Adapting three-dimensional (3D) cell culture into laboratory practises is a powerful option to enhance drug discovery process or understanding of disease biology. Culturing in 3D offers various benefits compared to culturing cells in two dimensions (2D).

A wide range of techniques for creating advanced 3D cell culture models are used. Generally, these are separated into scaffold-free and scaffold-based techniques. The main difference is that scaffold models utilize a three-dimensional material for seeding the cells. By varying the selected culture method, researchers can choose the suitable 3D culture model for their assay. In this overview, we are going to discuss commonly used 3D cell culture models and their pros and cons.


Scaffold free 3D cell culture models 


1. The hanging drop technique


In the hanging drop method, cell suspension drops of approximately 10 µl are placed on a flat surface of the culture vessel. After adding all cell suspension droplets to their determined positions, the surface of the droplets will be turned upside down. The surface tension of the cell suspension makes the droplet hang from the attached surface and gravity will drag the cells to the bottom of the droplet. In the bottom of the droplet, cells aggregate and form a spheroid. Formed spheroids are uniform and the size can be adjusted by changing the cell seeding density. 

In the hanging drop method, the change of the cell culture media without disturbing or discarding the spheroids is a difficult procedure. In addition, the technique can become very tedious when done on a large scale, which restricts its usage in high throughput screening (HTS) applications for example. Moreover, properties of the hanging drop method are not the most optimal for visualization. The droplets often fall whilst moving the plates to image the cells and the focal working distance can be a challenge for many microscopes since the droplets can be far from the bottom of the plate.

To overcome these challenges, some commercial hanging drop plates have been developed which are compatible with automated liquid handling robots and plate readers which enhance the HTS suitability. For down-stream analysis like RNA/DNA extraction and high content imaging, spheroids need to be transferred to a separate plate, increasing the likelihood of experimental variability.  Moreover, being a scaffold-free method, hanging drops lack cell-matrix interaction.


2. Ultra-Low Attachment plates


Ultra-low attachment (ULA) plates were created to produce large scale scaffold-free 3D-cell cultures. These plates are made using liquid overlay techniques where the bottom of a cell culture dish is coated with a non-adhesive material, which prevents cell adhesion and protein absorption. Commonly, ULA-plates are produced by covalently binding a hydrophilic and biologically inert material on the surface of a plate.

When the cell suspension is added to a well of an ULA cell culture plate, cells sink to the bottom of the well, but do not attach to the culture surface which facilitates their aggregation and spheroid formation. There are various well bottom shapes of ULA cell culture plates available, such as U bottom, Flatbottom, Spindle bottom and V-bottom.



U-bottom ULA plates have been created to form and analyse homogenous spheroid populations. Like the hanging drop technique, the U-shape uses gravity to pull down the cells to the bottom of the well. The round geometry will force the cells to aggregate and form a 3D cell complex. The size and shape of cell complex can be easily adjusted by calculating the wanted cell density in the well. 

Compared to U-bottom plates, Flat-bottom ULA plates form heterogenous spheroid populations. In each well, cells can freely move on the flat surface and randomly form a cell aggregate. Between plate wells, there may occur varying number of spheroids with different sizes.

ULA Plates are compatible with liquid robotic systems which increases their usage in HTS applications. Downstream analyses and visualization can be done in the same plate as cell culturing. However, in ULA-plate the spheroids float in suspension and are not physically in a fixed position. In live cell imaging, this can cause difficulties to obtain and maintain optical focus when small movements can get spheroids on the move. Together with this, biological relevance of ULA cell culture plates should be considered because this method lacks tissue-like stiffness and moreover cell-matrix interaction.


Hint: Biological relevance in flat bottom ULA cell culture can be increased applying hydrogel. Hydrogel usage provides cell-matrix interaction and suitable tissue-like stiffness. Enhanced biological relevance provides reliable results and helps avoid pitfalls.


Scaffold based 3D cell culture models 


1. Cells embedded in hydrogel 


Physiological relevance in 3D cell culture models can be increased using scaffold-based models. The Scaffold-based models provide in vivo-like tissue stiffness and Cell-Matrix interactions to support correct cell phenotype formation. 3D cell culture scaffolds consist wide range of biomaterials. Depending on their origin, Biomaterials can be divided into synthetic or natural materials. One sub-category of scaffold materials are hydrogels. Hydrogels are usually classified into three categories which are synthetic, animal-derived and plant-derived polymers. These polymers have high capacity to retain large amounts of water and are commonly used in 3D cell culture. 

A commonly used hydrogel 3D cell culture application is the embedding. Embedding process of cells in hydrogel consists of couple steps for ensuring the even distribution of cells. Cells are mixed with hydrogel and added to the culture vessel, such as 96-well plate, followed by dispensing the culture medium on top of the gel. Embedded cells produce heterogenous populations of spheroids. Depending on the origin of the hydrogel, preparation steps can slightly vary. 

The origin source of the hydrogel can bring its own advantages and disadvantages. Hydrogels that are temperature sensitive and need certain temperature for handling and polymerization are challenging for HTS applications because the temperature needs to be adjusted. Moreover, in animal-derived hydrogels exact compound content is not known and there can occur batch-to-batch variation. In long-term 3D cell culture, animal-derived hydrogels that contain collagen and hyaluronic acid can be degraded enzymatically by cultured cells, causing a structural change over time.

