Production of decellularized porcine lung scaffolds for use in tissue engineering

Abstract

There is a growing body of work dedicated to producing acellular lung scaffolds for use in regenerative medicine by decellularizing donor lungs of various species. These scaffolds typically undergo substantial matrix damage due to the harsh conditions required to remove cellular material (e.g., high pH, strong detergents), lengthy processing times, or pre-existing tissue contamination from microbial colonization. In this work, a new decellularization technique is described that maintains the global tissue architecture, key matrix components, mechanical composition and cell-seeding potential of lung tissue while effectively removing resident cellular material. Acellular lung scaffolds were produced from native porcine lungs using a combination of Triton X-100 and sodium deoxycholate (SDC) at low concentrations in 24 hours. We assessed the effect of matrix decellularization by measuring residual

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine

“…For pig lungs, the accessory lobe was dissected away and the lobar bronchus and artery were cannulated. The accessory lobe, which is the smallest of the seven lobes in the pig lung, makes for a convenient model system for decellularization studies due to its size (22-24). …”

“…The decellularization process is described previously, with the addition of a benzonase rinse at the end of the decellularization process (24). Briefly, tissues were perfused using a gravity feed at approximately 30-40 cm H 2 O pressure.…”

“…Samples were then stored overnight in 70% ethanol, embedded in paraffin, and sectioned at 5 μm. Tissue slides were stained for hematoxylin and eosin (H&E), Masson's Trichrome staining (Trichrome), or Verhoff's Van Gieson (EVG) as described previously (24). For immunofluorescence, section were blocked with PBS containing 3% BSA and 0.2% Triton X-100 for 45 minutes, and subsequently incubated in primary antibody against PCNA (Abcam, ab29, 1:1000) overnight at 4°C.…”

“…Absorption of the samples were read at 550 nm and run against a heparin standard (Sigma). Sulfated GAGs were quantified using the Blyscan GAG assay kit according to manufacturer's instructions (Biocolor) (24). Content of total and sulfated GAGs were normalized to tissue dry weight.…”

“…Fixation was done using 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2M Sorensen buffer at pH 7.4 for 30min at room temperature and 1hr at 4°C (24). Following dehydration, samples were infiltrated and embedded in Embed-81, polymerized, and sectioned at 70 nm.…”