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  • Writer's pictureMikayla Shelton

Maximizing Your 3D Cell Culture Success: Tips and Tricks for Harnessing the Power of Hydrogels


An in vitro 3 dimensional (3D) cell culture is an artificially produced environment where the cells grow and interact with other cells in a 3D environment, which, unlike traditional 2D methods, better mimics the in vivo circumstances and is generally more physiologically relevant [1]. In the field of 3D cell culture, hydrogels have emerged as a versatile biomaterial that offer benefits to researchers and clinicians in studying complex biological processes, modelling diseases, and screening potential therapeutics [2]. These gels are water-swollen networks of fibres that act as a scaffold to support cell growth, enabling cells to form a complex 3D tissue model that better recapitulates native cellular milieus found in vivo [3]. However, achieving optimal results using hydrogels for 3D cell culture requires careful attention and many considerations of the tissue/microenvironment the hydrogel is trying to mimic. In this blog post, we will delve into some of the best tips and tricks to enhance the effectiveness of your 3D cell culture experiments using hydrogels.

Optimisation of your gels – choosing the right scaffold

The choice of your gel scaffold material is one of the most important considerations before embarking on 3D cell culture, where a clear understanding of the properties of your material will allow you to be conscious of how they will affect cellular behaviour. The hydrogel’s mechanical properties, including stiffness, tensile strength and elasticity can influence cellular mechanotransduction (the process of converting microenvironmental mechanical cues into downstream signalling within the cell), influencing cellular and tissue model development in 3D [3]. By matching the hydrogel’s mechanical properties such as stiffness to the target tissue you are trying to emulate, cell behaviours like migration, invasion, differentiation, morphology and interactions with other cells better mimic those found in vivo. In order to understand the viscoelastic properties of your desired hydrogel material, it is recommended to use techniques such as atomic force microscopy (AFM) or rheological tests such as the stress relaxation test [4]. In addition, the scaffold material will also determine how biocompatible it might be with your desired cell types. In general, hydrogels are thought of as biocompatible due to their hydrophilic nature, but the chemical composition of the polymer or the type of crosslinking involved, if any, may cause cytotoxic effects or immunological responses in certain cell types [5]. In order to assess biocompatibility, it is important to conduct preliminary tests with your cells and the potential hydrogel to assess cell viability and function and detect any adverse cellular responses.

Much like steel scaffolding in a building site, hydrogels scaffolds provide structural support which allow the organisation and building of more complex cellular models.

Another consideration when choosing your hydrogel scaffold is to examine the kinetics of how the gel forms and degrades, as these can also greatly affect cellular properties. Whilst hydrogels are broadly described as 3D networks that swell from absorbing water, they can be categorised in many ways, including by composition, crosslinking, ionic charge, porosity, and how they are sourced, which all influence how the hydrogel is formed from liquid precursor solutions into a solid or semi-solid material [6]. For example, the porosity of the material (size of the gaps in the mesh structure) is incredibly important to consider when choosing a hydrogel, as this affects how the cells are encapsulated and how they interact spatially with their surroundings and other cells, as well as diffusion of nutrients through the gel architecture [7]. Characterisation of pore size or mesh microstructure can be investigated in a variety of ways, including scanning electron microscopy (SEM), fluorescence recovery after photobleaching (FRAP), or quantifying the diffusion of a fluorescent molecule through the hydrogel [3]. Another example for deliberation is the gelation kinetics that could affect how the cells are encapsulated, where more rapid gelation may cause cells to distribute unevenly, whereas slow gelation kinetics could affect cell settling and viability, especially if the gel requires cytotoxic crosslinking agents [3]. In addition to gelation kinetics, it may also be important to consider the degradation profile of the gel, including hydrolytic or enzymatic mechanisms, as this controls the spatiotemporal release of encapsulated cells or bioactive molecules if desired, depending on the goal of the experiment, for example controlled drug delivery [8].

Different scaffolds have different porosities, which determines what size of molecule can pass through the material. Selecting the material that allows for the free exchange of nutrients, whilst not compromising on the other mechanical properties, is critical.

Finally, it is important to consider the scalability and sterility of the gel when it is being prepared depending on the needs of the researcher. Scaling up of hydrogel-based methodologies, for example in a clinical testing or industrial setting, may present issues during the making of the hydrogel, which may affect consistency between batches. Factors such as dissolving and adequate mixing of the precursors, maintaining optimal temperature and pH, ensuring time and cost effectiveness and reproducibility are all issues that present challenges when gels are made on a larger scale [9]. Therefore, it is important to develop a robust hydrogel fabrication method that utilises quality control measures throughout the process of making the gel, such as accurate quantification of the precursors, pH and temperature, as well as testing for parameters like stiffness via rheology to ensure consistent performance characteristics. It is also important to ensure the gels are sterilised before the cell culture process to prevent contamination that will affect cellular responses. Common sterilisation methods include the use of autoclaving, gamma radiation, ethanol or ethylene oxide treatment, or filtering of the precursor materials if possible [3]. The method chosen to sterilise the hydrogel must carefully consider how to avoid affecting the gel’s structural integrity or bioactivity.