When considering hydrogel embedding in high content screening (HCS) applications easy degradation and transparency of hydrogels are essential properties. Retrieving cells for RNA/DNA extraction, the hydrogel embedding is noticed to be challenging for some hydrogels. Synthetic hydrogels cannot be degraded, and animal-derived matrix degradation affects cell surface proteins of cultured cells. 


2. Dome culture 


Dome culture is suitable for organoid and spheroid 3D cell model cultures and traditionally done with animal-derived hydrogels. These hydrogels are temperature sensitive and have a capacity to polymerize in 37⁰C. When preparing the dome 3D cell culture, the hydrogel material, pipet tips and cell suspension must be kept on 4⁰C. This will keep the matrix in a liquid form. Also, working temperature sets the requirement that the workflow must be kept rapid. Cell culture vessel where cold matrix-cell suspension is added is kept on 37⁰C. The temperature promotes polymerization of the matrix and formation of domes. In the end when domes are formed, culture media is added carefully on top of the domes to cover them. 

When recovering 3D cell models from the matrix, temperature needs to be adjusted to 4⁰C, to make the matrix a liquid state again and when using an enzymatic digestion, the cell surface of the cultured cells can be damaged. Because of working temperature changes, this method is not optimal for HTS applications. The dome 3D cell culture technique can minimize the amount of matrix material used, but when using animal-derived hydrogels there occurs batch-to-batch variability. Variability includes ingredient changes, thus affecting poor control of mechanical properties and cellular differentiation.


3. Seeding cells on top of hydrogel 


Usage of hydrogel is not only limited to the embedding solutions. Hydrogel can also be used as an underlay material for cell suspension and therefore cells can grow on top of the gel. This is suitable, for example, with endothelial and epithelial cells, which are not surrounded by extracellular matrix in physiological conditions.

When using hydrogel as an underlay material, the surface architecture has different kinds of shapes for cell growth and there is an opportunity for cells to invade inside the gel. Depending on the hydrogel used, cells can attach to the hydrogels surface proteins forming more 2.5D than 3D cell culture. This happens generally with animal-derived matrices, but there is a possibility to modify plant-based hydrogels to have binding proteins on the surface so that the matrix better mimics the normal extracellular matrix (ECM) composition. If cells are not adherent type and hydrogel does not have proteins on the surface for binding, cells prefer to form spheroids.

When cells are growing on top of the gel, they can produce their own ECM proteins and form a matrix. Moreover, gel is dividing cell culture into different layers which provides an opportunity to simultaneously grow some cells in the hydrogel and others in the media layer. If the cells are not migrating into the hydrogel or attaching to the hydrogel surface, it might be difficult to obtain and maintain optical focus similarly to other suspension cell models. 


Hint: Using plant-based natural hydrogels such as GrowDex®, batch to batch variability can be minimized while handling the hydrogel in the room temperature. Moreover, cell retrieving can be optimized without compromising imaging properties. The solution is suitable for automatization and opens HTS opportunities for researchers. Still enabling sustainable and ethic matters. 


4. Cell culture inserts 


Cell culture inserts provide the most complex 2D and 3D cell culture models. Cell culture insert can be used together with multi-well plates and together they are forming an apparatus which can be divided into two separate chambers. On the bottom of the insert is a microporous membrane which allows signalling molecules or cells go through. The permeability of the membrane can be adjusted by selecting suitable micropore size.


By chancing the pore size of the membrane, researcher can modify the purpose of the cell culture insert to be suitable for different applications. Smaller pore size is suitable for transport studies of drugs and bigger pore size is better for cell invasion studies. Moreover, culturing cells in different chambers is advantageous for HCS studies, because cells can be easily separated from each other for further analysis.

With cell culture inserts, there is a limited option to choose the material and size of the cell culture area. Also, inserts provide only one layer where cells can live, if not used together with matrix material. Matrix material helps to grow a 3D cell culture with an insert and opens an opportunity to use other surfaces of insert as well. The most complex cell models produced by inserts are not suitable for automatization which limits their usage in HTS applications. When compared to other 3D cell culture models the visualization with normal bright-field microscope has been detected to be difficult.


Hint: Using a hydrogel, the usage of cell culture insert can be revolutionized. For example, growing and imaging of lung cells can be enhanced restricting media and cells only to upper chamber. This gives the researcher freedom to modify cell culture insert apparatus in the way that fits aimed purposes. Moreover, cells can be embedded in hydrogel which provides a cell-matrix interaction and a tissue-mimicking stiffness for the cells, simulating more in vivo like environment.


Next generation solution for reproducible and scalable animal-free 3D cell culture models

GrowDex® is an animal-free hydrogel which helps researchers to take next steps towards reproducible assays and sustainable 3D cell culturing. Compatibility with HTS and HCS applications gives freedom to leave the tedious manual work for automatized liquid handling robots. Read more about how you can transfer your 3D cell culture matrices to animal-free GrowDex.

Transfer animal free with GrowDex


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