Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Abstract

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

Electrosprayed extracellular matrix nanoparticles induce a pro‐regenerative cell response

“…We expect a much faster degradation in the presence of enzymes produced by cells. The efficiency of turning milled PLECM into nanoparticles is over 30% (data not shown), making it possible to creatẽ 6 g of nanoparticles from pig lungs-estimated 10 g of decellularized ECM from one lung, two lungs per animal (Link et al, 2017).…”

“…We decellularized porcine lung according to methods published by Link, Pouliot, Mikhaiel, Young, and Heise (2017) and Pouliot et al (2016). In brief, we used Triton X-100 (0.1% v/v), followed by 2% sodium deoxycholate, then 1 M NaCl, and finally DNase (12,000 units/L with 0.156 g of MgSO 4 and 0.222 g of CaCl 2 ).…”

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

“…Milling is another important step that may lead to loss of material. Others have shown success with a cryogenic mill, but this protocol is based on milling via a handheld mortar and electric drill with a pestle attachment 8,17 . This has the advantage of lower capital costs and minimizing material loss but may introduce variability in the final product.…”

“…NOTE: This procedure was adapted from previously published methods focused on adipose decellularization, which included the sodium deoxycholate ionic detergent rather than sodium dodecyl sulfate to remove DNA efficiently 12,16,17 .…”

Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix

“…Male and female porcine lungs were obtained from Smithfield-Farmland slaughterhouse or euthanized research pigs to produce dECM coating solution as previously published by our laboratory [48,69]. Quality control of dECM powders from different donors is performed by picogreen dsDNA quantification to ensure each has below 50 ng of dsDNA per mg of dry tissue.…”