Starting a cell line - (Oct/08/2008 )
Well today during a lab meeting, my PI said he has always wanted to start a cell line and we apparently have all the equipment for it but nobody in the lab knows the first thing about getting it started. If anyone could either help me out or point me in the direction of some guide that would be appreciated. Right now in our lab we're working on free radicals and antioxidants. We're looking at avenanthramide mainly.
One study we're doing is an acute bout of downhill running to cause oxidative stress followed by a 2 month supplement of avenanthramide. Then they perform a second acute bout and we compare the results with the baseline and the control. Another experiment underway deals with mitochondrial signaling with ROS.
So here's my thoughts. We'd probably be looking at skeletal muscle or heart muscle tissues ideally. Now where would we start?
1. Do we need to purchase initial cell lines to work with or can we start our own? If either is possible, which is more time/cost effective?
2. What happens next? Am I missing anything broad or some small detail? Like I said before, we're all very green to working with cell cultures but would love to start something in our lab with it.
Thanks!
One study we're doing is an acute bout of downhill running to cause oxidative stress followed by a 2 month supplement of avenanthramide. Then they perform a second acute bout and we compare the results with the baseline and the control. Another experiment underway deals with mitochondrial signaling with ROS.
So here's my thoughts. We'd probably be looking at skeletal muscle or heart muscle tissues ideally. Now where would we start?
1. Do we need to purchase initial cell lines to work with or can we start our own? If either is possible, which is more time/cost effective?
2. What happens next? Am I missing anything broad or some small detail? Like I said before, we're all very green to working with cell cultures but would love to start something in our lab with it.
Thanks!
Starting a cell line? you mean establishing a cell line?
it is easy, you just need to get the tissue and cut it by blade (some people use tryspin to separate tissue cells but I don't) under laminar flow and in a very sterile condition and then add medium/serum to it. the only problem is that the risk of contamination is very high during establishment and you certainly need to take care of it during the first days of culturing.
and yes, you need control cell lines indeed. there are many ways to determine if the cell line you established is the cell line of your interest of not. one is Western Blot using antibodies specific those cells.
it is easy, you just need to get the tissue and cut it by blade (some people use tryspin to separate tissue cells but I don't) under laminar flow and in a very sterile condition and then add medium/serum to it. the only problem is that the risk of contamination is very high during establishment and you certainly need to take care of it during the first days of culturing.
and yes, you need control cell lines indeed. there are many ways to determine if the cell line you established is the cell line of your interest of not. one is Western Blot using antibodies specific those cells.
Sorry for the semantics. Establishing a cell line. Could you elaborate a little bit? Its something none of us have any experience with so its not exactly easy for us.
1. What would be the medium/serum? Is this specific to the type of tissue or is it something general you order?
2. How do we "take care of it" during the first few days? Antibiotics?
3. What is the advantage/disadvantage to using a blade versus trypsin to separate the cells? What is involved in the trypsin method?
I'm just an undergrad but I've been working in our lab for a year now and the PI said this could be a potential independent project for me. I really appreciate the help.
it is easy, you just need to get the tissue and cut it by blade (some people use tryspin to separate tissue cells but I don't) under laminar flow and in a very sterile condition and then add medium/serum to it. the only problem is that the risk of contamination is very high during establishment and you certainly need to take care of it during the first days of culturing.
and yes, you need control cell lines indeed. there are many ways to determine if the cell line you established is the cell line of your interest of not. one is Western Blot using antibodies specific those cells.
Sorry for the semantics. Establishing a cell line. Could you elaborate a little bit? Its something none of us have any experience with so its not exactly easy for us.
1. What would be the medium/serum? Is this specific to the type of tissue or is it something general you order?
2. How do we "take care of it" during the first few days? Antibiotics?
3. What is the advantage/disadvantage to using a blade versus trypsin to separate the cells? What is involved in the trypsin method?
I'm just an undergrad but I've been working in our lab for a year now and the PI said this could be a potential independent project for me. I really appreciate the help.
Apparently you are from the town that Butch Vig is from....so I will try to explain more to you:D
but i need to know if you have any cell culture facility in there?...I know university of Wisconsin has but in your lab?....laminar flow?....class 1-2 hood?
Media that we use are for cancer cells....we use human tumor samples...we take directly from the surgeon in the operation room, we put in PBS and keep on ice, we come to the lab....we wash the tissue to get rid of blood or red blood cells....we cut the tissue on petri dish....then we transfer the chopped tissue to a T75 flask containing DMEM medium, and 10% FBS, 1% Gentamycin.
Just to comment establishing a cell line is not easy.
What the prvious comments author did with the cancerous cell is not technically establishing a line as the cell is already immortalised, as it is a tumor.
To establish a cell line from square one:
First you need to figure out what cell it is you wish to immoratlise (form a cell line with)
You then need to obtain this tissue depending on your needs, ie if human tissue is available, otherwise maybe a rat or a mouse dissections.
You will then need to read many papers from various other people who have worked withthe same cell and see how they isolated the cell of interest as there are many cell types in each tissue section
You will then need to find a way to seperate or isolate the particular cell you want from all the surrounding cells. some people use percoll which sepereates different cells depending on their cell size and weight, there are other methods but i have not used them, you will need to do the research into these techniques.
When the cells of interst are isolated a medium which will maintain/feed them will have to be obtained (best way to do this is to see what other authours who have worked with either very similar cells from the same tissue or exactly the same cell type have done and use this), there are a very wide range of medias available all of which may need some extra growth factors specific for your cell type to grow in, which again you will need to identify from published work or by trial an error (which i dont recommend as may take years)
You will then need to immortalise this cell line - usually performed by a virus containing specific genes which can disrupt the oncogenes or some other area of the nuclear material (I am not an expert so i do not know the exact m.o.a.) to transfer this normal cell into a tumor cell and hence a cell line.
this step may take many many attempts and dissections (if using mice or rats) and can take a months to get right, you can also be lucky and immoratlise the cell on your first attemmpt but rarely happens
if you do mange to immortalise a cell you will then need to make sure the cell line that has been immortalised is the cell you reguired which you can do by using primers specific to that cell by PCR analysis and also by immunostaining with antibodies specific to markers found in your cell alone.
so to comment again on the development of a cell line, it is not easy, there are many techniques involved all of wich may have problemns whioch can delay the development.
As i said i am not an expert but i am in the middle of developing my own cell line, i had some experience with cell culture before i started and 10 months after starting i am still at immortalisation stage.
if you want my honest opinion, if you dont have the time money or resource to do the above work, buy in a cell line if you wish to work with one from one of the databases such as ATCC.
hope this helps.
What the prvious comments author did with the cancerous cell is not technically establishing a line as the cell is already immortalised, as it is a tumor.
true,....some cell lines are called TRANSFORMED cell lines....we have normal human kidney cells (293FT) that are transformed in order to last longer...because normal cells have a limited life time and they die after some passages....tumour or cancer cells normally shouldn't die.....I don't remember how exactly a 293Ft cell has been transformed but I think they have added 1-2 genes by specific plasmids to the genome to sort of fix them.
I suggest getting hold of a copy of R.I. Freshney's Culture of Animal Cells as an excellent starting point. It covers both establishing cell lines and culture of cells as well as the basics of cell culture and contamination etc.
One major point to consider is that cells in culture are usually very different from cells in the body, and as such can have quite different responses to the subject they are derived from.
Another thing to consider, is to find someone who routinely does cell culture and find out how they do it... it is much easier to learn off someone than trying it with no prior experience, especially for the things like contamination control, normal appearances of cells etc.