The answer to this question largely depends on what your experiment is. Each cell type will require different media, growth factors and/or extracellular matrix to maintain them in culture. Are you planning on deriving cell lines? If so, you will have to isolate stem cells. Or is the experiment shorter term whereby you only want to maintain differentiated cells without perpetuating them? Perhaps there is an established cell line that you can obtain/purchase and use for your experiment. My suggestion is that you focus on one cell type at a time. There should be establish protocols for collecting these cells and maintaining them in culture. 

I usually use RPMI 10% for mouse and human macrophage/lymphocyte. As for the cardiomyocyte culture media the best would be DMEM/F-12 high glucose supplemented with sodium pyruvate 1.65g/l, ascorbic acid 88mg/l, 1M HEPES (pH7.6 is very important), BSA fraction V 10g/l, Linoleic acid 25mg/l, Transferrin 20mg/l, 100 microL Selenite [20mM], NaHCO3 12.2g/l and Penicillin/Streptomycin.

As for cardiomyocyte extraction I used to use 0.12% collagenase type 2 freshly diluted in PBS in perfused heart to digeste it.

Thank you for your replays!

To Erica: im planing to test the toxicity of different compounds and measuring different enzymes and biomarkers of cell damage. Also short on funds and no lab of my own where i can deal with them all the time!

To Souhad: After digeston what do you do? Only seed them in plates? orcentrifugation on Percol? Why is HEPES so important? 

Hi Nikola,

HEPES is important to keep the pH stable it is very important for cardiomyocytes.

After digestion centrifugation on percol is recommended but not essential (depending on the age and the rodent strain; I used to work with rat heart). In all cases you have to plate cardiomyocytes to gelatin-coated culture dishes with cardiomyocyte culture media, cited before, supplemented with 5% FBS the first 24h then you can remove FBS. It is recommended to culture it at 0.6 million cell/1 well of a 6 well plate (3ml culture medium) or 0.25 million/well of a 12 well plate (1.5ml culture medium).

Hi Nikola,

I culture Bone Marrow Derived Macrophages in DMEM (High Glucose) supplemented with 10% FBS (heat inactivated) and antibiotics.

The protocol is pretty straight forward and the yield is pretty high (millions of cells)! Here there is a very nice video where you can check how to perform bone collection.

http://www.jove.com/video/50323/investigation-macrophage-polarization-using-bone-marrow-derived

After collection, I flush the bone marrow out the bones with cold PBS, pellet the cells, filter and plate them in DMEM10% plus M-CSF (extremely important for macrophage differentiation from progenitors!). I then collect the cells after 7 days. You can test the quality of your culture by checking the expression of F4/80 CD11b by FACS. Your cells of interest are the one double positive.

If you need to replate your cells do it in DMEM 10% + MCSF but replace this medium with DMEM10% or DMEM1% without MCSF during your treatment!

Hope this helps! Good luck!

Fede

Hi Nikola,

I can tell you about hepatocytes.. they are not  easy to isolate nor to maintain.. You can keep them in colture for about a week.. then they started to dedifferentiate..

I can suggest you the protocol described in the supplementary material of this paper

"Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450"  Vinchi F. et al 2014

The protocol works very well.. bare in mind that the isolation' step is critical.. after the islolation the cells are seeded on plates coated with collagen type I.!

Good luck!

Hello Nikola,

For each tissue/cell types/species mix you have to use a very specific protocol and conditions. I found the OptiTDS (from CHI Scientific) product line very interesting. Indeed they developed a enzymatic cocktail adapted to the species, tissue & cell types you need. This contains a mix of enzymes (up to 9) tested to be efficient. I am sure you can find this on the Internet.

Thank you all for your answers and long explanations!

To Federica La Russa: I can get access to JOVE :( so i cant wach video...can you pleas download it and send it to my e-mail  : [email protected] (via dropbox)? Also if you see some other interesting videos pleas do send me if you can?

As I sad at my first replay i need cell's for tracking the effects of compounds on their viability and function (24-48h after the application of substances) so no long cultivation.

Yes im aware the each cell line needs its own "special" conditions but some general thoughts...for example i can finde what amount of antibiotics should be added or present in well? People just write they are added and nothing else! Also what volume of antibiotics should be added to medium...i cant add 10ml of antibiotic solution to 40ml of medium!

