Objective: The aim of this study was to construct a multicompartment synchronous rotating bioreactor (MCSRB) for batch-production of homogenized adipose-derived stem cell (ADSC) microspheres and treat neurogenic erectile dysfunction (ED). background: Erectile dysfunction due to cavernous nerve injury is commonly associated with pelvic site surgery1, particularly radical prostatectomy, which is one of the most common first-line treatments for men with limited and locally progressive prostate cancer, with a probability of 14-82% of causing erectile dysfunction after surgery, severely affecting patients&#039; quality of life. For this neurogenic erectile dysfunction, the traditional first-line treatment with phosphodiesterase type 5 (PDE5) inhibitors is ineffective and has side effects such as headache, dizziness, and indigestion; thus, there is an urgent need to find a new alternative therapy. In recent years, with the rise of stem cell therapy, the application of stem cells to the treatment of erectile dysfunction has attracted extra attention. Zhang et al. showed that a cytokine secreted by adipose tissue-derived stem cells has a neurotrophic effect on cavernous nerve regeneration. It has also been shown that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) attenuates erectile dysfunction after cavernous nerve injury in rats. However, because of the loose pore-like structure of the penile corpus cavernosum, stem cells injected via the corpus cavernosum carry the risk of cell escape, leading to pulmonary embolism; thus, this issue of safety and limited treatment due to cell escape warrants intensive study. It has been shown that culturing MSCs into 3D stem cell microspheres can significantly activate their paracrine function, which is important for improving the therapeutic potential of MSCs, while Xu&#039;s study showed that stem cell microspheres can increase cell retention in the cavernous body and reduce the risk of pulmonary embolism. Previous studies have found that stem cell microspheres can enhance paracrine, anti-inflammatory, and anti-apoptotic functions. Currently, there are many methods for 3D sphere formation of stem cells, such as the suspension droplet method, micropatterning method and microfluidic method, but all of these methods have deficiencies, such as low yield, low recovery of culture medium and the need for special equipment. The multicompartment synchronized rotating bioreactor is a device designed and developed by our team to culture MSC microspheres in 3D. During our experiments, we found that the dynamic culture of 3D stem microspheres using the MCSRB is very simple and convenient, and the design of multiple compartments scales well, which can greatly improve the efficiency and reduce the time of determining the optimal conditions during culture, and significantly improves MSC paracrine function. Therefore, in this study, we investigated whether the dynamic culture of dry 3D microspheres in this bioreactor could effectively restore erectile function and pathological changes in rats with cavernous nerve injury. Methods: Firstly, an MCSRB was constructed using a centrifugal device and hinged trays. Secondly, influence factors (density, rotational speed) on the formation of ADSC-spheroids were explored. Finally, a neurogenic ED model was established to verify the effectiveness and safety of ADSC-spheroids for ED treatment. objective: To investigate the therapeutic effects of dynamically cultured rat adipose stem cell (ADSC) microspheres in a multicompartment synchronous rotating bioreactor (MCSRB) in rats with bilateral cavernous nerve injury-induced erectile dysfunction. Results: An MCSRB promoted ADSCs to gather microspheres, most of which were 90-130 μm in diameter. Supernatant from three-dimensional culture led to a significant increase in cytokine expression in ADSCs and migration rate in human umbilical vein endothelial cells (HUVECs) compared to control groups. The erectile function and pathological changes of the penis were improved in the ADSC-spheroids treatment group compared to the traditional ADSCs treatment group (p < 0.01). Conclusion: Efficient, batch, controlled and homogenized production of ADSC stem cell microspheres, and effective improvement of erectile dysfunction in neurogenic rats can be achieved using the MCSRB device. conclusion: Treatment with 3D ADSC microspheres dynamically cultured in MCSRBs significantly improved erectile dysfunction in neurogenic rats with erectile dysfunction and contributed to the recovery of tissue damage, demonstrating the value of scaled-up culture and the potential clinical application of stem cell microspheres.

