A serum-free medium supplemented with a few growth factors was devised to grow lymphocyte hybridomas. The medium was developed with the hybridoma line MPCll-BL, a fusion product between a mouse plasmacytoma cell line (MPCllTG70na3) and mouse (BALB/c) spleen cells. In the process of developing the-medium, ethanolamine was found to be an essential growth factor for the hybridoma. Phosphoethanolamine at 10-fold higher concentration could substitute for ethanolamine. Long-term cultivation of the cells was achieved in the defined medium supplemented with insulin, transferrin, ethanolamine, and selenium. The defined medium supported the growth of various other mouse hybridoma cell lines, mostly at a rate comparable to that observed in a serum-containing medium. After one-step ammonium sulfate precipitation of the spent medium, more than 95% of the protein recovered was immunoglobulin as shown by NaDodSO/polyacrylamide gel electrophoresis.Traditionally, tissue culture media have required serum supplementation in order to support long-term culture of cells. Serum is a complex mixture of components which may vary according to the age, sex, and species of its source. Sato and his co-workers have systematically investigated the hormone and growth factor requirements ofa large number ofcell lines. They described completely defined serum-free media that promote growth comparable to serum-containing media and in which cells maintain their differentiated characteristics (1, 2). Among the lines investigated 'are the mouse myeloma MPC11 (3), rat neuroblastoma B104 (4), and human colon carcinomas (5).Fusion of a plasmacytoma and a splenic plasma cell can produce a hybridoma that continues to secrete splenic antibody (6), and the monoclonal antibodies produced have been useful for immunology, molecular biology, and cell biology research (6, 7). Cultivation of these cells in a defined medium would facilitate such studies by eliminating serum, the major source of protein contamination.We have developed a hormone-and growth factor-supplemented, serum-free medium for culturing mouse plasmacytomas and hybridoma cell lines of MPC1l-BL and SP2/0-BL' origins. In the process of defining this medium we identified two compounds, ethanolamine and phosphoethanolamine, as necessary growth-promoting materials. These compounds, which had originally been found to be essential for growth of a rat mammary carcinoma (8, 9) have been required for growth of all hybridoma lines tested. Analyses of proteins precipitated at 50% ammonium sulfate saturation of the spent medium of hybridoma cell cultures indicated that in most cases >95% of the proteins were IgG or IgM. MATERIALS AND METHODSCells and Cell Culture. Most ofthe hybridoma cell lines used in these studies were obtained from W. Raschke (Salk Institute, La Jolla, CA). For NaDodSOipolyacrylamide gel electrophoresis of antibodies, hybridomas between SP2/0 and BALB/c splenocytes immunized against S100 protein were used. These cell lines were ordinarily maintained in a 1:1 mixture of Ham's F-1...
Plants often compete with closely related individuals due to limited dispersal, leading to two commonly invoked predictions on competitive outcomes. Kin selection, from evolutionary theory, predicts that competition between relatives will likely be weaker. The niche partitioning hypothesis, from ecological theory, predicts that competition between close relatives will likely be stronger. We tested for evidence consistent with either of these predictions by growing an annual legume in kin and nonkin groups in the greenhouse. We grew plant groups in treatments of symbiotic nitrogen fixing bacteria differing in strain identity and composition to determine if differences in the microbial environment can facilitate or obscure plant competition patterns consistent with kin selection or niche partitioning. Nonkin groups had lower fitness than expected, based on fitness estimates of the same genotypes grown among kin. Higher fitness among kin groups was observed in mixtures of N-fixing bacteria strains compared to single inoculations of bacteria strains present in the soil, which increased fitness differences between kin and nonkin groups. Lower fitness in nonkin groups was likely caused by increased competitive asymmetry in nonkin groups due to genetic differences in plant size combined with saturating relationships with plant size and fitness- i.e. Jensen's inequality. Our study suggests that microbial soil symbionts alter competitive dynamics among kin and nonkin. Our study also suggests that kin groups can have higher fitness, as predicted by kin selection theory, through a commonly heritable trait (plant size), without requiring kin recognition mechanisms.
The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9712-2) contains supplementary material, which is available to authorized users.
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