Mammalian Cell Media

Artificially prepared isotope-labeled mammalian media, like the ones based on DMEM formulation, require all amino acids to be exchanged with the isotope-labeled counterpart. This drives up costs and is often not economically feasible. Silantes has developed a medium that solves this problem:

Silantes’ stable isotope-labeled medium for mammalian cells is based on protein fractions from a biotechnologically fermented organism.

During fermentation, proteins are economically enriched with stable isotopes. The carefully hydrolyzed protein fractions constitute the source for the labeled amino acids and provide a cost-effective way for the culture of both adherent and suspension mammalian cells.

Bio-Based Stable Isotope-Labeled Media for Mammalian Cells

Silantes has developed media kits for both adherent and suspension mammalian cells:

• For the cultivation of adherent mammalian cells, we offer a stable isotope-labeled media kit that contains all the components necessary for growth and protein expression.
• For the cultivation of suspension mammalian cells, we also offer a stable isotope-labeled media kit. The kit contains higher amino acid concentrations and additional growth factors compared to the kit for adherent mammalian cells.

Both media kits are available for ¹³C, and ¹⁵N, and ¹³C,¹⁵N isotope labeling.

The following table shows the amino acid quantities exemplary from 15N-labeled protein hydrolysates that are present in the media kits for adherent and suspension mammalian cells. For comparison, the amino acid concentrations in a conventional DMEM/F12 medium are also shown in the table.

Amino acid	15N Silantes medium kit for adherent mammalian cells Conc. [µmol/L]	15N Silantes medium kit for suspension mammalian cells Conc. [µmol/L]	Reference (DMEM/F12) Conc. [µmol/L]
ALA	162.39	488.74	49.95
ARG	25.96	266.04	699.47
ASN	–	74.09	49.96
ASP	1173.52	857.87	49.96
CYS	–	–	99.98
CYS(2)	–	1.96	99.90
GLN	–	–	2.504.37
GLU	67.70	504.00	49.96
GLY	162.91	277.70	249.77
HIS	–	43.04	150.17
ILE	22.96	241.22	414.47
LEU	37.96	282.39	449.46
LYS	38.48	154.65	500.25
MET	–	87.13	115.54
PHE	17.74	126.87	214.78
PRO	44.87	136.74	150.00
SER	43.30	134.39	249.79
THR	33.26	132.26	449.55
TRP	–	12.26	44.17
TPR	–	93.39	213.68
VAL	34.04	236.35	449.51

The comparison shows that the Silantes media contain most amino acids in comparable concentrations to the DMEM/F12 medium.

L-glutamine, an important amino acid for the growth of mammalian cells, is not present in the Silantes media. Glutamine should therefore be added externally to the medium in its unlabeled or labeled form depending on the desired label incorporation of the expressed protein.

For all the following experiments, 2 mL of 200 mM L-glutamine was added to the Silantes medium.

Validation of cell growth

To validate the growth-performance of the Silantes media kits for mammalian cells, growth experiments were performed and compared to experiments with conventional artificial media.

Growth of adherent mammalian cells

To validate the growth-performance of the Silantes medium for adherent mammalian cells, the growth was compared to DMEM/F12 medium. Figures 1 and 2 show cellular growth and viability after 1 passage (figure 1) and multiple passages (figure 2), respectively.

Growth of suspension mammalian cells

To validate the growth-performance of suspension mammalian cells in Silantes medium kit it was compared to BalanCD medium. Figures 3 and 4 show cellular growth and viability after multiple passages, respectively.

Validation of protein expression

To validate protein expression in Silantes media for mammalian cells, eGFP (enhanced green fluorescent protein) and SOD1 were expressed in HEK293T cells and compared to reference media.

The following figures show the protein yields of eGFP after 1 passage in adherent (Figure 5) and suspension HEK293T cells (Figure 6).

The protein expression experiments show that the overexpressed protein yields from cells cultured in the Silantes Medium Kit for adherent mammalian cells and the Silantes Medium Kit for suspension mammalian cells exceed the protein yields achieved with DMEM/F12 and competitor medium.

The protein superoxide dismutase 1 (SOD1), an enzyme implicated in apoptosis, familial amyotrophic lateral sclerosis and Parkinson’s disease, was expressed in adherent HEK293T cells in different media. Figure 7 shows the SDS-PAGE comparing the protein yields in DMEM/F12 medium (“R”), Silantes Medium Kit for adherent mammalian cells (“SI”) and a comparable medium from a competitor (“X”).

The stained SOD1 band intensities indicate that the Silantes Medium Kit for adherent mammalian cells achieves comparable protein yields with artificial DMEM and competitor media.

The experiment also shows that the Silantes Medium Kit for adherent mammalian cells is not suitable for use with a DNA-transfection reagent complex.

Growth and protein expression were also validated for MDA-MB-231 cells (courtesy of Prof. Dr. Stefan Kalkhof,,Institute of Bioanalytics at Coburg University of Applied Sciences).

Validation of the isotopic enrichment

eGFP expressed in adherent and suspension HEK293t cells using ¹⁵N-labeled Silantes Medium Kit was analyzed for its isotopic enrichment by tandem mass spectrometry. The following figures show the results of the analysis in adherent culture (Figure 8) and suspension culture (Figure 9).

After just one passage in the Silantes Medium Kit for adherent cells supplemented with ¹⁵N-labeled L-glutamine an isotope enrichment of over 90 atom % was achieved in the overexpressed eGFP. When using unlabeled L-glutamine, a ¹⁵N isotope enrichment of over 85 atom % was achieved after the first passage using a starvation protocol.

The ¹⁵N isotope enrichment for valine reaches almost 80 atom % after 4 passages with Silantes Medium Kit for mammalian cells supplemented with unlabeled L-glutamine.

The analysis suggests that ¹⁵N isotope enrichment of over 90 atom % can be achieved in the Silantes Medium Kit for suspension mammalian cells by either supplementing it with isotope-labeled L-glutamine or using of the starvation protocol.

Both the passage protocol and the starvation protocol are provided by Silantes.