| Cryopreservation plays an important role in obtaining off-the-shelf availability for a variety of cells and tissues. Cryopreservation decouples cell culture and implantation and makes possible for long distance transportation and for long-term storage of cells. Bone marrow-derived mesenchymal stem cells (BMSCs) have become good candidates for cell therapy and tissue-engineering applications due to the capacity for self-renewal and multilineage differentiation into mesenchymal tissues such as osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. However, an essential prerequisite for successful applications of BMSCs is to cryopreserve cells under well defined conditions to satisfy requirements in clinical applications.Up to date, most successful cryopreservation is achieved by the slow freezing method, of which the cooling rate is around 1℃/min. In laboratories, the most common cryoprotectant agent (CPA) formulation consists of 10%(v/v) dimethylsulfoxide (DMSO) and up to 90% (v/v) foetal bovine serum (FBS). However, DMSO, at a higher concentration such as 10% (v/v) has been associated with the neurological toxicity, as well as possible gene mutations and other adverse effects. FBS is an undesirable additive to cells for therapeutic purposes in humans because it is not defined in composition and also the use of FBS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. Hence, for the clinical application of cryopreserved cells, the use of well defined CPA solutions containing only authority approved ingredients and the elimination of animal-derived biological ingredients and all possible sources of infectious diseases are necessary.In this thesis, the effect of cryopreservation on hBMSCs viability, apoptotic rate, metabolic activities, intracellular pH, mitochondria distribution and differentiation potentials has been assessed. The results demonstrated the possibility of developing a well defined, serum-free and reduced DMSO freezing solution for hBMSCs cryopreservation, and showed that post-thawing viability of hBMSCs in 7.5% DMSO (v/v),2.5% polyethylene glycol (PEG) (w/v) and 2% bovine serum albumin (BSA) (w/v) was comparable with that obtained in conventional 10% DMSO, that was,82.9±4.3% and 82.7±3.7%, respectively. FBS can be replaced with albumin and 2%(w/v) albumin in CPA solution can significantly enhance the cell survival. This is a significant outcome as clinically approved human albumin can be easily obtained. In addition, the growth kinetics and osteogenic and chondrogenic differentiation potentials of the cryopreserved hBMSC cells are quite similar to those of non-cryopreserved.For the same cell type of different species, the same CPAs may exert different cryoprotective efficacies. To date, MSCs from various species (e.g. mice, rats, rabbits, calves, humans) have been cultured, expanded in vitro and characterized. However, the roles of different cryoprotective agents in the cryopreservation of MSCs from different species have not been studied in detail. Here, we performed a comparative study of MSCs from mice, rats and calves to investigate effects of different combinations of cryoprotective agents (CPAs) including non-permeating CPAs, such as PEG, trehalose and another permeating CPA, 1,2-propanediol with reduced DMSO concentrations and the possibility of replacing bovine serum on MSCs cryopreservation. Post-thaw cell viability, proliferation capacity and differentiation potential of MSCs from different species were assessed after cryopreservation with the conventional slow freezing method. Although the post-thaw viabilities and metabolic activities varied among the different species, the satisfactory results were obtained with 5% (v/v) DMSO,2%(w/v) PEG,3%(w/v) trehalose and 2%(w/v) bovine serum albumin (BSA) as the freezing solution. Our results showed that mouse MSCs were least susceptible to cryopreservation compared with rat and bovine MSCs.During cryopreservation of cell seeded scaffolds (or cell-matrices), it is critical for hMSCs to remain their attachment to the scaffold surface. The effect of cryopreservation on hMSCs attached onto different substrates was investigated in this thesis. Substrates coated with gelatin and matrigel enhances cell attachment and improves potential of cell growth. In addition, cryopreservation results in the change of F-actin morphology which may further lead to cell death, and lead to alternation of mitochondria localization, which may ultimately result in affecting undifferentiated status. Cell viability after cryopreservation with the cooling rate of 1℃/min was significantly higher than those with the cooling rates of 5℃/min and 10℃/min for hMSCs attached onto gelatin coated surface. Moreover, cell viability after cryopreservation is significantly lower for the cells attached onto substrates than the cells in suspension.Osteoblasts are considered as the one of the suitable seeding cells for tissue-engineered bone constructs. In this thesis, osteoblasts from calvaria of 3-day Sprague-Dawley (SD) rats were successfully isolated, cultured in vitro and cryopreserved. It was found that the adherent osteoblast exhibited a much narrow tolerable osmotic limit compared to the cells in suspension, which were 240～710 mmol/kg and 228～1605 mmol/kg respectively. After exposure to the various hypo-and hyper osmotic solutions, cell recovery was not retained after returning to isotonic conditions or even further culture for the adherent cells. Postthaw viability of cells in monolayer was significantly lower than that of cells in suspension.For cells or tissue cryopreservation by vitrification method, the protocols of CPA addition and removal can greatly influence the outcome of cryopreservation. Toxicity and osmotic injury could be reduced by the suitable CPA addition and removal protocols. For CPA addition by stepwise method, the equilibration time of CPA is a key factor. In this thesis, the cell inactive volume (Vb) and membrance permeability parameters of osteoblasts were determined with the microscopic imaging analysis. Based on the Boyle van't Hoff plot, it was concluded that osteoblasts osmotically behave as ideal osmometers and the osmotically inactive volume fraction was 0.335. The tolerable limits for cell volume excursion were 45～115% of isotonic volume. A cocktail of vitrification solution was analyzed by using differential scanning calorimetry and cryomicroscopy system to identify the vitrification occurance. Normalized cell volume change during each step of CPA addition was calculated with the Kedem-Katchalsky model. Together with the toxicity test during each addition step, the appropriate equilibrium time for each intermediate step was determined as 90,90,90,60 and 60 s. The cell viability after step-wise addition was 90.6±2.5%. The experimentally determined osmotic tolerance limits of osteoblasts, the "safe" cell volume excursion and membrane permeability coefficients are helpful for the design of proper cryopreservation protocols for osteoblasts-seeded engineered tissues.
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