A three-dimensional culture-enabling, dual-surface, bi-axially rotatable co-culture system
The biaxially rotatable, double-sided co-culture system addresses the limitations of current systems by allowing simultaneous cultivation of different cell types on both surfaces with enhanced nutrient exchange and mechanical stimulation, achieving more accurate in vitro models for multilayered tissues.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- T C ISTANBUL MEDIPOL UNIVERSITESI
- Filing Date
- 2025-12-12
- Publication Date
- 2026-06-18
Smart Images
Figure TR2025051663_18062026_PF_FP_ABST
Abstract
Description
[0001] A THREE-DIMENSIONAL CULTURE-ENABLING, DUAL-SURFACE, BLAXIALLY ROTATABLE CO-CULTURE SYSTEM
[0002] Technical Field
[0003] The present invention relates to a biaxially rotatable, double-sided co-culture system that enables three-dimensional cultivation, particularly for use in fields such as biomedicine, biotechnology, pharmacology, regenerative medicine, organoid production, and lab-on-a-chip applications.
[0004] State of the Art
[0005] Nowadays, co-culture and 3D cell cultures are increasingly used in fields such as cancer research, regenerative medicine, neuroscience, and tissue engineering (Langhans, 2018; Sosniak & Opiela, 2021). Co-culture systems allow for more accurate modeling of cancer progression, immune response, and drug responses by studying communication and signal transduction pathways between different cell types. For example, studying the interaction of tumor cells with fibroblasts or immune cells using co-culture systems is critical to understanding the cancer microenvironment.
[0006] 3D cell cultures overcome the limitations of classical 2D systems by allowing cells to grow in an environment similar to living tissues. Cell morphology, function, and gene expression are preserved as in natural tissues, leading to more meaningful results, particularly for tumor models, drug trials, and tissue engineering. 3D systems such as multicellular spheroids, organoids, and biomimetic tissue scaffolds enable the examination of processes such as tumor growth, metastasis, and drug toxicity under realistic conditions (Sciendo, 2021).
[0007] In traditional methods, different cell types are either cultured together on the same surface, or one cell is added to the material surface and the other to the culture medium. Another method involves separately seeding the cells on the front and back surfaces of the material. However, these techniques are insufficient for realistically modeling multicellular and multilayered biological tissues. In current co-culture systems, natural cell-cell interactions cannot be fully achieved because cells grow either on the same surface or in separate compartments. Furthermore, multilayered tissues such as skin and bone contain more than one cell type. However, current methods are inadequate for modeling these tissues in the laboratory. In unidirectional or fixed culture systems, cells are also deprived of the mechanical and biochemical stimuli found in their natural environment.
[0008] Conventional co-culture methods and culture insert systems are commonly used to culture different cell types together in the prior technique. The three most commonly used methods and systems shown in Figure 1 are described below.
[0009] A. Single-Sided Co-Culture Method
[0010] In this method, two different cell types are cultured by being added to the same surface. However, it has the following disadvantages:
[0011] • Since cell-cell interactions are confined to a homogeneous structure, it is difficult for cells to exhibit their natural behaviors in different layers.
[0012] • Creating layered structures is not possible, leading to deficiencies in the engineering of tissues such as skin and bone.
[0013] B. Compartmentalized Co-Culture Systems
[0014] In culture insert systems offered by manufacturers such as Thermo Fisher and CellQart, one cell type is added to the insert surface, while the second cell type is grown in the culture medium beneath the insert. However, these systems have the following disadvantages:
[0015] • Limited cell interaction: Since cells are grown in different layers, dynamic cellular interactions similar to those in the in vivo microenvironment cannot be adequately achieved.
[0016] • Lack of mechanical stimulation: Since these systems are static, cells are deprived of mechanical stimuli found in their natural environment. C. Double-Sided Culture Systems
[0017] In this system, one cell is added to the front surface of the material and the other to the back surface. However, this system has the following disadvantages:
[0018] • Coordination of both surfaces is difficult: Simultaneous development and interaction of cells on two different surfaces are limited.
[0019] • Two different cell types cannot be seeded on the same surface. In a co-culture system, either the two surfaces must be identical, with different cells seeded on the front and back, or the same cell type must be used on two different surfaces. This is one of the limitations in inventing cultivation systems that most closely resemble the body.
[0020] • Deficiencies arise in the engineering of multilayered tissues. For example, these systems are insufficient for modeling hard tissues like bone together with surrounding soft tissues.
