NationStates Jolt Archive

this is a last-ditch effort, kind of a random shot in the dark:

does anybody around here know anything about primary cell cultures and why are primary culture might show different protein/mRNA expression than fresh cells from the given tissue?

if you do, and feel like lending a hand, i have a theoretical experimental design problem that i could use help with.

if you don't, and feel like laughing at my pain, feel free to do so...i would, if i weren't me.

Fraid not mate :(

this is a last-ditch effort, kind of a random shot in the dark:

does anybody around here know anything about primary cell cultures and why are primary culture might show different protein/mRNA expression than fresh cells from the given tissue?

if you do, and feel like lending a hand, i have a theoretical experimental design problem that i could use help with.

if you don't, and feel like laughing at my pain, feel free to do so...i would, if i weren't me.
Mmmm.... Maybe there were mutations during the culturing process? What about the culturing conditions? Are the cells exposed to the same growth factors* in the culture as in the tissue?

Bioprocess engineering is not anywhere near my field of expertise; I only took one graduate-level course in the subject, so I'm afraid I can't be of any more help than this.

* Am I even using the term "growth factors" correctly? I can't remember anymore.

--The Democratic States of Cogitation
"Think about it for a moment."

this is a last-ditch effort, kind of a random shot in the dark:

does anybody around here know anything about primary cell cultures and why are primary culture might show different protein/mRNA expression than fresh cells from the given tissue?

if you do, and feel like lending a hand, i have a theoretical experimental design problem that i could use help with.

if you don't, and feel like laughing at my pain, feel free to do so...i would, if i weren't me.

I'm not laughing, but that subject area is WAY out of my admittedly limited repitoir! Sorry. :(

what kind of help are you looking for? the husband isnt in the cell culture biz but he has designed many scientific experiments in his day and is VERY good with statistical analysis.

and hes not doing much of anything right now.

this is a last-ditch effort, kind of a random shot in the dark:

does anybody around here know anything about primary cell cultures and why are primary culture might show different protein/mRNA expression than fresh cells from the given tissue?

if you do, and feel like lending a hand, i have a theoretical experimental design problem that i could use help with.

if you don't, and feel like laughing at my pain, feel free to do so...i would, if i weren't me.

The only thing I could think of is external signalling, besides that couldn't help you, sorry :(

If the cells were stimulated to grow, i.e. in an agar gel plate, then mutation can occur in the cell replication (mitosis) process.
Mutations can be one of three things:
A substitution (or Point mutation), where one nucleotide is swapped with another (usually purine for a purine or a pyrimidine for a pyrimidine). This isn't usually a problem because DNA is degenerate (multiple triplet codes can code for a single amino acid so a single nucleotide change might not affect it).
A deletion, where one or more nucleotides are deleted. This is usually a serious mutation because it affects all the triplet codes on the mRNA strand that come after it. A completely different protein would be produced.
An insertion is where one or more extra nucleotides are inserted, this would have a similar effect to a deletion.
Basically - mutations during DNA replication (part of the interphase of mitosis) can lead to a different triplet code being transcribed to the mRNA strand during transcription, meaning a different protein could result.

well, just in case anybody is bored or masochistic enough to want to try and deal with this mess, here's the complete problem:

I've got a protein, call it X, which has been found to be produced in
human liver cells. It has been sequenced and found to be homologous but
not identical with a growth factor which acts on neuronal cells
modulating neurotransmitter expression. The half life of X in serum is
several hours, but studies on X secreted by CHO cells transfected with
the human X cDNA yeild a half-life of minutes, thus seriously reducing
the yield of the recombinant protein. Addition of serine protease
inhibitors to the cells in culture improves procrastin yield by
inhibiting activity of the enzyme Xase, an X-specific protease. It has
also been observed that the X-expressing CHO cells grow poorly compared
to mock-transfected control cells; the addition of protease inhibitors
does not affect the growth of the transfected cells in culture. While
hepatocytes isolated from fresh human liver possess abundant X by
immunoblot, primary cultures of human hepatocytes don't express any
detectable procrastin mRNA or protein.

My fictional lab chief has identified two problems that need to be
addressed, and I must pick one to answer:

1. Determine the mechanism behind the proliferation rate observed for
the procrastin expressing cells and to attempt to restore the
proliferation rate to that of the mock transfected cells.
OR
2. Modify the "standard" conditions for primary culture of hepatocytes
to maintain X expression when cells are placed in culture.

I have slightly more of a clue for answering the second, so that's where I have focused my energies so far, but if anybody has insight into either question it would be useful :).

