Hello Atchara A.

It seems that the extract may be interfering with the MTT assay, potentially reducing MTT directly, or affecting cellular processes that influence MTT reduction. This could lead to a higher absorbance value than expected even when the fibroblasts are not healthy (shrunk morphology). If an extract causes an increase in absorbance in MTT assay, it is essential to run a control with the extract and MTT reagent without cells present. This control will help determine if the extract itself is directly reducing MTT, which would lead to false-positive results indicating cell viability.

It seems that changes in morphology even with a higher MTT absorbance can indicate that the extract is affecting fibroblast in ways not fully captured by the MTT assay. So, to get a comprehensive understanding of the extract’s effect, it is essential that you perform other cytotoxicity assays like the alamar blue assay or MTS assay.

The very fact that fibroblasts have shrunk in morphology, means that the extract is cytotoxic to cells and has the potential to damage or kill cells. This shrinkage can be a visual sign of cellular damage or cell death pathways like apoptosis or necrosis being triggered.

So, in addition to the cell proliferation assay, you should also work on apoptosis assay.

Best,

Fibroblast cells can exhibit significant shape changes even while maintaining high survival rates due to their inherent plasticity and dynamic response to environmental cues. These morphological alterations are often part of normal cellular physiology rather than indicators of compromised viability. Several factors contribute to this phenomenon: fibroblasts naturally undergo cytoskeletal remodeling during processes like migration (where they elongate to move through extracellular matrix), adhesion (adjusting their spread area based on substrate stiffness), and cell division (temporarily rounding up during mitosis). The cellular microenvironment plays a crucial role, as variations in substrate stiffness, extracellular matrix composition, or soluble factors like growth factors can all trigger shape changes without affecting cell survival. Experimental conditions frequently induce morphological responses - trypsinization causes temporary rounding that typically reverses after replating, while culture confluence may force cells to elongate due to spatial constraints. Importantly, fibroblasts can transition between different functional states, such as differentiating into myofibroblasts with characteristic flattened, stress fiber-rich morphologies when exposed to factors like TGF-β, while remaining fully viable. Non-lethal stress responses, including mild oxidative stress or mechanical disturbances from culture handling, can also alter cell shape through cytoskeletal reorganization without reaching thresholds that compromise viability. To properly interpret these observations, researchers should employ live-cell imaging to track temporal patterns of morphological change, perform cytoskeletal staining to examine actin or tubulin organization, and confirm viability through multiple complementary assays. The key insight is that fibroblast morphology serves as a sensitive indicator of cellular state and microenvironmental conditions, with shape changes often representing adaptive responses rather than signs of toxicity or pathology, especially when viability remains high. This plasticity is fundamental to fibroblasts' biological functions in wound healing, tissue remodeling, and maintaining structural integrity throughout the body.