Agarose-Based Preparation of Human Precision-Cut Lung Slices

Published: November 30, 2023

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 tub…

Divulgazioni

The authors have nothing to disclose.

Materials

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

Play Video

Citazione di questo articolo
Agarose-Based Preparation of Human Precision-Cut Lung Slices. J. Vis. Exp. (Pending Publication), e21847, doi: (2023).

View Video