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.

Current possibilities and future opportunities provided by three-dimensional lung ECM-derived hydrogels

“…In addition, viscosity and Young's modulus of porcine-sourced lung hydrogels were reported using parallel Measuring the mechanical properties is not only useful for diseased environment characterization, but also for verification of the success of methodologies designed to alter such properties. While it is clear that the use of different concentrations of the starting ECM material (powder) (Pouliot et al, 2016) and adjusting the pepsin digestion duration (the essential step in generation of a pre-gel ECM-derived substrate) (Pouliot et al, 2020) influences the mechanical properties, one of the initial attempts to specifically modulate the mechanical properties of lung ECM-derived hydrogels was treating the porcine lung ECM with genipin to increase the stiffness (Link et al, 2017). This approach has been extended with thiol-functionalization (Petrou et al, 2020;Saleh et al, 2022), alginate-reinforcing (De Santis et al, 2021) or fibre crosslinking (Nizamoglu et al, 2022a) to allow greater control over mechanical parameters in the lung ECM-derived hydrogels.…”

“…Initial studies using porcine lung ECM-derived hydrogels reported successful growth of human and rat mesenchymal stromal (stem) cells (MSCs) in 2016 (Pouliot et al, 2016). Link et al (2017) then described successful culture of mouse MSCs, human alveolar epithelial cells (the cell line A549), human primary microvascular endothelial cells (HpuVECs), and human umbilical vein endothelial cells (HUVECs) in porcine lung ECM-derived hydrogels. The field is now rapidly expanding with additional cells types including murine fibroblasts (Petrou et al, 2020), rat lung MSCs (Falcones et al, 2021) and rat primary alveolar epithelial cells (Marhuenda et al, 2022b) being grown in porcine lung ECM-derived hydrogels.…”

Using extracellular matrix derived from sugen-chronic hypoxia lung tissue to study pulmonary arterial hypertension

“…We decellularized lung tissue based on the same procedure described in (Link et al, 2017). We obtained healthy and Su/ CHx left lung lobes from rats from a prior study (Bhagwani et al, 2019).…”

Characterizing the viscoelastic properties of different fibroblasts in 2D and 3D collagen gels

“…Hence, cell-to-cell interaction as well as with its surrounding is vital for cell mechanical properties development. Cell behavior assessment surrounded by its natural ECM is desirable; otherwise artificial hydrogels mimicking ECM composition and mechanics offer a good compromise 23,[26][27][28] . 3D hydrogels provide physical constraints forcing the cells to adapt morphologically as well as chemically and biologically to an environment similar to that in tissue.…”