CryoLetters 36 (5), 325-335 (2015) © CryoLetters, [email protected] CRYOPRESERVATION STRATEGY FOR TISSUE ENGINEERING CONSTRUCTS CONSISTING OF HUMAN MESENHYMAL STEM CELLS AND HYDROGEL BIOMATERIALS Yingnan Wu1,2,#, Feng Wen3,#, Sok Siam Gouk3, Eng Hin Lee1,2 and Lilia L. Kuleshova3,* 1Tissue Engineering Program; 2Department of Orthopaedic Surgery, YLL School of Medicine; 3Low Temperature Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore . #These authors contributed equally. *Corresponding author email: [email protected] Abstract BACKGROUND: The development of vitrification strategy for cell-biomaterial constructs, particularly biologically inspired nanoscale materials and hydrogels mimicking the in vivo environment is an active area. A cryopreservation strategy mimicking the in vivo environment for cell- hydrogel constructs may enhance cell proliferation and biological function. OBJECTIVE: To demonstrate the efficacy of vitrification as a platform technology involving tissue engineering and human mesenchymal stem cells (hMSCs). MATERIALS AND METHODS: Microcarriers made from alginate coated with chitosan and collagen are used. Conventional freezing and vitrification were compared. The vitrification strategy includes 10 min step-wise exposure to a vitrification solution (40% v/v EG, 0.6M sucrose) and immersion into liquid nitrogen. RESULTS: Confocal imaging of live/dead staining of hMSCs cultured on the surface of microcarriers demonstrated that vitrified cells had excellent appearance and prolonged spindle shape morphology. The proliferation ability of post- vitrified cells arbitrated to protein Ki-67 gene expression was not significantly different in comparison to untreated control, while that of post-freezing cells was almost lost. The ability of hMSCs cultured on the surface of microcarriers to proliferate has been not affected by vitrification and it was significantly better after vitrification than after conventional freezing during continuous culture. Collagen II related mRNA expression by 4 weeks post-vitrification and post-freezing showed that ability to differentiate into cartilage was sustained during vitrification and reduced during conventional freezing. No significant difference was found between control and vitrification groups only. CONCLUSION: Vitrification strategy coupled with advances in hMSC-expansion platform that completely preserves the ability of stem cells to proliferate and subsequently differentiate allows not only to reach a critical cell number, but also demonstrate prospects for effective utilization and transportation of cells with their support system, creating demand for novel biodegradable materials. Keywords: vitrification, hydrogel, microcarrier, mesenchymal stem cell, chondrogenic differentiation INTRODUCTION 4,7,8,11,12]. In the present work, we aim to prove the efficacy of vitrification as a platform Ascertaining the superiority of vitrification technology for tissue engineered constructs with for tissue engineering constructs is particularly human mesenchymal stem cells (hMSCs). of intrest [1-12]. Designing of biomaterials with Vitrification has great potential as a part of properties that will not be transformed during tissue engineering approach, yet, it has been interaction with cryoprotective agents, cooling to largely investigated in the application to native cryogenic temperatures and subsequent warming tissues and cells [13-29]. There are fundamental is a challenge. Vitrification for cell-biomaterial differences between vitrification and freezing to constructs, including bio-inspired nanoscale cryopreservation [14,19,25]. Vitrification avoids materials and hydrogels mimicking the in vivo ice formation during cooling and warming inside environment is an active area of our research [1- cells and biomaterial [1-12,25] while freezing 325 allows ice outside cells in the medium and with MSCs will involve much greater number of biomaterial as cryoprotectant concentration is cells for transplantations. usually low [13,14]. This is an advantage of Culturing stem cells on microcarriers that vitrification over freezing which provides provide suitable biomechanical properties and amorphous solidification, resulting in less biocompatibility is one of reliable methods to mechanical stress to the cells/tissues and cell- increase a number of cells in several folds [40]. carriers. Over the years we have provided The microcarrier system can be applied to scale- evidence that a vitrification strategy is vital for up the hMSCs expansion to multiply amount of successful preservation of engineered constructs cells required in certain clinical applications. [1-4,7,8,10-12,17]. It provides intactness of cell Cryopreservation of cells along with