Size-controlled human adipose-derived stem cell spheroids hybridized with single-segmented nanofibers and their effect on viability and stem cell differentiation

Lee et al. Biomaterials Research (2021) 25:14 https://doi.org/10.1186/s40824-021-00215-9 RESEARCH ARTICLE Open Access Size-controlled human adipose-derived stem cell spheroids hybridized with singlesegmented nanofibers and their effect on viability and stem cell differentiation Jinkyu Lee1,2, Sangmin Lee1,3, Sung Min Kim2,4,5* and Heungsoo Shin1,3,6* Abstract Background: Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation. Methods: We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/ μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s t-test (for two variables) (P < 0.05). Results: Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063 mm2–0.322 mm2). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA). (Continued on next page) * Correspondence: ; 2 BK21 FOUR, Human-Tech Convergence Program, Hanyang University, Seoul 04763, Republic of Korea 1 Department of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2021 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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lee et al. Biomaterials Research (2021) 25:14 Page 2 of 14 (Continued from previous page) Conclusion: The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications. Keywords: Single segmented fibers, Spheroid, Stem cell, Angiogenic factor, Differentiation, Background Stem cells have been evaluated in tissue engineering due to their multipotent ability to differentiate into diverse tissue lineages, for example osteoblasts, neuronal cells, hepatocytes, and adipocytes [1, 2]. Adult stem cells (ASCs) can be isolated from various organs and tissues including bone marrow, turbinate, periosteum, and synovial membranes, and have been widely utilized for cellbased therapeutic approaches [3–5]. In particular, human adipose-derived stem cells (hADSCs) are an attractive source due to their ability to secrete diverse paracrine factors and their rapid doubling time of less than 70 h [6–8]. hADSCs have been used with direct injection [9, 10], in combination with scaffolds [11, 12] and as cell sheets [13, 14] to target tissues, such as skin and muscles. However, directly delivering stem cells without any scaffolds or cell related interactions are easily cleared or trapped in capillaries, limiting their therapeutic efficacy [15, 16]. Also, use of scaffolds can often activate potential inflammation and may reduce cell-cell interactions, leading to limited stemness [17–19]. Furthermore, mono-layered cell sheets have shown to be difficult to handle because of weak mechanical properties, and limit control of spontaneous and direct stem cell differentiation [20–22]. In addition, thick, multilayered cell sheets limit oxygen and nutrient diffusion and the stem cells are unable to survive in vivo without blood vessels [23]. Alternatively, fabrication methods for three-dimensional (3D) cell spheroids have been studied as 3D microenvironment for cells. For instance, methods such as hanging drop, round micro-well, centrifugation, and a spinner flask with a bioreactor were mainly used for preparing spheroids [24–27]. Although these spheroids had improved stem cell function, there are still challenges in tissue engineering due to the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid. By the reason, most of the spheroid diameters were less than 200 μm because larger-sized spheroids showed severe hypoxia from diffusion limitations [26, 28]. Severe hypoxia induces apoptosis, and it may limit the spheroid viability and survival time. So it was hard to expect the long survival for differentiation such as osteogenic or chondrogenic lineage which were needing at least 14 or 21 days of culturing under differentiation medium. The dynamic cultures in bioreactors could attenuate the hypoxia in spheroids and improve their viability, however, the fabricated spheroids showed heterogeneous sizes [29]. Also, spheroids composed only of cells could maintain their stemness because of high cell-cell in (...truncated)