Research progress and application of three-dimensional cell culture technology and related vectors

Bone marrow mesenchymal stem cells cultured in 3D cell culture scaffolds

Since WillhelmRoux first isolated cells from chicken embryos in 1885, in vitro cell culture has been established. Monolayer cell culture technology has been in existence for more than a hundred years. For more than a century, monolayer cell culture has flourished, especially in the industrialization of pharmaceuticals or vaccine synthesis, through the rapid division of cells, thereby efficiently manufacturing products. However, in the field of basic research in life sciences, the in vitro culture of cells is not only concerned with their split growth, but more importantly, whether they can maintain their traits after passage. In many cases, the results obtained by monolayer cell culture techniques are not consistent with the in vivo conditions, as cells proliferate in an environment that changes in vitro and gradually lose their original traits. Animal experiments are carried out entirely in the body, but due to various factors in the body and the interaction between the body and the external environment, it is difficult to study a single process. In addition, the results we observe in animals are often the final presentation, not the intermediate process that researchers are most concerned about. Obviously, how to fill the gap between monolayer cell culture and animal experiments has always been a question for life scientists. Especially in the field of developmental biology, there is an urgent need to establish a cell culture technique that can not only grow and pass, but also maintain the body's traits to the greatest extent, and differentiate to produce new tissue structures in order to comprehensively study the development process. With the development of tissue engineering, three-dimensional cell culture technology has emerged.

1 What is a three-dimensional cell culture techniques

An important principle of in vitro cell culture is the need to model the in vivo cell growth environment. The most important core factor in this simulation system is the interaction between cells and the culture environment. Different from the traditional two-dimensional cell culture, three-dimensional cell culture (TDCC) refers to the co-culture of vectors with different materials of three-dimensional structure and various kinds of cells in vitro. It can migrate and grow in the three-dimensional spatial structure of the carrier to form a three-dimensional cell-carrier complex. Three-dimensional cell culture is the cultivation of cells in a certain extracellular matrix. The extracellular matrix (ECM) protein acts as a growth scaffold, enabling cells to differentiate to produce a certain three-dimensional tissue-specific structure, and the created cell growth environment. Maximize the in vivo environment. As a bridge between in vitro monolayer cell system and tissue and organ research, TDCC shows that it can not only retain the material and structural basis of the cell microenvironment, but also demonstrate the advantages of cell culture intuitiveness and conditional controllability. In recent years, three-dimensional cell culture technology has been widely used in the field of developmental biology such as tissue formation, vascular development and organ remodeling. At the same time, in the analysis of therapeutic effects and toxicological experiments of new drugs, three-dimensional culture was used to obtain two-dimensional culture. The completely different results of monolayer culture have aroused great interest among pharmacologists.

2 three-dimensional cell culture scaffold material

The three-dimensional space on which the cells depend is to provide a living space for the cells, to obtain sufficient nutrients for gas exchange, and to grow the cells in a pre-formed three-dimensional scaffold to become a three-dimensional interface for the cells to grow well. The ideal bio-scaffold material should have the following characteristics: good biocompatibility, degradability, sufficient pore structure, promotion of cell adhesion and proliferation, ability to carry growth factors, volume of the stent should remain unchanged, and support energy A layered structure that is integrated with the surrounding tissue, is not easily detached from the defect area, has a certain elasticity, and has articular cartilage. Commonly used scaffold materials are: natural biomaterials, synthetic polymer materials, inorganic materials, and composites of these materials.

2.1 natural polymer materials

Natural biomaterials mainly include: collagen, gelatin, fibrin, chitosan, agar, glycosaminoglycans (eg hyaluronic acid, chondroitin sulfate, etc.), alginate, silk fibroin, cancellous bone matrix, decellularization Matrix, etc. The advantages of natural scaffolds are that they are beneficial for cell embedding, low antigenicity, non-toxicity, no inflammatory, and can promote cell growth and adhesion. However, the disadvantage of natural materials is that they have poor mechanical properties. On the one hand, they do not meet the needs of plasticity. On the other hand, they cannot maintain the spatial configuration and absorb too fast during the hydrolysis process in the body, and their application is limited. Furthermore, natural materials vary from batch to batch and are difficult to process in large quantities.

