Amphotericin B (AmB) is a macrocyclic polyene compound derived from the bacterium Streptomyces nodosus and functions as a potent, broad-spectrum antifungal agent. This compound is a common additive in cell culture media, where it is used to guard against the proliferation of yeasts and molds. Its inclusion maintains the sterility and integrity of sensitive cell lines during prolonged in vitro cultivation.
Preventing Fungal Contamination
Maintaining an aseptic environment is crucial in cell culture, as contamination by fungi and yeast can rapidly destroy valuable experiments and cell lines. Fungal spores are ubiquitous, easily introduced into cultures from the laboratory air, contaminated reagents, or poor aseptic handling techniques. Once established, fungal contamination is problematic because the organisms often grow slowly at first, only becoming visible when the culture is already compromised.
Standard antibacterial agents, such as penicillin and streptomycin, are routinely added to culture media to prevent bacterial growth but have no effect against eukaryotic fungal cells. This leaves the culture vulnerable to yeast and mold proliferation, which can quickly consume nutrients and produce toxic metabolites. The addition of AmB, or a similar antimycotic, is a necessary measure to fill this protective gap, ensuring that the media remains inhospitable to all major microbial contaminants.
How Amphotericin B Targets Fungi
Amphotericin B is classified as a polyene antibiotic, characterized by a structure that gives it an amphiphilic nature. This allows the drug to interact with and disrupt the fungal cell membrane, the primary site of its action. The antifungal activity is initiated by AmB’s high affinity for ergosterol, the sterol molecule that provides structural stability to fungal and yeast cell membranes.
By binding specifically to ergosterol, AmB molecules aggregate to form transmembrane channels or pores within the fungal cell membrane. This pore formation compromises the membrane’s integrity, leading to an immediate increase in permeability. Monovalent ions, such as potassium (\(K^+\)), leak out of the cell, disrupting the osmotic balance and electrical gradient. The leakage of cellular contents results in irreversible metabolic dysfunction, ultimately causing the death of the fungal cell.
Standard Preparation and Usage
Amphotericin B is typically supplied as a liquid stock solution, often at a concentration of 250 µg/mL, which is solubilized using a detergent like sodium deoxycholate. This solubilizing agent is necessary because the compound is virtually insoluble in water at the neutral pH of standard culture media. The recommended working concentration for routine prevention typically falls within the narrow range of 0.25 to 2.5 µg/mL.
To maintain potency, the stock solution must be stored frozen, usually at a temperature between -5°C and -20°C, and protected from light, as the polyene structure is highly photosensitive. Once added to the culture medium, AmB is stable for approximately three days at the physiological temperature of 37°C.
For continuous culture maintenance, the media containing the antimycotic agent should be refreshed at regular intervals, ideally coinciding with routine feeding or passaging schedules. Higher concentrations, up to 5 or 10 µg/mL, may be used for short-term, curative treatment in cases of contamination. This heightened concentration mandates close observation of the cultured cells for signs of distress.
Assessing Cytotoxicity in Cell Lines
The primary limitation of AmB is its potential to exert a toxic effect on cultured mammalian cells, which is known as cytotoxicity. While the drug preferentially targets ergosterol in fungi, it can also interact with cholesterol, the sterol found in mammalian cell membranes. This interaction, especially at higher concentrations, can lead to pore formation in the mammalian cell membrane, mimicking the destructive effect seen in fungal cells.
Due to this risk, researchers must empirically determine the optimal concentration for each specific cell line before routine use. This involves performing a titration, exposing the cell line to a range of AmB concentrations. The goal is to identify the minimal effective concentration that inhibits fungal growth while remaining non-toxic to the mammalian cells and preserving viability.