In discussions of serum-free media formulations, albumin and transferrin often take center stage, while insulin — a factor that appears in virtually every serum-free media ingredient list — is rarely discussed on its own. This is a curious oversight. Regardless of which supplier's serum-free media you use, insulin is almost never absent. It is not only the "fuel switch" for cell metabolism, but also a critical bottleneck factor sustaining the continuous proliferation of multiple industrial cell lines (CHO, HEK293, Vero, MSC). This article systematically examines the biological functions of insulin and the role, selection strategies, and application considerations of recombinant insulin in serum-free culture.

01 What Insulin Actually Does in Cell Culture

Insulin is a polypeptide hormone composed of 51 amino acids. In vivo, it is secreted by pancreatic β-cells, regulating blood glucose and promoting anabolic metabolism. In the culture dish, insulin's role extends far beyond "lowering blood sugar."

Pro-proliferative Signal Transduction

In the cell culture environment, insulin acts primarily through two pathways:

First, by binding to the insulin receptor (IR), it activates the PI3K/Akt pathway, promoting glucose uptake and glycogen synthesis while upregulating amino acid transporter expression, providing sustained nutrient supply to cells.

Second, at higher concentrations, insulin can cross-activate the insulin-like growth factor-1 receptor (IGF-IR). IGF-IR activation triggers not metabolic signals, but mitogenic signals — the Ras/Raf/MAPK pathway is engaged, and cells enter the proliferative program.

This is why insulin supplementation concentrations in serum-free media are typically far higher than physiological levels (mg/L rather than ng/L): in serum-free systems, insulin is deployed simultaneously as both a metabolic factor and a growth factor.

Anti-Apoptotic Effects

Insulin, via the Akt signaling pathway, phosphorylates and inactivates the pro-apoptotic proteins Bad and Caspase-9, while upregulating expression of the anti-apoptotic protein Bcl-2. For production cell lines such as CHO and HEK293 in long-term suspension culture, insulin's anti-apoptotic function translates to higher viable cell density and extended culture duration.

Regulation of Glucose and Amino Acid Metabolism

Insulin promotes membrane translocation of the GLUT4 glucose transporter, accelerating glucose entry into cells. Simultaneously, it activates the mTORC1 signaling pathway, promoting protein synthesis. Both effects are particularly critical in serum-free media — in the absence of the natural growth factors provided by serum, cells depend even more heavily on efficient uptake mechanisms for the limited nutrients available in the medium.

02 Insulin in ITS Formulations: More Than Just "Adding It In"

ITS (Insulin-Transferrin-Selenium) is the most classic three-factor supplement combination in serum-free media. The division of labor among the three components is clear: insulin drives proliferative signals and metabolism; transferrin transports iron ions, maintaining the activity of key enzymes; and sodium selenite provides selenium, supporting glutathione peroxidase activity.

However, ITS is not a fixed formulation — insulin concentrations must be adjusted based on cell type and culture objectives:

• CHO cell protein expression: 5–10 μg/mL — supports high-density suspension culture

• HEK293 virus production: 5–10 μg/mL — supports post-transfection cell recovery

• MSC expansion: 1–5 μg/mL — synergizes with bFGF; avoids overstimulation of differentiation

• Hybridoma cells: 5–10 μg/mL — traditional supplementation concentration

• Vero cell vaccine production: 1–5 μg/mL — lower concentrations suffice for metabolic maintenance

Insulin concentration is not "the higher the better." In MSC culture, excessively high insulin concentrations may accelerate cell aging or trigger non-specific differentiation through sustained IGF-IR activation. In CHO cells, excessive insulin signaling may lead to abnormal lactate metabolism. Optimal concentrations should be determined through cell growth curves and metabolite analysis.

03 Key Practical Considerations

Instability of Insulin Solutions

Insulin in aqueous solution is prone to fibril formation and deamidation degradation products, especially under 37°C culture temperatures and repeated freeze-thaw conditions. Recommendations: store stock solutions at -20°C after aliquoting; avoid more than 3 freeze-thaw cycles; use complete media supplemented with insulin promptly after preparation and avoid prolonged storage.

Synergy with Transferrin

Insulin and transferrin exhibit a pronounced synergistic effect in serum-free media. Transferrin supplies iron ions for ribonucleotide reductase (a key enzyme in DNA synthesis), while insulin drives protein synthesis through mTOR signaling. When both are simultaneously absent, most cell lines show marked proliferative arrest within 48 hours. Upon restoration of both factors, cells typically resume growth within 24 hours.

Division of Labor with bFGF

In MSC serum-free culture, bFGF is responsible for maintaining stemness (sustaining Oct4 and Nanog expression through FGF receptor activation), while insulin provides basal metabolic support and broad-spectrum proliferative signals. bFGF is the "steering wheel"; insulin is the "accelerator." Without bFGF, MSCs differentiate; without insulin, MSCs come to a standstill.

04 SciproTech Recombinant Human Insulin Product

Catalog No.: CK1038S | Product Name: Recombinant Human Insulin | Format: Lyophilized powder, -20°C storage. Suitable for ITS formulations and serum-free media preparation; from the same system as ART202S Transferrin and ART103S Albumin.

05 Conclusion

Insulin is not a supporting player in serum-free media. It integrates three functions: metabolic driver, proliferative signal, and anti-apoptotic protection. As the CGT and biopharmaceutical industries continue to demand serum-free, animal-origin-free, chemically defined media, the role of recombinant human insulin will only grow in importance. Treating it as a "basic ingredient" while overlooking differences in quality is the cheapest mistake one can make — because when insulin fails, the entire culture system comes to a standstill.

References

1. Barnes, D., & Sato, G. (1980). Serum-free cell culture: a unifying approach. Cell, 22(3), 649-655.

2. Hayashi, I., et al. (1978). Hormone growth factor combinations for serum-free cell culture. In Vitro, 14(1), 23-30.

3. Zhang, J., et al. (2012). Insulin and IGF-I stimulate proliferation of human MSCs. Stem Cells International, 2012.

4. Taniguchi, C. M., et al. (2006). Critical nodes in signalling pathways: insights into insulin action. Nature Reviews Molecular Cell Biology, 7(2), 85-96.