Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.