KR20240020086A - Osteoarthritis mimetics comprising 3 dimension co-culture system - Google Patents

Abstract

The present invention relates to an osteoarthritis mimetic using a three-dimensional co-culture system, and the present invention's original co-culture system, which simulates the inflammatory environment of osteoarthritis patients, is used to conduct drug screening in the in vitro culture stage when developing a treatment for osteoarthritis. Among the methods, it can complement the limitations and problems of two-dimensional monolayer culture or single cell spheroid culture and has the effect of replacing the use of laboratory animals.

Description

Osteoarthritis mimetics using a 3-dimensional co-culture system {Osteoarthritis mimetics comprising 3 dimension co-culture system}

The present invention relates to osteoarthritis mimics using a three-dimensional co-culture system.

Due to traffic accidents and the spread of various sports activities, the frequency of cartilage damage increases, which progresses to osteoarthritis, and degenerative joint disease is increasing even in young patients. The proportion of the elderly population aged 65 or older among the total population in Korea reached 14.3% in 2018 and is expected to continue to increase (Ministry of Health and Welfare). Population aging is naturally leading to an increase in degenerative joint diseases.

Regarding osteoarthritis treatment, the osteoarthritis market formed a global market worth $40.6 billion in 2018, and the osteoarthritis drug market in seven major countries (US, France, Germany, Italy, Spain, UK, and Japan) as of 2017. It is expected to grow from $3.9 billion to about $10.5 billion in 2024. In addition, in the domestic market related to osteoarthritis treatment, the domestic osteoarthritis market in 2016 is estimated to be over KRW 1.5 trillion. From 2012 to 2016, the number of osteoarthritis patients increased at an average annual growth rate of 3%, and pharmaceutical costs also grew at an average annual growth rate of 4.8%. increased to

Cell therapy for cartilage regeneration began with the expectation that the administered cells would regenerate damaged articular cartilage, thereby improving the structure of the joint and largely replacing drug and surgical treatments. Initially, autologous cartilage cells were used by culturing and proliferating, but it was discovered that the proliferated cells lost the properties of cartilage cells, so research was conducted on the use of mesenchymal stem cells (MSC), which are adult stem cells obtained from bone marrow, fat, and umbilical cord blood. The focus shifted to . Initially, it was thought that for successful cell therapy, it was important to induce differentiation of MSCs into cells with properties such as chondrocytes. Therefore, research was conducted to create cartilage-like characteristics of adult stem cells by finding appropriate cell growth combinations or gene transfer of essential factors for cartilage differentiation. Some groups attempted to promote differentiation into chondrocytes by applying various mechanical pressures to MSCs.

The inflammatory environment of osteoarthritis is a three-dimensional environment in which chondrocytes of the articular cartilage, synovium cells of the synovial membrane, and macrophages react with each other. Cells administered for cartilage regeneration in osteoarthritis interact in this three-dimensional environment under the influence of various inflammatory factors secreted from surrounding cells and mechanical factors of the joint. The method of administering inflammatory cytokines such as interluekin-1 alpha to create an inflammatory environment for cells in two-dimensional monolayer culture is bound to be significantly different from the inflammatory environment in vivo. In addition, the effects of various factors that affect cell survival/engraftment on administered cells can be better simulated in the in vivo environment by evaluating them in a 3D co-culture system composed of spheroids of intraarticular cells. there is.

The inflammatory environment seen in osteoarthritis, which is the biggest indication for cartilage regeneration treatment, is an unfavorable environment for tissue regeneration, unlike the in vitro environment. In joint diseases, if stem cells produce only a certain secretory effect and disappear soon after, the rationale for using stem cell therapy, which is much more expensive than other injectable treatments such as hyaluronic acid or corticosteroids, will be lost. Therefore, it is necessary for successful cell therapy to understand the intra-articular death mechanism of MSCs, which will be used as cell therapy in the inflammatory joint environment, and to analyze and explore factors affecting survival to increase the survival period of administered cells and, in some cases, to promote engraftment. .

Accordingly, the present inventors established a culture method using a three-dimensional co-culture system composed of chondrocytes, synovial cells, and adipocytes to simulate the inflammatory environment in the joint space of osteoarthritis patients, and used two-dimensional monolayer culture or spheroid culture of chondrocytes. The present invention was completed by confirming that the in vivo environment of osteoarthritis can be better simulated compared to the simulation of the inflammatory environment.

