The aim of this study was to develop a technique to culture satellite cells from isolated intact fast or slow human muscle fibers. Previous studies have been carried out on small rodent muscles where the fibers run from tendon to tendon, but this is the first description of the modification of this technique for much larger human muscles. We have demonstrated that the human muscle fibers are in fact segmental, and we have also shown that it is possible to obtain very pure satellite cell cultures. We discuss the importance of this technique as a source of highly purified muscle cell cultures, which can be used for further studies on satellite cell behavior.

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One^®, Trident^®, MYX-1806, Teknar-HPD^®, and Thuricide^®) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15–35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid–ribonucleic acid (DNA–RNA) hybridization, and in 103 cell cultures by PCR and DNA–RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successfull alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1–3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66–85% of the cultures treated. Mycoplasma resistance was seen in 7–21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3–11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee^™ software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 ± 0.12, 1.08 ± 0.07, and 1.15 ± 0.14 (mean ± SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 μM).

The collagen gel contraction (CGC) assay is used frequently to study the cell-mediated reorganization of the extracellular matrix. In a typical CGC assay, cells embedded in a disk-shaped lattice (gel) of native type I collagen fibers compress the fibers and, consequently, reduce the diameter of the collagen disk within h or d. The degree to which the collagen is contracted is usually quantified by measurement of the diameter or the area of the disk. During CGC assays, friction or adhesion (or both) between gels and their culture containers can cause gels to be incompletely contracted or to acquire distorted shapes. Such occurrences degrade the reproducibility and reliability of measurements of gel dimensions. To address these problems, we developed an oil-supported collagen retraction (OSCR) assay that creates an environment of low friction and adhesion around the contracting collagen gel. The OSCR assay is accomplished with simple equipment and is easily performed, sensitive, and consistently yields fully contracted gels with minimal distortion.

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5–7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 μM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50–150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The β-adrenergic receptor (βAR) population was 4000–7000 βARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25–30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the βAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000–18,000 βARs per cell. Basal concentration of cyclic adenosine 3′:5′monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 μM isoproterenol, limited increases of 12–20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 μM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher βAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.

Anchorage independence is an important hallmark of the transformation that correlates with tumorigenicity. We have isolated a variant clone of HT1080 human fibrosarcoma cells (cl-2) that is specifically defective in anchorage-independent growth. Interestingly, 10⁻⁷ M dexamethasone (DEX) substantially rescued the anchorage-independent growth of cl-2 cells in semisolid culture. DEX also promoted the anchorage-independent growth of parental HT1080 cells. However, the agent had no effect on the anchorage-dependent growth of cl-2 and parental cells in ordinary liquid culture. Cell cycle analysis demonstrated that the population of G₀/G₁ cells increased, whereas that of S and G₂/M cells decreased in growth-arrested cl-2 cells in suspension culture. However, such an effect of anchorage loss on cell cycle progression was alleviated by adding 10⁻⁷ M DEX. In cl-2 cells in semisolid culture, DEX suppressed the expression of P27^Kip1, whereas it stimulated the expression of cyclin A and hyperphosphorylated retinoblastoma (Rb) proteins. On the other hand, DEX had no effect on cyclin D1 and P21^Cip1 expression. These effects of DEX, except for the suppression of P27^Kip1, were blocked by an antimicrofilament drug, cytochalasin D. Our results suggest that the stimulation of anchorage-independent growth by DEX involves at least two regulatory mechanisms, i.e., one that leads to the suppression of P27^Kip1 protein without requiring cytoskeletal integrity, and another that requires cytoskeletal integrity, leading to stimulation of cyclin A and hyperphosphorylation of Rb protein.

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80–87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in 1g cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.