Roswell Park Memorial Institute (RPMI) 1640 is a liquid growth medium used in biological research to cultivate specific types of cells. Developed in 1966 by George E. Moore and his colleagues at Roswell Park, it was initially created to support the growth of human leukemic cells. Gibco, a brand under Thermo Fisher Scientific, is a major commercial producer of RPMI 1640, providing standardized formulations for research. The medium is a frequent choice for studies in immunology, cancer biology, and drug discovery.
Defining the RPMI 1640 Formulation
RPMI 1640 is an aqueous solution containing a mixture of amino acids, vitamins, inorganic salts, and glucose as the primary energy source. The formulation is rich in certain components absent in other common media like Eagle’s Minimal Essential Medium (EMEM). It contains high concentrations of inositol and choline, the reducing agent glutathione, and the vitamins biotin, vitamin B12, and para-aminobenzoic acid (PABA).
A key feature of RPMI 1640 is its sodium bicarbonate buffering system (2.0 g/L), which maintains a physiological pH around 7.2-7.4. To work correctly, the medium must be used in a humidified incubator with 5-10% carbon dioxide (CO2). The CO2 dissolves in the medium to form carbonic acid, which interacts with the bicarbonate to resist pH changes.
To provide a visual cue of the pH, most RPMI 1640 formulations include phenol red. This pH indicator gives the medium its pink-red color at physiological pH. If the medium becomes too acidic from cellular metabolism, it turns orange or yellow. If it becomes too alkaline from CO2 loss or contamination, it shifts to a fuchsia or purple color.
Primary Cell Culture Applications
RPMI 1640 is particularly well-suited for growing cells in suspension, meaning they do not attach to the culture flask surface. This makes it the medium of choice for many hematopoietic cells, which are related to blood and the immune system. Examples of cell lines routinely grown in RPMI 1640 include human lymphocytes, Jurkat cells (an immortalized line of T-lymphocyte cells), and K-562 cells (a chronic myelogenous leukemia line).
The medium is also widely used in hybridoma technology for generating monoclonal antibodies. Hybridomas are created by fusing antibody-producing B cells with myeloma cells, and these hybrid cells grow well in suspension. While designed for suspension cells, RPMI 1640 can also support the growth of some adherent cells, which require a surface to attach to.
In comparison, Dulbecco’s Modified Eagle Medium (DMEM) is generally favored for adherent cell types like fibroblasts and endothelial cells. DMEM typically has higher concentrations of certain amino acids and vitamins than RPMI 1640. The specific balance of components in RPMI 1640 creates an environment more suitable for lymphoid and other non-adherent cell lines.
Standard Supplementation and Variations
The base formulation of RPMI 1640 lacks proteins, lipids, and growth factors, so supplements are added to create a “complete” growth medium. The most common addition is Fetal Bovine Serum (FBS), a mixture containing a wide array of growth factors and hormones. FBS is typically added to RPMI 1640 at a final concentration of 10%, though this can vary depending on the cell type.
Another routine supplement is a combination of antibiotics to prevent microbial contamination, which can quickly ruin a cell culture. The most common cocktail is a mixture of penicillin and streptomycin, often called “pen-strep.” This combination is effective against a broad range of common bacterial contaminants.
Gibco offers several variations of the RPMI 1640 formulation. Some substitute the standard L-glutamine with the more stable dipeptide, L-alanyl-L-glutamine (GlutaMAX). Another modification is the addition of HEPES buffer, which provides extra buffering capacity to maintain a stable pH outside of a CO2 incubator. For experiments where phenol red could interfere with data collection, such as fluorescence-based assays, a formulation without the indicator is available.
Handling and Storage Protocols
Proper handling and storage are necessary to maintain the quality of RPMI 1640. Liquid RPMI 1640 must be stored refrigerated between 2°C and 8°C. It should also be protected from light, as exposure can degrade light-sensitive components like vitamins and riboflavin, reducing the medium’s effectiveness.
Powdered RPMI 1640 has a longer shelf life and is stored in a cool, dry environment at room temperature. Once reconstituted into a liquid, it should be stored under the same refrigerated and light-protected conditions as the pre-made version. The shelf life for an unopened bottle of liquid RPMI 1640 is around 12 months, but once opened, it should be used within a few weeks.
Strict aseptic technique is paramount when working with the medium to prevent microbial contamination from bacteria, yeast, or mold. This involves working in a sterile environment, such as a biological safety cabinet, and using sterilized equipment. A breach in sterile procedure can introduce microorganisms that outcompete cultured cells for nutrients and release toxic byproducts, leading to the loss of the culture.