Yes, the morphological change you're observing in 3T3-J2 cells, from a more epithelial-like to a more elongated, fibroblastic appearance over passages, is a known and common phenomenon with this cell line, and several factors could be contributing to it in your case.

Firstly, 3T3-J2 cells are fibroblasts by origin, and under certain conditions, especially after thawing or during early passages, they may initially appear more flattened or epithelial-like due to stress or adaptation to culture. As they recover and proliferate through subsequent passages, they often revert to their typical spindle-shaped, fibroblastic morphology, which is actually their more natural phenotype.

Secondly, enzymatic detachment conditions, especially the prolonged 15-minute TrypLE exposure without PBS washing, could have stressed the cells or affected surface proteins involved in cell adhesion and morphology. TrypLE is gentler than trypsin/EDTA but still can be overly harsh if used too long or without pre-washing to remove serum inhibitors. This initial stress might have temporarily altered their morphology. When you switched to trypsin/EDTA with a PBS wash, you likely provided a more optimal and quicker detachment, allowing cells to recover and spread normally, displaying their characteristic elongated form.

Lastly, cell density and confluency also influence morphology. At low density, 3T3-J2 cells tend to spread out more and appear elongated. At higher confluence, they may look more compact. So, passage-related changes in seeding density could also explain part of the visual shift.

In summary, the elongated, more fibroblastic morphology you're now seeing is likely the normal phenotype of healthy, actively proliferating 3T3-J2 cells, emerging after the cells recovered from the thawing stress and suboptimal initial detachment conditions.

Hi Muhammad Waqas Thank you for your sharing.

I seeded 1.3 × 10⁶ 3T3-J2 cells in a T75 flask. After 7 days, the culture reached approximately 80% confluency, but the cell count was only 1.8 × 10⁶. Is it normal for the cells to not even double in number over a week?

It seems that they are not fast growing in my case.

Dear Nur Mohd Faizal ,

Given your seeding of 1.3 × 10⁶ 3T3-J2 cells in a T75 flask and only reaching 1.8 × 10⁶ cells after 7 days (despite ~80% confluency), it's understandable to be concerned about the slow growth. However, this behavior isn't entirely unusual under certain conditions, especially with 3T3-J2 cells, which are known to be contact-inhibited and sensitive to culture conditions.

Several customized factors likely explain your observation. First, 3T3-J2 cells have a strong tendency to plateau in proliferation as they approach confluency, due to contact inhibition, even if they don’t fill the entire surface area. This means that at around 70–80% confluency, growth may slow significantly or stop altogether, which could explain the minimal increase in total cell number. Second, the apparent discrepancy between confluency and cell number could be due to cell morphology changes, as you've observed, the cells become more elongated and fibroblastic over passages, spreading more and covering more surface area per cell, which gives the illusion of higher confluency without a proportional increase in cell number.

Additionally, the initial recovery from thawing could have led to a lower proliferation rate during the first passages. Slow growth is often seen in early passages post-thaw, especially if the cells experienced stress during detachment or suboptimal enzymatic treatment, as in your first passage with prolonged TrypLE exposure. Also, if the serum quality, medium freshness, or passage technique wasn't optimal, that could further suppress growth.

In summary, yes, it can be normal for 3T3-J2 cells to show minimal doubling over a week under your described conditions. It's a combination of contact inhibition, post-thaw adaptation, and possibly cell spreading affecting perceived confluency. Monitoring medium pH, ensuring high-quality serum, optimizing seeding density (perhaps slightly lower), and limiting time near confluency can help improve proliferation in future passages.

Hi, Muhammad Waqas , I have seen that after few passaging, the cells started to grow in patches. and the morphology changed. Have you ever encountered something like this before?

Hi, Nur Mohd Faizal

Yes, based on the image you shared, I can confirm that I have also observed similar changes in 3T3-J2 cultures after a few passages, where the cells begin to grow in uneven patches with noticeable morphological variation. In your image, the culture shows regions of dense clustering alongside areas with sparse or no growth, and the cells exhibit a mixed population of elongated, fibroblast-like cells and more flattened, irregular forms, suggesting cellular stress or heterogeneity. This patchy growth and shift in morphology are often signs of phenotypic drift or culture instability, commonly seen in fibroblast lines after extended passaging or suboptimal culture conditions.

Muhammad Waqas what media and serum did you use in your culture? Does this mean the cells are not healthy and should not be further expanded?