KR20230059456A - Muscle cell culture using plant-derived 3D scaffold - Google Patents

Abstract

The present invention relates to a method for manufacturing a plant-derived scaffold for three-dimensional cell culture and to muscle cell culture using the same. A process of manufacturing the plant-derived 3D cell culture scaffold of the present invention is simple and can be manufactured in large quantities, which is advantageous in terms of cost and time, and the 3D cell culture scaffold manufactured through the method is structurally suitable for cell culture due to the extracellular structure of the plant, that is, a microtubule structure of the cell wall.

Description

Muscle cell culture using plant-derived 3D scaffold {Muscle cell culture using plant-derived 3D scaffold}

The present invention relates to a method for manufacturing a support for plant-derived three-dimensional cell culture and muscle cell culture using the same.

The field of tissue engineering is an applied science for clinical treatment by using the main elements of engineering and life sciences, that is, biological cells, engineering, materials, and appropriate biochemical/physiological-chemical factors in a complex way. In particular, in tissue engineering, studies on the regeneration of damaged tissues and the restoration of organs are focused on using cells, scaffolds on which cells can attach and grow, and various factors that can control the growth and differentiation of cells. .

Cell culture, which is most fundamentally performed in life science, is the basic basis for research on signal transmission between cells and cell differentiation, and is an essential part not only for understanding living things but also for experiments to conquer diseases. These days, cell culture is being used very widely to grow edible cells, expanding from the scope of research for the functions of living things or human diseases. Cell culture is the most basic research method in the bio field, and it is It is widely used not only for functional research but also for studying human diseases. The most commonly used method to date is culturing cells on a two-dimensional surface made of a substrate made of polystyrene or glass. However, a major problem in culturing cells on a two-dimensional surface is that it cannot accurately reflect the physiological environment of a living body in which cells grow on a three-dimensional surface, and interactions between cells can be considered through conventional two-dimensional cell culture There is a problem in that many complex life phenomena, such as receptor expression, transcriptional regulation of genes, cell migration and apoptosis, are different from phenomena occurring in an actual biological tissue environment.

Therefore, research and development of a 3-dimensional cell culture method considering the spatial organization of cells in order to solve problems in 2-dimensional cell culture has very important significance. A method mainly used to create a 3-dimensional living tissue-like cell culture environment is to culture cells using a porous biocompatible scaffold. Natural polymers are widely used as a material for producing a hydrogel, which is a major form of biocompatible scaffold, and these natural polymers are materials separated from natural materials and are widely applied as clinical materials.

Supports for 3D cell culture must meet basic requirements such as biocompatibility, bioactivity, and biomechanics. That is, adhesion between the support for cell culture and the cells should occur well, the support for cell culture should sufficiently support the growth of cells, there should be no restriction in the supply of oxygen and nutrients by the support for cell culture, and the support for cell culture should be It is a basic requirement to have an open structure for discharging waste. The scaffold for cell culture prepared in this way is commercially available in the form of a hydrogel using an artificially synthesized polymer as a main component. However, the relatively expensive support for cell culture is used very limitedly because it inevitably becomes an economic burden factor in a research institute or a corresponding field where it is essential to use it. In addition, many attempts have been made to develop various hydrogel scaffolds for 3D cell culture using natural polymers alone or composite materials, but due to the characteristics of the natural materials, the efficiency of 3D cell culture is not high. there has been

Currently, the field of use of support for cell culture goes beyond a specialized laboratory for researching life sciences to foodtech, that is, the field of making cultured-meat grown through cell culture without sacrificing animals. situation. In particular, this food tech technology is a near-future food technology that will solve ethical problems caused by animal sacrifice and environmental pollution generated during the livestock breeding process. This is an area that requires continuous R&D for reduction.

An object of the present invention is to provide a method for manufacturing a support for 3-dimensional cell culture.

Another object of the present invention is to provide a support for plant-derived three-dimensional cell culture.

Another object of the present invention is to provide a composition for tissue repair or regeneration.

In addition, an object of the present invention is to provide a method for producing cultured meat.

In order to solve the above problems, the present invention provides a method for manufacturing a support for 3-dimensional cell culture.

In addition, the present invention provides a support for plant-derived three-dimensional cell culture prepared by the above method.

In addition, the present invention provides a composition for tissue repair or regeneration comprising the scaffold and cells seeded thereon.

In addition, the present invention provides a method for producing cultured meat of livestock.

