Cryopreserved clumps of mesenchymal stem cell/extracellular matrix complexes retain osteogenic capacity and induce bone regeneration

Motoike et al. Stem Cell Research & Therapy (2018) 9:73 https://doi.org/10.1186/s13287-018-0826-0 RESEARCH Open Access Cryopreserved clumps of mesenchymal stem cell/extracellular matrix complexes retain osteogenic capacity and induce bone regeneration Souta Motoike, Mikihito Kajiya* , Nao Komatsu, Manabu Takewaki, Susumu Horikoshi, Shinji Matsuda, Kazuhisa Ouhara, Tomoyuki Iwata, Katsuhiro Takeda, Tsuyoshi Fujita and Hidemi Kurihara Abstract Background: Three-dimensional (3D) cultured clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. C-MSCs can regulate cellular functions in vitro and can be grafted into a defect site without an artificial scaffold to induce bone regeneration. Long-term cryopreservation of C-MSCs, which can enable them to serve as a ready-to-use cell preparation, may be helpful in developing beneficial cell therapy for bone regeneration. Therefore, the aim of this study was to investigate the effect of cryopreservation on C-MSCs. Methods: MSCs isolated from rat femurs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make a round clumps of cells. The C-MSCs were cryopreserved in cryomedium including 10% dimethyl sulfoxide. Results: Cryopreserved C-MSCs retained their 3D structure and did not exhibit a decrease in cell viability. In addition, stem cell marker expression levels and the osteogenic differentiation properties of C-MSCs were not reduced by cryopreservation. However, C-MSCs pretreated with collagenase before cryopreservation showed a lower level of type I collagen and could not retain their 3D structure, and their rates of cell death increased during cryopreservation. Both C-MSC and cryopreserved C-MSC transplantation into rat calvarial defects induced successful bone regeneration. Conclusion: These data indicate that cryopreservation does not reduce the biological properties of C-MSCs because of its abundant type I collagen. More specifically, cryopreserved C-MSCs could be applicable for novel bone regenerative therapies. Keywords: Artificial scaffold free, Bone regeneration, Cryopreservation, C-MSC, MSCs Background Mesenchymal stem cells (MSCs) are self-renewing multipotent progenitor cells that have attracted considerable scientific and medical attention for many years as an effective tissue regenerative cell therapy [1–4]. In particular, bone marrow-derived MSCs are highly investigated stem cells for bone regeneration in basic and clinical studies [5]. * Correspondence: Department of Periodontal Medicine, Applied Life Sciences, Institute of Biomedical & Health Sciences, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, Japan It is widely accepted that the implantation of bone marrow-derived MSCs promotes bone regeneration at the sites of defects. However, there still remain obstacles to be overcome in applying these cells for established bone regenerative medicine. At present, MSCs isolated from patient bone marrow are expanded ex vivo and then mixed with biocompatible artificial scaffold to graft the cells into the defect site. This process requires a prolonged culture period which results in increased contamination risks and culture costs. In addition, despite recent advances, clinical application of artificial scaffolds still harbors several © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Motoike et al. Stem Cell Research & Therapy (2018) 9:73 limitations, including biodegradability and unfavorable host inflammation and immunological reaction [6, 7]. To address these problems, we have recently generated three-dimensional (3D) clumps of MSC/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM [8]. C-MSCs can be grafted into bony lesions without artificial scaffolds to induce successful bone regeneration [8, 9], suggesting the avoidance of the problems regarding the usage of artificial scaffolds described above. Moreover, we have also reported that xenografts of human C-MSCs treated with interferon (IFN)-γ induced bone regeneration in a mouse calvarial defect model because of its highly regulated immunomodulatory capacity [10]. This fact indicated the availability of allogenic C-MSCs for clinical bone regenerative cell therapy, which can eliminate the autologous MSC isolation and expansion process. However, even though C-MSCs seem to be promising for clinical bone regenerative cell therapy, their preparation process is inevitably time consuming. Cryopreservation, which maintains the cell viability and function of bioengineered cellular constructs, is a significant research avenue for successful tissue engineering in regenerative medicine [11]. The development of cryobanked materials will enable us to supply the cellular product at the time when the patient needs it. Moreover, the materials can provide adequate quality control and standardization of the same cell preparation at different times when the cellular product is needed. Briefly, if cryopreserved C-MSCs retain their 3D structure, cell viability, and osteogenic properties, C-MSCs will take an important step toward their clinical application for bone regenerative medicine because the C-MSC preparation process can be omitted immediately before its transplantation and we will have standardized material on demand. This novel cell therapy using cryopreserved C-MSCs could be implemented through optimized easy cryopreservation procedures. In general, cryopreservation of cells or tissues is carried out by two techniques, either vitrification or slow freezing in the presence of a cryoprotectant, such as dimethyl sulfoxide (DMSO). Although vitrification is well known to show beneficial cytoprotective effects in various types of cells, its inherent problems include the difficulty of large-scale processing and risk of contamination from liquid nitrogen [12, 13]. On the other hand, slow freezing is a well-established traditional approach for cryopreservation of MSCs as cell suspensions [14–16]. Importantly, this procedure can handle a large number of samples easily, which makes it more clinically relevant. Moreover, a recent study revealed that this slow freezing is an effective technique for (...truncated)