Have you done a quick comparison between the morphologies of your fibroblasts and published images of dividing HFFs?

From your description, I would guess that your cells may be senescent or at least near enough to senescence to require over a week before a division. You could try a beta-galactosidase assay (See Debacq-Chainiaux F. et al, Nature Protocols, 2009) on your cells to check.

The problem might also be with your serum, or seeding density. In my experience, fibroblasts are quite prone to contact inhibition, so a high population at the start will not grow much if at all.

Good luck.

Dear Anthony Ovie Abraham,

I appreciate your response. But, your answer to this problem is not satisfying.

1. "Quick comparison between the morphologies of your fibroblasts and published images of dividing HFFs. "

A. The morphological comparisons of the Human fetal fibroblast (Dermal) was performed. Time elapse photography was also done. No morphological dissimilarities were observed.

2. "I would guess that your cells may be senescent or at least near enough to senescence."

A. I have clearly mentioned that: a) The cells were cultured after freeze thaw from Working Bank. b) Moreover, the counting performed with Trypan Blue did not show dead cells. Hence, your statement do not match with my observations.

3. " You could try a beta-galactosidase assay"

A. I think I should not waste my time because, the cells do not die in my culture. They just refuse to grow.

4. " The problem might also be with your serum, or seeding density "

A. I have clearly mentioned in my problem that the media and its ingredients were evaluated (a battery of methods). Moreover, the seeding density was appropriate because I had my pipettes and hemocytometer calibrated and standardized prior to each test as mentioned in my problem.

5. "High population at the start will not grow much."

A. I cannot agree with this statement.

The cells that refused to grow was seeded only at 3000 cells/cm2. Whereas I had expected yields with cells seeded at 5000 and 10000 cells/cm2. I had observed and experienced that, the lower the cell seeding density, the lower the cell yield. This is due to non efficient cell signalling because the cells are far apart.

But, if low cells are seeded in low surface area flasks, the normal is their yield rate (due to less space in flask, cells grow adjacent to each other with high cell signalling).

In my case, the lower the cell seeding density, the yield is similar to the cell seeding density.

I humbly request you to first understand the problem in every bit of its concern before giving suggestions.

Dear K. Amaldev,

before you accuse people of not understanding the problem, you should maybe carefully read/check what they are saying!

2. "I would guess that your cells may be senescent or at least near enough to senescence."

A. I have clearly mentioned that: a) The cells were cultured after freeze thaw from Working Bank. b) Moreover, the counting performed with Trypan Blue did not show dead cells. Hence, your statement do not match with my observations.

"Cellular senescence is one phenomenon by which normal cells cease to divide. In their seminal experiments from the early 1960's, Leonard Hayflick and Paul Moorhead found out that normal human fetal fibroblasts in culture reach a maximum of approximately 50 cell population doublings before becoming senescent."

[Hayflick, L., and Moorhead, P. S. (1961). "The serial cultivation of human diploid cell strains." Exp Cell Res 25:585-621.]

There is absolutely NO link between cellular senescence and cell death. Therefore, your statements about the viability of your cells are not related to the suggested problem at all.

In conclusion, you can not exclude what Anthony Ovie Abraham suggested.

Dear Philip Wolfgang,

Thank you for the suggestion. I have already gone through the above mentioned literature prior to posting the problem here.

I understand the terminologies.

I beg pardon to you and Anthony Ovie Abraham to not get offended in my criticism. Afterall, science is to criticize and I criticize science and not persons.

Your suggestion to the problem seems doubtful to me for the above mentioned reasons:

1. Primary Human fetal fibroblasts are obtained from repository after their first passage (P1)- two doublings.

2. These cells were then subjected to banking (P4)- 12 doublings.

3. It is from the working bank (P4) which when subjected to revival doesn't grow.

Cell doublings were always considered. Hence, all the working banks were maintained below 12 doublings.

When the working banks were utilized in the experimental setup, cells were always maintained below 20 doublings to get optimal results and to omit the cellular senescence (Hayflick and team).

So, in my experimental design, cells had a maximum of 20 doublings in total.

If your proposition on cellular senescence is agreeable, "then the cell repositories may have provided cells at higher passage level with high doublings" This is a crime of fraud and forgery. If it is so, the cells should lose its primary cellular qualities and show differentiation. But, there were no differentiated cells as visually observed.

f your proposition on cellular senescence is not considered, "then the cells have not reached senescence phase" as per my doubling calculation at each step.

Now, what really went wrong? I am breaking my head to troubleshoot this. Please give alternate suggestions if any.

Thank you all for your suggestions. I have cracked and pinned down the problem. Thanks for the discussions.