The process of manufacturing the plant-derived 3D cell culture scaffold of the present invention is simple and can be mass-produced, which is advantageous in terms of cost and time. Due to the microtubule structure of the cell wall, it is structurally suitable for cell culture.

1 is a diagram showing a process of manufacturing a cell culture support.
Figure 2 is a diagram showing the process of freeze-drying the cell culture support produced.
Fig. 3 is a microscopic view of the fabricated cell culture support before and after decellularization.
Figure 4 is a diagram confirming the porous microstructure of the cell culture support produced by scanning electron microscope.
5 is a diagram showing a process of attaching and culturing chicken muscle stem cells to the fabricated cell culture support.
6 is a diagram confirming the differentiation and proliferation of muscle cells and their muscle cells attached to the surface and inside of the fabricated cell culture support.
Figure 7 is a diagram confirming the differentiation into muscle fibers after attaching and culturing muscle cells to the fabricated cell culture support by histofluorescence staining analysis.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

In one aspect, the present invention comprises the steps of a) removing the outer membrane of a plant; b) decellularization; and c) washing and sterilizing.

In one embodiment, decellularization may be performed by treatment with 0.5 to 3% SDS for 48 hours.

In one embodiment, further comprising the step of lyophilizing after step c)

In one embodiment, a step of cutting into a size suitable for use may be further included prior to the step of decellularizing.

In one embodiment, the plant can be a fruit, more preferably a grape.

Conventional animal tissue decellularization for animal tissue engineering requires a complicated process, yield is low, and cost is high, but improvement is required. It can be produced in large quantities, so it has superior effects in terms of cost and time, and is structurally suitable for cell culture due to the extracellular structure of plants, that is, the microtubule structure of the cell wall. In particular, the tissue of grapes has a rich porous structure and has an efficient form for tissue composition with a maximum surface area to volume ratio.

The SDS used in the present invention is an anionic surfactant that is inexpensive and is suitable for efficiently removing cell membranes and removing intracellular substances other than cell walls of plant tissues. However, since SDS adversely affects muscle cells when remaining, residual chemicals must be completely removed, and the sterilization step is essential because various adverse effects are caused to cell growth when external bacteria are introduced into muscle cell culture.

In one aspect, the present invention relates to a support for plant-derived three-dimensional cell culture prepared by the method of the present invention.

In one embodiment, the scaffold of the present invention has a plant cell wall structure and may have a porous microstructure.

In one embodiment, the plant can be a fruit, more preferably a grape.

In one embodiment, the scaffold of the present invention may be used for tissue engineering through 3-dimensional cell culture or for therapeutic use.

In one aspect, the present invention may relate to a support for plant-derived 3-dimensional cell culture of the present invention and a composition for tissue repair or regeneration comprising cells seeded on the support for plant-derived 3-dimensional cell culture.

In one embodiment, the cells are chondrocytes; fibrochondrocyte; osteocytes; osteoblast; osteoclast; synovial cells; bone marrow cells; nerve cells; fat cells; mesenchymal cells; epithelial cells, hepatocytes, muscle cells; stromal cells; vascular cells; Stem Cells; embryonic stem cells; mesenchymal stem cells; progenitor cells derived from adipose tissue; peripheral blood progenitor cells; stem cells isolated from adult tissue; induced pluripotent stem cells (iPS cells); cancer cells derived from these cells; And it may be any one selected from the group consisting of combinations thereof, more preferably muscle stem cells, most preferably chicken-derived muscle stem cells.

In one aspect, the present invention comprises the steps of isolating the muscle tissue of livestock; treating the muscle tissue with a proteolytic enzyme; Separating muscle stem cells and mixing them with a culture solution containing CEE (chick embryo extract); And it relates to a method for producing cultured meat of livestock comprising the step of culturing and differentiating by dispensing to a support for plant-derived three-dimensional cell culture prepared by the method of the present invention.

In one embodiment, the livestock may be cattle, pigs, horses, sheep, ducks or chickens, more preferably chickens.

In one embodiment, the proteolytic enzyme is elastase, papain, pepsin, pronase, proteinase K, subtilisin and thermolysin. It may be one or more selected from the group consisting of, more preferably a pronase.

