A Novel Multi-compartment Rotating Bioreactor for Improving ADSC--Spheroid Formation and its Application in Neurogenic Erectile Dysfunction

Abstract

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.