Cultivating cells in 3D as Spheroids and organoids have emerged as a more physiologically relevant model for in vivo cellular responses. These more complex models improve upon traditional monolayer culture of cell-lines and primary cell-based methods with their ability to mimic native cytoarchitecture and to display physiological attributes of the native tissue There are many different 3D culture strategies with varying strengths and challenges, in this article we will explain six popular techniques together with their strengths and weaknesses.
Low binding plates
Low binding plates are similar in format to, e.g., 24 or 96-well plates making the transition easy. Often the low binding well has an additional covalently bound hydrogel layer, preventing cellular attachment to its surface.
Low binding plates can be used to create spheroids (spherical cell aggregates that can be created due to 3D connections of membrane proteins such as integrins and ECM proteins produced by the cells) This simple and affordable technique can be used without any specialized equipment, it has been used for spheroid creation from human mesenchymal stem cells.
Transwell culture
Another plate-based culture is performed by adding porous membrane bottom inserts into existing well creating a trans well cell culture. Cells can be cultured in both chambers, on the insert membrane as well as well bottom. This type of culture is particularly useful for studying effect of secreted analytes which are shared through the membrane between culture chambers. Trans well cell culture can be utilized to investigate processes such as migration abilities95, cytokine secretion or blood-brain-barrier passage.
Hanging drop
A third method using the plate format is the hanging drop 3D culture strategy, where one can use a standard petri dish or specialized plates for hanging drop culture. The basics of the culture work by creating a single cell suspension which is placed in drops on a surface. The surface is inverted, creating drops kept in place by liquid capillary tension forces, The cells are collected at the bottom curve of inverted capillary drop and this way small spheroids are created. The hanging drop technique has been used in drug screening, cancer research and clinical studies.
Gel embedding
If cells are unable or slow to produce extracellular matrix to create spheroids, they can be embedded in hydrogels to create the artificial 3D environment. Hydrogels can have various origins such as animal, algae or synthetic and be permanent, biodegradable or decayable. Gel embedding can be used to produce spheroids of cells that cannot produce aggregates on their own. Biodegradable and decayable hydrogels allow for gradual elimination of artificial support through progression of spheroid culture. One of the most common hydrogels used is Matrigel, which consists of basement membrane extracts, an ECM isolated from a tumor cell line. It has been used as a scaffold for many different 3D cultures, e.g.: for brain organoids originating from induced pluripotent stem cells.
Microfluidics/organ-on-a-chip
Many variations of the microfluidics-based organ-on-a-chip strategy for 3D culture exists. In general, cells are culture in culture chambers often embedded in some type of hydrogel and an adjacent capillary-like media flow is facilitated to mimic an in vivo microenvironment. Additionally, media flow, electric current or oxygen gradient between different compartments can be applied. Microfluidics allows for the spatial influence of fluids in micron-sized channels to control perfusion flow and gradients of signaling molecules, nutrients etc. This enables mimicry of interactions between numerous organs such as the liver, intestine, heart and kidney. Chips of different organs can be interconnected to create a human-on-a-chip. Organ-on-chip 3D cell culture strategy can be used for drug toxicity assessment, studying inflammatory stimuli effects, drug screening and drug efficacy.
Clinostats
Cell culture in a clinostat is facilitated by the gravitational stimulus to enhance cluster formation and nutrient supplementation. The rotation of the clinostat need to be adapted to spheroid size to facilitate uniform distribution of cultured construct. Several different methods for 3D conglomerates creation can be utilized together with clinostat-based culture method. Constructs can be made via spontaneous aggregation from single-cell suspension, can be created by force aggregation using micropattern plates, scaffold material, microcarrier bead and hydrogel-based conglomeration methods can also be used. Clinostat based 3D model strategy has been used for cancer research, developmental research, tissue modelling and drug discovery and toxicology.
Considerations
Depending on the experimental strategy and prerequisites it is essential to choose a well-thought-out 3D culture strategy. It is also necessary to be mindful of the selection of cell types and their origin and decide what results one would like to obtain.
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