Arthur E. Greene

SPECIES IDENTITY OF INSECT CELL LINES

SummaryThree mosquito cell cultures designated as Suitors clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells.

Characterization and Identification of Insect Cell Cultures

During the past two decades we have witnessed an increasing accumulation of biological knowledge in cytogenetics, biochemistry, immunology and biology related to experimental techniques developed in mammalian cell cultures. Comparable achievements are being reported from research studies in poikilothermic cell cultures. It is now obvious that insect cell cultures pioneered by many of the authors in this volume and by the late Dr. Suitor will also allow us to gain further insight into many biological phenomena associated with insects.

Differential susceptibility of epithelial cells and fibroblasts of human skin to freeze injury: Report on an improved method for storage of human skin at −196°C*

Summary Fresh human skin placed in plasma clot culture shows outgrowth of epithelial cells within 3 days followed 3 to 6 days later by fibroblasts. Plasma clot culture of human skin pretreated in medium containing 10% glycerol at 4°C, frozen, and thawed showed outgrowth of fibroblasts only. Using this increased susceptibility of epithelial cells to freeze injury as a criterion, we developed a freezing method in which skin is pretreated with 20 to 30% glycerol at 4°C for 1 to 2 hrs before freezing. Human skin preserved in liquid nitrogen by this method grew out in plasma clot culture very much like fresh skin.

PRESERVATION OF CELL CULTURES BY FREEZING IN LIQUID NITROGEN VAPOR.

Summary Nine continuous, 2 diploid and one primary cell line were successfully preserved by freezing in liquid nitrogen vapor at cooling rates of 1.4°C/min to 43°C/min, but poor survival was observed when cells were frozen by immersion directly in liquid nitrogen. The cells were frozen directly in a liquid nitrogen refrigerator by placing the ampuls at different distances below the lip, demonstrating that elaborate and expensive cell preserving equipment is not required. The plating efficiency technique was shown to be the most sensitive indicator of cell damage.

Viability of Cell Cultures Following Extended Preservation in Liquid Nitrogen

Summary Twenty-two frozen batches of 16 different cell lines certified by the Cell Culture Collection Committee were successfully stored in liquid nitrogen over an interval of 2.5 to 4.5 years. Confluent cell growth obtained in milk dilution bottles and roller .tubes prepared from thawed aimpules of frozen cells clearly demonstrated the high viability of these cell lines. The studies indicated that cells harvested and frozen under optimal conditions retained high viability during prolonged storage in liquid nitrogen, but that damaged cells as shown by initial low viability may be expected to show more variable results.

Historical development of cell and tissue culture freezing

Abstract In conclusion, the use of glycerol or dimethyl sulfoxide has permitted successful freezing, storage, and thawing of most, but not all, animal cell cultures. Storage in liquid nitrogen without detectable change in observable characteristics appears to be indefinite. Methods are empiric, understanding of the basic physics and chemistry involved are fragmentary, and results cannot always be exactly duplicated within one laboratory or in different laboratories. These problems will be solved in the future by the combined application of many disciplines with overlapping interests in molecular biology. In the meantime, present methods permit indefinite cold storage of most cell cultures with a great saving in labor and inconvenience, while avoiding accidental loss, contamination, or mutation of the cells during repeated serial passage.

Characterization of a new human diploid cell line—IMR-91

SummaryA human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.

The effect of prolonged storage of cell cultures in dimethyl sulfoxide and glycerol prior to freezing

Summary Two tissue culture cell lines, HeLa and WI-38, were stored in the presence of 5% DMSO or 5% glycerol for intervals of 24 to 168 hrs at 4 and 37°C to test the toxic effects of these additives on the cells. The studies demonstrated the superiority of storage at 4°C. Viability was reduced after 72 hrs of storage of HeLa cells at 4°C and 24 hrs at 4°C was found to be the maximal storage time without toxic effects. During this time interval no demonstrable loss of viability was observed as monitored by the cell permeability and cell adhesion tests in either the frozen or unfrozen cells. Cell damage of the more delicate WI-38 cells was observed after 24 hrs at 4°C in both the prefreeze and post-thaw cultures; 6 hrs was the maximal storage period at 4°C without loss of viability. We recommend from the results of these studies that the cell lines be stored at 4°C when they cannot be immediately frozen after ampulizing. It is suggested that the more delicate diploid and primary cell cultures must be frozen within 3 to 6 hrs of ampulizing. Although the more hardy established cell lines may be left overnight at 4°C and successfully frozen on the next day with little loss in viability, it is advisable to freeze them within several hours after ampulizing to minimize cell damage.

Human Biopsy Material from Genetic Abnormalities

Publisher Summary This chapter focuses on human biopsy material from genetic abnormalities. Among the most useful tissue cultures available to the geneticist are those derived from human skin. Skin biopsies are easily obtained, the cultures derived from them are easy to grow, they can be frozen for years in liquid nitrogen, and recovered in a highly viable state. The skin specimen may be collected by simple punch biopsy. In the method described in the chapter, stainless steel punches together with forceps and scissors are cleaned, wrapped, and autoclaved. The skin is swabbed with alcohol, rinsed with sterile saline, and dried with sterile gauze. Almost any area of the body can be used including the buttocks, the back medial to the upper border of the scapula, the flexor surface of the forearm, and the deltoid region. The procedure is simple, rapid, and an anesthetic is usually not required. If it is considered essential to use one, an intradermal injection of several milliliters of 285 procaine solution will anesthetize the skin.

In vitro preservation of functioning rabbit hearts in a depolarized state at 4°C*†

Summary Tissue culture media of various types have been devised in the past few years to support the growth of living cells for many generations vitro . We have made modifications in one tissue culture medium (Eagles minimum essential medium, MEM) so that it will support the functional viability of rabbit hearts stored for 48 hrs at 4°C. Rabbit hearts perfused with Eagles MEM with added KCl to give a final concentration of 13 to 15 mEq per liter of K + stop contracting within 1 min. Perfusion with the high K + medium is then continued at 4°C. After a series of 40 experiments it has been established that rabbit hearts perfused with Eagles MEM containing 143 mEq of Na + per liter, 13 to 15 mEq of K + per liter, 9.8 mEq of Ca ++ per liter, and 0.2% glucose equilibrated with 95% O 2 -5% CO 2 can be preserved at 4°C for 24 to 48 hrs with 100% recovery of function as measured by the force of contraction.