Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34(+)-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%-91%. The total number of CD34(+)-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4)+/-4.7, 3.49 x 10(4)+/-6 and 6.31 x 10(4)+/-12.27 cells, respectively, and 31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively). CONCLUSIONS: This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells

Number of CD34+ cells before freezing and after thawing and lyophilization.

<p>Results are from three different experiments (i.e. different donors); each individual number represents a different sample.</p

Viability percentage of MNC derived from HUCB after freeze thawing with one of the following solutions: IMT-2, EGCG solution, Trehalose solution.

<p>The samples were either immediately evaluated or underwent a washing process and then evaluated again. The data is presented as the mean±SE, different letters represent statistical differences between groups (p<0.05). The means were calculated as the number of viable cells after thawing divided by the number of viable cells before freezing. (N = 48).</p

Viability percentage of MNC derived from HUCB after freeze drying with one of the following solutions: IMT-2, EGCG solution, Trehalose solution.

<p>The samples were either immediately evaluated or underwent a washing process and then evaluated again. The data is presented as the mean±SE, different letters represent statistical differences between groups (p<0.05). The means were calculated as the number of viable cells after thawing divided by the number of viable cells before freezing. (N = 48).</p

Photographs showing colonies formed after lyophilization and rehydration with DDW of samples frozen in IMT-2 solution.

<p>Photographs showing colonies formed after lyophilization and rehydration with DDW of samples frozen in IMT-2 solution.</p

The number of colonies grown, before freezing and after thawing and rehydration.

<p>Results are from three different experiments (i.e. different donors); each individual number represents a different sample and the colony type is given in brackets (erythrocytic- E/BFU; granulocytic – GM).</p

Percentage membrane integrity after freeze-thawing and freeze-drying with different concentrations of EGCG.

<p>This experiment was performed on HUCB units from three different donors, in duplicate (n = 60). Data are presented as mean±SE.</p

Percentage membrane integrity after freezing at different ice interface velocities.

<p>The solution used in this experiment was 12.5% (w/v) HSA and 0.1 M trehalose in PBS (Ca²⁺- and Mg²⁺-free). This experiment was performed on HUCB units from three different donors, in triplicate (n = 54). Data are presented as mean±SE.</p

Percentage membrane integrity after freeze-thawing and freeze-drying in different solutions.

<div><p>All solutions are based on PBS (Ca²⁺- and Mg²⁺-free). This experiment was performed on HUCB units from three different donors, in duplicate (n = 60). Data are presented as mean±SE.</p> <p>donors, in duplicate (n = 60). Data are presented as mean±SE.</p></div

A photo of dry MNCs derived from HUCB after freeze-drying, before (right) and after (left) rehydration with DDW.

<p>A photo of dry MNCs derived from HUCB after freeze-drying, before (right) and after (left) rehydration with DDW.</p