I suggest you read some papers from the 70's and 80's when allot of this work was done. It is straightforward to generate an immortalized cell line from fibroblasts. For example, you can simply overexpress SV40 Large T antigen to immoralize the cells, and/or certain combinations of oncogenes, such as c-myc and E1a. Typically, H-ras will also immortalize fibroblasts. Check out this reference - Woo and Poon, Genes & Dev. 2004. 18: 1317-1330

Hallo Sohrab

Cell lines are something and primary cells are something else. you can not establish a cell line from primary cells as far as it would be a healthy cell, not require some kind of activator to continuously grow and divide. the cell lines are mainly from cancer, or transformed with some sort of genes which enables the cell behave like cancer cell, to grow without stop as fare as there is food and service. so if you isolate fibroblast from mouse they will grow nicely for few rounds.. after 5 or 6 splits the cell start to slow down and eventfully die, you have to carefully spit the cells to not let them become dense and out growth.

This what I know. probably others know more

hello Soharb, I agree with Hemin that it's difficult to use primary cell as a cell line that for cell line you need a cancer cell that have the same character to grow without stop for ever as you are supplying it with its medium while the primary cell line is a normal cells that you already isolated it from healthy tissue and need it for a short time that you can make passage for it for about 6 times and as you are continuing passage for the cell you will find that the cell lose some of its physiological characters. and its growth and division will be slow

Im-mortalization of primary cells is possible, but then they become cancer cells or cell lines !!! From your question --- I am not sure if you want to grow them? 3T3 assay, transformation through viruses or loss of p53 ... are few ways ..if you want!!!

Hope this will help,

Rajesh

Hello,

I just finished a work about myoblast immortalization. If you want to obtain fibroblast cell lines, you can immortalize them. It will take you some time if you want to get several mice fibroblast cell lines. You just have to find out in the litterature what would be the best infection process.

Good luck

Dear Soab, to my knowledge primary cultures derived directly from excised tissue while continuous cultures derived from subculture (or passage, or transfer) of primary culture and can be serially propagated in culture for several passages. There are two types of continuous cultures:1) Cell lines which have finite life and that reach the senescence after approximately thirty cycles of division. Cells are usually diploid and maintain some degree of differentiation and 2) Continuous cell lines that can be propagated indefinitely. Generally cells have this ability because they have been transformed (tumor cells, viral oncogenes, chemical treatments). Thus if you have performed some passages of the fibroblasts that you have derived from some tissue mouse you have a Cell line and you can plan your experiments until ten or twelve passages. You must characterize the fibroblasts for the expression of specific markers. Bye!

Hi,

I don't have any experience about this but I was reading the book

" Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications

By R. Ian Freshney" Ed.6th

I believe that it has almost everything you need about to isolation cell tissue and cell cullture.

or you can probably look in this website to get some idea.

http://www.jove.com/search?q=isolation+cell+tissue

You can look up references on the internet that closely match your needs. A couple of articles I read are as follows:

1. Chapter 3-Immortalisation of Primary Cells (Cell Biology and Toxicology. 2001; 17: 231–246.) (Glyn Stacey and Caroline MacDonald)

2. Review: Immortalization protocols used in cell culture models of human breast morphogenesis (T. Gudjonsson, R.Villadsen, L. Rønnov-Jessen and O.W. Petersen)

Thanks to all for sharing your experiences, honestly.

Long time ago people sometimes observed spontaneous immortalisation from primary cultures. One example I know of is the OLN-93 oligodendroglioma cell line which was derived from primary rat brain glial cultures (Richter-Landsberg C, Heinrich M. OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. J Neurosci Res. 1996 Jul 15;45(2):161-73.). You may ask Professor Richter-Landsberg on her experiance. The OLN-93 have proved valuable for many studies on oligodendroglial research questions and more than 20 papers I now of used them since.

I suggest you read some papers from the 70's and 80's when allot of this work was done. It is straightforward to generate an immortalized cell line from fibroblasts. For example, you can simply overexpress SV40 Large T antigen to immoralize the cells, and/or certain combinations of oncogenes, such as c-myc and E1a. Typically, H-ras will also immortalize fibroblasts. Check out this reference - Woo and Poon, Genes & Dev. 2004. 18: 1317-1330

Dear, if are you in Iran, I suggest you read my book entitled"Cell Culture Biotechnology". I hope this book of help.

Good luck

HI,

I think the following book should be helpful

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications

we have the 4th edition but it must be a 5th or 6th out now

good luck

Dear Sohrab.

I can tell you the protocol that I usually follow to isolate fibroblasts from mice ears. Give it a chance. Good luck

Actually there is no specific and precise method or protocol that can guarantee obtaining long term continuous cell line from fibroblasts. Although fibroblasts are actively dividing cells but they do not divide forever, they just go for division for short term and then differentiated for fibrocytes. However, many fibroblast cell line have been established yet but all of them must be considered as transformed cell but not normal cells as they must have activated oncogene tio let them continuously divide. You can get skin fibroblast cell line by keeping culture skin cell and adding some growth factors. However sometimes sporadically cell lines can be obtained without adding any factor.

A number of methods for the derivation of cell lines from primary cell cultures have been well documented : spontaneously or with viral and cellular genes. It has well known that spontaneous immortalization of rodent primary celle has been frequently achieved. However, it needs to be immortalize with viral or cellular genes in most cases..

There appears to be some confusion. Most of the answers to date assume that a cell line is something that has been immortalised. In fact there are many unimmortalised cell available from cell repositories such as the ATCC and characterised by having a finite life span, generally assumed to about 60 cell cycles from initiation and known as the Hayflick limit. If the primary cultures are expanded quickly and efficiently, sufficient low passage cells of a given line can be frozen to last the rest of your scientific career. However, beware of confusing passage number with cell divisions. The Hayflick limit of ~60 divisions can be a small number of passages depending on whether the cells were confluent at the time of subculturing and the cell density used for reseeding the culture dishes. In my experience normal diploid mouse cell lines frequently spontaneously transform in culture, unlike human cells that require help from one of the many transforming agents mentioned in previous responses. The main question to be addressed on whether or not to use immortalised or non-immortalised cells is whether you require genetically normal cells (diploid) for your experiments. For example, gene expression studies are fraught with problems using immortalised cell lines due to the high level of aneuploidy and differences in gene copy number frequently present.

Dr. Pearson's point is right. Immortalization of cells essentially change their physiology because of introduction of extraneous factors. For example human lymphocytes can be immortalized by Epstein-Bar Virus. But this process changes these cells biochemically and functionally.

Mouse primary fibroblasts can be immortalized by the 3T3 protocol (see the paper from Todaro and Green @ Pubmed; PMID: 13985244). The protocol is quite simple (split the primary cells every three days and replate them). After some time spontaneous immortalization occurs.

If you want to establish your cell lines in a shorter time frame I recommend to use the SV40 large T antigen preferably transduced by viral transfer.

We did both procedures and they work fine.

Whatever the method you use to prolong cell proliferation, you select for cells that are able to grow on plastic in a 2D environment devoid of a dynamic micro environment. Although cell lines have learned us a lot, research has clearly shown us their limitations. Even short-term culture of primary cells induces changes that are not a reflection of their in vivo characteristics.

Dr. Pearson’s answer should also be taken into account. Additionally, heterogeneity of the primary source may need to be considered.