If the cells were stimulated to grow, i.e. in an agar gel plate, then mutation can occur in the cell replication (mitosis) process.
Mutations can be one of three things:
A substitution (or Point mutation), where one nucleotide is swapped with another (usually purine for a purine or a pyrimidine for a pyrimidine). This isn't usually a problem because DNA is degenerate (multiple triplet codes can code for a single amino acid so a single nucleotide change might not affect it).
A deletion, where one or more nucleotides are deleted. This is usually a serious mutation because it affects all the triplet codes on the mRNA strand that come after it. A completely different protein would be produced.
An insertion is where one or more extra nucleotides are inserted, this would have a similar effect to a deletion.
Basically - mutations during DNA replication (part of the interphase of mitosis) can lead to a different triplet code being transcribed to the mRNA strand during transcription, meaning a different protein could result.
ahhh, i was not aware that was a significant consideration with primary cultures (i've never worked with them, so i am clueless). do you know if there is anything that can be done to reduce this problem for primary cultures?

oh, and thank you very much to all of you for even being willing to read/respond to this. i swear, if they weren't paying me to go to grad school i would never put up with this :P. who's stupid idea was it to make a neurophysiologist study non-neuro cell culturing, anyhow?!

well, just in case anybody is bored or masochistic enough to want to try and deal with this mess, here's the complete problem:
-snip-
Oh, Goddess above, it burns!
This reminds me of why I didn't continue along the route I was taking in school

Sorry, can't help you.
:(

who's stupid idea was it to make a neurophysiologist study non-neuro cell culturing, anyhow?!
What?
You're right, that makes no sense...

Oh, Goddess above, it burns!
This reminds me of why I didn't continue along the route I was taking in school

Sorry, can't help you.
:(
lol, yeah, i'm starting to wonder about my choice of career path...

though the second year students assure me that this first year is mostly a kind of academic hazing ritual, and the profs are just giving our mental buttocks a good paddling. i wish they could just get us to drink until we pass out and then leave us sleeping pantsless on the lawn.

Primary cell cultures are pretty much always going to demonstrate different expression profiles from those in vivo. The reason is pretty basic - they aren't in the same environment anymore. A cell culture plate is really nothing like the body.

Some suggestions (and I can provide some resources for similar things if you need them):

Research the type of ECM (extracellular matrix) in the liver. Perhaps coating the culture dishes with the appropriate ECM would provide better signals to the cells.

Research the oxygen tension in the liver. Most of the research in my lab (my advisor is an anesthesiologist on liver transplants btw) has to do with oxygen tension and its effects on cells. Your standard cell culture is kept at 20% oxygen, but there is not a single place in the body with that type of oxygen tension. High oxygen can be *very* damaging to cells. There are many ways to grow cells at lower oxygen tensions, and just about every cell type we have placed in low oxygen conditions has proliferated much more. (Since you're a neuro type, growing neural progenitors in low oxygen also greatly increases the percentage of dopaminergic cells).

Growing in a 3-D matrix might help as well, but would be rather difficult.

Edit: Hope this helps!

I'm afraid by basic knowledge of what a cell is, and knowing what RNA stands for will not be adequate here.

I'm afraid by basic knowledge of what a cell is, and knowing what RNA stands for will not be adequate here.
Hee, probably not.
:D

Most cultures are made so the cell adapts to it, and are also made to accelerate growth. This changes the conditions and thus changes the mRNA to adapt. Your best bet is to do a 3D growing area (I suggest taking cell samples putting them into a culture gel in a pitri dish and taping the dish shut) so there are some that have the adaptations so they grow on one another. I did a lab in my HS biology class (harvard professor teaches it too) were we took air samples from various places in the school and examined. It's kind of sad how bad some of the stuff we god. But the space of actual air enclosed in the pitri dish (what its adapted to) made it grow as if it were just in the air, save the cells touching the gel. We found plasmodium in two places where I am a lot. Plasmodium can cause serious bacterial disease. Ah.

man, there are some WICKED smart people on this forum! thank you guys so much, these are very useful leads...if any of you ever need help with something physiological or systems-neuro in nature, don't hesitate to press me into service. i swear i'm not a total moron, i just am clueless when it comes to cultures (as most neuroscientists are :P).

man, there are some WICKED smart people on this forum! thank you guys so much, these are very useful leads...if any of you ever need help with something physiological or systems-neuro in nature, don't hesitate to press me into service. i swear i'm not a total moron, i just am clueless when it comes to cultures (as most neuroscientists are :P).

Little known secret: We are *all* cluless when it comes to cell culture, especially with mammalian cells. No matter how careful you are, some primary cultures will always end up thrown away because they do strange things. On top of that, half of the additives work and we have absolutely *no* clue why. However, the cells die without them.

well, just in case anybody is bored or masochistic enough to want to try and deal with this mess, here's the complete problem:
<snip>
I almosty, almost understood that. :(

who's stupid idea was it to make a neurophysiologist study non-neuro cell culturing, anyhow?!
How different are neural and non-neural cultures? Maybe they're just making sure you understand basic culturing techniques?

Keep in mind that neurons do not exist in a vacuum. Neural cells have to interact with non-neural cells somewhere to control muscles and gather sense information. I'll have to defer to Myrth and Dempublicents, here, as they seem to be much better-versed in this field than I, but I wouldn't be surprised to be told that non-neural cell cultures are of interest because neural cells have to interact with them.

--The Democratic States of Cogitation
"Think about it for a moment."