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Name: Jasmine
Status: Other
Grade:  Other
Location: AL
Country: United States
Date: September 2007


Question:
There is a limit to the number of some cells that can be grown in culture; what are the conditions that cause these limitations?



Replies:
Hi Jasmine,

If there was unlimited food and space on Earth, you could imagine that our population would continue to grow forever, since our children could always find a new place to live with food to eat. Earth, just like cells growing in a culture, has a limited amount of space and resources. For very similar reasons that our population can't continue to grow forever, the population of cells growing in a culture will reach an upper limit and eventually die off as nutrients in the media are depleted and toxic cellular waste builds up (for example, some bacteria release acidic metabolic byproducts which will lower the pH of the media solution).

In fact, the growth of bacteria in culture follows a predictable curve. Initially, after a lag phase in which the cells are adjusting to their new environment, nutrients are plentiful and the cells experience an exponential growth phase. This is followed by a stationary phase where environmental conditions are no longer as supportive and the rate of cell death is similar to the rate of growth. Finally, when nutrients become depleted and toxic waste products build up, cell deaths will exceed cell divisions in what is known as a death phase.

Ethan Greenblatt
Ph.D. Candidate
Stanford University


As cells grow up, assuming you have an 'immortal' cell line (some cell lines can't keep dividing indefinitely), there are physical limitations that occur.

All cells need nutrients of some kind, and most industrial cultures need oxygen as well. Large scale cell cultures are grown in large stirred tanks called 'fermentors'. Fermentors typically have some kind of mixing paddle along with inlets for air/oxygen and nutrients and outlets for off gases like CO2.

Normally, you add a small number of cells to the tank (these are called 'seed' cells) along with food/nutrients and water (the food, nutrients, and water are called the 'growth media', or 'media' for short). Hopefully, if things go right, the cells will divide, and the culture will grow and become more dense. As the culture grows, you have to keep adding air, and in many cases, keep adding food as well.

As the culture gets more dense, it's harder to stir, and as a result, it's harder to get the food and air into the cells. Cell near the air inlets might be fine, but there might be places where the air and food don't reach, and you can get 'dead zones' full of dead cells, or where the cells are alive, but don't get enough food or air to grow and be healthy. This problem is called "mass transfer limitation".

"Mass transfer" refers to how materials like food and air move around. Imagine you are stirring food coloring in water. You give it a few stirs and it's all evenly mixed; easy. However, what if you were mixing coloring into paint? Paint stores have special machines to do this, but even with the machines it takes a long time. If you tried to do it by hand it would take a very long time (and give you sore arms too)! That's because the thicker paint doesn't mix as easily as the thinner water. The same thing is true for cells. As they grow, and the culture gets thicker, it gets harder to mix, and harder to get nutrients into the cells.

Another consideration is that some cells actually make poisons that prevent their own growth. Yeasts that make alcohol are a great example. When you ferment beer or wine, if there is enough sugar, the yeasts can make so much alcohol that they kill themselves. This is just one example, but the same thing can happen with acids and other substances as well.

Both of these examples refer to well-mixed cultures of single cells. In industry, this is the most common type, and the most dense kind of cell culturing. It is not the only type of culture, though. Many scientists are working on making structured groups of cells -- like might be used for an artificial tissue or organ. In these cases, the rules are completely different. You have very dense cultures (the cells are all touching one another), and you have to find away to get nutrients and oxygen to them without being able to stir them. Scientists are working on artificial vessels to feed the cells, while others just immerse the clump of cells in a tank full of medium. This is an ongoing research problem that scientists are still trying to solve.

Each type of cell and each type of culture is different -- some cells can be very dense, while others cannot be. There's no single number that applies to all cultures.

Hope this helps,
Burr


Mammalian cells are limited to approximately 50 cell divisions. This number is known as the Hayflick number, named after the scientist who observed this phenomenon. This number obviously does not apply to germ (sex) cells, otherwise we wouldn't be here. This number also does not apply to cancer cells. HeLa cells (a human cervical cancer cell line) has been growing in tissue culture since 1950. The currently accepted theory to explain this limit is that every time a chromosome duplicates, some DNA from the tips of hromosomes (the telomeres) is lost which eventually prevents the chromosome from duplicating.

Ron Baker, Ph.D.



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