Background

In prior research we demonstrated that freeze-dried stem cells stored for 3 years at room temperature were able to 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 this study (Natan, et al. 2009) , we improved the cells’ viability and functionality after freeze-drying.

Methodology

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. As part of this research, six samples were sent to two leading US pharmaceutical companies – 3 to each facility – where the cells were reconstituted with distilled water, placed in culture, and then tested for viability and functionality (colony formation).

Principal findings

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 (5.4×104±4.7, 3.49×104±6 and 6.31×104±12.27 cells, respectively, and 31±25.15, 47±45.8 and 23.44±13.3 colonies, respectively).

Conclusions

This was the first report of nucleated cells which were dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells.

Source

Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation
Y. Natan, A. Nagler, A. Arav
Plos One, vol. 3, e5240 (2009)
For more information click here.

Supporting information

Figure 1a. Freeze-dried stem cells

Figure 1b. Freeze-dried stem cells

Sample Total WBC (106/ml) % CD34+ CD34+ Total
(cell number/ml)
Fresh UCB 5.8 0.21 12400
Fresh MNC 2.3 0.68 15600
Lyo 1 2.5 0.69 17300
Lyo 2 2.5 0.70 17600

Table 1. Determination of CD34+ cell number within the MNC population (FACS)

Figure 2. MNC survival after freeze-drying with solution

Experiment Fresh (x104 cells) After thawing (x104 cells) After rehydration (x104 cells)
1 10.4 1.9 2.3 1.6 5.7
2 1.05 15.6 0.25 36.4 0.2 2
3 4.85 0.7 0.2 1.7 2.9 0.15
Mean ± SD 5.4 ± 4.7 3.5 ± 6 6.3 ± 12.3

Table 2. Number of CD34+ cells before freezing and after thawing and lyophilization
Note: Results are from 3 different experiments (i.e. different donors) with each individual number representing a different sample

Experiment Fresh After thawing After rehydration
1 60
(33 – E/BCFU)
(27 – GM)
12
(4 – E/BCFU)
(8 – GM)
53
(31 – E/BCFU)
(22 – GM)
9
(2 – E/BCFU)
(7 – GM)
30
(1 – E/BCFU)
(29 – GM)
13
(1 – E/BCFU)
(12 – GM)
2 18
(6 – E/BCFU)
(12 – GM)
21
(3 – E/BCFU)
(18 – GM)
29
(8 – E/BCFU)
(21 – GM)
42
(11 – E/BCFU)
(31 – GM)
28
(1 – E/BCFU)
(27 – GM)
43
(4 – E/BCFU)
(39 – GM)
3 15
(5 – E/BCFU)
(10 – GM)
10
(0 – E/BCFU)
(10 – GM)
108
(1 – E/BCFU)
(107 – GM)
27
(3 – E/BCFU)
(24 – GM)
12
(0 – E/BCFU)
(12 – GM)
6
(0 – E/BCFU)
(0 – GM)
Mean ± SD 31 ± 25.15 38.83 ± 37.3 23.3 ± 13.9

Table 3. Number of colonies grown, before freezing and after thawing and rehydration
Note: Results from 3 different experiments (i.e. different donors) with each individual number representing a different sample; Colony type is given in brackets (erythrocytic E/BFU; granulocytic – GM)

Figure 3a. Human umbilical cord blood stem cells after thawing and rehydration

Figure 3b. Human umbilical cord blood stem cells after thawing and rehydration