Role of Bovine Serum Albumin in Blastoid Transformation of Lymphocytes by Phytohemagglutinin

Abstract

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...

Long term growth of factor-producing lymphoid and myeloid cells in serum-free medium.

“…Previously, Uittenbogaart et al (24) reported failure to maintain long-term, serum-free cultures of Mo cells, which may have been due to the use of different supplements in the serum-free medium : They used delipidated BSA and did not add lipids and insulin. Lipids in particular, appear to be essential for the growth of Mo cell because we previously demonstrated the essential role of albumin-bound lipids in human T lymphocyte growth (1). Cohn frantion V BSA recently has been reported to be required for mouse erythropoietic burst production in vitro (19) and the same authors speculated that Cohn fraction V BSA might facilitate the production of burst-promoting activity by bone marrow T cells (19).…”

“…circumvent these problems when culturing human lymphoid (1,3,4,15,17,21,24), erythroleukemic (14,23), promyelocytic (2,20) and murine lymphoid (11,13) cell lines as well as murine bone marrow cells (6,10,12,19). We previously reported the development of a serum-free medium which supports the growth of human B lymphocytes that are stimulated by pokeweed mitogen to produce immunoglobulines (21).…”

Enhancement of mitogen-induced human lymphocyte transformation by Diiron enneacarbonyl in serum-free medium.

“…In addition to 0.5% BSA (Sigma Chemical Co., St. Louis, Mo. ), Eagle's minimum essential medium (MEM) was supplemented with sodium pyruvate, insulin, putrescine, zinc sulfate and various nucleosides, as described previously (Yamane et al 1976; Arai et al 1977). RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS; Grand Island Biological Co., Grand Island, N.Y.) was used as serum containing medium.…”

Enhancing effect of saccharated ferric oxide on human lymphocyte transformation in serum-free medium.

“…The cells (5 X 105/ml) were incubated in microplates (Falcon 3040) in serum-free medium (Arai et al 1977) with or without PHA-P(Difco) and saccharated ferric oxide (`Fesin', Yoshitomi Pharmaceut.…”

“…In the course of our investigation of the factors supporting lymphocyte growth in serum-free medium (Arai et al 1977), we found that saccharated ferric oxide enhanced the [3H]thymidine incorporation into the phytohemagglutinin (PHA)-stimulated lymphocytes in serum-free culture medium supplemented with bovine serum albumin and insulin.…”