At present, the three-dimensional scaffold products on the market are mainly Matrigel and collagen complexes. Collagen is a kind of natural protein, which is widely found in the skin, bone, cartilage, teeth, spleen ligament and blood vessels of animals. It accounts for more than 30% of the total protein of the human body. It is a very important structural protein of connective tissue and plays a supporting role. Organs, protect the function of the body. Collagen is generally a white, clear, unbranched fibril surrounded by a matrix of polysaccharides and other proteins. The collagen molecule has a variety of reactive groups on the peptide chain, such as hydroxyl, carboxyl and amino groups. It is easy to absorb and bind a variety of enzymes and cells to achieve immobilization. It has good affinity with enzymes and cells, and is highly adaptable. Features. Collagen has important biological properties - high mechanical properties, promote cell growth, hemostasis, biocompatibility and biodegradability. It has been reported that collagen can be used as a carrier for the carriage of the bone interstitial protein, bone morphogenetic protein 2 (rhBMP2). Collagen is now widely used as a carrier for the delivery of cultured skin cells and drugs for skin replacement and burn treatment. In addition, collagen is a film-forming substance with biocompatibility and can be gradually absorbed in the body. Therefore, collagen immobilized enzyme is particularly suitable for artificial application materials. However, it also has certain disadvantages, such as difficulty in scale preparation, poor mechanical strength, and ineffective treatment of infected sites. Commercially available collagen composite scaffolds are available from cowhide, chicken feet, and the like. Matrigel is an extract extracted from mouse tumor tissue, which poses a risk for three-dimensional culture-mediated clinical treatment of stem cells.

2 .2 Inorganic materials

Currently used inorganic materials are hydroxyapatite, calcium phosphate cement, tricalcium phosphate, etc. These materials are mainly used in bone tissue engineering.

Hydroxyapatite (HA) is a major component of human and animal bones, and this material has a long history of research. HA has high brittleness and compressive performance compared with bone tissue. Although it is implanted in the body, its strength will gradually increase to reach the cancellous bone strength due to the invasion of tissues and cells. However, some researchers believe that HA absorbs slowly in the body, Bone-healing bones withstand stress and new bone formation. Similar to hydroxyapatite, tricalcium phosphate (TCP) has good biocompatibility and absorbability, but lacks pores, small particles, easy decomposition (6 weeks), brittleness and no pressure resistance. , limiting its application. Using calcium phosphate, calcium carbonate and tricalcium phosphate as raw materials, a series of polymerization reactions at room temperature are carried out to synthesize calcium phosphate cement (CPC). The crystal structure of this chemical compound is similar to that of natural bone, and can be shaped and has good Bone morphology and mechanical properties provide mechanical integrity. Its porosity is about 50%, similar to hydroxyapatite, but it is brittle and its degradation time is longer, which takes about 2 years. On the other hand, the pore diameter of the calcium phosphate cement material is less than 1 mm, and the osteogenesis rate is slow. The current research focus is how to combine it with macroporous materials to increase the absorption speed and accelerate the osteogenesis.

In general, such materials have high compressive strength, abrasion resistance, and chemical stability, and can be degraded in living organisms, absorbed and replaced by new bone tissue, and are commonly used materials for bone tissue scaffolds. However, there are disadvantages such as poor strength of the porous body, difficulty in processing, low porosity of the stent formed, large brittleness, and incomplete growth of the tissue.

2 .3 synthetic polymer materials

Synthetic polymer materials can be precisely controlled by molecular design and other means, and products of substantially the same nature can be obtained by chemical production. Compared with natural materials, it is more conducive to standardized production, and the mechanical strength is also good, but the biocompatibility needs to be improved. At present, the more common method is to introduce bioactive factors on the surface of the material through surface modification. Synthetic polymer materials include polylactic acid (PLA), polyglycolic acid (PGA) and copolymers thereof (PLGA), polycaprolactone and other aliphatic polyesters. These polymers have good plasticity and can be molded and extruded. Pressure, solvent casting and other techniques are processed into various structural shapes, but there are also disadvantages: 1 poor hydrophilicity, weak cell adsorption. 2 easy to cause the occurrence of aseptic inflammation, while polymer degradation is likely to cause local pH decline. 3 mechanical strength is insufficient. 4 Others: Cytotoxic effects of residual organic solvents, as well as possible fibrosis and immune response to surrounding tissues.

At present, PLGA is the most widely used and researched in synthetic polymer materials. It has good biodegradability and bioabsorbability. Its structural formula is [-OCH(R)CO-]. The final metabolites of degradation in vivo are C02 and H20. They do not accumulate in the body and have no toxic side effects. The shape of new tissues and organs provides an environment for cells to obtain nutrients, gas exchange, and waste elimination, and provides space for cell proliferation and reproduction. It has been approved by the FDA in the United States and applied to the clinic. PGA has high degradation rate, PLA has high strength and PLGA has low degradation rate and high drug permeability. Therefore, a series of polymers with different degradation rates and mechanical properties can be synthesized on the basis of polymer design. The control of the material composition, composition ratio, molecular weight, molecular weight distribution, etc., can adjust the biodegradation rate of the material to vary from several weeks to several years.

