Fat-free Cultured Cells: No Need For PUFAS; Where Are The Studies ?

[burtlancast]

Can anyone point to a study proving cultured cells don't need fats/PUFAS into their medium to grow ?

I know Ray's right about that, but i still would like an example, and can't seem to find one.

 

[haidut]

Can anyone point to a study proving cultured cells don't need fats/PUFAS into their medium to grow ?

I know Ray's right about that, but i still would like an example, and can't seem to find one.

I think the studies below settle that question pretty well - Ray is right again.

http://www.ncbi.nlm.nih.gov/pubmed/8301228

"...Adult human epidermal keratinocytes grow rapidly in medium that is essential fatty acid (EFA)-deficient. In this medium they exhibit decreased amounts of the fatty acids, 18:2, 20:3, 20:4, and contain increased amounts of monounsaturated fatty acids. [14C]- and [3H]acetate and radiolabeled fatty acids, 16:0, 18:2, and 20:4 were used to study the fatty acid metabolism of these cells."

"...Hypothetically, a keratinocyte growing in serum-free medium can adapt to the EFA-deficient growth condition in a number of ways.
I) The cell can develop the capacity to desaturate beyond the 9-position, thus acquiring the ability to synthesize essential fatty acids;
2) the keratino-cyte can synthesize unusual fatty acids that can functionally compensate for the EFA-deficient state;
3) the cell can adjust and flourish in a classically defined EFA-deficient state."

"...We have studied keratinocyte de novo fattyacid synthesis, and elongation and desaturation of long chain essential and nonessential fatty acids using a number of radiolabeled substrates of fatty acid metabolism. Our studies have allowed kinetic descriptions of lipid metabolism in intact normal human cells...The results are consistent with the hypothesis that the loss in essential polyunsaturated fatty acids is balanced by a gain in the monounsaturated fatty acids... Our previous studies reported that the essential fatty acids are not “essential” for the optimal growth of normal adult human epidermal cells (keratinocytes) in vitro (8). This present report describes a relatively large and complicated set of experiments on fatty acid metabolism in the EFA-deficient keratinocyte.
To summarize our conclusions:
I) These experiments demonstrate a method to study enzyme kinetics in lipid biochemistry in living human cells.
2) These experiments provide clear-cut verification that the biochemistry of EFAs and non-EFAs as described by the schemes in Fig. 2 are valid for keratinocytes
cultured in an EFA-deficient medium.
...
7) Our data show that EFA-deficient keratinocytes exhibit the same fatty acid pathways as normal mammalian cells (no unusual fatty acids)."

And here is the study the authors mention in reference [8].

http://www.ncbi.nlm.nih.gov/pubmed/1469285

"...The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA)-deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty acids 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85-90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid-deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure."

 

[burtlancast]

Awesome find, Haidut.

From your article:

Both classical and newer techniques for culturing primary epidermal basal cells (keratinocytes) use 10-15% serum in standard medium formulations (1-4). In culture
medium, serum is usually the only source of essential and nonessential fatty acids. One system (5-7) uses serum-free, low-calcium growth medium MCDB 153 that allows the rapid growth of keratinocytes in vitro. Because the rapidly growing keratinocytes are in an essential fatty acid (EFA)-deficient environment, they show altered fatty acid compositions (8).

Googling "MCDB 153" , one finds:

MCDB-153 (Modified) is a defined serum-free basal medium for the growth of normal keratinocytes and squamous epithelial cells. It is a basal media with growth factor supplementation. MCDB media were uniquely designed and formulated not only quantitatively but also qualitatively to provide a defined and optimally balanced cell culture milieu that selectively promotes the growth of specific cell types. These types of media were designed by Ham and colleagues specifically for the low-protein or serum-free growth of specific cells utilizing growth factors (e.g. EGF, Insulin), hormones, trace elements and or low levels of Dialyzed Fetal Bovine Serum Protein. This series of media originated out of the Department of Molecular, Cellular and Developmental Biology at the University of Colorado.
MCDB 153 is a modification of Ham's nutrient mixture F-12 designed for the growth of non-transformed cell types in a serum-free formulation

Googling then HAM F-12:

"Ham’s F-12 medium has been used for carcinoma cells, rat skeletal myoblasts, Chinese hamster lung cells and rat, chicken and rabbit embryos. "

So, in summary, rats, hamsters, chicken and rabbits cells can be grown in PUFA-free growing medium .
In humans, i've only found epidermal keratinocytes, and carcinomas.