their membrane [12], attachment ability of cells to the culture support system has its own advantages. carrier [8] and cell-cell interactions [1,2,15,16]. Clinical treatments are frequently altered or In a biomaterial context, we have provided delayed. Cryopreservation of cells with their evidence that properly calculated composition of culture support system adds to the flexibility of vitrification solutions and the cooling-warming clinical scheduling and facilitates continuous cycle do not impair the integrity and quality of cell expansion. The detachment and dissociation the materials involved, allowing free migration of MSCs may trigger unwelcome behaviour in and aggregation of cells if necessary [1,2]. It cells, such as differentiation. The re-introduction was also demonstrated by us that vitrification is of hMSCs into their original culture system superior in the maintenance of viability and again with the purpose of perpetual culture metabolic function of cells [1-4,7,8,11,12, and requires special research facilities which proliferation and differentiation potential of 3D becomes a rather challenging task in the clinical clusters of neuronal stem cells [15,16] and swine setting. Finally, cryopreservation of detached mesenchymal stem cells (MSCs) cultured on cells is much less effective compared to that for 2D-, 3D tissue engineered constructs for bone attached cells [12]. A cryopreservation strategy regeneration [7,8]. coupled with a MSC-expansion platform permit In cartilage regeneration, the development not only attaining a critical cell number, but also of effective cryopreservation protocols is a effective utilization and transportation of MSC- useful tool to preserve human MSCs from a hydrogel cultures at any time point or location, variety of sources for future clinical application. creating the demand for novel biodegradable Owing to its intrinsic property of being a materials. A vitrification strategy accompanies nonvascularized tissue, damaged articular the biomaterial platform and is applicable to cartilage severely lacks the capacity to heal or other similar cultures, in particular, a growing regenerate. The research is of high significance. number of novel hydrogel culture systems will Despite recent advances in surgical and non- benefit from a fully developed vitrification surgical interventions, treatment of cartilage strategy. In recent time, it was proven in swine lesions still remains a problem [30]. Cell-based model that combination of a gel scaffold with therapy has shown promising results in treating MSCs can be used successfully for the purpose damaged cartilage [31,32]. Since the 1970s, of new formation of cartilaginous tissue (41). MSCs have been used as chondrocyte progenitor Articular cartilage defects were repaired through cells for cartilage healing [33] but it was not transplantation of MSCs/scaffolds in a primate until the end of 1990s that intense investigation model (42). The collagen scaffold improves the into the regenerative potential of proliferated repair of cartilage (43). Therefore, developing a MSCs [34-36]. MSCs are multipotent cells reliable strategy for preservation of allogeneic present in the bone marrow in low quantity (1 in hMSCs on microcarries involving bioabsorbable 104-105 mononuclear cells), which are capable of materials, serving as permissive substrates for differentiating into chondrocytes, osteocytes, cell growth is an important issue in the field of myocytes, and adipocytes [37]. MSCs have low regenerative medicine. or little immunogenicity when transplanted into another host [38,39]. This advantage makes MSCs more amenable to cell therapy. Treatment 326 MATERIALS AND METHODS was coated on the alginate bead surface followed by conjugation of collagen type I to the chitosan layer (Figure 1.1). All steps were performed at (A))…… … (B))…… … (C) Figure 1.1 Scanning electron microscopy of microcarriers. (A) alginate microcarrier, (B) chitosan-coated alginate microcarrier, (C) collagen-conjugated chitosan/alginate microcarrier.Scale bares are 20µm. room temperature. Microcarriers coated by chitosan and collagen type I were tested for process of introduction to and removal of cryoprotectant as well as cooling to -196°C and warming to 38°C as described in section 2.2 to confirm the integrity. Phase contrast microscopy was used to assess the integrity of microcariers during optimization. Microcarriers can withstand vitrification procedure described in Figure 1.2 without compromising the structural integrity. Seeding of hMSCs on the microcarrier surface hMSCs were harvested from T175 culture flask at P3 and mixed with microcarrier system in a 15mL conical tube and cultured in growth Figure 1.2 Schematic representation