Effect of AAGP™ on Preserving Cell Survival Number in Human and Mouse Neural Stem Cells During Cryopreservation

Hussein, August 2007

Introduction

Cryopreservation is a technique commonly used to preserve human tissue and cells during long-term storage for their use in transplantation and research. One of the largest problems with stem cell cryopreservation is maintaining cell number and viability - previously we found AAGP™ to maintain cell stem viability at 96% (assessed using an OCT4 stem cell marker). Thus, the aim of this investigation was to address the effectiveness of AAGP™ in maintaining human and mouse stem cell number during cryopreservation.

Method

In order to test AAGPs™ effectiveness in maintaining stem cell numbers in human embryonic stem cells four samples were prepared. The first two being embryonic stem cells at high densities of 2x106 and 3x106 cells/ml which were exposed to AAGP™ and the last two being embryonic stem cells at densities of 2x106 and 3x106cells/ml which had not been exposed to AAGP™. All cell populations were cryopreserved at -80°C in 20%FBS growth serum containing 10%DMSO. After 2 days of cryopreservation cells were thawed at 37°C. Population cell number was calculated using Trypan blue (4 counts per population). Cell viability/number was calculated as a percentage of cells present prior to cryopreservation.

The effectiveness of AAGP™ in maintaining cell number as a percentage of cells prior to Cryopreservation was measured in mouse embryonic neural stem cells in the same way. We used both high and low densities of mouse embryonic stem cells in one cell line and a high density of mouse neural stem cells in another cell line.

Results

Cell viability tests showed exposure of cells to AAGP™ to increase the cell viability (as a percentage of cells initially cryo-preserved) by 36% and 38% in the two high-density human embryonic stem cell populations and by 36% and 26% in the two high density mouse embryonic stem cell populations.


Figure 1: Effect or AAGP™ on human ES cell Cryopreservation.


Figure 2: Effect or AAGP™ on mouse ES cell Cryopreservation.


Figure 3: Effects of AAGP™ on low cell density mouse ES cell Cryopreservation.

Conclusion

Thus it can be concluded that in both high and low density cell populations of mouse embryonic stem cells and in high density populations of human embryonic stem cells the presence of AAGP™ during Cryopreservation gives the cells an advantage in terms of cell survival whilst maintaining cell function/competence. It could be said that this makes AAGP™ an attractive preserver in the storage of stem cells for transplants where only a low density of cells can be extracted. Though these experiments will need to be completed in other cell lines. The mechanism of this survival signal is yet to be established.