Preparation and analysis of cutaneous primary myofibroblast three-dimensional culture: an experimental system for myofibroblast deactivation studies.

Myofibroblasts are activated fibroblasts involved in tissue repair and cancer that are characterized by de novo expression of alpha smooth muscle actin (?-SMA), increased secretion of growth factors and immunoregolatory fenotype. It is known that myofibroblasts form clusters during cancer, fibrotic tissue and wound healing. At the end of wound healing, myofibroblasts undergo apoptotic cell death, whereas in vitro fibroblasts undergo a necrosis-like programmed cell death, called nemosis, associated with inflammatory response. As primary myofibroblasts better resemble the tissue environment in vivo, in this work we generated spheroids from human primary cutaneous myofibroblasts to evaluate apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The study showed that spheroid formation does not trigger apoptosis, necrotic cell death and cyclooxygenase-2 (COX-2) protein induction associated with inflammatory response. The significant decrease of ?-SMA in protein extracts of spheroids, compared to myofibroblasts monolayer, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is supported by cytostatic effect exerted by spheroid-conditioned medium on both normal and cancer cell lines, by anti-migratory effect on normal cells and also by the low number of nuclei positive for the proliferation marker Ki-67. The analysis of spheroid reversion to monolayer growth demonstrated that fibroblasts cultured as aggregates preserved their proliferation capability. Surprisingly, we obtained spontaneous clusters by seeding myofibroblasts on plastic and glass substrates: thus the cluster formation could be a physiological feature of cutaneous myofibroblasts. This work represents an experimental model to study myofibroblast deactivation and highlights the possible relevance of clusters as reservoir of myofibroblasts for regulating myofibroblast and tissue turnover.