Agarose-Based Preparation of Human Precision-Cut Lung Slices

Abstract

Source: Neuhaus, V. et al., Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices. J. Vis. Exp. (2018)

In this video, we describe a procedure to inflate isolated human lungs with agarose and prepare human precision-cut lung slices. The prepared ex vivo lung slices can be used for further immunologic experiments.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. General Preparation of Human PCLS and Subsequent Exposure to Chemicals

NOTE: Two persons are required to fill a lung. An up-to-date vaccination record for hepatitis A and B is recommended. Patients are routinely screened for HCV and HIV prior to lung transplantation. If an active infection with Mycobacterium tuberculosis is diagnosed or suspected, the lung should be rejected. Nevertheless, all fresh human lung tissue and samples derived from it must be treat....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon hose 3.0 x 5.0 mm	A. Hartenstein (Leipzig, Germany)	SS04
Syringe	Faust Lab Science (Klettgau, Germany)	9.410 050
Coring tools	custom-made	Custom-made
Trimming Blade Handle (FEATHER)	pfm medical ag (Cologne, Germany)	205530001
Trimming Blades (FEATHER)	pfm medical ag (Cologne, Germany)	205500000
Microtome: Tissue Slicer	Alabama R&D (Bad Homburg, Germany)	303400-ADPT	Krumdieck Tissue Slicer (MD6000)
Microtome blade	Wilkinson Sword (Solingen, Germany)	ENR-4027800011506
Tissue culture dishes	Sigma (München, Germany)	Z666246-420EA
Inoculation loop	Copan Diagnostics (Murrieta, USA)	CD176S01
TPP Tissue culture plates 24wells	Sigma (München, Germany)	Z707791-126EA
Cassette	Thermo Scientific (Schwerte, Germany)	1000957
Nunc MaxiSorp flat-bottom	Fisher Scientific GmbH (Hannover, Germany)	44-2404-21
Nunc MicroWell 96-Well	Thermo Scientific (Schwerte, Germany)	260836
Reagents
Agarose, low gelling temperature	Sigma (München, Germany)	A9414-100G
Balanced Salt Mixtures and Solutions, Cell Culture, Classic Media and Salts, Earle's Balanced Salts (EBSS)	Sigma (München, Germany)	E2888-500ML
Penicillin and streptomycin	Lonza (Verviers, Belgium)	17-602E
Dulbecco´s Modified Eagle´s Medium Nutrient Mixture F-12 Ham (DMEM F-12)	Gibco (Darmstadt, Germany)	11039-047	Culture medium
Dulbecco´s Phosphate with Ca and Mg (DPBS)	Lonza (Verviers, Belgium)	BE17-513F