[0021] In summary, the general shortcomings of current systems are a lack of dynamism and limited biomimetic design. Since most systems currently in use are static, sufficient nutrient exchange and mechanical stimulation between cells cannot be achieved. In the field of multicellular tissue engineering, current techniques involving the seeding of cells to a single surface or layer are insufficient for more complex, multilayered structures. In particular, in organ and tissue models (e.g. skin, bone, vascular structures), multiple cell types work together to maintain tissue homeostasis and function. This makes it difficult to create realistic in vitro models, which are critical for drug testing and regenerative medicine studies. For tissues with multiple cell types and structural layers, such as bone and skin, systems capable of cultivation both surfaces with cells of different structures are needed.
[0022] The patent document CN111876329A, which is part of the prior art, relates to an immunoisolation dynamic co-culture bioreactor for creating in-vitro cultures for hematopoietic stem cells. The present invention involves a culture chamber that can rotate axially via a motor. It also includes rotating inner and outer cylindrical structures. However, the planes on which the cells are seeded are different in this system. Furthermore, the bioreactor is currently a three- dimensional environment, requiring cell seeding followed by placement in the reactor. of the Invention
[0023] The object of this invention is to provide a biaxially rotatable, double-sided co-culture system that enables the cultivation of at least two different cell types on different surfaces.
[0024] Another object of the invention is to provide a solution to the shortcomings of existing 2D cultures and classical co-culture methods, enabling the growth of cells in a more realistic and dynamic environment.
[0025] Furthermore, the invention aims to go beyond classical cell culture methods by enabling multilayered and multicellular tissue engineering studies and providing a new infrastructure for the successful simulation of layered structures of biological tissues, such as skin and bone, in a laboratory environment.
[0026] Figure 1: A representative view of three systems in the prior art. (A: Single-Sided Co-Culture Method, B: Compartmentalized Co-Culture System, C: Double-Sided Culture System).
[0027] Figure 2: A representative disassembled perspective view of an implementation of the co-culture system of the invention.
[0028] Figure 3: A representative side (a), front (b) and top (c) view of an implementation of the coculture system of the invention.
[0029] 1. Co-culture system
[0030] 1.1. Upper compression module
[0031] 1.2. Lower compression module 1.3. Rotation mechanism
[0032] 1.4. Compartment
[0033] 1.5. Pm
[0034] 1.6. Hole
[0035] D. Tissue scaffold
[0036] Y. Height
[0037] Detailed Description of the Invention
[0038] The co-culture system (1) of the invention is a biaxially rotatable, double-sided system that enables three-dimensional cultivation, designed for use in a wide range of applications including biomedicine, biotechnology, pharmacology, regenerative medicine, organoid production, and lab- on-a-chip applications.
[0039] The co-culture system (1) of the invention includes at least one upper compression module (1.1) designed to fit perfectly onto the top of a tissue scaffold (D), gripping the upper surface of the tissue scaffold (D) at a predetermined height (Y), thereby enabling the seeding of cells at a suitable depth and stabilization in the culture medium. This upper compression module (1.1) provides a stable environment for the uniform growth of cells.
[0040] Furthermore, the co-culture system (1) of the invention includes at least one lower compression module (1.2) that fits perfectly onto the lower part of the tissue scaffold (D), allows the scaffold (D) to be fixed in the correct position, supports the growth of cells on the lower surface of the tissue scaffold (D), and has the same height as the upper compression module (1.1), thus enabling the cells to be seeded at a suitable depth on this surface. The size of the lower compression module (1.2) is designed in a way that the upper compression module (1.1) fits perfectly inside the lower compression module (1.2).
[0041] In an implementation of the invention, the system (1) includes an upper compression module (1.1) and / or a lower compression module (1.2) containing at least one compartment (1.4) that divides the surfaces inside the upper and / or lower compression modules (1.1, 1.2) into at least two. This compartment (1.4) allows for the simultaneous cultivation of more than one cell type (e.g. stem cells, immune cells, muscle cells) on the same surface. Thus, since different cells can be seeded on both the upper and lower surfaces of the tissue scaffold (D), as well as in the parts of these surfaces separated by compartments (1.4), it becomes suitable for indirect co-culture by seeding at least 2 to 4 different types of cells.