中文翻译：

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一种改善ADSC球体形成的新型多室旋转生物反应器及其在神经源性勃起功能障碍中的应用

目的：本研究的目的是构建多室同步旋转生物反应器（MCSRB），用于批量生产均质脂肪干细胞（ADSC）微球并治疗神经源性勃起功能障碍（ED）。背景：海绵体神经损伤引起的勃起功能障碍通常与骨盆部位手术1相关，特别是根治性前列腺切除术，它是患有局限性和局部进展性前列腺癌的男性最常见的一线治疗方法之一，概率为 14-82%导致术后勃起功能障碍，严重影响患者的生活质量。生活质量。对于这种神经源性勃起功能障碍，传统的一线治疗5型磷酸二酯酶（PDE5）抑制剂效果不佳，并且有头痛、头晕、消化不良等副作用；因此，迫切需要寻找新的替代疗法。近年来，随着干细胞疗法的兴起，干细胞在治疗勃起功能障碍中的应用引起了格外的关注。张等人。研究表明，脂肪组织来源的干细胞分泌的细胞因子对海绵体神经再生具有神经营养作用。研究还表明，静脉输注骨髓间充质干细胞 (MSC) 可减轻大鼠海绵体神经损伤后的勃起功能障碍。但由于阴茎海绵体具有疏松的孔状结构，经海绵体注射的干细胞存在细胞逃逸的风险，导致肺栓塞；因此，由于细胞逃逸而导致的安全性和有限治疗问题值得深入研究。研究表明，将MSC培养成3D干细胞微球可以显着激活其旁分泌功能，这对于提高MSC的治疗潜力非常重要，而Xu的研究表明干细胞微球可以增加细胞在海绵体中的保留并降低肺栓塞的风险。此前的研究发现，干细胞微球可以增强旁分泌、抗炎、抗凋亡功能。目前，干细胞3D球体形成的方法有多种，如悬浮液滴法、微图案化法、微流控法等，但这些方法均存在产量低、培养基回收率低、需要特殊设备等不足。设备。多室同步旋转生物反应器是我们团队设计开发的用于3D培养MSC微球的装置。在我们的实验中，我们发现利用MCSRB进行3D茎微球的动态培养非常简单方便，并且多隔室的设计具有良好的扩展性，可以大大提高效率并减少培养过程中确定最佳条件的时间，并显着改善MSC旁分泌功能。因此，在本研究中，我们研究了在该生物反应器中动态培养干3D微球是否能够有效恢复海绵神经损伤大鼠的勃起功能和病理变化。方法：首先，使用离心装置和铰接托盘构建 MCSRB。其次，探讨了ADSC球体形成的影响因素（密度、转速）。最后，建立神经源性ED模型，验证ADSC球体治疗ED的有效性和安全性。目的：探讨多室同步旋转生物反应器（MCSRB）动态培养大鼠脂肪干细胞（ADSC）微球对双侧海绵神经损伤所致勃起功能障碍大鼠的治疗作用。结果：MCSRB促进ADSCs聚集微球，大部分直径为90-130μm。与对照组相比，三维培养上清液导致 ADSC 中的细胞因子表达和人脐静脉内皮细胞 (HUVEC) 的迁移率显着增加。与传统 ADSC 治疗组相比，ADSC 球体治疗组的勃起功能和阴茎病理变化均得到改善（p < 0.01）。结论：利用MCSRB装置可实现ADSC干细胞微球的高效、批量、可控、均质化生产，并有效改善神经源性大鼠勃起功能障碍。结论：在MCSRB中动态培养的3D ADSC微球治疗可显着改善神经源性勃起功能障碍大鼠的勃起功能障碍，并有助于组织损伤的恢复，证明了放大培养的价值和干细胞微球的潜在临床应用。使用MCSRB装置可以控制和均质化生产ADSC干细胞微球，并有效改善神经源性大鼠的勃起功能障碍。结论：在MCSRB中动态培养的3D ADSC微球治疗可显着改善神经源性勃起功能障碍大鼠的勃起功能障碍，并有助于组织损伤的恢复，证明了放大培养的价值和干细胞微球的潜在临床应用。使用MCSRB装置可以控制和均质化生产ADSC干细胞微球，并有效改善神经源性大鼠的勃起功能障碍。结论：在MCSRB中动态培养的3D ADSC微球治疗可显着改善神经源性勃起功能障碍大鼠的勃起功能障碍，并有助于组织损伤的恢复，证明了放大培养的价值和干细胞微球的潜在临床应用。