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Background: A significant limitation to the study of stem cells from human adipose is the ability to isolate them from fresh tissue in a timely manner. Unlike those derived from animal models, human adult stem cells are often isolated from tissue that is stored frozen secondary to limited specimen availability. Frozen human tissue banks are frequently utilized by our laboratory and others for the study of adipose derived stem cells in translational and basic science applications. Consequently, it is important to examine the effects of cryopreservation on stem cell isolation and viability. Here, we specifically characterize the roles of cryopreservation duration and patient age on adipose derived stem cell isolation and viability.
Methods: Subcutaneous abdominal fat was excised during abdominoplasties between November 2010 and January 2014. On the day of the procedure, the tissue was divided in 50mL tubes and frozen at -70°C. At defined dates, the tissue was thawed and stem cells were isolated from 10g of tissue using standard a collagenase protocol. The cells were grown in Dulbecco’s Modified Eagle Medium with F12 for 17 days. The total number of cells, live number of cells, and viability of the cells was measured using the Countess automated cell counter. The cells were then re-plated to a density of 1x105 on a 12 well plate and counted again on days 9, 18, and 28. The data was analyzed respective to the cryopreservation time and the age of the patient. The time frozen was divided into 3 groups: >2 years, 1-2 years, and <1 year frozen. The isolated cells were further analyzed using FACS analysis for receptors CD34, CD43, CD105.
Results: Stem cells were isolated from 33 patients. The number of days the tissue was frozen ranged from two to 1159 days. The patients varied in age from 26 to 62 years old. There was a significant difference found in the number of live cells initially isolated from tissue frozen less than 1 year (mean =4.06x105) compared to tissue frozen greater than 2 years (mean=1.75x105)(p<0.005). In contrast, there were no significant differences found in the number of live cells, the change in number of cells, or cell viability on all subsequent counting days when comparing both the number of days frozen and the age of the patient.
Conclusions: Cryopreservation time decreases the number of adipose derived stem cells that are able to be initially isolated from tissue, irrespective of patient age. However, once isolated, the growth and viability of the stem cells is not affected by the number of days the tissue was frozen or the age of the patient. Consequently, cryopreservation of human adipose tissue at -70°C is an effective means to store and build human tissue banks for human adipose stem cell isolation, while avoiding the potential mitigating effects of lipoaspirate-induced cellular trauma and the use of preservative media. Future studies will address the effects of cryopreservation on stem cell differentiation and cellular functions.