[0042] The co-culture system (1) of the invention includes at least one rotation mechanism (1.3) that allows the system (1) to rotate in the x-y and / or y-z planes at a predetermined degree, preferably 360 degrees, perpendicular and / or parallel to the ground, thus rotating the tissue scaffold (D), enabling the cells to be fed from different directions on both surfaces of the tissue scaffold (D) and, in one implementation, on the parts of these two surfaces separated by compartments (1.4), optimizing oxygen and nutrient exchange while allowing the cells to be naturally exposed to mechanical stimuli, enabling the seeding of different cells on these surfaces and increasing the flexibility of the system (1).
[0043] The system (1) includes at least two interlocking units positioned opposite each other for each module (1.1, 1.2), which allow the upper compression module (1.1) and the lower compression module (1.2) to fit perfectly into each other. In addition, the system (1) includes at least two pins (1.5) that allow the upper compression module (1.1) and the lower compression module (1.2) to be attached to the rotation mechanism (1.3). Furthermore, the system (1) includes at least two holes (1.6) in the lower compression module (1.2) and the rotation mechanism (1.3), positioned opposite each other, for the insertion of the pins (1.5).
[0044] The system (1) can adapt to different tissue scaffold (D) structures thanks to the ability to design the upper and lower compression modules (1.1, 1.2) in all geometries (e.g. square, rectangular, circular, spherical, or hexagonal). The compartments (1.4) dividing the surfaces inside the upper (1.1) and lower (1.2) compression modules into at least two allow for the simultaneous cultivation of multiple cell types (e.g. stem cells, immune cells, muscle cells, etc.) on the same surface. This design enables different cell types to interact via signaling and allows for the realistic modeling of multilayered tissues (e.g. skin, bone, blood vessels, etc.) in a laboratory setting. Thus, three-dimensional cultivation and cell-cell interactions are achieved in a more natural environment.
[0045] In an implementation of the invention, the co-culture system (1) of the invention is suitable for placement in well-plates. The present system (1) can be manufactured in different sizes and used in all well-plates comprising 6 wells, 12 wells, 24 wells, etc. Furthermore, when placed in these well-plates, the rotation mechanism (1.3) can easily rotate the tissue scaffold (D).
[0046] This system (1) is designed for use not only in tissue engineering but also in pharmacology, regenerative medicine, organoid production, and lab-on-a-chip applications. Supporting the dynamic interactions of cells and their responses to mechanical stimuli, this structure allows for the development of more accurate and reliable biological models in vitro. The system (1) contributes to the reduction of animal experiments while offering advantages such as modeling multilayered tissues in vitro and providing more precise results in drug testing.
[0047] The developed co-culture system (1) enables cells to grow in a dynamic environment with mechanical and biochemical stimuli. This allows for the production of tissues such as skin, bone, and blood vessels in a laboratory setting. System (1) allows for three-dimensional cultivation closest to the body in a well-plate system (such as the simultaneous seeding of preosteo, stem cells, immune cells, and muscle cells). Three-dimensional cultivation contributes to obtaining more accurate and reliable results for regenerative medicine, cancer research, and drug development processes.
[0048] The system (1) of the invention can be manufactured from any type of plastic and / or resin. For example, glass, PP, PE, ABS, PET, PLA, PVA, PC, PVC, PS and / or their derivatives; epoxy resins, polyurethane, phenolic resins, melamine resins, silicone resins, acrylic resins and / or their composites; natural fiber reinforced composites made from sustainably recyclable materials, carbon fiber, glass fiber, aramid fiber, basalt fiber, cellulose fiber and / or hybrid combinations thereof; bioplastics (e.g. PLA, PHA, PBAT, PBS, PHB, PHBV); mixtures of natural and synthetic polymers and / or nanocomposites, hybrid materials and bioactive composites using these polymers; lignin, starch-based polymers, chitosan, gelatin, alginate, cellulose derivatives (CMC, MCC, nanocrystalline cellulose), polyesters (e.g. PCL, PET, PHBV), polyamides, polyimides, poly(ether ketone) (PEEK), thermoplastic elastomers, poly(lactic acid)- hydroxyapatite composites, poly(methyl methacrylate) (PMMA); natural polymers (e.g. collagen, fibroin, elastin) and / or biocompatible mixtures thereof with synthetic polymers; organic- inorganic hybrid materials and / or biodegradable or non-biodegradable synthetic polymer derivatives or any mixture thereof can be used for its production.
[0049] Industrial Applicability of the Invention
[0050] The present invention relates to a biaxially rotatable, double-sided co-culture system (1) that enables three-dimensional cultivation, particularly for use in the biomedical and biotechnology sectors, and is industrially applicable.
