# Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis

> **NIH NIH F31** · MAGEE-WOMEN'S RES INST AND FOUNDATION · 2020 · $45,520

## Abstract

Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis
Spermatogonial stem cells (SSCs) have a tremendous capacity to regenerate spermatogenesis under proper
conditions. Chemotherapy or radiation treatments for cancers or other conditions can destroy the stem cell
pool and cause permanent infertility. The only option to preserve the fertility of prepubertal male patients is
testicular tissue cryopreservation. When those tissues are thawed, testicular tissue organ culture is an
approach to mature those tissues to produce sperm outside of the body. Takehiko Ogawa’s lab produced
mouse sperm and offspring from cultures of fresh neonatal testes that were maintained for several months in a
pump-driven microfluidic (MF) device. To overcome the bulkiness of the MF device, the same laboratory
invented a pumpless microfluidic (PL) device. However, sperm and offspring have not been produced using the
PL devices; spermatogenesis was not reported from cryopreserved tissues in the PL system, and the system
has not been tested in any other species. This study aims to fill the gaps in knowledge by generating mouse
sperm and offspring using the PL devices in cultures of fresh and cryopreserved testes, and to translate this
technology to non-human primate and/or human testicular tissues. In addition, to overcome several pitfalls in
the PL design, I developed and tested a novel PDMS-roof transwell (PRT) culture system, comprised of a
polycarbonate-membrane-transwell and a polydimethylsiloxane (PDMS) roof, to ease device production and
simplify sample loading and culture maintenance. The efficiency of in vitro spermatogenesis using the PRT
device was similar to the PL device in cultures of fresh neonatal mouse testes (>70% tubules showed Acrosin-
GFP+ post-meiotic cells within 1 month in culture). In the PRT system, I confirmed VASA-positive germ cells in
81.6±6.63%, SALL4-positive undifferentiated spermatogonia in 70.1±10.21%, STRA8-positive differentiating
spermatogonia in 50.0±14.01%, SYCP3-positive spermatocytes in 75.9±9.2%, and SOX9-positive Sertoli cells
in 99.08±0.9% of tubules. A similar experiment using cryopreserved neonatal mouse testes in both culture
systems is underway. In a pilot study on immature rhesus macaque testicular tissues using the PRT system, I
observed VASA-positive germ cell retention in 90.3% of tubules after 3 months in culture. More detailed
analyses are in process. Unlike mouse, monkeys and humans have a prolonged prepubertal period before the
initiation of spermatogenesis and there is limited information about the mechanisms that regulate prepubertal
to pubertal to adult developmental transitions in testicular somatic cells or germ cells. I will exploit our unique
pipeline of healthy rhesus and human testicular tissues from prepubertal, pubertal and adult individuals and
perform high-throughput single-cell RNA sequencing to identify a subset of genes with differential expression
at these devel...

## Key facts

- **NIH application ID:** 10068649
- **Project number:** 1F31HD101254-01A1
- **Recipient organization:** MAGEE-WOMEN'S RES INST AND FOUNDATION
- **Principal Investigator:** Kien T.D. Tran
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $45,520
- **Award type:** 1
- **Project period:** 2020-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10068649

## Citation

> US National Institutes of Health, RePORTER application 10068649, Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis (1F31HD101254-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10068649. Licensed CC0.

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