Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

Frederickx, Veerle; Michiels, An; Goossens, Ellen; Block, Gert De; Steirteghem, A.C. Van; Tournaye, Herman

doi:10.1093/humrep/deh154

Cited by 87 publications

(76 citation statements)

References 27 publications

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“…A study in rats already highlighted the differential sensitivity of immature tissue to the selection of the cryoprotectants and their concentrations (Unni et al, 2011). Furthermore, functional integrity evaluation of cryopreserved tissue is fundamental to validate a freezing -thawing/cooling -warming procedure, since previous studies concluded that a high survival rate of morphologically normal SG does not necessarily imply that those SG are actually functional (Frederickx et al, 2004;Jahnukainen et al, 2007;Zeng et al 2009). The opposite may also be true; our previous study showed that cryoinjured tissue retained its function and could recover after transplantation (Baert et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

What is the best cryopreservation protocol for human testicular tissue banking?

et al. 2013

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study question: Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking? summary answer: Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue.what is known already: Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue. study design, size, duration: Fragments (n ¼ 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (2S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV). materials, setting, methods: Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types.main results and the role of chance: The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 + 5.6 in control tissue to 4.9 + 2.1, 8.2 + 5.4, 11.6 + 5.1, 8.8 + 3.9, 12.6 + 4.4 and 11.7 + 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO 2S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P , 0.001).limitations, reasons for caution: Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue.wider implications of the findings: An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.

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Section: Discussionmentioning

confidence: 99%

What is the best cryopreservation protocol for human testicular tissue banking?

et al. 2013

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“…62 In this study, the cryopreservation method of Izadyar et al 43 was used to isolate neonate testicular cells as well as to fragment seminiferous tubules. Goossens et al 63 cryopreserved mouse tissue pieces in ethylene glycol and DMSO as cryoprotectant while their protocols had already been used for freezing testicular stem cell suspensions by Frederickx et al 64 They obtained the best morphology of the basal compartment when the cryoprotective medium contained DMSO. 63 Honaramooz et al 65 investigated the effects of cooling or cryopreservation with DMSO on the testis fragments of pigs before grafting and observed complete spermatogenesis.…”

Section: Discussionmentioning

confidence: 99%

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

Eslahi¹,

Hadjighassem²,

Joghataei³

et al. 2013

IJN

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Introduction A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. Methods The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers ( Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6 , and Itgβ1 ), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. Results The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) ( P ≤0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. Conclusion Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.

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“…It is suggested that a number of stresses during slow freezing, including osmotic stress and, stress to cell-junction and cell-transport systems (63,64), can contribute to the loss of pluripotency. Using straws as a container, programmed cell freezer and/or a high concentration of FBS/KO-SR, the survival rate of hESCs after slow-freezing and thawing was found to increase nearly 80%, with no apparent increase in differentiation (65,66).…”

Section: Cryopreservation Conditions For Human Embryonic Stem Cellsmentioning

confidence: 99%

Human Embryonic Stem Cells: Derivation, Maintenance and Cryopreservation

Lee

Lee²

2011

Int J Stem Cells

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Human embryonic stem cells (hESCs) are the most powerful candidate for the treatment of incurable diseases through the replacement of damaged cells and/or tissues in patients, although there are some obstacles to overcome for the clinical application of hESCs such as the assurance of guided differentiation and control of the immune response following cell therapy or tissue grafting. To obtain genetically stable hESCs and use them clinically, it is important to develop appropriate culture conditions. Additionally, the establishment of a hESC bank with a large number of hESC lines will be required for their clinical application because each hESC line is directed to have a different differentiation ability and immune characteristics such as HLA type. In this review, we describe the derivation and culture conditions of hESCs based on recent advances. Then, we will introduce several cryopreservation methods for hESCs, which is important for the development of cell bank.

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Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

Abstract: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.

Cited by 87 publications

References 27 publications

What is the best cryopreservation protocol for human testicular tissue banking?

What is the best cryopreservation protocol for human testicular tissue banking?

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

Human Embryonic Stem Cells: Derivation, Maintenance and Cryopreservation

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