Table 1.

Technical Differences in the Generation of Precision-Cut Lung Slices

Step	Property	Variation	Notes/Examples
Lung filling	Agarose (%)	• 0.4–1.5 (36, 57, 72)	• Lower percentages are used in rodents, and larger ones are used in human tissue and bigger animals
		• 1.5–3 (15, 46)
			• The percentage of agarose may affect the mechanical properties of the filled tissue
	Media	• Salt solutions: PBS, EBSS, and HBSS (33, 73, 74)	• Some protocols add more supplements to the filling media, such as HEPES, penicillin/streptomycin, and Amphotericin B
		• Cell culture medium: (D)MEM, DMEM/F-12, RPMI) (48, 72)	• The choice of filling media may influence the quality and survival of specific cell types within the PCLS
Lung slicing	Media	• Salt-buffered solutions (16, 73, 75, 76)	• Most protocols use culture media for slicing with no alterations
		• Cell culture medium (15, 48, 57, 77)	• Some protocols supplement media with CaCl2, MgSO4, KCl, NaCl, NaH2PO4, glucose, NaHCO3, HEPES, penicillin/streptomycin, insulin, hydrocortisone, retinyl acetate, gentamicin, or Amphotericin B
	Devices	Tissue slicers (rotary chopping) (29)	• Krumdieck Slicer
			• Compresstome VF-300
			• Brendel Vitron Tissue Slicer
		Vibratome (74)	• Leica Vibratome VT1000
			• Zeiss Hyrax Vibratory Microtome V55
			• Campden 7000 SMZ-2
	Thickness	• 150–225 μm (12, 74)	• Thinner PCLS are generally obtained from rodent tissue and allow for easier nutrient perfusion
		• 250–500 μm (24, 48, 72, 73, 78)
			• Thicker PCLS are generally obtained from larger animal tissues or human tissue
			• No perfusion/diffusion problems have been reported for PCLS 500 μm thickness or less
			• Thicker PCLS allow for visualization of full alveolar spaces, improving the modeling aspects of specific diseases
			• The thickness of PCLS can vary for different CLDs
			• Slice thickness is usually optimized differently in each laboratory based on the research question
Culture	Preparation	Agarose removal (79)	• Some protocols attempt to remove agarose from PCLS by repetitive washes within the first few hours after slicing
			• Agarose removal may allow for easier access to nutrients
		Pretreatment	• Most protocols replace media with varying initial incubation periods
			• Washing may be important for removal of secreted factors that may have been caused by the slicing (“cytokine storm”)
	Media	(D)MEM, Ham’s F-12, RPMI, Medium 199, EBSS, HBSS	• Several protocols use the same culture media during the procedure
			• Additional supplements may include CaCl2, MgSO4, KCl, NaCl, NaH2PO4, glucose, NaHCO3, HEPES, penicillin/streptomycin, insulin, hydrocortisone, retinyl acetate, gentamicin, or Amphotericin B
			• The media needs to be optimized depending on the modeled disease
	Serum	• None	• Most protocols do not add serum to cultured PCLS because it might alter cell proliferation
		• 0.1% (48)
		• 10% (72)
	Length	Minutes to a few hours	• Earlier studies used only short incubation times to study metabolites
		1–7 d	• Most current PCLS models have shown viability across the different cell types for up to 7 d. Most of the discussed models use durations within this range
		>21 d (68, 80)	• Few studies have reported PCLS culture for up to 28 d. More recently, embedding in hydrogels was shown to extend culture times to >21 d

Definition of abbreviations: CLDs = chronic lung diseases; DMEM = Dulbecco’s modified Eagle medium; EBSS = Earle’s balanced salt solution; HBSS = Hanks’ balanced salt solution; PCLS = precision-cut lung slices.