Cellular transplantation for the nervous system: impact of time after preparation on cell viability and survival.
OBJECTIVE
Cell transplantation has shown promise for the treatment of various neurological disorders, but the factors that influence cell survival and integration following transplantation are poorly understood. In fact, little is known regarding how simple but potentially critical variables, including the method of cellular preparation and administration, might affect transplant success. The goal of the present study was to determine the impact of time between tissue preparation and implantation on cellular viability. Time can vary with cell preparation, delivery to the operating room, and surgical technique. This study was also designed to evaluate the sensitivity of various methods of assessing implant viability.


METHODS
Cell lines of neural progenitor cells and bone marrow stromal cells were generated from healthy adult mice. On the day of experimentation, the cells were collected, suspended in a balanced salt solution, and sequentially assessed for viability for up to 3.5 hours based on their appearance under phase-contrast microscopy, their ability to retain a fluorescent dye, and their attachment to a cultivation surface for 24 hours.


RESULTS
When viability was measured based on the ability of cells to retain a fluorescent dye, there was a decrease in viability of 10-15% each hour. Based on the ability of the cells to attach to a culture surface and grow for 24 hours, viability decreased more rapidly at approximately 20% per hour. In addition, only about one-third of the cells judged viable based on phase-contrast microscopy or acute dye retention were found to be viable based on plating, and only 10% of the cells initially judged as viable were still capable of survival after 3 hours in suspension.


CONCLUSIONS
The authors' results indicate that that there can be significant losses in viability between preparation and implantation and that more sophisticated methods of evaluation, such as the ability of cells to attach to a substrate and grow, may be required to detect decreases in viability. The time between preparation and implantation will be an important factor in clinical trial design.
Gobbel
Glenn T
GT
Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. gobbelg@upmc.edu
Kondziolka
Douglas
D
Fellows-Mayle
Wendy
W
Uram
Martin
M
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D016026:Bone Marrow Transplantation / Q000379:methods; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival; D005260:Female; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008858:Microscopy, Phase-Contrast; D009474:Neurons / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D013997:Time Factors
D005456:Fluorescent Dyes