Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures

Abstract

Standard cell cultures are performed in aqueous media with a low macromolecule concentration compared to tissue microenvironment. In macromolecular crowding (MMC) experiments, synthetic polymeric crowders are added into cell culture media to better mimic macromolecule concentrations found in vivo. However, their effect on cultured cells is incompletely understood and appears context-dependent. Here we show using human gingival fibroblasts, a cell type associated with fast and scarless wound healing, that MMC (standard medium supplemented with Ficoll 70/400) potently modulates fibroblast phenotype and extracellular matrix (ECM) composition compared to standard culture media (nMMC) over time. MMC significantly reduced cell numbers, but increased accumulation of collagen I, cellular fibronectin, and tenascin C, while suppressing level of SPARC (Secreted Protein Acidic and Cysteine Rich). Out of the 75 wound healing and ECM related genes studied, MMC significantly modulated expression of 25 genes compared to nMMC condition. MMC also suppressed myofibroblast markers and promoted deposition of basement membrane molecules collagen IV, laminin 1, and expression of LAMB3 (Laminin Subunit Beta 3) gene. In cell-derived matrices produced by a novel decellularization protocol, the altered molecular composition of MMC matrices was replicated. Thus, MMC may improve cell culture models for research and provide novel approaches for regenerative therapy.

Novel decellularization method to generate cell-free extracellular matrices from three-dimensional human gingival fibroblast cultures

“…Cultures were then trialed with various concentrations (0.2-5 μM) and incubation times (1-4 hours) of latrunculin B, before subsequent incubations with sodium deoxycholate and DNase (see below). Phase contrast microscopy, staining of DNA by DAPI, and immunostaining of actin and b-tubulin showed that incubation with 0.6 μM latrunculin B for 3 hours 15 followed by standardized treatment with deoxycholate and DNase 19 were effective in removing cellular and cytoskeletal elements and DNA from the cultures 15 . The following decellularization protocol has been tested with gingival broblasts cultured up to 14 days as above.…”

“…This decellularization method has been used in a variety of contexts including primary human dermal and lung broblast cultures, cancer-associated broblast cultures from esophageal, ovarian, renal, pancreatic, prostatic and lung tumors, and murine mammary broblasts 10,12,13 . In addition, we have used this method to decellularize 3D cultures of human gingival and skin broblasts 14,15 . To this end, primary broblasts were seeded at a high density (25,000 cells per cm 2 ) and cultured for 7 days, as originally described for mouse NIH-3T3 cells, to generate 3D cultures 11 .…”

“…Analysis of the cultures showed that the cells had formed 3-5 cell layers and were surrounded by their own ECM at day 7 14,15 . However, immunostaining of the decellularized cultures for cytoskeletal actin and b-tubulin showed that the decellularization protocol was unable to remove these cytoskeletal elements completely 15 . Therefore, decellularization methods need to be tailored distinctly for different cell types.…”

“…Functionally, rat aortic artery CDM yielded via the LAT-D method demonstrated accelerated repopulation following transplantation compared to the DET-D method 16 . Therefore, we developed a novel decellularization method of human gingival broblast 3D cultures that uses sequential incubations with latrunculin B as an actin destabilizing agent 18 , deoxycholate as a mild detergent 19 , and DNase to remove cellular elements, including cytoskeletal actin and b-tubulin, and DNA from the cultures 15 .…”

Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

“…Use of Ficoll 70 and 400 at 37.5 mg/mL and 25 mg/mL, respectively, results in an estimated fractional volume occupancy (Ψ) of 17% in medium of cultured human broblasts or MSCs 7,13 , and in uences composition and organization of the broblast ECM niche in a tissue-type speci c manner 7,8,[13][14][15][16][17][18][19] . We have recently adapted the use of MMCs (Ficoll 70/400) to cultures of human gingival broblasts 20 . The following describes the detailed methods to generate these cultures.…”