This is surely the difficult issue in Biochemistry and Biotechnology.

I have been doing a part time job at Dr. Takeaki Nagamine's laboratory (Graduate School of Health Sciences, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8514, Japan) from 2014 to 2015. Cell culture study has been performed from 2002 as a technical official, Ministry of Health, Labour and Welfare, Japan at Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan; i.e., I have studied biochemistry using cultured cells for c.a. 13 years as a Ph.D. researcher.

1) Greiner's advanced TC Flasks T25 (Greiner Holding AG, Kremsmünster, Österreich) is good. This may be due to the Flasks' surfaces, which give least stress to the cells as compared to another Flasks. This good result has personally informed to Greiner Holding AG.

2) Addition of D-Biotin (5.0 μg/mL) is good. In this case, addition of Amphotericin B is also necessary in order to prevent contamination by Fungi (please see file; JCB Fucoidan Transport). This idea of addition of Biotin has been due to the finding that Human children's serum contains c.a. 3.0 μg/mL of Biotin (obtained by HPLC method), which is surely higher c.a. 2,000-fold than USA's value obtained from Microbiological and Immunological methods (please see files; Wide Range of Biotin and Netherlands Biotin).

2') D-Aspartic acid is usually highly contained in cancer cells (please see file; D-Asp Avidin 1). However, addition of D-Aspartate in the medium is not successful. I am now considering that D-Aspartic acid in the cancer cells may be a precursor of D-Biotin. Fucan derived from Okinawa Futo-Mozuku is effective to child's liver cancer cell HuH-6, but not effective to adult cancer cells HuH-7 and HepG2. On the contrary, Fucoidan derived from Noto-Mozuku/Kinu-Mozuku is effective to adult cancer, but not effective to HuH-6 (please see file; Rat DEN Np-Fuco). This may be related to the different D-Aspartic acid metabolism between child and adult Humans.

I am very grateful to Dr. Marijana Pavlovic (Department of Experimental Oncology, Institut za onkologiju i radiologiju Srbije, Belgrade, Serbia) for leading me to this important conclusion.

3) Addition of Lipoic acid (50 μg/mL) is good for Human cells but not good for Rodent cells. This is due to the our article about Lipoic acid (please see file; Lipoic acid Avidin). Lipoic acid synthetase is present in Rodents, but adult Humans does not have it (Species differences with age is present in lipoate metabolism).

Another famous example of the Species differences with age.is present; i.e., Chymosin/Rennin is present in child bovine, but absent in adult bovine and in Humans.

4) Addition of Fe (0.02 mg/L) is good for AdMEM, which contains no Fe but contains Transferrin (7.5 mg/L).

5) Addition of Cu to DMEM is good. AdMEM has Cupric Sulfate at 0.00125mg/L.

6) Addition of L-Glutamine (4 or 10 mM) is usually good. Since L-Glutamine seems to be used as an energy source, I am now considering that Glucose metabolism (energy obtaining mechanism) has Species differences among Humans, Rodents, Porcine, and Bovine.

7) Addition of Oligosaccharide (0.1 mg/mL) is good. This effect seems not limited by Species differences. Effect of Oligosaccharide on bacterial growth (Lactobacillus) has been already reported (please see files; Oligosaccharide growth 1 and Oligosaccharide growth).

8) Addition of IGF may be good to human cells, since human cells have no Insulin and Insulin receptor.

9) Increase of FCS (to 35% v/v) in AdMEM (but not in DMEM) is good for Caco-2 (please see file again; JCB Fucoidan Transport). Further, I must sadly say that Williams’s Medium E is not good for rat TRL1215, HuH-7, and HuH-6. RPMI is not good to Caco-2 and JMSU1.

10) I also am considering that Hg may be effective at c.a. 50 nM concentrations, but I have been sadly gotten fired (please see file; Thiol-type BIN).

Further, I must say that Breast cancer is occurring in male. Therefore, I am now estimating that Breast cancer in Humans is occurring by the co-infection of HIV-1 and Dengue virus/DENV (Flavivirus; +ssRNA；with envelope) or Sindbis virus/SINV (Togavirus; +ssRNA；with envelope). Further, Chikungunya virus/CHIKV (Togavirus) may be possible (please see file; HepG2 Fucoidin).

It is a characteristic of the cell line: ATCC says "The line grows as large epithelial cells with a tendency to float at high cell densities"

http://www.lgcstandards-atcc.org/products/all/CRL-2336.aspx?geo_country=it

My suggestion is that you use cell culture plates that have specific chemical properties to be the most adherent ones among the available ones. In this way you could compensate.

What about your serum concentration? My medium consist of RPMI 1640 with 15% fetal bovine serum.

Yang Huanhuan I'm using RPMI with 10%FBS. I have already tried to increase serum to 15%, but cells started to differentiate into gigantic multivacuolated cells, and epithelial cells became smaller population of this heterogenic cell line. Do you use any special conditions, culture flasks or medium components when cultivating this cells? I'm using RPMI with 4.5 g/l D-glucose. What is the population doubling time in your case?

Thanks