Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug:77:14-18.
doi: 10.1016/j.cryobiol.2017.06.004. Epub 2017 Jun 9.

Methodology for reliable and reproducible cryopreservation of human cervical tissue

James M Fox  1 , Rebecca C Wiggins  2 , John W J Moore  2 , Christine Brewer  3 , Bill Hunter, Alison C Andrew  3 , Fabiola Martin  4
Affiliations

Methodology for reliable and reproducible cryopreservation of human cervical tissue

James M Fox et al. Cryobiology. 2017 Aug.

Erratum in

Abstract

Background: In order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required.

Methods: An active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to -50 °C then 10 °C/min to -120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time.

Results: Repeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage.

Keywords: Bio-banking; Cervical tissue; Cervix; Cryopreservation; Ex vivo; Freezing; Storage; Thawing.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
MTT assay comparison of fresh and freeze-thawed cervical tissue explant viability. The mean viability of seven fresh cervical explants (each performed in triplicate at every time point) compared to that of freeze-thawed cervical explants from three separate donors (in triplicate) at days 0, 1, 7 and 14 days assessed by MTT assay is shown. Average viability over time is expressed as a percentage of the respective explant's viability at day 0 ± SEM.
Fig. 2
Fig. 2
Representative cervical tissue H&E staining of freeze-thawed cervical tissue. Composite representation of sequential images of an H&E stained section of cervical tissue that had been frozen, cryopreserved, thawed then cultured to d1.
See this image and copyright information in PMC

References

    1. Best B.P. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Res. 2015;18:422–436. - PMC - PubMed
    1. Cowan M.E., Buchan A., Skinner G.R. Synthesis of immunoglobulins by human endocervix in organ culture. Br. J. Exp. Pathol. 1982;63:125–132. - PMC - PubMed
    1. Cummins J.E., Jr., Guarner J., Flowers L., Guenthner P.C., Bartlett J., Morken T., Grohskopf L.A., Paxton L., Dezzutti C.S. Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture. Antimicrob. Agents Chemother. 2007;51:1770–1779. - PMC - PubMed
    1. Gupta P., Ratner D., Patterson B.K., Kulka K., Rohan L.C., Parniak M.A., Isaacs C.E., Hillier S. Use of frozen-thawed cervical tissues in the organ culture system to measure anti-HIV activities of candidate microbicides. AIDS Res. Hum. Retroviruses. 2006;22:419–424. - PubMed
    1. Guven S., Demirci U. Integrating nanoscale technologies with cryogenics: a step towards improved biopreservation. Nanomedicine (Lond) 2012;7:1787–1789. - PMC - PubMed