Since its inception, in vitro cell culture technology has been widely used in biology and medicine. Common in vitro culture models include 2D and 3D cell culture.
2D culture involves the growth of cells in a monolayer on a two-dimensional plane, simulating cell-cell interactions. However, this method has some limitations, such as cellular heterogeneity, uneven nutrition, and unnatural cell-substrate interactions, resulting in low cell survival, impaired morphology, and lack of normal tissue structure.
In contrast, 3D cell culture mimics the in vivo cell growth environment by constructing three-dimensional structures from bioscaffolding materials, allowing cells to exhibit behaviors and functions that are closer to those under physiological conditions.
It compensates to some extent for the shortcomings of 2D cultures and animal models, and is particularly advantageous in terms of cost reduction, cycle time, and reduction of species variability.
1. 3D cell culture methods
Currently, commonly used 3D cell culture methods include sphere suspension culture, microfluidic culture, bionic scaffold culture, 3D bioprinting and hydrogel scaffold culture.
Each method has its unique advantages and limitations.
Sphere suspension cultures do not rely on external scaffolds, but may affect cell viability;
Microfluidic culture enables precise analysis of cell behavior, but it is expensive;
Bionic scaffolds and hydrogel scaffolds provide an ideal growth space for cells, but their mechanical properties and parameters are more difficult to control;
Three-dimensional bioprinting, as an emerging technology, has strong tissue-forming ability and a promising application.
Advantages and limitations of common culture methods for 3D cells
| designation | Methodological details | Advantages | limitations |
| spherical suspension culture method | Applying buoyancy or agitation to bring cells together into spheres | No bracket material support required | The turbulence and shear of the fluid can cause some damage to the cells, causing them to die or become less viable |
| Microfluidic system culture method | 3D culture of cells by creating nanoscale channels on a microfluidic chip and precise manipulation of fluids in the channels | Improved accuracy in analyzing cell behavior, integrated miniaturization, high throughput | Higher costs |
| Bionic scaffolding model culture method | Application of biomimetic materials to mimic the extracellular matrix and construction of three-dimensional scaffolds | Provide suitable growth space and sufficient mechanical support for nascent tissues, and can mediate intercellular signaling and interactions to induce tissue formation | The internal structural properties of the stent cannot be evaluated prior to manufacturing, and parameters such as porosity and connectivity are difficult to control during the manufacturing process |
| Hydrogel scaffold culture method | Use of hydrogel systems as scaffolds for 3D cell culture and also as delivery vehicles for bioactives | Room temperature stable, pH neutral, transparent, water permeable, compatible with different imaging systems, easy and convenient cell recovery, can be injected into animals for in vivo studies | Single-component gel stiffness is affected by gel concentration and influences cell displacement and deformation under mechanical action |
| 3D bioprinting technology | Three-dimensional bioprinting utilizing bioink as a carrier for directly piggybacking cells or bioactive factors | The ability to print bio-ink loaded with one or more types of cells and growth factors into bionic tissues or organs with complex three-dimensional structures and certain functions | Further exploration is still needed to achieve the functionalization of human organ tissues |
2. 3D cell culture scaffold materials
In 3D cell culture, scaffold materials not only provide the microenvironment necessary for cell proliferation and differentiation, but also act as carriers of growth factors, which play a crucial role in tissue engineering regeneration.
Therefore, it is crucial to choose the right stent material to better mimic the in vivo environment.
Currently, the commonly used 3D culture scaffold materials are mainly categorized into two types: natural materials and synthetic materials.
Natural materials are derived from plants, animals or the human body and include hyaluronic acid, chitosan, alginate, gelatin and collagen.
They have good biocompatibility and low immunogenicity, but are difficult to mass produce.
Synthetic materials, on the other hand, are made by compounding two or more substances with specific integrated properties, such as polycaprolactone, polyethylene glycol, polylactic acid and polyvinyl alcohol.
While synthetic materials can precisely modulate the performance of scaffolds, certain materials may be toxic to cells and have adverse effects.
Advantages and disadvantages of natural and synthetic materials
| vantage | drawbacks | |
| natural material | Mainly from human, as well as animal and plant organisms, many endogenous substances on the growth of cells with the promotion of good biocompatibility, very little toxicity to the cell, easy to degrade, low immunogenicity. For example: ① hyaluronic acid has a promotional effect on cell movement, adhesion, proliferation and tissue structuring; ② chitosan, on the other hand, has good biocompatibility, minimal immunogenicity and antimicrobial properties, and has been widely used in wound healing and soft tissue engineering; ③ gelatin is widely used in cell culture because of its ease of gel formation and good interaction with cells; ④ collagen has a very good coagulant effect and can be Collagen has a good coagulation effect and can be used for wound hemostasis, and has been widely used in biomedical fields in different forms; ⑤ Agarose, filipin, chondroitin sulfate, etc. are widely used in the construction of extracellular matrix by virtue of their own unique advantages. | Large lot-to-lot variation, making mass production difficult |
| synthetic material | The hardness, porosity and microstructure of the scaffolds can be precisely regulated according to different applications. For example: ① polycaprolactone has good biodegradability, biocompatibility and non-toxicity, and has been widely used as medical biodegradable materials; ② polyethylene glycol has the advantages of good biocompatibility, non-toxicity, low immunogenicity, etc., which is often used in the study of the interaction between the cells and the micro-environment; ③ poly(lactic acid) has good biocompatibility, degradation rate, high mechanical strength, good chemical stability, and is one of the commonly used materials in bone tissue engineering; ④ poly(vinyl alcohol) hydrogel formed by repeated freezing-thawing method at room temperature. Poly(vinyl alcohol) is one of the commonly used materials in bone tissue engineering; (4) Hydrogel formed by repeated freezing and thawing is stable at room temperature and highly elastic; (5) Poly(vinyl alcohol) has good biocompatibility and degradation rate. | Acidic degradation by-products of polylactic acid cause inflammatory reactions, and cross-linking of polyvinyl alcohol chemicals makes the gel toxic and in most cases non-degradable, limiting its in vivo application |
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