On-Chip Cryopreservation: A Novel Method for Ultra- Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Abstract
Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated
cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at
exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on
polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip
cryopreservation with different micro-channel height (group A: 10 mm height, group B: 50 mm height, group C: 100 mm
height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay
and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed
spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly
decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed
spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet
assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and postthawed
spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and
group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of
micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the
highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 mm
height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.
Citation: Zou Y, Yin T, Chen S, Yang J, Huang W (2013) On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small
Amounts of Human Spermatozoa. PLoS ONE 8(4): e61593. doi:10.1371/journal.pone.0061593
Editor: Wei Yan, University of Nevada School of Medicine, United States of America
Received November 8, 2012; Accepted March 11, 2013; Published April 30, 2013
Copyright:  2013 Zou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (No. 30973196, 20975077, 31070995), the Science Fund for Creative
Research Groups (No. 20921062), and the Program for New Century Excellent Talents in University (NCET-10-0611). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.