Frederick H. Kasten

A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials.

SummaryA model experimental culture system and protocol are described to screen polymerized dental materials for diffusible toxic products. The system employs cultures of human gingival fibroblasts grown in plates containing immobilized samples of polymerized resins. Comparative cytotoxicity is evaluated by counting viable cells with the aid of phase optics at several time periods up to 48 h. To achieve adequate statistical sampling, multiple counts are made in four different zones at 90° angles from each sample and at three distances from the centers of samples. The most significant data were generated during a 24 to 48 h test period in culture. This cytotoxicity test measured cell death as a function of time of exposure and distance from the sample (24 h, 0 to 3 mm; 48 h, 3 to 6 mm) and permitted a calculation of the relative cytotoxicity for each material, which is termed the viability index (VI). This can be expressed as a percentage related to the control, which is called the time-distance cytotoxicity index (TDCI). This method is simple to carry out because it uses basic laboratory equipment, is rapid, and has a sound scientific basis. It focuses on times and distances when or where, or both, the greatest cellular changes are taking place. Some data illustrated are based on the screening of eight different restorative resins. The literature of cell culture testing of dental materials is reviewed.It is concluded that biotoxicity studies ideally should employ diploid human target cells from the oral cavity because the cells retain specialized features. Secondary cultures or strains of human diploid gingival fibroblasts, which are relatively easy to obtain and maintain, are recommended as cells of choice for screening dental restorative materials in vitro.

The Origins of Modern Fluorescence Microscopy and Fluorescent Probes

This review of the history of fluorescence microscopy and fluorescent probes emphasizes the roots of modern work in the field and contemporary lines of research. It centers on significant accomplishments and the pioneers involved. The contribution is presented as a survey rather than an exhaustive review. To conserve space, bibliographic citations are restricted largely to a small number of significant articles and reviews. Further details and references are given in “The Development of Fluorescence Microscopy up through World War II” ( 21 ). Other fundamental sources of information are De Ments volume on Fluorochemistry ( 5 ) and Radley and Grants Flourescence Analysis in Ultra-Violet Light ( 34 ). The volumes by Wang and Taylor ( 41a , 42c ) give a modern perspective of fluorescence microscopic technology applied to living cells.

T-tubes in cultured mammalian myocardial cells.

SummaryT-tubes are among the last structural elements of the mammalian myocyte to develop in vivo. We were able to identify T-tubes in early cultures of neonatal rat myocytes. Ventricles were excised from 3- to 4-day-old neonatal rats, incubated overnight in cold trypsin, and treated with sequential changes of collagenase-hyaluronidase. Fractions of cells isolated in this manner were pooled and cultured in plastic petri dishes. In cells prepared for transmission electron microscopy, T-tubes were observed at the cell periphery of cultured myocytes, but were more difficult to identify as the cultures aged and became overgrown by fibroblasts. T-tubes were identified by virtue of their continuity with the sarcolemma, their relatively large diameter, and their regular entry at the level of the Z line. Even at optimal culture ages, T-tubes were not present in every myocyte. At the times T-tubes could be located, myocytes were beating and had begun to establish intercalated discs and gap junctions. The de novo formation of T-tubes in cultured myocytes of neonatal rat heart reflects a duplication of in vivo differentiation by the cultured myocyte. The appropriateness of cultured myocytes in the study of the development and physiology of the heart is emphasized by the in vitro formation of T-tubes.

Functional Capacity of Neonatal Mammalian Myocardial Cells during Aging in Tissue Culture

Human aging accompanied by a logarithmic increase in death rate from disease, especially from cardiovascular disorders and decreased efficiency in homeostatic mechanisms. Comfort (1) speculates that, is throughout life man had the same resistance to stress and disease as at age 20, one-half of us could expect to live to the age of 700. Heart diseases are the leading causes of death in people over the age of 55. Since the best way to prolong life in humans is to remove the pathologic causes of death, it would be the most effective course ofaction. The heart is responsive to a multiplicity of external influences impinging on it through the circulation and by the nervous system. Within the heart, self-regulation occurs by means of pacemaker tissue and the Purkinje fiber system (2). Because of these factors which complicate studies of the cellular changes of myocardial cells during aging, we have chosen to work with ventricular newborn rat heart cells in culture as an experimental model. The initial studies on this material were made by Harary and Farley (3) and later by Mark and Strasser (4). We find that the individual myocardial cells are self-contracting units which rapidly link up in vitro to form beating networks. In long-term primary cultures which are kept as long as 100 days, these networks aggregate during this period and produce “mini-hearts” and fibers which are visible to the eye. Cultured cells can be sucessfully stored at liquid nitrogen temperature and recultured several times. These and other results were presented at the meeting, partly in the form of two 16 mm movie films --- “Mitosis and Differentiated Properties of Mammalian Myocardial Cells in Culture”, and “Contractile Behavior of Myocardial Cells In Vitro”. For the purpose of documentation, still photomicrographs, which are based on the same biological material as that employe- in the films, are used in the manuscript.

