retinal organoids to linker spheres. Figure 4d shows the result of simple connection between retinal organoids and linker spheres. Figure 4e shows the result of double connection. Astrocytes were labeled with GFP and stained using a similar method.

[0097] Figure 5 shows a dual connection of a linkersphere, with a neurosphere connected to one side and a retinal organoid connected to the other side. The neurosphere is connected to the retinal organoid via a neural pathway extending through the linkersphere. Figure 6 shows GFP-labeled cells of retinal organoids projected into the neurosphere by axons. These GFP-labeled cells are positive for NEFM, a ganglion cell marker.

[0098] Figure 7 shows a dual connection of the astrolinker, with a neurosphere connected to one side and a retinal organoid connected to the other side. The neurosphere is connected to the retinal organoid via neural pathways extending through the linker sphere.

[0099] Figure 8 shows a schematic diagram of a composite model of the optic nerve. This composite model of the optic nerve is used, for example, to model glaucoma and consists of retinal organoids containing retinal ganglion cells, two linkers filled with oligodendrocytes (myelinated parts of the optic nerve), astrocytes (intraretinal parts of the optic nerve), and patterned brain organoids, such as those in the diencephalon or posterior thalamus.

[0100] Figure 9 shows a schematic diagram illustrating a method of supporting angiogenesis using linker spheres, for example, by connecting tissue organoids and vascular organoids.

[0101] Figures 10a and 10b illustrate the concept of multiple connections. Using linker spheres makes it possible to combine multiple organoids with various connection concepts (e.g., neuronal connections and angiogenesis). Linker spheres have the potential to facilitate the growth and assembly of complexes consisting of multiple organoids and multicellular tissues (such as nerve growth and angiogenesis).

Claims

1. A method for producing biocompatible structures ("linkerspheres") for connecting and culturing biomaterials, 1) A step of providing a nonpolar and non-aqueous miscible biocompatible liquid; 2) A step of introducing a solution of a biocompatible matrix material into the biocompatible liquid to obtain an emulsion; 3) The step of incubating the emulsion; and 4) A step of forming a three-dimensional structure from the biocompatible matrix material by incubation of the emulsion to obtain a linker sphere. Includes, The nonpolar and non-aqueous miscible biocompatible liquid is mineral oil. The biocompatible matrix material is a basement membrane-like matrix, In step (2), the biocompatible matrix material solution is introduced into the mineral oil as fluid droplets. method.

2. The method according to claim 1, wherein in step (2), a solution of a basement membrane-like matrix with reduced growth factors is introduced.

3. The method according to claim 1 or 2, wherein in step (3), the emulsion is treated to solidify the biocompatible matrix material.

4. The method according to claim 3, wherein the treatment includes exposure to heat at 37°C, and the heat exposure is performed for 15 to 30 minutes.

5. The method according to claim 1 or 2, wherein the solution of the biocompatible matrix material includes a cell culture medium.

6. The method according to claim 1 or 2, wherein the solution of the biocompatible matrix material contains living cells.

7. The method according to claim 1 or 2, wherein the solution of the biocompatible matrix material contains a dye.

8. The method according to claim 1 or 2, wherein the introduction of the solution of the biocompatible matrix material as a fluid droplet into the mineral oil is performed using a pipette tip.

9. The method according to claim 1 or 2, wherein after step (4), step 5) is performed to isolate the linker sphere from the emulsion.

10. The method according to claim 9, wherein after step (5), step 6) washing the isolated linker spheres is performed, and the washing is carried out using an aqueous solution.

11. The method according to claim 9, wherein after step (4) and before step (5), step 4') is performed to introduce an aqueous solution into the emulsion to form an aqueous phase and move the linker spheres into the aqueous phase.

12. The method according to claim 9, wherein the isolated linker spheres, which may be washed, are transferred to a culture vessel.

13. The method according to claim 9, wherein the isolated linker spheres, which may be washed and moved, are cultured at 37°C under 20 vol% O₂ and 5 vol% CO₂ for at least 12 hours.

14. A method for culturing aggregates of biomaterials, 1) A step of bringing aggregates of biomaterials into contact with biocompatible structures ("linkerspheres") for linking and culturing biomaterials in a suitable culture medium to obtain a composite of the aggregates of biomaterials and the linkerspheres, and 2) A step of culturing the complex of the biomolecular aggregate and the linker sphere. Includes, A method wherein the linker sphere is obtained by the method described in claim 1 or 2.

15. The method according to claim 14, wherein the aggregate of the biomaterial is cut before it comes into contact with the linker sphere.

16. The method according to claim 15, wherein the aggregate of the biomaterial is brought into contact with the linker sphere at its cross-section.

17. The aforementioned culture was carried out at 37°C, under 20 vol% O₂ and 5 vol% CO₂. 2 The method according to claim 14, as described below.

18. The method according to claim 14, wherein the aggregate of the biomaterial is an organoid and / or spheroid and / or assemblyroid.

19. The method according to claim 18, wherein the aggregate of the biomaterial is selected from the group consisting of retinal organoids, vascular organoids, brain organoids, and neurospheroids.

20. The method according to claim 14, wherein the aggregate of the biomaterial comprises astrocytes derived from induced pluripotent stem cells (iPSCs).

21. The method according to claim 14, wherein after step (2), a step (3) is performed in which steps (1) and (2) are repeated.

22. Use of linker spheres obtained by the method of claim 1 or 2 for linking and culturing biomaterials.

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