A review of co-culture models to study the oral microenvironment and disease

Figures & data

Figure 1. (a) Common bacterial species present in pathogenic oral biofilms and their communication between species (adapted from Parashar et al. [92]). (b) Cells and tissue types present in the oral mucosa, demonstrating complexity of 3D structure.

Table 1. Summary of co-culture methodologies, common protocols employed, and the advantages and disadvantages of each model system.

Method	Summary of protocol	Advantages	Disadvantages	References
2D monospecies co-culture with planktonic bacteria	Seed eukaryotic cells into well plate. Culture until confluent monolayer formed, with media changes every 1–2 days. Prepare bacteria overnight culture. Centrifuge overnight and re-suspend bacteria in eukaryotic cell culture media to achieve desired concentration. Add media containing bacterial suspension to monolayers and perform assays at desired time points.	Can use simple assays to investigate Can attribute direct cellular responses from interactions with bacteria Reproducible with reduced batch-to-batch variation Supports homogenous growth All cells have equal access to nutrients	Not representative of in vivo tissue structure Does not account for immune cells Does not account for many cues found in vivo, including mechanical signalling Cannot monitor interaction between cell types, in particular, the immune system Does not represent the complex bacterial biofilms present in the oral cavity	[15–17,19,33,34]
2D multispecies co-culture with planktonic bacteria	Seed appropriate ratio of eukaryotic cells into well plate. Culture until confluent, with media changes every 1–2 days. Prepare bacteria overnight culture. Centrifuge overnight and re-suspend bacteria in eukaryotic cell culture media to achieve desired concentration. Add media containing bacterial suspension to cell culture and perform assays at desired time points.	Can monitor the interaction between cell types Reproducible with reduced batch-to-batch variation Supports homogenous growth All cells have equal access to nutrients	May require optimisation due to different nutrient requirements Not representative of in vivo tissue structure Traditional assays cannot always determine between cell species Does not account for many cues found in vivo, including mechanical signalling Does not represent the complex bacterial biofilms present in the oral cavity	[29,30]
2D co-culture with biofilm	Seed appropriate ratio of eukaryotic cells into well plate. Culture until confluent, with media changes every 1–2 days. Prepare bacteria overnight culture. To form biofilm, seed overnight culture onto coverslips placed in the bottom of a well plate. Change media every 1–2 days. At chosen time point, once biofilm has formed, remove media and attach coverslip to base of transwell insert. Place insert into cell-culture plate. Perform assays at desired time points.	Can monitor the interaction between cell types Reproducible with reduced batch-to-batch variation Supports homogenous growth All cells have equal access to nutrients More clinically relevant, as biofilms show increased antibiotic resistance to planktonic cultures.	Bacteria can overrun eukaryotic cells if co-culture system is not carefully designed	[46,55–57,66,67]
3D tissue engineered co-culture with planktonic bacteria	For collagen-based system, mix fibroblasts with collagen gel and pipette into transwell inserts. Set gel in incubator at 37°C for 1 hr. Seed epithelial cells onto surface of gel. Seed monolayer of epithelial cells into separate well plate to monitor confluence. Culture cells until confluent monolayer formed. Raise cells to air-liquid interface and culture for 7–10 days to allow stratified epithelium to form. Prepare bacteria overnight culture. Centrifuge overnight and re-suspend bacteria in eukaryotic cell culture media to achieve desired concentration. Add media containing bacterial suspension to 3D cell culture and perform assays at desired time points.	More representative of in vivo environment Can study cell-cell signalling Two mucosa models well established in literature – collagen-based and decellularised matrix	Can be challenging to achieve cell numbers required for multiple models Require specifically enriched media Significant optimisation may be needed More resource-intensive More difficult to produce replicates Models may not be fully representative of native tissue structure Does not represent the complex bacterial biofilms present in the oral cavity	[41–44,46,47]

Figure 2. Common co-culture systems reported in the literature (a) monospecies 2D cell culture with planktonic bacteria applied; (b) multispecies 2D cell culture with planktonic bacteria applied; (c) multispecies 3D cell culture, typically a collagen-based or decellularised matrix containing fibroblasts, with planktonic bacteria applied; and (d) monospecies 2D cell culture with biofilm applied, typically suspended from a well insert.

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