INTRODUCTION TO 3D CELL CULTURE

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A general overview of 2D cell culture

2D culture has been used over the past decades not only to study different cellular types in vitro (Figure 1) but also to conduct drug screening and testing. Eventually, this monolayer system allows cell growth over a polyester or glass flat surface, presenting a medium that feeds the growing cell population [4]. 

Countless biological breakthroughs occurred thanks to 2D cell culture. However, due to its simplicity, this model can’t accurately depict and simulate the rich environment and complex processes observed in vivo, such as cell shape, signaling, differentiation, and chemistry. Consequently, data gathered with 2D cell culture methods could be misleading and non-predictive for in vivo applications [5].

That’s why scientists have recently been working on 3D biomimetic cell cultures, a technique that represents more precisely the microenvironment in which cells thrive in vivo.

A general overview of 3D cell culture

The first 3D cell culture model was provided in 1992 to describe 3D organotypes that mimic breast structures in cancerous and non-cancerous cases. Figure 2 shows a recent image of a 3D cell culture of HER2-positive breast cancer cell line [3]. Soon, 3D cell culture evolved to have broader applications [2]. 3D cell culture mimics in vivo cell behavior and organization – morphologically and physiologically – imitating the structure of the extracellular matrix (ECM) of cells, recreating their natural microenvironment [6].

Comparison between 2D and 3D cell culture

A 3D cell culture model is an attractive method to overcome many limitations of 2D cell culture. 3D cell culture facilitates cell differentiation and tissue organization using micro-assembled structures and complex environmental parameters [5]. In this context, cells are more subject to morphological and physiological changes than those grown in a 2D environment [6]. Moreover, genes promoting undesired cell proliferation can be repressed, avoiding the anarchic proliferation encountered in 2D cell cultures [1].

Using 3D cell culture, it is possible to simultaneously grow two different cell populations with co-cultures [7]. Finally, combining 3D cell culture with micro-engineering (microfluidics) makes it easier to control and monitor the growing cells’ micro-environment parameters (temperature, chemical gradients, oxygen rate, pH, etc.) [7].

However, with this new technology come some notable downsides. Firstly, scaffolds that are micro-organized cell supports, incorporate macromolecules isolated from animal tissues that could interfere with cell culture [8]. Some other matrices provide good cell adherence, making cell removal all the more difficult. Moreover, while 3D cell culture could be a less expensive technique to replace animal drug testing, developing applications remain very costly [5]. 

Despite 3D cell culture limitations, a survey found that most of the participants have plans to switch or have already switched from 2D to 3D cell culture [9]. Thus, we will now look at the different 3D culture systems currently available.

3D culture systems

Depending on the conditions and intended goals, culture systems can use scaffold-based (on natural or synthetic solids) or scaffold-free techniques.

Scaffold-based 3D culture

The role of scaffolds is to support the elements in 3D cell culture (Figure 3). Due to their porosity, scaffolds facilitate the transport of oxygen, nutrients, and waste. Thus, cells can proliferate and migrate within the scaffold web to eventually adhere to it [7]. Maturing cells interact with each other and eventually turn into structures similar to those they initially originated from. In addition, assembling multi-layer 3D cells is possible using scaffolds. Researchers have found that the geometry and composition of scaffolds influence gene expression [11], and enhance cell-cell communication [12].

The scaffold layout should match the tissue of interest in structure, scale (macro, micro, nano), and function. It must support cellular growth and be biocompatible. However, it is important to keep in mind that, as the scaffold gets bigger and more complex, extracting the cells becomes harder [13]. 

Different materials can be used as scaffolds, such as polymers, metals, ceramics, glass, and composites. Each one provides various advantages and shortcomings and should be selected according to your experimental requirements.

Natural scaffolds

Hydrogels are composed of hydrophilic polymer chains, covalent or non-covalent bonded. Natural or synthetic polymers form gels when crosslinked through covalent or noncovalent bonds. Natural polymers are biodegradable and bioactive materials that can be classified into proteins, polysaccharides, and polynucleotides [14], such as alginate, gelatin, hyaluronic acid, agarose, laminin, collagen or fibrin [5, 7]. In contrast to gels that are more solid-like than liquid-like, hydrogels absorb large amounts of water and swell without dissolving [14]. 

Hydrogels are compatible with a range of biological tissues and processes. They present a tissue-like stiffness and perfectly mimic ECM, allowing soluble factors such as cytokines and growth factors to navigate through the scaffold (Figure 4) [15, 16]. However, scaffolds comprising only natural hydrogels present the issue of poor mechanical properties incapable of maintaining the desired 3D shape [14]. Composites can be a solution for such issues, which will be discussed later on.

Different scaffold features are needed for 3D tissue engineering and cell culture. Synthetic scaffolds are widely used since they propose almost complete control over mechanical properties and construct architecture. Commonly used materials are synthetic polymers, metals, ceramics, glass, and a myriad of composites [14].

