Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues

Schneider, Florian; Redmαnn, K.; Wistuba, Joachim; Schlatt, Stefan; Kliesch, Sabine; Neuhaus, Nina

doi:10.1016/j.fertnstert.2015.05.001

Cited by 15 publications

(5 citation statements)

References 29 publications

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“…Therefore, we concluded that the Medimachine II approach is optimally suitable for the testis disaggregation. Our data are in strong agreement with other groups [19,69,70].…”

Section: Cell Response Of Differently Isolated Testis Cells To Cryopr...supporting

confidence: 94%

“…As a final consideration, mechanical procedures were set for the "minimal manipulation" to avoid regulatory restrictions established by the Food and Drug Administration (FDA) in the US and worldwide, as previously stated by De Francesco and coworkers [19]. The FDA, particularly for the regenerative medicine field, considers enzymatic procedures to be "more than minimally manipulated" and have set heavy restrictions on them, while non-enzymatic methods are considered to be "minimally manipulated" by the FDA.…”

Section: Discussionmentioning

confidence: 99%

“…The mechanical disaggregation from CTSV (and, from other Companies [18]) was previously adopted and compared to enzymatic digestion for some specific tissues [19].…”

Section: Introductionmentioning

confidence: 99%

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Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

et al. 2022

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The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.

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“…Therefore, we concluded that the Medimachine II approach is optimally suitable for the testis disaggregation. Our data are in strong agreement with other groups [19,69,70].…”

Section: Cell Response Of Differently Isolated Testis Cells To Cryopr...supporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

et al. 2022

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“…Mechanical tissue disaggregation is an efficient and time-effective alternative to enzymatic digestion. High cell numbers, viability and spermatogonial enrichment were reported after mechanical disaggregation of TT (Schneider et al ., 2015). However, the cell viability in the samples was highly variable among suspensions, suggesting the need for further optimization of this method.…”

Section: Resultsmentioning

confidence: 98%

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

Onofre

Baert

Faes

et al. 2016

Hum. Reprod. Update

162

116

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BACKGROUNDGerm cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful.OBJECTIVE AND RATIONALEThis review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed.SEARCH METHODSAn extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy.OUTCOMESThe feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS.WIDER IMPLICATIONSFertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration.

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“…To assess the characteristics of cell types in testicular tissues of trans women, immunohistochemical stainings were performed. The use of immunohistochemical markers (lutropin-choriogonadotropic hormone receptor (LHCGR), smooth muscle actin (SMA), vimentin (VIM), sal like protein 4 (SALL 4), melanoma-associated antigen 4 (MAGE A4), DEAD-box helicase 4 DDX4/VASA) revealed that the expression patterns in trans women were identical and in agreement with the staining pattern in samples from non-transgender men [48]. This finding was confirmed by others [21,22].…”

Section: Testicular Tissues and Steroidogenesis In Our Cohort Of Tmentioning

confidence: 97%

Options for Fertility Treatments for Trans Women in Germany

Schneider¹,

Scheffer²,

Dabel³

et al. 2019

JCM

Self Cite

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Fertility preservation in trans women is a crucial but thus far neglected component in the gender confirming treatment in Germany. It is difficult for trans women to access reproductive health care because centers offering treatment, psychological guidance, gender confirming surgery, as well as reproductive health services are scarce in Germany. Legal, social, or financial issues as well as individual patient comorbidities prevent trans women from receiving appropriate counselling. This review provides an overview on options of fertility preservation in trans women. We consider recent publications on testicular regression at the time of gender confirming surgery demonstrating presence of sperm or at least spermatogonia in the majority of tissues. This may open options for cryopreservation of sperm or testicular stem cells in trans women even at the final stage of transition. Hence, standardized urological procedures (i.e., sperm cryopreservation after masturbation or sperm extraction from the testicular tissue) and experimental approaches (cryopreservation of testicular tissue with undifferentiated spermatogonia) can be offered best at the initiation but also during the gender confirming process. However, counselling early in the gender confirming process increases the chances of fertility preservation because gender confirming hormone therapy has an impact on spermatogenesis.

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Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues

Abstract: Medimachine dissociation of human testicular tissues is comparably fast and leads to an enrichment of SALL4-positive spermatogonia. The use of this method may therefore constitute an advantage for the establishment of human spermatogonial cell cultures.

Cited by 15 publications

References 29 publications

Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

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

Options for Fertility Treatments for Trans Women in Germany

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