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Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

ABSTRACT: Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5%/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P ≤ 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5%/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the %0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications.

SUBMITTER: Asgari F

PROVIDER: S-EPMC8065005 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

Asgari Fatemeh F Asgari Hamid Reza HR Najafi Mohammad M Eftekhari Behnaz Sadat BS Vardiani Mina M Gholipourmalekabadi Mazaher M Koruji Morteza M

Journal of materials science. Materials in medicine 20210423 5

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellu ...[more]

PMID: 33891169

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Project description:Study questionCan human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia?Summary answerGerm cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures.What is known alreadyThe development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures.Study design, size, durationTesticular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures.Participants/materials, setting, methodsImmunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4).Main results and the role of chanceUTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected.Limitations, reasons for cautionDifferent groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences.Wider implications of the findingsThe ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy.Study funding/competing interestsThe experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests.Trial registration numberNot applicable.

Dataset Information

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

Publications

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.

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