Table 2. Co-culture paradigms used to model non-cell autonomous aspects of neurological disorders.

Various co-culture systems and media transfer paradigms can be used to model non-cell autonomous aspects of neurological disorders. The utility as well as advantages and disadvantages are listed for each system. Considerations for each system and exemplary references are provided.

	System	Applications	Advantages	Disadvantages	Considerations	Ref.
Co-cultures	Classic	Assess contact-dependent and contact independent interactions	Easy, affordable, well established	Not possible to discern contact dependent and independent interactions	Density of both cell types needs to be optimized, media composition needs to be permissive for both cell types; media composition, starvation, contact inhibition can alter the outcome of the experiment	86,141,145,158,160,206,207
	Sandwich co-culture		Allows separation of cell types for consequent RNA/protein isolation	More complicated to set-up, increased risk of contamination due to additional equipment. Not possible to discern contact dependent and independent interactions	Glass coverslip and paraffin need to be properly sterilized; glass coating substance can influence gene expression; density of both cell types needs to be optimized, media composition needs to be permissive for both cell types; media composition, starvation, contact inhibition can alter the outcome of the experiment	162,208
	Transwell or Insert	Assess contact-independent interactions	Allows separation of cell types for consequent RNA/protein isolation; continuous interaction/secretion of factors	Transwells and Inserts are expensive and usually not re-usable	Density of both cell types needs to be optimized, media composition needs to be permissive for both cell types; media composition, starvation, contact inhibition can alter the outcome of the experiment	164,206,209
	2-sided seeding Transwell	Assess permeability of soluble factors through a barrier forming cell type	Easy to set up	Transwells and Inserts are expensive and usually not re-usable	Proper density is crucial for the success of the experiment	164,209
	Microfluidic chambers	Assess axonal growth or transport, myelination, neuromuscular junctions, permeability of barrier forming cells	Fewer cells needed and reduced reagent consumption, real time analysis possible, automation possible	Complicated setup, less standardized, expensive	Medium turnover, proliferation rate and gas exchange are different in these devices and need to be monitored and optimized	164–168,209–213
	Multi-cell co-culture	Assess interaction of multiple cell types in combination	Easy to set up, closer to in vivo situation	Not possible to discern contact dependent and independent interactions. Difficult to determine responsible cell type	Density of individual cell types need to be optimized, media composition needs to be permissive for each cell type. Media composition, starvation, contact inhibition can alter the outcome of the experiment	170,214–216
Conditioned Media Transfer	From mono-culture	Assess contact-independent effects of soluble factors	Easy to set up, well established straight-forward interpretation of origin of influencing factors.	Does not recapitulate real physiological state, many effectors are only released upon activation or cell-cell contact and will be missed in this setup	Cell density and coating is crucial; media composition can alter the outcome of the experiment; depending on the transfer-protocol and receiving cell-type, supplementation with additional growth factors or serum should be considered	62,80,86,207
	From co-culture		Easy to set up, well established, more physiological	Cell type of interest is needed in larger amounts (2 parallel co-cultures) Interpretation more difficult since different cell types can be responsible for the secretion of molecules	Cell density and coating is crucial; media composition can alter the outcome of the experiment; depending on the transfer-protocol and receiving cell-type, supplementation with additional growth factors or serum should be considered	126,208
	Microfluidic chamber		Fewer cells needed and reduced reagent consumption, real time analysis possible, automation possible	complicated setup, expensive	Medium turnover, proliferation rate and gas exchange are different in these devices and need to be monitored and optimized	164–168,209–213