Republic of Korea Patent Publication No. 10-2021-0062128

One aspect of the present invention aims to provide a three-dimensional co-culture system for three-dimensional co-culture of chondrocytes, synovial cells, and adipocytes derived from osteoarthritis patients.

Another aspect of the present invention is a first compartment in which cartilage cells derived from osteoarthritis patients are cultured, a second compartment in which adipocytes derived from osteoarthritis patients are cultured, a third compartment in which synovium cells derived from osteoarthritis patients are cultured, and a second culture medium while distinguishing the above compartments. The purpose is to provide a shared co-culture structure for the cytokines secreted through.

One aspect of the present invention provides a three-dimensional co-culture system for three-dimensional co-culture of chondrocytes, synovial cells, and adipocytes derived from osteoarthritis patients.

The co-culture system can better reflect the microenvironment of the osteoarthritis patient's body, especially the bone or cartilage exposed to the inflammatory environment, by co-culturing osteoarthritis patient-derived chondrocytes, synovium cells, and adipocytes in three dimensions.

The 'Osteoarthritis (OA)' is characterized by hypertrophic differentiation of chondrocytes followed by chondrocyte death and damage to the extracellular matrix (ECM) of cartilage in response to excessive mechanical stress. It is the most common degenerative joint disease.

The terms “derive” and “derived” mean ingredients obtained from the mentioned source by any useful method.

Conventionally, the method of administering inflammatory cytokines such as Interlukin-1b was mainly used to create the inflammatory environment of osteoarthritis. However, this method is bound to be significantly different from the in vivo inflammatory environment, and the present invention can more accurately simulate the in vivo microenvironment by replicating the expression of various inflammatory factors by using cells derived from osteoarthritis patients.

The 'chondrocytes' are cells located in cartilage and are specially differentiated cells distributed between the cartilage matrix. Additionally, chondrocytes play a role in creating and maintaining joint cartilage. Cell division occurs in chondroblasts, but once growth stops, chondrocytes no longer divide under normal circumstances. Since the chondrocytes of the present invention are derived from osteoarthritis patients, they can better reflect the body microenvironment in which osteoarthritis disease occurs.

The term 'synovial cells' refers to a group of cells present in the synovial membrane, and is used interchangeably with synovium-derived cells, unless otherwise specified. Since the synovium cells of the present invention are derived from osteoarthritis patients, they can better reflect the body microenvironment in which osteoarthritis disease occurs.

The ‘adipose cell’ refers to a cell derived from adipose tissue.

The adipocytes of the present invention may be derived from patients with osteoarthritis. The adipocytes can simulate the osteoarthritis environment in the body by interacting with cartilage cells derived from osteoarthritis patients, inflammatory factors expressed in synovial cells, and/or inflammatory factors derived from external sources.

The 'co-culture' refers to cultivating two or more different types of target cells in the same culture space with the same culture environment. Co-culture includes direct co-culture, which allows contact between multiple different cells in the same culture space, and indirect co-culture, where individual cells interact through secreted proteins or metabolites while being blocked from physical contact. There is (indirect co-culture). In the present invention, it means indirect co-culture. Also, depending on the co-culture environment, it can be divided into two-dimensional co-culture or three-dimensional co-culture. Two-dimensional co-culture means that heterogeneous cells are cultured in a single layer, and three-dimensional co-culture means that they are multi-layered.

The three-dimensional co-culture system of the present invention can better simulate the microenvironment of the body of an osteoarthritis patient by three-dimensionally co-culturing chondrocytes, synovial cells, and adipocytes derived from osteoarthritis patients, and can be cultured in a structure described later.

One aspect of the present invention includes a first compartment in which cartilage cells derived from osteoarthritis patients are cultured, a second compartment in which adipocytes derived from osteoarthritis patients are cultured; A third compartment in which synovial cells derived from osteoarthritis patients are cultured and a co-culture structure in which the compartments are distinct are provided.

The co-culture structure implements the three-dimensional co-culture system described above.

In one embodiment of the present invention, the chondrocytes, adipocytes, and synovium cells may be derived from patients with osteoarthritis.

Details of the chondrocytes, adipocytes, and synovium cells are the same as described above.

In one embodiment of the present invention, the first compartment, second compartment, and third compartment may be connected so that the secreted substances of the cells can move. This structure allows interaction between cells in each compartment to be expressed, and the first, second, and third compartments are respectively chondrocytes, adipocytes, and synovial cells so that each cell can be stably positioned. It may further include a well where is located.

