Mammalian Cell Media

Mammalian cells are an important resource for the expression of proteins in their native structure including posttranslational modifications. If the protein of interest is to be studied by NMR spectroscopy, the cells must be cultured in isotope-labeled medium.Mammalian cell culture refers to the in vitro cultivation of cells derived from mammals. Cell culture experiments such as these are designed to minimize contamination and ensure reproducible results. This technique involves growing cells outside their native tissue environment using specialized growth media designed to mimic natural conditions and support cell proliferation, and enhance cell growth.

Mammalian cells are an important resource for the expression of proteins in their native structure, including post-translational modifications. To obtain properly folded and post-translationally modified proteins, mammalian expression systems are essential. Labeled proteins from mammalian cells are especially valuable when bacterial systems don’t yield functionally relevant proteins.

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.

Silantes has established itself as a reliable cell culture media supply partner. 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.

Stable isotope-labeled L-glutamine and dFBS are not included in the kit. These can be ordered separately from Silantes.

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 viability (figure 1) and cell count (figure 2) after several passages, respectively.

From Figures 1 and 2, it can be deduced that the isotope-labeled Silantes medium for adherent mammalian cells shows similar viability and growth characteristics to the more expensive DMEM/F12 medium when tested on various mammalian cell lines.

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 viability and cellular growth after multiple passages, respectively.

Figures 3 and 4 also show that similar viability and growth characteristics are obtained for the isotope-labeled Silantes medium for mammalian suspension cells compared to a more expensive artificial medium.

Validation of protein expression

To validate protein expression in Silantes media for mammalian cells, eGFP (enhanced green fluorescent protein) and SOD1 (superoxide dismutase 1) were expressed in adherent HEK293T and suspension HEK293sus.2 cells and compared to reference media.

The following figures show the protein yields of eGFP after 1 passage in adherent HEK293T (Figure 5) and suspension HEK293sus.2 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, 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). These results demonstrate the capability of the Silantes media formulations to support complex biology studies that involve intricate cellular pathways and protein interactions.

Validation of the isotopic enrichment

eGFP expressed in adherent HEK293T cells and suspension HEK293sus.2 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.

Although our media formulations have been optimized using established cell lines, they could potentially be adapted for primary cell cultures as well.

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

FAQ

1. How do cell culture media formulations support cell growth?
   Cell culture media supply essential nutrients, growth factors, vitamins, amino acids, and often serum or serum-free supplements that are critical for maintaining cell viability and proliferation. Optimized formulations ensure that mammalian cells, such as CHO, HEK293 or primary cells, achieve optimal cell growth and function.
2. What are the advantages of using serum-free media in mammalian cell culture?
   Serum-free media provide a chemically defined environment, reducing batch-to-batch variability and risk of contamination. This is especially beneficial for applications like recombinant antibody expression, where regulatory compliance and consistency are paramount.
3. What are the common types of mammalian cells used in culture?
   Researchers culture a wide range of mammalian cells including immortalized cell lines like CHO, HeLa, and HEK293 cells, as well as primary cells and stem cells. Each cell type may require specific formulations and supplements tailored to its unique nutrient and growth factor needs.
4. What types of labels are typically incorporated in cell culture media?
   Common labels include stable isotopes such as ¹³C or ¹⁵N. These are added to the nutrient components of the medium so that during cell growth and protein synthesis, the stable isotopes get incorporated into the proteins and the labels can be traced through metabolic processes in mammalian cells.
5. How can labeled medium enhance cell culture experiments?
   By using labeled medium, researchers can analyze the structures of the labeled biomolecules by NMR or quantitatively measure metabolic changes and nutrient utilization in real-time. This allows for a more detailed analysis of cell culture applications such as recombinant antibody expression and other protein production studies.