Subaqueous 3D stem cell spheroid levitation culture using anti-gravity bioreactor based on sound wave superposition

(2023) 27:51 Park et al. Biomaterials Research https://doi.org/10.1186/s40824-023-00383-w RESEARCH ARTICLE Biomaterials Research Open Access Subaqueous 3D stem cell spheroid levitation culture using anti‑gravity bioreactor based on sound wave superposition Jung Hwan Park1†, Ju‑Ro Lee2†, Sungkwon Park3†, Yu‑Jin Kim1, Jeong‑Kee Yoon4, Hyun Su Park1, Jiyu Hyun1, Yoon Ki Joung2,5, Tae Il Lee6* and Suk Ho Bhang1* Abstract Background Recently, various studies have revealed that 3D cell spheroids have several advantages over 2D cells in stem cell culture. However, conventional 3D spheroid culture methods have some disadvantages and limitations such as time required for spheroid formation and complexity of the experimental process. Here, we used acoustic levitation as cell culture platform to overcome the limitation of conventional 3D culture methods. Methods In our anti-gravity bioreactor, continuous standing sonic waves created pressure field for 3D culture of human mesenchymal stem cells (hMSCs). hMSCs were trapped and aggerated in pressure field and consequently formed spheroids. The structure, viability, gene and protein expression of spheroids formed in the anti-gravity bioreac‑ tor were analyzed by electron microscope, immunostaining, polymerase chain reaction, and western blot. We injected hMSC spheroids fabricated by anti-gravity bioreactor into the mouse hindlimb ischemia model. Limb salvage was quantified to evaluate therapeutic efficacy of hMSC spheroids. Results The acoustic levitation in anti-gravity bioreactor made spheroids faster and more compact compared to the conventional hanging drop method, which resulted in the upregulation of angiogenic paracrine factors of hMSCs, such as vascular endothelial growth factor and angiopoietin 2. Injected hMSCs spheroids cultured in the anti-gravity bioreactor exhibited improved therapeutic efficacy, including the degree of limb salvage, capillary formation, and attenuation of fibrosis and inflammation, for mouse hindlimb ischemia model compared to spheroids formed by the conventional hanging drop method. Conclusion Our stem cell culture system using acoustic levitation will be proposed as a new platform for the future 3D cell culture system. Keywords Acoustic levitation, Anti-gravity bioreactor, Human mesenchymal stem cell spheroid, Hindlimb ischemia † Jung Hwan Park, Ju-Ro Lee, and Sungkwon Park contributed equally to this work. *Correspondence: Tae Il Lee Suk Ho Bhang Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Park et al. Biomaterials Research (2023) 27:51 Page 2 of 14 Graphical Abstract Introduction Three-dimensional stem cell spheroids have gained a growing attention as cell therapies and biomaterials. So far, spheroids have been fabricated by numerous methods including pellet culture, hanging drop, spinner flask, magnetic levitation, and culture in space [1–4]. The conventional 3D culture methods can change the phenotypes of cultured cells such as gene expressions of proliferation, cell–cell interaction, and paracrine factors compared to 2D stem cells [1, 2]. The time for spheroid formation is basically long in most methods. From 2 to 7 days, there is a large deviation depending on the culture method and process. Even in the culture process, complex or labor-intensive processes are included. Pellet culture and hanging drop methods require separate preparation for each spheroid [1, 5], and spinner flask method require continuous rotational control [6, 7]. Methods using magnetic levitation require additional steps such as mechanical operation or nanoparticles that enter the medium for help levitation [8, 9]. To solve problems such as time and labor, the acoustic levitation was applied in this study. To solve these limitations, we cultured human mesenchymal stem cells (hMSCs) with the acoustic levitation method to fabricate spheroids. Several studies have developed an anti-gravity bioreactor equipped with transducer and reflector for acoustic levitation [10, 11]. The anti-gravity bioreactor only needs a simple process, one injection of the medium and cells before the operation. After that, the anti-gravity bioreactor does not require any additional control. Then, standing wave is generated into the liquid medium through the vibrator. The energy of the standing wave is transferred into the medium, creating a pressure field. The hMSCs in the medium were located in the node of the standing wave. The hMSCs are arranged and cultured while floating in the anti-gravity bioreactor. Spheroids are formed when cultured for more than 12 h in the anti-gravity bioreactor. (Fig. 1). The anti-gravity bioreactor takes shorter time to formulate spheroids with compact and uniform morphologies compared to those of conventional methods without cytotoxicity. As previously reported, compact spheroids exhibited higher metabolic activity, viability, and proliferation compared to spheroids with voids and uneven morphologies [12]. As a result, after 12 h of incubation, the gene expression levels of angiogenic, senescent and proliferative factors were improved in the spheroids from the anti-gravity bioreactor compared to the conventional hanging drop method. Also, in in vivo experiment, spheroids formed in anti-gravity bioreactor showed remarkably improved therapeutic efficacy for the hindlimb ischemia mouse model compared to conventional Park et al. Biomaterials Research (2023) 27:51 Page 3 of 14 Fig. 1 Schematic diagram of 3D stem cell spheroid culture by acoustic levitation. During incubation with acoustic levitation, hMSCs were aggregated near the node and incubated as spheroids. These spheroids showed the characteristics of spheroids such as improved expression of angiogenic factors and proliferation factors. By injecting these spheroids, the treatment efficiency of hind limb ischemia was observed spheroids formed by hanging drop (...truncated)