The above compartments are distinguished, and while restricting the movement of cells, cell secreted substances may be able to move. This excludes the effects of direct contact between cells and allows cellular secreted substances, i.e. inflammatory factors, to move between compartments, preventing osteoarthritis in the body. The microenvironment can be more accurately simulated. The structures of the above compartments can be appropriately changed and used depending on the purpose.

Another aspect of the present invention provides a three-dimensional co-culture method, which includes preparing isolated chondrocytes, adipocytes, and synovial cells derived from patients with osteoarthritis and proliferating them by a monolayer culture method; And cultivating the cartilage cells, adipocytes, and synovial cells separately in each space separated by a compartment (wall) within the co-culture structure, and preventing different types of cells from mixing.

Another aspect of the present invention is an in vitro or in vitro condition, comprising the steps of preparing isolated chondrocytes, adipocytes, and synovial cells derived from patients with osteoarthritis and proliferating them by a monolayer culture method; Culturing the cartilage cells, adipocytes, and synovial cells separately in each space separated by a compartment (wall) within the co-culture structure, and preventing different types of cells from mixing; After the cells are cultured in the form of spheroids in each compartment, they are treated with an inflammatory response-inducing substance, and the volume of the cell culture medium in each compartment is ensured to be the same, but not mixed with each other; A method for evaluating the viability of cells associated with osteoarthritis, a method for evaluating proliferation of cells associated with osteoarthritis, or inflammation of cells associated with osteoarthritis, comprising collecting the culture medium from each compartment and evaluating cell viability, cell proliferation, or inflammatory response. Provides a response evaluation method.

In the present invention, we construct a three-dimensional co-culture system that can culture intraarticular cells (chondrocytes, synovial cells, adipocytes, etc.) of osteoarthritis patients as spheroids, and when using this system, an inflammatory environment is induced by drug treatment. It was confirmed that the in vivo environment could be better simulated than the two-dimensional monolayer culture or single cell spheroid culture environment. Through a three-dimensional co-culture system that mimics the inflammatory environment of osteoarthritis patients, the limitations and problems of two-dimensional monolayer culture or single cell spheroid culture are complemented among the drug screening methods currently performed in the in vitro culture stage when developing osteoarthritis treatments. It can be done and has the effect of replacing the use of laboratory animals.

Through the original co-culture system of the present invention, which simulates the inflammatory environment of osteoarthritis patients, the limitations and problems of two-dimensional monolayer culture or single-cell spheroid culture among the drug screening methods currently performed in the in vitro culture stage when developing osteoarthritis treatments are addressed. It can supplement and have the effect of replacing the use of laboratory animals.

Figure 1 shows a spheroid three-dimensional co-culture system of intra-articular cells isolated from an osteoarthritis patient of the present invention, and Figure 1A shows the structure of the purchased three-dimensional co-culture construct, a schematic diagram and an actual photo of the three-dimensional co-culture system. 1B shows a photograph of spheroids of each cell formed in a 3D co-culture system.
Figure 2 shows the results of inflammatory environment simulation evaluation using a 3D co-culture system of spheroids derived from osteoarthritis patients. Specifically, Figure 2A shows the results of confirming cell viability (left) and nitrite production (right) after inducing an inflammatory environment (IL-1β treatment) on spheroids formed from cells derived from each osteoarthritis patient, and Figure 2B shows the results of confirming cartilage derived from osteoarthritis patients. This is the result of analysis of cell survival rate (top) and inflammation-inducing factors (bottom) after inducing an inflammatory environment by two-layer monolayer culture of cells, spheroid culture of chondrocytes, and co-culture of cells derived from osteoarthritis patients.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1: Production of a 3D co-culture system

Example 1-1. Preparation of co-culture constructs

We purchased and prepared a three-dimensional co-culture structure in which polydimethylsiloxane (PDMS), a silicon-based material, was implemented as a micro hemisphere pattern and wall (Figure 1A).

Example 1-2. Fabrication of co-culture system

Chondrocytes, adipocytes, and synovial cells derived from osteoarthritis patients were each proliferated using a monolayer culture method before being cultured as spheroids, and then grown in each space of the co-culture structure in Figure 1 with ^4x10 cells contained in 400 ㎕ culture medium. Cultivate the cells. At this time, be careful not to mix cells from the three different spaces. At the same time, for the monolayer culture cell control group, chondrocytes are cultured in a total of 3 wells (to match the number of cells in the co-culture structure) so that 4x10 ⁴ cells are cultured in each well of a 6-well plate culture vessel.

