Nishida Hidetaka, Matsuura Haruhi, Yoshizaki Karin, Sakaguchi Yusuke, Izawa Takeshi, Sonoda Hikaru, Edamura Kazuya, Tabata Yasuhiko
Small Animal Clinics, Department of Veterinary Medicine, School of Veterinary Medicine, Azabu University, 1-17-71, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan.
Department of Veterinary Surgery, Graduate School of Veterinary Science, Osaka Metropolitan University, 1-58, Rinku Orai Kita, Izumisano, Osaka 598-8531, Japan.
Regen Ther. 2025 Jun 12;30:164-172. doi: 10.1016/j.reth.2025.05.014. eCollection 2025 Dec.
Extracellular vesicles (EVs) are expected to be a novel promotes in regenerative therapy. EVs derived from canine mesenchymal stromal/stem cells (MSCs) have been shown to play an important role in tissue repair. For clinical application of EV therapy, a major challenge is to find scalable methods for EV collection and to facilitate continuing local EV action. The aim of this study is to develop collection methods for EVs derived from canine MSCs using anion exchange chromatography (AEX), and to determine the therapeutic effects of sustained release of EVs from cationized gelatin hydrogels in rats with spinal cord injury (SCI).
Canine MSC-derived EVs were isolated from the culture supernatant by AEX. Lipopolysaccharide-stimulated BV-2 cells were used to determine the immunomodulatory effect of EVs. Distinct cross-linked cationized gelatin hydrogels were created by thermal dehydration. The amount of EVs captured by and released from hydrogels were measured using ELISA for CD63. The effects of sustained release of EVs on SCI in rats were evaluated via motor function and cavity volume.
EVs were collected from the culture supernatant of canine MSCs using AEX. Canine MSC derived EVs collected by AEX significantly reduced the expression levels of IL-1β and IL-6 in BV-2 cells induced by LPS. Sustained release of EVs from cationized hydrogels led to significant improvement of locomotion in SCI rats relative to the control group at 14- and 28-days post-injury. Cavity formation in the spinal cord with sustained release of EVs was significantly less than in the control group.
Canine MSC derived EVs cultured in serum-free medium using AEX were successfully collected. Sustained release of canine MSC derived EVs suppresses cavity formation in the spinal cord after SCI and promotes improvement in motor function.
细胞外囊泡（EVs）有望成为再生治疗中的一种新型促进剂。源自犬间充质基质/干细胞（MSCs）的EVs已被证明在组织修复中发挥重要作用。对于EV治疗的临床应用而言，一个主要挑战是找到可扩展的EV收集方法，并促进EV在局部的持续作用。本研究的目的是开发使用阴离子交换色谱法（AEX）从犬MSCs中收集EVs的方法，并确定阳离子化明胶水凝胶中EVs的持续释放在脊髓损伤（SCI）大鼠中的治疗效果。
通过AEX从培养上清液中分离犬MSC来源的EVs。使用脂多糖刺激的BV-2细胞来确定EVs的免疫调节作用。通过热脱水制备不同的交联阳离子化明胶水凝胶。使用针对CD63的ELISA法测量水凝胶捕获和释放的EVs量。通过运动功能和空洞体积评估EVs持续释放在大鼠SCI中的作用。
使用AEX从犬MSCs的培养上清液中收集到了EVs。通过AEX收集的犬MSC来源的EVs显著降低了LPS诱导的BV-2细胞中IL-1β和IL-6的表达水平。与对照组相比，在损伤后14天和28天，阳离子化水凝胶中EVs的持续释放使SCI大鼠的运动能力得到显著改善。EVs持续释放组脊髓中的空洞形成明显少于对照组。
成功使用AEX收集了在无血清培养基中培养的犬MSC来源的EVs。犬MSC来源的EVs的持续释放可抑制SCI后脊髓中的空洞形成，并促进运动功能的改善。
