Herndon B. Lehr

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.

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.

Successful preservation of canine small intestine by freezing

Abstract Five of fourteen canine intestinal autografts frozen at −196°C for 7 days were surviving 2 wk after reimplantation. The mucosal epithelium sloughed immediately, but repopulated from surviving crypt cells over the ensuing 3–5 days. Absorptive and peristaltic functions were present at 2 wk after reimplantation.

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.

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.

VIABILITIES OF FROZEN MAMMALIAN CELLS FOLLOWING SLOW THAWING.

Summary 1. Under the conditions described, good viability of frozen mammalian cell cultures was often obtained following slow thawing in the presence of added di-methylsulfoxide. 2. Variation of pH of the freeze medium had no effect on viabilities of frozen HeLa, SJ. cells following slow or rapid thawing. 3. Dimethylsulfoxide is superior to glycerol in preventing damage of the cells tested during freezing. This is particularly evident when slow thawing is employed.

Ultrastructural changes in the mucosa of frozen-preserved canine ileum*†

Summary Serial ultramicroscopic observations were made on four segments of canine ileum frozen to −50°C after use of 10% glycerol solution as a cryoprotectant. The observed alterations in cellular structure were progressive in degree during the perfusion, frozen and thawed, and revascularized stages. Normal fine structure was evident in the cells studied 1 month after freezing. There was a wide variation in the response of individual cells to the freezing process. Changes observed in the epithelial cells included swelling of mitochondria, distention and vesiculation of the endoplasmic reticulum, and rounding with dilation of the cisternal profiles of the Golgi apparatus. In the terminal stages prior to cell disruption, fragmentation of the microvilli was observed. Consistent or typical changes in the nuclei were not identified in this study. In the lamina propria, the capillary endothelium retained its integrity during perfusion but showed mitochondrial swelling. Late changes following freezing included disruption of endothelial cytoplasm. Minimal changes in smooth muscle cells were observed but included decrease in peripheral myofibrills and increase in condensed nuclear chromatin. Plasma cells were relatively unaffected by perfusion but showed vesiculation of the endoplasmic reticulum after freezing and thawing. It would appear that the morphological changes observed after freezing and thawing first appear after perfusion. The one ultrastructural change which may prove most useful in evaluating improved storage procedures is the gradual alteration in the rough endoplasmic reticulum, which develops over a broad range of experimental conditions.