Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation

Abstract

Objective To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. Design In vitro human testicular tissues. Setting Academic research unit. Patients Adult testicular tissues (n = 4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n = 3). Intervention(s) Testicular tissue vs. single cell suspension cryopreservation. Main Outcome Measures Cell viability, total cell recovery per milligram of tissue, as well as, viable and SSEA-4+ cell recovery. Results Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Conclusions Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient age, type of samples cryopreserved, and end points of therapeutic applications.

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

“…Pacchiarotti et al . (2013) and Yango et al . (2014) stated that TT cryopreservation should be recommended.…”

“…2007, (2012)HumanMature1.5 M DMSO + 0.15 M sucrose + 10% HSAInsulated container in −80°C, LN2Maintenance of tissue integrity, preservation of cell dynamics in propagation cultureBaert et al . (2013), (2015)1.28 M DMSO + 25% FBSInsulated container in −80°C, LN2Preservation of SSCsYango et al . (2014)VitrificationMouseImmature2.8 M DMSO + 2.8 M EG + 25% HSAStraw, LN2Maintenance of tissue integrity and activity in organotypic tissue cultureCuraba et al .…”

“…Some of these present interesting and promising results on post-thaw cell and tissue integrity, and show the ability to purify viable SSCs for further use (Milazzo et al ., 2008; Gouk et al ., 2011; Wu et al ., 2011, 2014; Unni et al ., 2012; Gholami et al ., 2013; Pacchiarotti et al ., 2013; Yango et al ., 2014; Zhang et al ., 2015). However, these evaluations are merely indicative for the quality of the cryopreservation protocol.…”

“…(2007)VitrificationHumanAdult(1)1.1 M DMSO,1.34 M EG, 10%HSAopen pulled straw (OPS) vitrification56 ± 24No functional assay performedSá et al . (2012)(2)0.67 M S,2.3 M DMSO, 3.0 M EG, 10%HSAN/AHumanAdult1.28 M DMSO 25% FCS−1°C/min, LN 2 75 ± 4No functional assay performedYango et al . (2014)Fetal67 ± 4

DMSO: dimethyl sulfoxide, EG: ethylene glycol, FCS: fetal bovine serum, HSA: human serum albumin, N/A: information not available, LN 2 : liquid nitrogen.

a Sexual reassignment

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Stem Cell Therapies for Male Infertility: Where Are We Now and Where Are We Going?

“…Two studies tested this question directly on human samples. Yango and colleagues reported that recovery of SSEA4 + cells from cryopreserved fetal testicular tissue was similar to cryopreserved testicular cells, but recovery of SSEA4 + cells from cryopreserved adult testicular cells was greater than cryopreserved testicular tissue [ 142 ]. Pacchiarotti and coworkers reported that cryopreservation of testicular tissue was comparable in most aspects to cryopreservation of a cell suspension, except the viability of cells from the cryopreserved tissue was higher.…”