Optimisation of your cells – creating the highest quality in vitro model

In addition to developing a deep understanding of the hydrogels properties, it is equally important to consider the complex culturing conditions your cells require to best mimic the in vivo environment you are trying to replicate. In order to do this, it is important to research the structure of the in vivo tissue and consider how the cells grow and what other types of cells interact with them. For example, cells can grow as simple spheroids that can be different shapes and sizes depending on the cell type, such as rounded, grape-like, or stellate morphologies [1]. Cells could also grow in more complex organoid or co-culture systems with other cell types to better recapitulate the complex organisation and interactions of cells you would find in tissues and organs in the body [10]. It is therefore important to carefully consider the cell types required for the model and determine the costs and benefits of using multiple cell types that may require longer culturing time and have more complex growth conditions. The density of the cells being seeded into the hydrogels is also important to optimize, as cells may need to be encapsulated at certain densities to allow cell interactions to occur, but need to also avoid excessive cell densities that does not allow adequate diffusion of nutrients, oxygen and waste in and out of the gel [1]. In order to ensure proper cell growth, it is therefore important to try a range of cell densities to work out which one is optimal for your cell type, as well as how many days the cells are growing in the gel to allow enough time for the cells to interact with their environment, but not too long that they go quiescent or develop necrotic cores due to depletion of oxygen and nutrients.

Optimisation of seeding density of acute myeloid leukaemia cells encapsulated in peptide hydrogels (Source: J. James et al. (2023)).

In order to best mimic physiological conditions found in vivo, it is crucial to consider the types of proteins and bioactive molecules that are found in the tissue and adjust the composition of the cell media or incorporation into the hydrogel accordingly. In addition to choosing the best media type for your cells, it is also imperative to consider which supplements to add that are necessary for the growth of your cell type, and also the differentiation of the cells if desired [11]. Bioactive molecules such as growth factors or components of the extracellular matrix can be directly added to the media, or can be immobilised on the hydrogel scaffold matrix, or chemical/amino acid motifs of the molecule could be added to the hydrogel’s polymer composition to mimic certain structures. For example, hydrogels can be functionalised with an RGD- amino acid motif that has the ability to bind cell surface integrin receptors and act to promote cell adhesion to the scaffold [12]. However, it is also important to consider that introduction of a wide variety of proteins and biomolecules introduces another source of variability, as composition may differ between batches if isolated from animal sources, affecting reproducibility and reliability [13].

Finally, once the cell encapsulation and growth parameters have been optimised, it is necessary to validate your model’s relevance to compare the characteristics with in vivo models. In order to do this, various parameters must be measured, including cell/spheroid morphology, gene expression, differentiation markers, ECM deposition and cell migration and invasion [3]. These can be measured using a wide variety of methodologies, such as immunofluorescence staining where the cells can remain in the gel, or removing the cells from the gel for protein/RNA extraction. For studying the cells in situ, cells can be fixed, stained and imaged for desired markers using fluorescent antibodies, where cell morphology and ECM deposition can be visualised in 3D [3]. If this is your desired end-point assay, then it is important to choose a hydrogel that is optically transparent and allows good diffusion of the antibodies within the gel, as well as using great care when washing to avoid disturbing the gel. For most molecular analytical methods, isolating cells from the gels is required, such as isolation of the whole cell for flow cytometry, or isolation of a biochemical component of the cells to study gene/protein expression. How you decide on removing your cells from the gel is important to prevent damaging of the cells, or disrupting features such as cell surface receptor expression that may alter the properties of the cell and distort results. Cells can be isolated from gels using a variety of methods, including diluting out the gel to form a liquid again, spinning the cells gently, photodegradation of gels, or enzymatic degradation of the gels [3, 14, 15]. Once these functional and biochemical assays have been carried out, the relevance of the 3D model can determined by comparing to data obtained from in vivo experimentation to identify key differences and similarities and adjust experimental conditions accordingly.

MCF7 breast cancer cells encapsulated in PeptiMatrix™ hydrogels assessed for viability (left) or matrix deposition and morphology (right) (Source: M. Shelton (unpublished)).


In conclusion, it is important for the researcher who is looking to start 3D cell culture using hydrogels to consider a variety of parameters regarding the hydrogel properties itself and the cells that will grow in/on the hydrogel before commencing with experiments. Properties such as the hydrogel’s material, stiffness, biocompatibility, porosity, gelation kinetics and sterility to name a few must all be factored in when making a decision on the type of gel that is best for the tissue model. Cellular considerations such as the types and combinations of cells growing, cell density, growth time, media and supplements and types of end point assays for validation must all be carefully optimised to create a robust and biologically relevant model. For both the hydrogel and the cell deliberations, it is imperative that you have a good understanding of the tissue/organ you are trying to emulate, and in depth research of how the cells act and interact in their environment in vivo will help you choose the best conditions that suit your experimental objectives and requirements. Overall, hydrogels offer an amazing model technology which can be tailored to specific experimental requirements over a wide range of applications, such as drug discovery and understanding cellular interactions involved in disease processes, and offer many benefits over 2D cell culture models currently used.

About the Author

Mikayla is a cancer research scientist who recently completed her PhD at Leeds Beckett University. Her PhD project focused on the bidirectional crosstalk between melanoma and cells of the tumour microenvironment via secretion of extracellular vesicles. This project involved study of a wide variety of cellular phenotypic and expression changes, as well as determination of cargo within the vesicles. She also has a background as a bioassay scientist in industry in a multitude of client projects.



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