To Letizia De Chiara: are plates with collagen type I needed for cultivation? Or i can use regular plates?

For primary cultured astrocytes, i used a mechanical dissociation to break the tissu with a syringe and a needle. The medium i used, was DMEM with 10% calf serum foetal and to clone this cells i used a cloning ring. I cultivated cells with conditioned medium.

Maybe you could have a try with the L15 medium, as I done with it for macrophages.

Independent from the tissue culture medium, which you are using, I would suggest to test different batches of FCS independent from each other. There can be marked differences between batches depending on the cell type in the culture. It is also advisable to use small amounts of frozen serum and thaw these one by one just before use. 

Hi Nicola,

it is truth that every cell lineage requires different conditions. So you should find the protocols that are most suitable and convenient for you. What about antibiotics - we use penicilin at final concentration 100U/ml and streptomycin at final concentration 0,1 mg/ml. They are usually sold as a 100x concentrated mixture, so you add 1 volume of antibiotics to 99 volumes of medium.

About FCS/FBS ro any other medium - we use heat-inactivated medium routinely, to get reproducible results. However, I'm not convinced that it is necessary. The inactivation of complement is important only for immunological analyses. I recommend to read this article:

http://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/Cell_Culture/Application_Notes/heat_inactivation.pdf?&storeId=10652.

Good luck!

I have experience qith isolatin of fibroblast amd myoblast from fresh bioptic tissue. I always used inactivated FCS. I do not use collagenase but I fragmented the tissue in small pieces and I cultured with medium supplemented with 30% FCS for 8-10 days (I usually see single adherent cells in 6-8 days), afterwards I incubate the cells with 10% FCS.

Good luck

I have used collagenase IV to isolate fibroblasts from mouse fetuses and testicular cells. Typically you need to do a second step with 0.25% trypsin-EDTA as well as DNase I. Testing different types and batches of FCS is a good idea but I would definitely heat inactivate it.

The first question to answer from the literature is if cell separation and/or culture is the right experimental tool to test your hypothesis.

If so, the conditions for separation and culture are tissue specific.

The devil is in the details. For example, lots of enzyme may be inconsistent in terms of contaminants and ma need to be QC'ed. The type of culture flask makes a difference - plastic or teflon, as an example. The percent of serum can make a difference; too much can be inhibitory in some cases. In short, one needs to thoroughly research the experience with any given cell type to be most efficient. 

The key search words for you to find good classic references are "primary cell culture." When I trained at the W. Alton Jones Cell Science Center (Tissue Culture Assoc.) in Lake Placid, we did outgrowth from almost every type of tissue--  fibroblasts from adults were least problematic, but getting cells to crawl out from minced fetal tissues like liver and heart is really amazing.

   It's complex, from mince or dissociation to attachment techniques and media  and treatment in the dishes or microwells-- you need a chapter for each tissue type. Embryonic liver is relatively easy, so why not start with that. Biggest error you can make is to overtrypsinize. Find a procedure for a tissue you'd like to try, and be really fastidious.

    Mouse is easier than rat!  Just sayin'....

Good Luck, Nicola.

Hi Nicola,

I agree with most of the previous suggestions. The best conditions depend from the cell-line you are culturing. You have to check on papers or manuals. For sure you have to use heat-inactivated FCS. After inactivation, my advice is to filter FCS and to make aliquots that you can freeze and taw when you have to use them .

Good luck! 

Heat inactivation of Fetal calf serum is generally only necessary for cells of the immune system (bone marrow MSCs, macrophages, microglia etc).  Heat inactivation does not generally hurt other cell cultures but it is an time consuming extra step.  Please note that all batches of fetal calf serum, even from the same supplier do not work equally well. Try to stick to a single lot of fetal calf serum for any given study to avoid variability in cell growth.   As far as enzymes for cell dissociation, it all depends on the type of tissue and age of the donor.  Embryonic cells can often be dissociated with low dose papaine in calcium free media.  Adult organs with a lot of connective tissue may need to be incubated with collagenase and/or dispase for several hours before trypsinization.   

Heat inactivation is to avoid complement mediated cell lysis.

If this is not an issue for your cell type, not needed.

If you are unsure, may be best to err on the side of inactivation.

Be careful not to overcook the serum, and I agree with the observation that lots vary and you may need to test a few at the onset of your study. Then buy enough to last a while and aliquot as appropriate.