Vancouver Prostate Centre, Vancouver, British Columbia, Canada.
Aspect Biosystems, Vancouver, British Columbia, Canada.
F S Sci. 2022 May;3(2):130-139. doi: 10.1016/j.xfss.2022.02.004. Epub 2022 Feb 17.
To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA).
A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded in vitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted in vitro control.
Academic medical center.
PATIENT(S): A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome.
INTERVENTION(S): Three-dimensional bioprinting and in vitro culturing of patient-derived testis cells.
MAIN OUTCOME MEASURE(S): Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of in vitro culture.
RESULT(S): Testicular cultures were expandable in vitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold.
CONCLUSION(S): This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of in vitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.
研究从非梗阻性无精子症（NOA）患者中分离的 3 维（3D）生物打印个性化人睾丸细胞的可行性和生精潜能。
从一位 31 岁的非梗阻性无精子症患者的睾丸活检中分离出单细胞，在体外扩增，并使用 AGC-10 生物墨水和 Aspect Biosystems 的 RX1 生物打印机和 CENTRA 共轴微流控打印头打印成类似于精小管的管状结构。三维类器官培养用作非生物打印的体外对照。
学术医疗中心。
一位 31 岁的非梗阻性无精子症患者，睾丸活检显示为支持细胞综合征。
患者来源的睾丸细胞的 3D 生物打印和体外培养。
打印后细胞活力的测定，以及体外培养 12 天后表型和生精功能遗传标记的表达。
睾丸培养物可在体外扩增，生物墨水每毫升可产生足够数量的细胞用于 3D 生物打印，达到 3500 万个细胞。打印后 24 小时的存活率为 93.4%±2.4%。12 天后的免疫荧光染色显示，SRY-Box 转录因子 9、胰岛素样 3、肌动蛋白 alpha 2 平滑肌和联会复合体蛋白 3 的表型标记物呈阳性，证实存在支持细胞、莱迪希细胞、小管周围肌样细胞和减数分裂生殖细胞。逆转录定量聚合酶链反应分析显示，在生精培养基中培养 12 天后，生物打印在与非生物打印对照相当的水平上显著上调了生精基因的表达，并特别显著改善了与精原干细胞维持相关的基因：脱氧核糖核酸结合抑制因子 4 上调 365 倍；成纤维细胞生长因子 3 上调 94152 倍；干细胞生长因子受体 KIT 上调 2 倍；维甲酸刺激蛋白 8 上调 125 倍；无精子症样缺失蛋白 114 倍；联会复合体蛋白 3 上调 7 倍；透明带结合蛋白上调 2 倍；过渡蛋白 1 上调 2908 倍；和顶体蛋白 2 上调 11 倍。
本研究首次证明了成人睾丸细胞 3D 生物打印的可行性。我们表明，生物打印过程与高睾丸细胞活力兼容，并且不会丢失睾丸组织内的主要体细胞表型。我们在体外培养 12 天后，在 3D 生物打印的小管中观察到生殖细胞标记物的增加。该平台可能在个性化医学框架内为疾病建模和再生机会提供未来的潜力。
