The Viability of Lyophilized HeLa Cells

Introduction: There are numerous applications of human cells in the treatment of trauma and disease. The most prominent being the need of blood for transfusions and skin cells for burn victims. In the United States, fresh blood and skin cells are easily accessible; however, in many other countries, including countries where the US military is stationed and on spacecraft sent to outer space, blood is in limited supply. Consequently, blood must be preserved by cryopreservation due to the tremendous need for blood by military and civilians alike. Skin cells on the other hand must be stored in liquid media and are not preserved by freezing. In places where technology and resources are limited, the traditional methods of storing and preserving these cells makes them virtually unavailable. Therefore it is of interest to preserve cells in a way that does not include freezing or liquid suspension. A dry powdered form of cells would allow blood and skin to be transported effortlessly around the globe and stored without hassle in third world countries, military bases, and on NASA spacecrafts. The aim of the research was to determine if cells could be sprayed onto a thin layer of plastic, frozen, and lyophilized while remaining viable.

Materials and Methods: HeLa cells were cultured, trypsinized, and resuspended in CrySOfree DMSO-free freezing media (Sigma-Aldrich). The cell suspension was deposited into a 2mL spray bottle that dispensed a constant concentration of 40,000 cells/spray. The HeLa cells were sprayed into a 48-well plate at a density of 40,000 cells. In addition, a control was prepared by pipetting cells at the same density as the sprayed cells. The plate was then placed in a styrofoam container and frozen at -80oC with a cooling rate of -1oC/min. After freezing, the cells that had been sprayed into the wells were lyophilized while the control was immediately incubated. The sprayed cells were incubated after being dry frozen. A cell proliferation assay (MTS, Promega) was preformed on the cells, after a 24hr incubation period, using a UV-Vis Spectrometer set to an absorbance of 490nm. After a 5 day incubation period, photographs were taken using a confocal microscope and a manual count of the cells was preformed. The cells were again trypsinized, sprayed, frozen, and dry frozen. The day immediately following the dry freezing, the cells were photographed using SEM.

Results: The cell proliferation assay showed that the control had an absorbance of
0.802 while the freeze dried cells had an absorbance of 0.413. The absorbance,
which is directly proportional to the viability, indicated that the sprayed and dry
frozen cells remained viable. Although the number is 50% absorbance compared to
the control it is important to note that the traditional method of freezing cells will
occasionally result in 0% viability. Therefore, it is a significant finding that the
cells remained viable after the great amount of shock the freeze dried cells
underwent. The confocal microscope photograph of the cells showed elongated
cells that remained connected together, demonstrating that the cells had not only
successfully gone through the G1 phase but were also able to reproduce. The
manual cell count gave roughly 2,000,000 cells. Before freezing and dry freezing
there had been 320,000 cells. Therefore, through a manual cell count it was also
demonstrated that the cells were viable and reproducing. The SEM photographs taken
showed an intact cell membrane and nucleus indicating that no damage to the individual cells had occurred.

Conclusions: Based on the preliminary results, it can be stated that spraying cells onto plastic and freeze drying does not kill the cells. They can successfully pass through the G1 phase and go through the entire cell cycle. Reproduction was not inhibited and the cells continued to maintain all the characteristics they had had before freeze drying had occurred. Future work can continue the process with other cell lines to decide if this will be the preferred method of dry powdered packaging of cells. Increasing the viability of the freeze dried cells as well as additional research into the structure of the cell after undergoing multiple rounds of freeze drying should be continued as well.

Acknowledgments: Beu Oropeza, Luis Solis, Julio Rincon, and Dr. Renato Aguilera, UTEP Biological Sciences Dept. for supplying the HeLa cells used in this project.
Research reported in this paper was supported by the National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.