Although the synthetic degradable polymer material itself has weak affinity for cells, it is often necessary to introduce an appropriate amount of active groups, growth factors or adhesion factors which can promote cell adhesion and proliferation. However, its degradation rate and strength can be adjusted, and it is easy to construct a high-porosity three-dimensional stent. At the same time, compared with natural biological materials, it can be stored at room temperature, which is simpler and easier to operate.

Recently, a biodegradable three-dimensional scaffold synthesized by polycaprolactone (PCL) has been developed by 3D Biotek in the United States. This material has been used in many surgical implants approved by the US FDA. Compared to other synthetic polymer three-dimensional scaffolds, this three-dimensional cell culture product has 100% pore connectivity and precisely controlled pore size and pore structure, resulting in a high degree of consistency across batches. On the other hand, this product has good pressure resistance, and cells can be well proliferated and differentiated on it. It is an easy-to-use 3D culture product suitable for market demand.

3 three-dimensional cell culture application

3 .1 Tissue Engineering

Tissue Engineering is a new subject emerging in recent years. The term "organizational engineering" was first formally proposed and determined by the National Science Foundation in 1987, namely: the application of principles and techniques of life sciences and engineering. On the basis of correctly understanding the relationship between structure and function in the normal and pathological states of mammals, research and development are used to repair, maintain and promote the functional and morphological biological substitution of various tissues or organs after injury. The core of tissue engineering is to establish a three-dimensional complex of cells and biological materials, that is, living tissue with vitality, to reconstruct the morphology, structure and function of the damaged tissue and achieve permanent replacement.

Mature chondrocytes and stem cells are widely used in three-dimensional cell culture to regenerate damaged cartilage, bone, ligaments, tendons, and knee meniscus. Some growth factors are often added to the culture system to stimulate differentiation and produce tissue. Spitzer cultured rabbit osteogenic precursor cells in fibrin with 7.5% tricalcium phosphate (alpha-TCP) for 53 days, using the alpha-free group as a control. The results showed that this system can benefit bone in vitro. form. Quarto et al reported successful use of autologous bone marrow mesenchymal stem cells and tissue engineered artificial bone constructed with hydroxyapatite to repair bone defects. Three-dimensional scaffolds suitable for tissue engineering skin construction have been prepared from type I collagen and chitosan in China, and dermal fibroblasts (Fbs) with stable biological traits are isolated from rat skin. In the combined culture, it was found that the interaction between the seed cells and the three-dimensional porous scaffold was similar to the dynamic interaction between the cell-extracellular matrix in normal tissues in vivo during tissue engineering skin construction.

Using 3D technology to simulate the natural complex structure of the heart remains a medical problem. In the latest study, Professor Doris Taylor of the University of Minnesota Cardiovascular Rehabilitation Center and colleagues took a decellularization approach using the acellular matrix of the natural heart. The platform creates an artificial heart. The researchers first removed all the cells in the rat and porcine hearts, leaving only the extracellular matrix as a three-dimensional scaffold, injecting neonatal rat cardiac "progenitor cells" into the laboratory for in vitro culture. The artificial heart of rats and pigs was successfully made. Four days later, contraction was observed; eight days later, the new heart began to pulsate. There are tens of thousands of people with severe heart disease such as heart failure worldwide. A large number of patients die every year because they don't get a suitable donation heart. This result is expected to provide a new method for human artificial heart preparation, and because of the new heart. Filled with the recipient's cells, the chances of a rejection reaction will also decrease. The study also means that three-dimensional culture techniques may be used to artificially manufacture any organ, kidney, liver, lung, and pancreas.

3. 2 tumor model

Tumor cells have a specific phenotype under certain time and space conditions, showing a special structure and growth behavior, which is closely related to the occurrence and development of tumors. In the 1970s and 1980s, scientists began to realize that the microenvironment plays an important role in tumor phenotype. The relationship between specific genetic alterations and the malignant phenotype of tumor cells can be recognized by studying the microenvironment of tumor cell growth.

Traditional two-dimensional cell culture can only perform genetic manipulation on cells in a two-dimensional plane, and can understand the effects of specific genetic changes on cell biological behavior, but can not accurately reflect the morphological characteristics of tumorigenesis. Although the animal model can accurately reflect the morphological characteristics of tumorigenesis, it is difficult to conduct large-scale genetic manipulation research, and it is also impossible to observe the middle occurrence process in real time. Three-dimensional cell culture combines the advantages of two-dimensional cell culture and animal models to construct a cell developmental structural system similar to that in vivo. Therefore, in addition to simulating the growth of tumor cells in vivo, three-dimensional cell culture can also study the relationship between specific gene changes and cell phenotype and the changes of biological behavior in a high-throughput manner.

