We have developed two serum-free chemically defined media (RITC 78-6 and RITC 80-7) that support the growth in culture of human diploid fibroblasts to the same extent as Eagle's basal medium (BME) supplemented with 10% fetal bovine serum (FBS). These two media contain modified Eagle's minimum essential medium (MEM) supplemented with nonessential amino acids, various trace metals, organic compounds and growth factors [insulin, mouse epidermal growth factor (m-EGF), transferrin and triiodothyronine (T3)]. RITC 80-7 medium differs from RITC 78-6 in that it contains thymidine, hypoxanthine, and vitamin B12 and supports the long-term serial cultivation of human diploid cultures. The addition of commercial bovine serum albumin (BSA, 5 g/liter) to the medium enhances cell growth. This effect is not observed if BSA is first delipidized, but reconstitution of BSA with certain lipids restores its ability to promote growth. BSA has an inhibitory effect on cellular attachment but this is overcome when fibronectin (FN, 10 mg/liter is added to the medium.

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Human peripheral lymphocytes are activated to. undergo blast transformation by specific and nonspecific stimulants in vitro (1)(2)(3). Supplementation with autologous serum or fetal calf serum to the medium is generally required for the transformation of lymphocytes. However, the factor(s) in serum which cause blast transformation of the stimulated lymphocytes are still unknown.The study of such factors in serum may reveal the mechanism of activation and proliferation of the stimulated lymphocytes. Polet and Spieker-Polet and others demonstrated that serum albumin itself was indispensable to the medium component for the blast transformation of lymphocytes by mitogens (4-6).Recently, Yamane et al. have formulated a serum-free culture medium (SF medium) in which various mammalian tumor cells including a human lymphoid cell type can grow well (7). With this SF medium, albumin-bound fatty acids were found to play an important role in cell culture. Therefore, we undertook to investigate mitogen-induced transformation of human lymphocytes in this medium to see whether the albuminbound fatty acids play a significant role in the transformation of lymphocytes. As a result, an important role of fatty acids in the p h yt ohemaggl ut in in (PH A)-st im ulated transformation of the lymphocytes was established.Materials and methods. Basal medium and BSA. As described previously by Yamane (7) for the preparation of basal SF medium, Eagle's minimum essential medium (MEM) was supplemented with sodium pyruvate, insulin, putrescine , zinc sulfate, and various nucleosides. All components were dissolved in deionized distilled water and then sterilized through Millipore membrane filters. BSA (Cohn Fr.V) and defatted preparations of BSA were purchased from Armour Pharmaceutical Co. (Chicago, Ill.), Pentex Inc. (Kankakee , I11 .), and Sigma Chemical Co. (St. Louis, Mo.). These BSAs were dissolved in the basal medium, neutralized with 1 N NaOH, and then sterilized through the membrane filters. Oleic and linoleic acids were first dissolved in a small amount of ethanol and then diluted to a suitable concentration with culture medium.Preparation of lymphocytes. Heparinized peripheral blood was obtained from healthy adults. The blood was mixed with one-third volume of 3% gelatin (Difco, Detroit, Mich.) in phosphate-buffered saline (PBS) as described by Coulson and Chalmers (8). The mixture was kept for 45 min at 37" to sediment the erythrocytes. The leukocyteenriched plasma was collected and washed twice with Hanks' balanced salt solution containing 0.1 % gelatin and then suspended in the culture medium. The viable cells were counted by trypan blue dye exclusion tests and adjusted to 5 X lo5 cells/ml. These cell preparations usually contained 60-70% lymphocytes as detected by Giemsa staining and had more than 96% viability.Cell culture. Lymphocytes were cultured in 6 X 100-mm round-bottomed glass tubes with loosely fitted aluminum caps. Each tube contained 1 ml of tissue culture medium with 5 x lo5 lymphocytes. These suspensions were culture...

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A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterin-thymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.