In one embodiment, the culture medium may contain 3 to 7% of CEE.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

Example 1. Cell culture support fabrication

In order to prepare a decellularization cell culture scaffold capable of culturing muscle cells by removing cells from edible grape tissue, the outer membrane of edible grapes is removed, and cells are removed from grape tissue by treatment with 1% SDS for 48 hours, In order to remove residual chemicals and sterilize the scaffold, purified water was treated for 24 hours and 70% ethanol was treated for 24 hours to finally prepare a dietary fiber-based decellularized grape scaffold (FIG. 1). The completed scaffold can be used as such, but freeze-drying was performed at -80 ° C. at 0.133 MBAR for 8 hours to prevent deformation of the shape and enable long-term storage (FIG. 2).

Example 2. Cell culture scaffold analysis

Before and after decellularization of the decellularized grape scaffold prepared in Example 1 was compared and observed under a microscope to confirm decellularization, and a porous microstructure for nutrient and gas exchange for cell survival was confirmed with a scanning electron microscope. . As a result, decellularization was well performed in the decellularized grape scaffold (Fig. 3), and a sufficient porous structure was observed (Fig. 4). It was confirmed that the decellularized grape scaffold of the present invention had a cell-attachable surface.

Example 3. Cell culture scaffold-based muscle stem cell culture and confirmation

In order to culture muscle stem cells on the decellularized grape scaffold prepared in Example 1, the tibialis anterior muscle, gastrocnemius muscle, and leg muscles were isolated from chicken embryos, minced to increase the surface area, and the obtained muscle tissue was treated with Pronase enzyme 5,000,000 chicken muscle stem cells obtained by disassembling muscle tissue were mixed with a culture medium containing 5% CEE (chick embryo extract) to decellularized grape scaffolds (decellularized cellulose scaffolds or lyophilized scaffolds) of the present invention. After injection using a micropipette, it was maintained in a cell culture medium for 30 minutes for cell attachment. After transferring the cell-attached scaffold to a 50ml tube, the culture medium containing 10ml of CEE was replaced every 2 days and cultured. At this time, a 22um filter was placed on the lid of the 50ml tube so that the gas exchange required by the cells could be achieved. was installed to enable circulation and prevent the inflow of microorganisms (FIG. 5). In this way, when a tissue was formed by culturing for 30 days, a scaffold was obtained and confirmed with an optical dissecting microscope and the naked eye. As a result, muscle cells are attached to the surface and inside of the decellularized scaffold, proliferate and differentiate, and proliferate into muscle cells in the form of myofibers. It was confirmed that tissue analogues were formed on the surface (FIG. 6), and as a result of histofluorescence staining with F-actin, it was confirmed that the muscle cells grew in the form of fibers, that is, in the form of muscle fibers (FIG. 7).

Claims (15)

a) removing the outer membrane of the plant;
b) decellularization; and
c) a method for manufacturing a support for 3-dimensional cell culture comprising the steps of washing and sterilizing. The method of claim 1, wherein the decellularization is performed by treating with 0.5 to 3% SDS for 48 hours. The method of claim 1, further comprising the step of freeze-drying after step c). The method of claim 1, wherein the plant is a fruit. The method according to claim 1, wherein the plant is grapes. A support for plant-derived three-dimensional cell culture prepared by the method of claim 1. The support for plant-derived three-dimensional cell culture according to claim 6, which has a plant cell wall structure. The support for culturing plant-derived three-dimensional cells according to claim 6, which is porous. The scaffold for culturing three-dimensional cells derived from plants according to claim 6, wherein the plant is grape. A composition for tissue repair or regeneration comprising the support for plant-derived 3-dimensional cell culture of claim 6 and the cells seeded on the support for plant-derived 3-dimensional cell culture. The composition for tissue repair or regeneration according to claim 10, wherein the cells are muscle stem cells. a) isolating livestock muscle tissue;
b) treating the muscle tissue with a proteolytic enzyme;
c) isolating muscle stem cells and mixing them with a culture solution containing CEE (chick embryo extract); and
d) A method for producing cultured livestock meat comprising the step of culturing and differentiating by dispensing onto a plant-derived 3-dimensional cell culture support prepared by the method of claim 1. 13. The method of claim 12, wherein the livestock is cattle, pigs, horses, sheep, ducks or chickens. 13. The method of claim 12, wherein the proteolytic enzyme is elastase, papain, pepsin, pronase, proteinase K, subtilisin and thermo A method for producing cultured meat of livestock, which is at least one selected from the group consisting of lysine. 13. The method for producing cultured meat of livestock according to claim 12, wherein CEE is contained at 3 to 7%.

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