Bissell, one of the pioneers of three-dimensional cultured cell technology, used this technology to successfully construct a breast cancer epithelial cell culture model and made significant progress in breast cancer research. The study found that the extracellular matrix and its integrin family receptors determine the phenotype of mammary epithelial cells, which exceeds the effect of cell genotypes on cell phenotype. Under the two-dimensional culture conditions, normal breast epithelial cells S-1 and tumor-forming cells T4-2 derived from the same line showed little difference in morphology and growth rate. However, when cultured on a three-dimensional reconstructed basement membrane, S-1 cells exhibited a complete acinar-like structure, while T4-2 cells formed large, loosely arranged, disorderly invasive clones. When the B1-integrin antibody intervention experiment was performed on the T4-2 cell clonal population, it was found that the malignant phenotype of the T4-2 cell clone was completely reversed, and the phenotypic change was reversible, and the results were not observed in the two-dimensional culture. It can be seen that the three-dimensional cell culture model can assist in the determination and selection of specific inhibitors and combinations thereof, in order to most effectively inhibit the growth of specific types of cancer cells, and can be applied to the design of tumor-specific chemotherapy drugs to kill cancer cells or Stop the progression of the tumor.

The microenvironment in which tumor cells are located plays an important role in tumorigenesis and development. In vivo, tumor cells maintain tumor growth through signal transmission between cells and cells, cells and extracellular matrices. Three-dimensional cell culture technology can simulate the micro-environment of signal transmission between cells and cells in cells and between cells and extracellular matrix. It can also be used to improve the co-culture of various cells and their relationship with extracellular matrix. Although the three-dimensional culture model is mature in breast cancer research, in theory every tissue and every organ has its own unique microenvironment.

3 .3 Stem Cell Research

Stem cells are a group of cells with self-renewal and differentiation potential. In recent years, biologists have come to realize that in terms of the physiological and pathological effects between cell populations, it is very likely that some valuable biological information will be lost through a two-dimensional culture system. At present, many studies have focused on three-dimensional culture, using tissue engineering and genetic engineering techniques to induce stem cells in vitro and manual manipulation, and research on alternative treatment of tissue cell injury diseases has attracted much attention. However, it is worth noting that the research systems used in many reports are limited to two-dimensional planar culture systems. In fact, the growth and differentiation micro-environment of stem cells in vivo should be three-dimensional. A large amount of data indicates that the differentiation and development of stem cells and their development direction depend on the microenvironment in which they are located. More and more studies have shown that only in vitro three-dimensional culture systems can better mimic the in vivo growth pattern of stem cells, which may provide more scientific and complete experimental data for tissue engineering research.

Wei Guofeng et al. attempted to construct a three-dimensional cell model that can realize the growth and differentiation of embryonic stem cells in vitro. In this model, ESCs-collagen complex was constructed by using liquid collagen as a scaffold and mouse embryonic stem cells (ESCs) as a cell model. . The results showed that ESCs not only maintained a good growth and proliferation state in the three-dimensional culture system provided by the collagen strip, but also established cell connections with each other to form a structural and functional unity. It also preliminarily detected whether the ESCs in the collagen band spontaneously differentiate into cardiomyocytes. The results showed that ESCs can differentiate into cardiomyocytes in the collagen band. The sarcomere structure of these ES-derived cardiomyocytes has matured, close to the structure of neonatal rat cardiomyocytes. It can be seen that the three-dimensional culture system can not only support the proliferation and differentiation behavior of ESCs, but also facilitate the establishment of cell connections between the differentiated cells, thereby promoting its tissue development.

The three-dimensional culture system can more realistically simulate the growth mode of the stem cells, and the relevant information obtained will provide more scientific and detailed experimental data for the related research of mammalian developmental biology.

4 summary

In the development of three-dimensional culture technology, it is important to explore better biological materials. The growth, transplantation and internal growth rate of cells on the scaffold are directly dependent on the porous structure of the scaffold, the porosity, the diameter of the pores and the shape of the pores. The high porosity provides sufficient space for cell growth and can promote the formation of engineered tissue capillary networks to meet their nutritional needs. Connected holes are also necessary for tissue growth, blood vessel formation, and nutrient supply. The connecting channel of the hole must be greater than 100um, otherwise the tissue can only invade the surface of the implant and it is not easy to get sufficient nutrients in the tissue. To date, there is still no stent that fully satisfies the adhesion, proliferation, infiltration, and continuous differentiation of the target cells and provides sufficient mechanical properties to meet the requirements of stent degradation and matrix formation in vivo. In order to promote the development and application of three-dimensional culture technology, in October 2003, the National Cancer Institute set up a special fund to study the cell micro-environment, and plans to invest tens of millions of dollars to develop three-dimensional culture technology. For different organizations and different applications, what kind of spatial structure is more effective, and whether there are better biological scaffold materials remains to be further studied.