Cholesterol requirement of P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro.

Abstract

Since the original demonstration by Kohler and Milstein (1) of the feasibility of using cell fusion to produce antibody-secreting hybridomas, several improved mouse myeloma parent cell lines have been developed. In recent years NS-1-Ag4-1 (NS-1) (2), Sp2/0-Ag-14 (Sp2/0) (3), and X63-Ag8.653 (X63.653) (4) have been commonly used to produce hybridomas . All three of these cell lines were independently derived from the original myeloma parent line P3-X63-Ag-8 (X63) (1), and all three cell lines differ from X63 with respect to Ig gene expression . Sp2/0 cells were obtained by subcloning Sp2/3-3, an X63 hybridoma (1, 3), while NS-1 and X63.653 were subcloned in several steps from X63. An examination of the growth requirements of NS-1 cells in serum-free medium revealed that NS-1 had absolute requirement for low density lipoprotein (LDL), which was not shared by NS-1 hybridomas (5). The lipid requirement of NS-1 cells was subsequently found to be a requirement for cholesterol (6). As cholesterol auxotrophy is an exceedingly rare phenotype in cultured mammalian cells, it was of interest to determine whether NS-1 cells were unique among X63 and the other derivatives of the MOPC21 cell line P3 (7). We have found that Sp2/0 cells require neither LDL (6) nor cholesterol for growth, and here we demonstrate that X63 and X63.653 cells, like NS-1 cells, are cholesterol auxotrophs . In addition, we report that cholesterol-independent variants of X63 and X63.653 can be selected . Materials and Methods Brief Definitive ReportMaintenance of Cells and Medium Preparation. P3-X63-Ag8, X63-Ag8 .653, Sp2/0-Ag14, and U937 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in a humidified atmosphere of 5% C02 at 37°C in RD + 5F (factor) medium supplemented with 1% or 5% FCS (HyClone Laboratories, Logan, UT). RD + 5F medium consisted of a 1 :1 mixture (by volume) of RPMI 1640 (Gibco Laboratories, Grand Island, NY) and DME (high glucose; Gibco Laboratories) with 5 ug/ml crystalline bovine insulin (Sigma Chemical Co., St. Louis, MO), 5 'Ug/ml human transferrin (Fe"-free), 10 juM 2-ME, 10 AtM 2-aminoethanol, and 10 nM sodium selenite (all from Sigma Chemical Co.). The 5F supplements were made as sterile 200X concentrates and stored at 4°C. RD medium included 15 mM Hepes buffer, 2 MM Lglutamine, 0 .01% sodium pyruvate (all from Sigma Chemical Co.), 2.2 g/L sodium bicarbonate (J.T. Baker Chemical Co., Phillipsburg, NJ), 20 mg/L penicillin G (Sigma Chemical Co.), 45 mg/liter streptomycin sulfate and 40 mg/L ampicillin (both Sigma

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