I will try to find other types of human cells grown in this manner.

 

[haidut]

Awesome find, Haidut.

From your article:

Both classical and newer techniques for culturing primary epidermal basal cells (keratinocytes) use 10-15% serum in standard medium formulations (1-4). In culture
medium, serum is usually the only source of essential and nonessential fatty acids. One system (5-7) uses serum-free, low-calcium growth medium MCDB 153 that allows the rapid growth of keratinocytes in vitro. Because the rapidly growing keratinocytes are in an essential fatty acid (EFA)-deficient environment, they show altered fatty acid compositions (8).

Googling "MCDB 153" , one finds:

MCDB-153 (Modified) is a defined serum-free basal medium for the growth of normal keratinocytes and squamous epithelial cells. It is a basal media with growth factor supplementation. MCDB media were uniquely designed and formulated not only quantitatively but also qualitatively to provide a defined and optimally balanced cell culture milieu that selectively promotes the growth of specific cell types. These types of media were designed by Ham and colleagues specifically for the low-protein or serum-free growth of specific cells utilizing growth factors (e.g. EGF, Insulin), hormones, trace elements and or low levels of Dialyzed Fetal Bovine Serum Protein. This series of media originated out of the Department of Molecular, Cellular and Developmental Biology at the University of Colorado.
MCDB 153 is a modification of Ham's nutrient mixture F-12 designed for the growth of non-transformed cell types in a serum-free formulation

Googling then HAM F-12:

"Ham’s F-12 medium has been used for carcinoma cells, rat skeletal myoblasts, Chinese hamster lung cells and rat, chicken and rabbit embryos. "

So, in summary, rats, hamsters, chicken and rabbits cells can be grown in PUFA-free growing medium .
In humans, i've only found epidermal keratinocytes, and carcinomas.

I will try to find other types of human cells grown in this manner.

I remember seeing a study long time ago showing that cells in a medium of pure glucose lose their so-called Hayflick limit for division - they become immortal, but not cancerous. It may have been a study by Alexis Cartel, but I can't find it right now.

http://en.wikipedia.org/wiki/Hayflick_limit

"...Prior to Hayflick's discovery, it was believed that vertebrate cells had an unlimited potential to replicate. Alexis Carrel, a Nobel prize-winning surgeon, had stated "that all cells explanted in culture are immortal, and that the lack of continuous cell replication was due to ignorance on how best to cultivate the cells".[4] He supported this hypothesis by having cultivated fibroblasts from chicken hearts, and to have kept the culture growing for 34 years.[5] This indicated that cells of vertebrates could continue to divide indefinitely in a culture. However, other scientists have been unable to repeat Carrel's result."

Here are some more studies on cell senescence.
http://jem.rupress.org/content/34/6/599

http://gerontologist.oxfordjournals.org ... 2/297.full
"...For the sake of simplicity, the views in these two quotations will be referred to as the “Hayflick hypothesis” in this review essay. Those of us who were trained as mammalian physiologists found this hypothesis to be outlandish and not to be taken seriously. We felt that it was highly unlikely that cells of a specific lineage in an artificial medium would undergo senescence in a manner similar to that of cells residing in organisms, which are influenced by neighboring cells of various types and by the modulating actions of the nervous, endocrine, and other systems. Surprisingly, however, the hypothesis was enthusiastically embraced, at first by cell biologists and later by molecular biologists. Indeed, studies on the loss of proliferative ability of cells in culture rapidly became a major focus of research in biological gerontology. Moreover, the term “cell senescence,” which one might expect to describe the vast array of age-associated cellular deterioration occurring in an organism, has come to denote this permanent loss of proliferative ability."

"...Significantly, however, the results of studies published during the past few years have challenged some of the key evidence in support of the “Hayflick hypothesis.” Cristofalo, Allen, Pignolo, Martin, and Beck (1998) studied cultures of fibroblasts from healthy donors and found no relationship between the age of the donor and the replicative ability of the fibroblasts. They suggest that the finding of an inverse relationship, as reported in earlier studies, could have been due to the inclusion of subjects suffering from diseases. In addition, Lorenzini, Tresini, Austad, and Cristofalo (2005) have shown that the body size of a species, rather than its longevity, is the primary correlate of proliferative potential of its cells in culture. Finally, there is growing evidence that SA-β−gal staining is not a reliable biomarker of cell senescence, and that its use for this purpose may potentially lead to spurious conclusions."