Synthetic scaffolds

Synthetic scaffolds are made of synthetic polymers or non-polymer scaffolds. 

Synthetic polymers are different from natural polymers and comprise synthetic hydrogels and hard polymers. Synthetic polymers exhibit excellent biocompatibility with negligible inflammatory response, which makes them a convenient choice in clinical practice [17].

Synthetic hydrogels are made of synthetic polymers (polyester, polyethylene glycol (PEG), polyamide, polylactic acid (PLA)). They provide higher consistency, reproducibility, and customization properties than natural hydrogels [2]. However, synthetic polymers show low cell affinity due to hydrophobicity and lack of cell recognition sites [14]. Figure 4 shows two hydrogel-based scaffolds, (a) constituted of natural polymers that provide cellular support by enabling the cells to bind with a variety of integrin-binding sites and growth factors, and regulate cell behavior through activating signaling cascade; (b) composed of synthetic polymers, that lack growth factors and integrin-binding sites that usually provide mechanical support for cell growth [16].

Hard polymeric material-based support is made of polystyrene (PS) or polycaprolactone (PCL) biodegradable polyester. It is an important tool in studying cell-to-ECM interactions due to the scaffold’s ability to replicate the structure of the ECM. These materials also show high cell recovery properties. Furthermore, hard polymeric scaffolds are extremely useful in studying tissue regeneration as well as testing tumor cell treatments [2, 5].

However, the use of polymers for 3D scaffold engineering requires further improvement, due to their insufficient mechanical strength, and inappropriate degradation rate [17].

Non-polymer synthetic scaffolds can be composed of metals, ceramics, or bioglass.

Porous 3D metallic scaffolds mostly made of titanium (Ti) and tantalum (Ta), are commonly used for load-bearing applications because of their high mechanical strength, fatigue resistance, and printing processability. However, these metallic scaffolds lack metabolization over time, are non-biodegradable, thus need repeated surgery, and present low tissue adherence and prolonged recovery time [14].

Bioceramic or bioglass, is a bioresorbable material that improves the regeneration activity of a nascent tissue. Ceramic biomaterials usually include inorganic calcium or phosphate salts. They are bioactive, biocompatible, biodegradable, less elastic, and brittle. The use of bioactive glasses has demonstrated the ability to enhance bone cell growth, bond to both hard and soft tissues, and control degradation rate in vivo [18]. 

Lately, composites have been used to build scaffolds (Figure 5). They are made of two or more different materials as a remedy to cover each material’s issues and provide the required mechanical and physiological properties [17]. Recent studies have shown that alginate combined with synthetic polymers provides biomechanical support optimized cell attachment conditions, and hydrophilicity [7]. The addition of ceramic materials, hydroxyapatite (HA) and β-tri-calcium phosphate (TCP), to polymeric PCL scaffold, enhanced mechanical properties and cell proliferation rate [19].

Scaffold-free 3D culture

Scaffold-free 3D cell culture techniques, such as forced-floating, hanging drop, and agitation-based methods (Figure 6) can be used to generate heterogeneous-sized spheres called spheroids.

The forced-floating method uses low-adhesion polymer-coated well plates. Spheroids are generated by filling well plates with a cell suspension after centrifugation [3].

The hanging drop method allows a cell suspension aliquot inside micro trays to aggregate and fabricate spheroids in the form of droplets. By controlling the volume of the drop or density of cell suspension, it is possible to control the spheroid size [20]. 

Agitation-based approaches reconstruct microgravity using a constantly rotating bioreactor. A cell suspension gradually turns isolated cells into aggregates that cannot adhere to the container wall due to continuous stirring. As a result, a broad range of non-uniform spheroids are eventually generated [20].

Cell interactions in a 3D culture

Cells cultured in 3D are characterized by cell-ECM and cell-cell interactions (Figure 7); these interactions influence cell organization and cell regulatory pathways [8].

Communication is implemented by cell junctions which are direct intercellular passageways made of protein that form the channel connecting one cell to its neighbors (or one cell to the matrix). Soluble factors secreted such as cytokines or growth factors, are transported to neighboring cells and ECM via direct contact or streamflow [21].

Cell-to-matrix interaction is crucial since some properties gained by the growing cells are only obtainable via the scaffold’s influence on gene expression and its supporting role in tissue organization [11]. ECM biochemical composition, made of different signaling biomolecules, modulates several adhesion-related cell functions, such as cell cycle, adhesion, and proliferation [8].

3D spheroid and organoid models are good representatives of in vivo cell interactions.

Spheroid and organoid models

Spheroids and organoids are 3D cell structures formed by gathering many cells. They differ in their fabrication method, cell origin, and type. 

