Biosample storage in biobanks is constantly under scrutiny to ensure that stored samples are usable and protected from degradation. This is especially true for human umbilical cord (UC) storage. As Fong et al. explain, UCs are a source of mesenchymal stem cells (MSCs), which are desired for cell-based therapies.1
To preserve UCs, some cord blood banks freeze entire pieces of UC and thaw them as needed. This practice leads to mixed heterogeneous populations of MSCs since some cells are derived from the UC compartments, which are not as useful for clinical studies. Fong and colleagues warn that this method of freezing often results in suboptimal post-thaw stem cell recovery because of poor cryoprotectant diffusion and intracellular ice formation, heat and water transport issues, and damage to intercellular junctions.
To get a better idea of the plight facing UC storage, the authors compared the post-thaw yields of frozen UC tissue, looking at pure gelatinous Wharton’s jelly harvested fresh from the UC without any cell separation (WJ), MSC populations separated from the Wharton’s jelly (WJSC) and entire UC segments.
The researchers collected fresh UC in sterile containers containing Hank’s balanced salt solution (Thermo Scientific). They mixed the samples with Recovery cell culture freezing medium (Thermo Scientific) and then transferred them to cryovials for freezing. They brought the samples from room temperature to 30°C at a rate of 1°C/min, held them there for 10 minutes, then brought them to 150°C at a rate of 15°C/min, held them there for 10 minutes, and then ﬁnally plunged them into liquid nitrogen at 196°C.
After 30 days, the team thawed samples from the three groups by placing them in a 37°C water bath. They diluted each vial with fresh stem culture media and centrifuged each sample at 300 g for five minutes. The researchers then examined the post-thaw morphology, cell viability and proliferation, cluster of differentiation (CD) markers, cell cycle (flow cytometry), and apoptosis rates, using an Annexin V-FITC assay. To see how the frozen samples responded to differentiation, the team exposed post-thaw cells to either adipogenic, osteogenic or chondrogenic differentiation media. They evaluated the gene expression levels of these lineages using quantitative real-time polymerase chain reaction (qRT-PCR) on a 7500 Fast RT-PCR system (Thermo Scientific).
The researchers found that the mixed cord (MC), WJ and WJSC samples produced high post-thaw stem cell recovery rates (93.52 ± 6.12% to 90.83 ± 4.51%). During culture, the WJ and WJSC samples produced epithelioid monolayers within 24 hours, whereas post-thaw MC explants showed slow growth with mixed epithelioid and fibroblastic cell outgrowths after several days.
Looking at the Annexin V-FITC analysis, they saw significantly greater apoptosis signals for the cells from the post-thaw MC group (6.93 ± 1.26%) compared to cells from the post-thaw WJ and WJSC groups (1.46 ± 0.67% to 1.71 ± 0.55%). All three post-thaw groups showed normal cell cycle proﬁles. Despite this, they found that the post-thaw MC samples revealed higher percentages of cells at sub-G1 (9.76 ± 1.14%) compared to cells from the post-thaw WJ and WJSC samples (6.83 ± 0.91% to 7.04 ± 0.69%).
They also found that the viability and proliferation rates of the post-thaw WJ and WJSC samples were significantly greater than in the MC samples. Post-thaw WJ and WJSC samples also produced significantly greater CD24(+) and CD108(+) fluorescence intensities and significantly lower CD40(+) contaminants.
Post-thaw WJ and WJSC samples produced significantly fewer Annexin V-FITC–positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation compared to MC samples. Looking at qRT-PCR analysis, the post-thaw MC samples showed significant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK1) compared to the WJ and WJSC samples.
The researchers concluded that freezing fresh WJ is a simple and reliable method of improving stem cell recovery, generating large numbers of clinically utilizable MSCs for cell-based therapies. The quality and numbers of cells obtained post-thaw require much less time and labor to obtain MSCs post-thaw.
1. Fong, C.-Y., et al. (2016) “Freezing of fresh Wharton’s jelly from human umbilical cords yields high post-thaw mesenchymal stem cell numbers for cell-based therapies,” Journal of Cellular Biochemistry, 117(4) (pp. 815–827), doi: 10.1002/jcb.25375.