Table 2.

Overview of the various types of co-culture approaches employed to investigate cardiovascular diseases.

Type of Co-Culture	Description	Advantages	Limitations	References
Direct co-culture	Various cell types are seeded in the same culture dish, which allows cell-to-cell communication via gap junction, adherens, and paracrine signaling.	Able to analyze contact-based and non-contact-based cellular interactions. Simple, easy, and cost-effective way to culture.	Difficult to achieve equal amounts of cellular densities of both the cells studied. One cell type could grow fast/slow and might not mimic the exact vascular environment. Culture media used should be adaptable for both the cell types used.	[123,124]
Direct co-culture with trans-well	Different cell types are seeded on the upper and lower sides of the porous trans-well inserts.	Direct cell–cell contact allows for study of the physical contact interactions between the cell types. Can demonstrate cell adhesion, permeability, and migration towards the other cell type. Different culture media can be used for the different cell populations across the trans-well.	Trans-well membranes are expensive, and they cannot be reused. There is no difference whether the pathological condition developed is based on contact-dependent or contact-independent signals.	[125,126]
Indirect co-culture, trans-well based	Two cell types are cultured in different chambers of the trans-well membrane, and the distance between them allows communication only through soluble factors in the culture media.	Can be used to study cell–cell interactions, drug permeability, and drug transport.	Cellular communication restricts to soluble secretions (growth factors, cytokines, and extracellular vesicles) alone and lacks signaling through physical contact to mimic in vivo environment. Expensive.	[98,127,128]
Conditioned media-based indirect co-culture	Cell secretions of one cell type (conditioned media) when transferred to another cell type, which can modulate the cell behavior.	Easy to establish and provides secretory factors to modulate the other cell type. Conditioned media can be frozen and can be used on other cell type later.	Unidirectional. It is used to study only secretory factor-based signaling and lacks contact signaling.	[129,130]
3D co-culture, scaffold based	Encapsulating different cell types in a 3D scaffold, which can provide topography and mechanical stimulus needed reflecting physiological microenvironment.	Mimics more of an in vivo condition and allows for the study of cell morphology, behavior, function, cell–cell contact signaling, and paracrine signaling. Recapitulates the vascular microenvironment realistically. Multiple cellular interactions (both physical and secretory) are feasible.	Expensive. Needs more time to optimize the culture. Proteolytic separation of a single layer of cells is difficult. Repeatability of the experiments is difficult.	[131,132]
Microfluidics-based co-culture	Dynamic fluid manipulation system designed for micrometer sized channels. It mimics physiological microenvironment, which can culture multiple cell types.	Regulation of signal gradients and perform simulation of physiological microenvironment such as shear stress. Reliable platform for drug screening and vascular modeling.	Needs external devices like pumps, connectors, and valves to function. Difficult to optimize and repeat the experiments. Expensive.	[133]
Organoids-based co-culture	Self-organizing 3D cellular structures that can recapitulate organs (cardiac organoid–endothelial cells, cardiomyocytes, fibroblasts, etc.).	Modeling cardiogenesis, drug screening, and testing and also in tissue engineering.	Difficult to optimize. Hard to reproduce. Less or insufficient vascularization limits the applicability of cardiac organoid.	[134,135]