Spheroids loosely describe 3D structures or multi-layered aggregates that may self-organize without recapitulating a tissue-like behavior. Spheroids are spherical cell units that are cultured as free-floating aggregates, such as tumor tissues, embryonic bodies, hepatocytes, neural tissues, and mammary glands. They show relatively low structural complexity, but as they cannot self-assemble or regenerate, they are less biocompatible than organoids [23].

An organoid is a miniaturized version of an organ produced in vitro in 3D. Organoids have self-differentiation capabilities and evolve from self-organizing stem cells to reproduce the targeted organ’s key structural, and functional properties [24]. Organoids can be cultured from embryonic stem cells (ESC), induced pluripotent SC (IPS), and adult SC [23]. They aggregate into spheroids by forming ECM fibers that link single cells together. Researchers can grow tumor models using patient-derived tissue cancer cells [5]. However, organoids present some challenges, such as microenvironment control, automation, and interaction modeling between different organs [24]. 

Stem cells are characterized by their ability to proliferate and differentiate into specific cell types in vitro, producing mature cells with constant quality. Stem cells can be classified as IPS generated in vivo from differentiated cells. They can also be isolated from an array of tissues such as the pancreas, cardiovascular system, brain, lung, liver, adipose tissue, and bone marrow [25].

We will now have a look at 3D cell culture’s main applications.

3D cell culture’s main applications

Being a tool to study cell behavior in an environment reflecting in vivo conditions, 3D cell culture has many applications that we will overview in this section.

3D cell culture for tissue engineering

Cell culture mimicking 3D tissues can be exploited for studying disease propagation and progression, drug discovery, and compound screening [26]. 

Instead of using biomaterial, human tissues can be generated with micro-structured fiber scaffolds in a 3D culture (e.g., epithelial-dermal sheets for skin reconstruction). Unfortunately, tissue engineering can be quite expensive, and regulations around this application are not well-defined in some countries [1].

3D cell culture for drug testing

Drug development involves both non-clinical (animal trials) and clinical trials. It remains a time-consuming and expensive process with a very low success rate of drugs reaching the market (13.8%) [23, 27]. In addition, nearly 200 million animals are used in biomedical research worldwide, a number that could be reduced substantially by alternative technologies [28]. Since then, 3D culture emerged as a cost-effective, time-saving, more precise culture technique for drug screening [23]. For instance, using micro-engineering applications (organ-on-a-chip), cancer therapeutics are getting better, improving the benefit-risk balance by targeting more precisely a specific cell type, a defined bio-mechanism, a precise receptor, etc.

However, some studies showed that cells grown in 3D culture are more resistant to drug treatments while showing promising results using other culture methods, mainly due to changes in membrane properties after 3D encapsulation [29]. Moreover, many drug tests fail due to their inability to provide progression-free survival. Indeed, there is still a gap between in vitro and in vivo cellular drug responses.

Microfluidics in 3D cell culture

With the development of microfluidic technologies, long-term, and controlled 3D cell culture models were created using biocompatible microfluidic chips that facilitate tissue manipulation and study. Organ-on-a-chip can be defined as microfabricated cell culture devices modeling the functions of living organs by mimicking their microstructures, dynamic mechanical properties, and biochemical functionalities [7, 24]. It relies on engineering artificial constructs in which cells and their microenvironment are precisely controlled [24].

Microfluidics combined with organ-on-a-chip (Figure 8) added a precise, controlled flow, introducing a hallmark of the native cell environment currently missing from most static organ-on-a-chip (Figure 9). It enables real-time microenvironment control (nutrient and gas exchange, molecular gradients, fluid flow, and mechanical phenomena). Fluid flow is accurately regulated by microfluidic output devices (flow control systems).

Thanks to this time-saving biomimetic model, drug development research is easily conducted to study human physiological responses on an organ scale or a systemic scale (multiple organ-on-a-chip). Researchers are now exploring the possibility of synergistically combining organoids and organ-on-a-chip best features to develop a more powerful in vitro technology [24].

Conclusion− 3D cell culture is the future of translational biomedicine

3D cell culture models can adequately mimic in vivo cell behavior and organization. 3D cell culture resulted in significant improvements in drug screening and tissue engineering applications. Scaffold-based techniques, using natural or synthetic biomaterials, are essential for tissue engineering, while scaffold-free techniques are used for generating spheroids. 

The number of animal models that are genetically engineered each year for breeding and not actually used, is increasing each year. Not to forget all the harm undergone by animals that are killed in the end. Implementing the 3Rs, reduce, refine, and replace, many countries are aiming to stop animal testing by the end of 2035. Switching to 3D cell culture and organ-on-a-chip is a relevant alternative. The possibility of controlling pressure and flow rate using microfluidics makes these translational techniques even more exciting. However, more accurate guidance is needed to better replicate cell and tissue conditions in the future.

Acknowledgment: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 690876

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