Ortimization of cryopreservation protocols for adipose tissue and adipose tissue-derived mesenchymal stem cells and their potential use in tissue engineering

According to American Society of Plastic Surgeons, in 2012, approximately 300.000 procedures have been performed for soft tissue repairs including breast and nipple reconstruction (after mastectomy), hand and face rejuvenation and this number continues to increase annualy. In Turkey, although the application of these procedures have usually been performed in many clinics, such information is not available for the public yet. Contemporary research in soft tissue engineering indicates that, numerous soft tissue defects can be efficiently repaired when appropriate biomaterials, cells and growth factors can be delivered together to the wounded location. For this reason, novel approaches and works are currently directed to the creation of artificial tissues by integrating stem/progenitor cells in natural scaffolds. Adipose tissue, which can be obtained after lipoaspiration, has a high poteintial in repairing and healing of soft tissue defects via its rich content of adipose mesenchymal stem cell (AD-MSC)/progenitor cell populations. Every year, milions of lipoaspirations and adipose tissue resections for cosmetic purposes are performed worldwide. Effective cryopreservation of these tissues can be very important natural autologus tissue sources for tissue engineering aplications other than soft tissue defects or reconstructive surgery applications. Successful cryopreservation of adipose tissue, which is mostly obtained during lipoaspiration procedures with the aim of using in the near future, and effective warming and transplanting it into the defective site when needed has often been applied in soft tissue engieering and plastic/reconstructive surgery. Although cryopreservation of fat tissue have long been a preferred approach in several clinics undergoing liposuction procedures, problems associated with their clinical use after warming (low cellular viability upon warming, high reabsorbtion rates on recipient sites etc.) have not been fully resolved. There is still a lack of successful and efficient adipose tissue/AD-MSC cryopreservation protocol in the world. Moreover, the number of studies on the ability and effectiveness of cryopreserved adipose tissue or AD-MSCs in repairing a defect in the required site are scarce and the results are conflicting. Based on current knowledge, our aim in this study is, to analyze the structural and functional integrity of human adipose tissues and adipose tissue-derived mesechymal stem cells during conventional cryopreservation procedures, to investigate the potential role of vitrification regimes and approaches in solving current problems associated with adipose tissue/ stem cell cryopreservation, and by optimizing the human adipose tissue and adipose tissue-derived mesenchymal stem cell cryopreservation protocols, to establish a tissue and cell cryobank that is suitable or compatible for clinical use. In accordance with the objectives set our study, 10 different tissue samples that were obtained from different donors after lipoaspiration (n=8) and surgical excision (n=2) were used in the study. In 6 of these samples, enzymatic dissociation method was used in order to isolate mesenchymal stem cells. At this stage, magnetic-based cell separation technique was also applied. Stem cell properties of these freshly isolated cells were measured by in vitro culture and morphological analysis, flow cytometry (CD90, CD105, CD29, CD34, CD45) and in vitro expansion/differentation tests. For tissue and cell cyryopreservation, staged slow freezing (10% DMSO; 10% DMSO+0.2M sucrose; 0.5M sucrose) and vitrfication (15% DMSO+15% Ethylene glycol+0.5M sucrose; 30% DMSO+30% ehylene glycol+0.5M sucrose) techniques that consists of DMSO, ehylene glycol (EG) and sucrose in different concentrations and coctails were used. Upon warming, tissue integrity was measured by immunohistochemistry and structural and functional integrity of cells were measured by trypan blue dye exclusion test, TUNEL test for DNA fragmentation and in vitro differentiation. Cryopreserved cells and cells that were isolated upon warming of tissues were also investigated according to their in vitro morphologies and in vitro expansion characteristics. Quantitative data obtained during these investigations were evaluated by SPSS 10.0 for windows software programme and the p value of <0.05 was taken as statistically significant. According to the study plan, the experiment had been performed in three parts. In the first phase of the study, cell isolation by enzymatic dissociation method was performed on 6 of the tissue samples (4 samples after lipoaspiration and 2 samples after surgical excision). Their stem cell properties were confirmed by their in vitro culture behaviours, morphologies, results of flow cytometry analysis and in vitro differention tests that shown their potential of differentiating in a variety of different mesodermal cells. In the second phase of the study, by using the cells that were isolated and characterized in the previous phase, cytotoxic or genotoxic effects of different cryoprotectant concentrations (1% - 30%), different combinations and different incubation durations (10 -40 minutes) of DMSO and ethylene glycol were investigated. In the presence of 10% and higher concentrations of DMSO and EG and upon more than 20 minutes of incubation durations significant decrease in cellular survival was observed (p<0.05). On the other hand, simultaneous analysis of DNA fragmentation on the same cell populations did not show any statistically significant increase. In the third phase of the study, adipose tissue samples obtained (n=4) and adipose tissue-derived stem cells (n=4) were cryopreserved by conventional slow freezing and vitrification techniques. In both cryopreservation groups, samples were subgrouped according to different incubation durations as well as different storage temperatures. In the cellular-level cryopreservation experiments, the groups of 10% DMSO and 10% DMSO+0.2M sucrose in the conventional slow freezing arm and VS1 and VS2 in the vitrification arm shown significantly higher viability compared to control groups (80.2%, 88.9%, 85.4% and 68.2% vs. 12.2% respectively; p<0.01). In the tissue-level cryopreservation experiments, significantly higher cellular survival values were observed in VS1 and VS2 vitrification groups as compared to 10% DMSO and 10%DMSO+0.2M sucrose groups in the conventional slow freezing arm and controls (90.2%, 82.0% vs. 50.0%, 62,8% and 10.2% respectively). It was observed that, samples that were cryopreserved and stored at +4°C and -20°C in the conventional slow freezing group did not show any signs of viability after long term storage conditions. In groups showing comparable survival rates upon warming in both cellular and tissue-level experiments, no significant differences were observed in in vitro expansion and in vitro differentiation results. As a result, in this study, both the conventional staged slow freezing protocols and, to our knowledge for the first time in the literature, vitrification protocols were simultaneously investigated for their effectivity on human adipose tissue and adipose tissue-derived mesenchymal stem cell cryopreservation. Our results have shown that, both the high concentration and longer incubation periods of cryoprotectants negatively and dramatically affect the survival and functions of the adipose tissue as well as mesenchymal stem cells in the same tissue. The results also confirmed that, in order to set up a clinically acceptable cryopreservation programme, one needs to optimize the cellular and tissue-level cryopreservation programmes separately. In addition to the above results, our data also clarified and confirmed that conventional storage temperatures (4°C and -20°C) are clearly ineffective in storing the functionality of the tissue and the cells. In conclusion, this study showed that, staged slow freezing is the most optimal route for cryopreservation of AD-MSCs whereas vitrification protocol is the most convenient for cryopreservation of adipose tissues. Based on this information, simultaneous cryopreservation of adipose tissue and AD-MSCs has been developed and optimized for future use. The findings of this study also helped us to generate a tissue and a mesenchymal stem cell cryobank from the donated materials. Therefore the resuls obtained in this study can be considered as a valuable source for experimental, preclinical or clinical studies that can potentially be involved in stem cells and tissue engineering. Keywords: Adipose tissue, AD-MSC, Soft tissue engineering, Cryopreservation, Slow freezing, Vitrification.