I don't have access to bovine insulin which used add the protocol of cell culture. I want to use human insulin for the culture so I have three question: first, has anyone used human insulin for culturing before? Also, would you think that any effect would be cell line specific? and what type of human insulin do you suggest? There are six different kind of human insulin and I don't know which of them I could use.
I have used it, you can try to use Sigma I3536-100MG, it is suitable for cell culture and it look cheaper in comparison of the other types. I'm sending one of my paper also regarding media composition.
Exp Cell Res. 1999 Mar 15;247(2):399-409.
Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells.
Zangani D1, Darcy KM, Shoemaker S, Ip MM.
We frequently use insulin as a supplement to grow breast cancer cells like MCF7 or T47D. I never worked with HS578T cells.
A lot of breast cancer cells grow better with insulin in the culture medium, however they won't die withouth it. Insulin stimulates proliferation via the insulin receptor and receptor expression and activation can vary in different cell lines.
To ensure optimal growth of your cells I would recommend to add ca 10ug/ml insulin to the culture medium. Both human insulin or bovine insulin are commonly used for this. I would just select one and stick to it for your experiment.
We use human insulin solution expressed in yeast from Sigma (I9278-5ml).
thank you for your replay and your good information. but i can't provide human insulin from sigma because my project is in time restriction and it takes me about 5-6 weeks long to provide it from sigma. we prefer to use one of the insulin types which is easy to find in Iran: Humalog,Novolog,Apidra which are rapid acting,Regular which is short acting, NPH which is intermediate acting and Lantusor Levemir which are long acting. could you pleas advise me which of these insulin type i could use for my cells?which of them has the most similarity to Sigma's insulin?
I guess all of them are for medical use and already in solution and you obtain them by the pharmacy. In this case, in my opinion, the best choice is regular because all the others have additives to allow for different release time. In this case you have to be careful because they are sold on the basis of activity for unit and you have to check how much of the injectable solution will correspond to 10 microgram/ml. Do not worry, regular is produced in recombinant bacteria or yeast as Sigma's and it is completely human, but be very careful in doing your calculation: activity does not correspond to weight and you have to adjust for that
dear dr.Danilo Zangani
i'm really thankful because of your help. your answer completely removed my worriness.
best regards.
Amended on 4 June 2018
This is an interesting question. I have recently found that dimer Insulinase has 6.2 μM of Km (please see file; The Fascio effect).
Therefore, you should add Insulin at 36 μg/mL or 36 mg/L to your culture medium.
Please, do not believe the notorious RIA and/or ELISA values, which are false.
By the way, I have searched for Insulin receptor in my protein database derived from PDMD (Protein-Direct-Microsequencing-Deciphering) method in vain.
Only a hepatoma HepG2 (cultured wothout fucoidan) has Insulin receptor substrate 2/IRS-2 at 15.0 μg/mg of cell protein (please see file; HepG2 fucoidan). This protein is unexpectedly resided in the membrane sub-fraction (precipitated by the ultra-centrifugation in 100,000 x g, 90 min, at 4 C).
Therefore, this protein may be the receptor actually working in humans.
IRS-2 is realy acting instead of putative insulin receptor in mouse has been recently reported by Spanish Researchers; i.e.,
"Roncero I, Alvarez E, Acosta C, Sanz C, Barrio P, et al. (2013)
Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver. PLoS ONE 8(4): e58797. doi:10.1371/journal.pone.0058797".
Interestingly, cancer portions has hormone receptors with JAK-STAT cascade (our unpublished observation); i.e.,
HCC tissue (named as No. 6) has Prolactin receptor type 2 at 6.4 μg/mg of tissue protein.
HCC tissue (with complication of primary biliary cirrhosis (PBC)) has Growth hormone receptor/GH receptor at 12.4 μg/mg of tissue protein.
By the way, insulin and insulin degrading enzyme (IDE) are also present in plant. Therefore, I am waiting for the report on how plant insulin and plant IDE are working similarly or differently to animal.
With respect to growth supplements, we have recently found that biotin (at 5 μg/mL), lipoic acid (at 50 μg/mL), and an oligosaccharide (at 200 μg/mL) are growth supplements for Caco-2 (please see file; JCB fucoidan transport), Hc, HepG2, HuH-7, HuH-6, and T24 (lipoic acid kills rat cells) (our unpublished observation at Gunma University, Maebashi, Gunma, Japan). Please add Amphotericin B to biotin-containing medium to prevent the fungal contamination.
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