Amphotericin B is an antifungal agent frequently used in biological research. It prevents and eliminates fungal contamination in cell cultures, maintaining sterile conditions for reliable experimental results and healthy cell growth.
Primary Purpose in Cell Culture
Amphotericin B prevents and treats fungal and yeast contamination in cell culture. These contaminants consume nutrients, alter media pH, and produce toxic byproducts, leading to cell death and compromised experiments. Fungal spores are ubiquitous, entering cultures via airborne particles, contaminated reagents, or improper aseptic techniques. Amphotericin B acts prophylactically to minimize fungal growth and therapeutically to eradicate existing infections, restoring cell line health.
How Amphotericin B Acts
Amphotericin B, a polyene antifungal, interacts with sterols in cell membranes. Its primary target in fungi is ergosterol, a sterol unique to fungal cell membranes. When it binds to ergosterol, it forms pores within the fungal membrane, disrupting its integrity. This leads to the leakage of essential intracellular components, causing the fungal cell to die.
Mammalian cells, in contrast, primarily contain cholesterol, not ergosterol, in their membranes. While Amphotericin B can interact with cholesterol, its affinity is considerably lower than for ergosterol. This accounts for its selective toxicity towards fungi.
Practical Usage Guidelines
Amphotericin B is added to media at concentrations ranging from 0.25 to 2.5 µg/mL. It is supplied as a 250 µg/mL stock solution, prepared in deionized water with excipients like sodium deoxycholate and sodium phosphate for solubility. Stock solutions should be stored frozen at temperatures between -5°C and -20°C for stability, remaining stable for up to 12 months.
Once thawed, working solutions are kept at 2-8°C for a few weeks, but stability in culture at 37°C is limited to about three days. Avoid repeated freeze-thaw cycles, as they can degrade the compound. Amphotericin B is light-sensitive; protect stock solutions and media from light during storage and handling. Use aseptic techniques when preparing and adding Amphotericin B to prevent contamination.
Effects on Cultured Cells
While Amphotericin B targets fungal cells, it can be toxic to cultured mammalian cells, particularly at higher concentrations or with prolonged exposure. This is due to its interaction with cholesterol in mammalian cell membranes, though weaker. Signs of cellular stress or toxicity include changes in cell morphology (e.g., rounding, loss of confluency) or vacuole formation.
To minimize adverse effects, use the lowest effective concentration of Amphotericin B. Some cell lines are more sensitive than others, so empirical testing for optimal dosage is beneficial. If contamination is eliminated, reducing or discontinuing Amphotericin B use promotes cell health and prevents long-term toxicity.