Hormonal secretion of cultured rat ovarian follicles isolated at various hours of proestrus

SummaryRat ovarian follicles were isolated at nine different times during proestrus and cultured for 3 d. During the period of cultivation, estrogen and progestagen secretion was measured. Other follicles were dissected out at the same time periods and analyzed directly for these hormones. The most intense hormone release was observed during the first 24 h in culture. The concentration of steroids secreted in vitro by follicles previously collected on the morning of proestrus was relatively low. The secreted estrogen concentration increased gradually from follicles previously isolated at later times during proestrus, with a maximum at 1800. The peak secretion of progestagens in culture was from follicles isolated at 2000. There was a marked decrease in secretion of both hormones from follicles isolated past their optimal times. The same secretion pattern of estrogens and progestagens was shown by follicles in vitro at 24 h as with similar follicles in vivo, according to their time of isolation. From these results it is evident that the normal rhythm of steroid secretion, predetermined at the time of follicle excision, was maintained during 24 h in organ culture. After a longer period in culture, the dynamics of hormone synthesis became altered, although follicles appeared morphologically normal up to 48 h.

Chapter 15 A Simple Device and Procedure for Successful Freezing of Cells in Liquid Nitrogen Vupor

Publisher Summary Cell preservation at low temperatures has become an essential practice in cell biology, particularly in laboratories working with tissue cultures. Sufficient experience has been gained in the practical technology of cryobiology to permit highly successful preservation of cell suspensions. Some of the freezing equipment that has been used for cell freezing includes sophisticated controllers. For the laboratory that only occasionally is required to freeze cells, the use of elaborate and expensive equipment would likely not be justified. Alternative approaches involve the placing of ampoules for timed periods in Dry-Ice chests or Dry-Ice-ethanol mixtures. Other freezing methods involve direct cooling in vapor or liquid nitrogen. In another approach, a mechanical freezer or liquid nitrogen is used to cool an ethanol bath containing the ampoules. While many of the various methods mentioned are reasonably effective, the recovery rate varies largely, according to the rate of initial freezing. The methods also differ in cost, sophistication and automation, amount of handling, and reproducibility of results. The cell freezer to be described has a number of attractive features that appeal to cell biologists and others working with isolated cell suspensions, especially where there is an infrequent need to freeze cells.

Reanimation of cultured mammalian myocardial cells during multiple cycles of trypsinization-freezing-thawing

SummaryIsolated newborn rat heart cells were cultured for several days, then subjected to a standard procedure of trypsinization, slow freezing in 10% dimethylsulfoxide, storage at −180° to −190°C for 1 to 3 days, rapid thawing, and recultivation. The same cells were recycled two more times in identical procedures. Morphological observations were made by phase-contrast optics and cinematography between each cycle and at the end of every experiment. After comparing the cellular morphology and contractile patterns of treated cells with control cultures, it was shown from the results of more than 15 experiments that most myocardial cells survived the standard procedures of trypsinization, freezing, and thawing and regained the ability to contract normally and form synchronized networks. Evidence was obtained which indicates that a cycle of the standard trypsinization-freezing-thawing procedure permits a recovery rate of 83 to 91% viable cells, with myocardial cells surviving to the same extent as endothelial cells. Of the cells which were nonviable, approximately half the deaths were a result of prior damage by trypsin and half were due to the freezing-thawing procedures. The same proportion of spontaneously contracting myocardial cells was observed after a cycle of trypsinization-freezing-thawing as before. Occasionally, there was a delay of 24 hr after thawing before myocardial cells began contracting spontaneously in vitro. An experiment using Viokase (in place of trypsin) and glycerol (in place of dimethylsulfoxide) excellent results after one cycle of freezing-thawing. It was concluded that myocardial cells exhibited a remarkable recovery from the toxic effects of trypsin and the traumatic influences of multiple freezing-thawing procedures. Endothelial cells in the cultures survived the same procedures and proliferated normally in vitro.

Twenty-Eighth Annual Meeting of the Tissue Culture Association

Presidents Welcome--EDWIN H. L ENNETTE Joseph F. Morgan: The Man and His Contributions. H. J. MORTON, Dept. Health and Weffare, Ottawa, Canada. Development of Improved Media for Normal Diploid Cells. R. G. HAM and W. L. McKeeban, University of Colorado, Boulder, Colorado Hormonal Growth Control of Cells in Culture. I. HAYASHI, S. Hutchings, J. Mather, J. Larner, G. Sato, University of California, San Diego, California. Coffee Break Hormonal Regulation of Differentiation in Plant Cells. T. L. SHININGER, The University of Utah, Salt Lake City, Utah. A Tissue Culture Analysis of the Steps in Limb Chondrogenesis. M. SOLURSH, University of Iowa, Iowa City, Iowa.