[0051] The developed double-sided co-culture system (1) can be used as a consumable in the in vitro tissue engineering studies. In industrial applications, these systems (1) can be produced as singleuse materials in laboratories and widely used. It is expected to be used particularly in fields such as drug development, cancer research, tissue engineering, and regenerative medicine. The present invention can be supplied as packaged consumables for biomedical research laboratories and centers conducting cell culture studies.
[0052] The present invention can be integrated into the existing product range of companies producing cell culture consumables, creating a new market opportunity. Furthermore, the production of high-quality, multilayered tissue scaffolds will become a critical tool for laboratories.
[0053] The present invention is not limited to the above descriptions, and a person skilled in the art can easily develop different implementations of the invention. These should be considered within the scope of the protection sought by the claims of the invention.
Claims
CLAIMS1. A co-culture system (1) characterized in that it comprises at least one upper compression module (1.1) designed to fit perfectly onto the upper surface of a tissue scaffold (D), having a predetermined height (Y), firmly gripping the upper surface of the tissue scaffold (D), enabling the cells to be seeded at a suitable depth and stabilized in the culture medium due to its height (Y), and which can be designed in all geometries; at least one lower compression module (1.2) designed to fit perfectly onto the lower surface of the tissue scaffold (D), enabling the scaffold (D) to be fixed in the correct position, supporting the growth of cells on the lower surface of the tissue scaffold (D), and having the same height (Y) as the upper compression module (1.1), thus allowing the cells to be seeded at a suitable depth on this surface and having a size designed in a way that the upper compression module (1.1) fits perfectly inside it, and which can be designed in all geometries; at least one rotation mechanism (1.3) that allows the system (1) to rotate in the x-y and / or y-z planes at a predetermined degree perpendicular and / or parallel to the ground, thus rotating the tissue scaffold (D), enabling the cells to be fed from different directions on both surfaces of the tissue scaffold (D), optimizing oxygen and nutrient exchange while allowing the cells to be naturally exposed to mechanical stimuli, allowing different cells to be seeded onto said surfaces and increasing the flexibility of the system (1); at least two connecting units positioned relative each other for each module (1.1, 1.2) that allow the upper compression module (1.1) and the lower compression module (1.2) to fit completely into each other; at least two pins (1.5) that allow the upper compression module (1.1) and the lower compression module (1.2) to be attached to the rotation mechanism (1.3); and at least two holes (1.6) in the lower compression module (1.2) and the rotation mechanism (1.3) that are positioned opposite each other for the insertion of the pins (1.5).
2. A co-culture system (1) according to claim 1, characterized in that it comprises an upper compression module (1.1) and / or a lower compression module (1.2) containing at least one compartment (1.4) that divides the surfaces inside the upper and / or lowercompression modules (1.1, 1.2) into at least two sections, allowing for the simultaneous cultivation of more than one cell type on the same surface.
3. A co-culture system (1) according to claim 1 or 2, characterized in that it is suitable for placement in well-plates and it is produced in different sizes to fit and be used in all wellplates such as 6- well, 12- wells, 24- well, etc., and that it can easily rotate the tissue scaffold (D) of the rotation mechanism (1.3) when placed in said well-plates.
4. A co-culture system (1) according to any one of the preceding claims, characterized in that it comprises a rotation mechanism (1.3) that allows the system (1) to rotate 360 degrees.
5. A co-culture system (1) according to any one of the preceding claims, characterized in that it is manufactured from any type of plastic and / or resin.
6. A co-culture system (1) according to any one of the preceding claims, characterized in that it is manufactured from glass, PP, PE, ABS, PET, PLA, PVA, PC, PVC, PS and / or their derivatives; epoxy resins, polyurethane, phenolic resins, melamine resins, silicone resins, acrylic resins, or any composites thereof; natural fiber reinforced composites made from sustainably recyclable materials, carbon fiber, glass fiber, aramid fiber, basalt fiber, cellulose fiber, and / or hybrid combinations thereof; bioplastics; natural and synthetic polymer mixtures and / or nanocomposites, hybrid materials, and / or bioactive composites made using these polymers; lignin, starch-based polymers, chitosan, gelatin, alginate, cellulose derivatives, polyesters, polyamides, polyimides, poly(ether ketone), thermoplastic elastomers, poly(lactic acid)-hydroxyapatite composites, poly(methyl methacrylate); natural polymers and / or their biocompatible mixtures with synthetic polymers; organic-inorganic hybrid materials and / or biodegradable or non-biodegradable synthetic polymer derivatives, or any mixture of these materials.