Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 ± 0.45 μm and 2.84 ± 0.14 μm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 ± 0.21 μm × 2.00 ± 0.11 μm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.

A mammary tumor cell line, designated MTCL, was successfully established from a mouse primary mammary tumor (MTP). The MTCL cells retain cytokeratin and both estrogen receptor (ER) and progesterone receptor (PR) in vitro. In vitro exposure of MTCL cells to progesterone causes a decrease in the cellular ³H-thymidine uptake, indicating an inhibition by progesterone on MTCL cellular deoxyribonucleic acid synthesis, whereas exposure of the cells to a high dose of estrogen (15 pg/ml) for 48 h causes an increase of ³H-thymidine uptake. We inoculated both MTP or MTCL tumor cells into normal cycling female C₃HeB/FeJ mice and demonstrated that the post-resection metastatic recurrence of MTCL tumors, like the original MTP tumors, depends on the time of tumor resection within the mouse estrous-cycle stage. Both MTCL and MTP tumors have similar histological appearances with the exception of less extensive tumor necrosis and higher vascularity in MTCL tumors. Equivalent levels of sex hormone receptors (ER α, ER β, and PR), epithelial growth hormone receptors (Her2/neu, EGFR1), tumor suppressors (BRCA1, P53), and cell apoptosis–relevant protein (bcl-xl) were found in these in vivo tumors by immunohistochemistry. Cyclin E protein, however, was significantly higher in MTP tumors compared with MTCL tumors. Our results indicate that MTCL cells retain many of the biologic features of the original MTP primary tumor cells, and to our knowledge, it is the first in vitro cell line that has been shown to maintain the estrous-cycle dependence of in vivo cancer metastasis.

In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3–4, and, thereafter, declined over the next 5–10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (−/−)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (−/−) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50–75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo–like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.

HeLa was the first human cell line established (1952) and became one of the most frequently used lines because of its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast-growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these “HeLa contaminants” prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without being aware of their true identity and how many were not using available means to ensure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines; yet, only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969–2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using HeLa contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations.

Retinoic acid (RA), bromodeoxyuridine (BrdU), and the Δ205 mutant polyoma middle T antigen affect the expression of a common ensemble of proteins in HL-60 human myeloblastic leukemia cells. Each of these agents is known to be able to prime HL-60 cells and accelerate subsequently induced myeloid or monocytic differentiation and G0 cell cycle arrest, suggesting that they have equal or identical cellular targets relevant to the early stages of inducing cell differentiation and G0 arrest. As a test of this possibility, a survey of protein expression changes induced by RA, BrdU, or Δ205 transfection was performed. Retinoic acid induced numerous changes within h. Bromodeoxyuridine caused larger numbers of changes, whereas Δ205 caused a more limited number. Among the hundreds of affected proteins detected, there were comparable numbers of up- or downregulated proteins. A small number changed between undetectable and detectable expression. The affected proteins were not restricted to a single functional class and included transcription factors, receptors, signaling molecules, cytoskeletal molecules, and effectors of various cellular processes such as deoxyribonucleic acid replication, transcription, and translation. The intersect of the sets of proteins affected by RA, BrdU, and Δ205 was identified to determine if these agents regulated a common subset of proteins. This ensemble contained the commonly upregulated proteins AF6, ABP-280, ENC-1, ESE 1, MAP2B, NTF2, casein kinase, IRF1, SRPK2, Rb2, RhoGDI, P47phox, CD45, PKR, and SIIIp15. The commonly downregulated proteins were SHC, katanin, flotillin-2/ESA, EB 1, p43/EMAPIIprecursor, Jab1, FNK. The composition of the ensemble suggested three apparent themes for cellular processes that were affected early. The themes reflected the ultimate fate of the treated precursor cells as a mature myeloid cell, namely a cell whose hallmarks are (1) motility to migrate to a target and phagocytize it, (2) inducible oxidative metabolism to reduce the target with superoxide from a respiratory burst, and (3) biosynthetic slow down consistent with conversion from cell proliferation to quiescence. Interestingly, RA appears to induce aspects of an interferon-like response of potential significance as part of a biosynthetic slow down leading to cell cycle arrest. In conclusion, three biologically disparate ways to prime cells to differentiate were used to filter out a small ensemble of commonly regulated proteins that group as either microtubule associated, oxidative metabolism machinery, or effectors of cellular responses to interferon.