Figure 1B is a photograph confirming through a microscope that cells were divided into structures and monolayer culture vessels in the same manner as above and were cultured in a spheroid form immediately and 24 hours later.

Example 2: Evaluation of inflammatory environment simulation

The culture method for the construct and monolayer culture control group was the same as the cell culture method in Figure 1 above.

Figure 2A shows the results confirming that cells originating from different tissues (cartilage, fat, synovial membrane) have different measures of inflammatory response. Cells are placed in the structure using the above method, and after one day, when the cells cultured in the structure form a spheroid shape, the cells are treated with IL-1β (10 ng/ml) to induce an inflammatory response. At this time, be careful not to mix the cultures of each construct. After 48 hours of IL-1β treatment, the culture media of chondrocyte spheroids, adipocyte spheroids, and synovial cell spheroids were collected from each construct, and the secretion amount of nitric oxide (NO) was measured, and each spheroid was a cell MTT assay was performed to evaluate proliferation and viability.

As a result, there was no significant difference in cell proliferation and cell viability for each cell (left graph in Figure 2A), but in the case of chondrocyte spheroids, it was confirmed that the secretion amount of NO was significantly higher than that of synovium cells or adipocyte spheroids. It was confirmed through the NO assay that the response in the inflammatory environment varies greatly depending on the tissue of origin.

Figure 2B shows that since the scale of the inflammatory response of cells is different depending on the cell's origin tissue and culture method, in order to properly simulate the inflammatory environment of osteoarthritis, a sample of chondrocytes, synovial cells, and adipocytes isolated from the main constituent tissues of articular cartilage must be used. This result confirmed that Lloyd co-culture was necessary. To culture the co-culture construct, seed ^4x10 cells in each space using the cell culture method shown in Figure 1A, wait for 10 minutes for the cells to settle inside the well of the construct, fill the space above the construct with culture medium, and incubate for 24 hours. Incubate for a while. However, in the case of spheroid culture using only chondrocytes, cartilage cells are added to all of the structure spaces a, b, and c on the right side of Figure 1A. For the monolayer culture cell control group, only chondrocytes were cultured as a monolayer in the same manner as the culture method in Figure 1B. After 24 hours of culture, IL-1β (10 ng/ml) is treated to induce an inflammatory response. At this time, it is important to keep the volume of the culture medium equal to 3 ml. In other words, since the total number of cells in the structure is (4x10 ⁴ x 3) and the culture medium volume is 3 ml, in the case of monolayer cell culture, 4x10 ⁴ cells per well are cultured in 1 ml of culture medium, and the 3 wells cultured in this way are cultured. Collect as one sample. 48 hours after IL-1β treatment, culture media were collected from the constructs in which chondrocyte spheroids, adipocyte spheroids, and synovial cell spheroids were co-cultured, the constructs in which only chondrocyte spheroids were cultured, and the monolayer cell culture plate, respectively, to evaluate the inflammatory response. The secretion amount of inflammatory cytokines (IL-6, PGE 2, and MMP 13) secreted during induction was measured, and MTT analysis was performed using each spheroid and cell.

As a result, there was a significant difference in cell proliferation and cell viability depending on the cell culture method (monolayer or spheroid culture) and cell type (spheroids of chondrocytes only or co-culture of spheroids of chondrocytes, adipocytes, and synovium cells). However, it was confirmed that there was a difference in the amount of inflammatory cytokines secreted (upper graph of Figure 2B).

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (5)

Chondrocytes and synovial cells derived from osteoarthritis patients; and
A 3D co-culture system for 3D co-culture of adipocytes.
A first compartment where osteoarthritis patient-derived chondrocytes are cultured;
A second compartment where adipocytes derived from osteoarthritis patients are cultured;
A third compartment where synovial cells derived from osteoarthritis patients are cultured, and
A co-culture structure in which the compartments are distinct.
According to paragraph 2,
A structure in which the first compartment, second compartment, and third compartment are connected so that the secreted substances of the cells can move.
The structure of claim 2, wherein the first, second, and third compartments further include wells in which chondrocytes, adipocytes, and synovial cells are located, respectively.
According to paragraph 2,
A structure in which the permeability network restricts the movement of cells while allowing the movement of cell secreted substances.