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134. Preservation of engineered bone tissue
Abstracts / Cryobiology 55 (2007) 324–378 made after freezing and thawing. In the aortic media, continuous elastic lamellae in the circumferential direction of the artery were arranged periodically in the radial direction, and complicatedly intertwined smooth muscle cells and collagen fibers fill the gap between the elastic lamellae. The freezing behavior was dependent on the histological structure of the aortic media and the cooling rate used. At a low cooling rate, ice crystals formed and grew outside the smooth muscle cells between the bundles of several elastic lamellae. Growth of the ice crystals pushed the elastic lamellae out on both sides of the crystals, enlarging the gap between the bundles tearing the media, and the elastic lamellae in the bundle were packed closely together. The smooth muscle cells in the gaps were not only dehydrated and shrunk, but also torn and destroyed, so that a bridge of broken smooth muscle was built across the gap. In the bundles of elastic lamellae that were packed closely, the dehydrated and flattened smooth muscle cells and collagen fibers were packed together between the elastic lamellae. After the freezing and thawing, formation of cracks by the ice crystals, destruction of the smooth muscle cells, fragmentation of the cytoplasm, and distension of the nuclei were all found. Also, fine elastic fibers distributed around the smooth muscle cells were fragmented, and collagen fibers around the cells were pushed away and unevenly distributed on the surfaces of the elastic lamellae. At a high cooling rate, bundles of two or three elastic lamellae packed closely were not common. Around almost every elastic lamella, intra- and extracellular freezing had happened. The histological change of constituent elements was mitigated but was qualitatively similar to that seen at a low cooling rate. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.135
133. Improvement of cell survival in TE products after cryopreservation. Inga Bernemann, Birgit Glasmacher, Leibniz Universitaet Hannover, Hannover, Germany Effective cooling and thawing of tissue and tissue engineered products (TEP’s) pose a specific challenge in cryopreservation for the purpose of medical application. 293T-cell seeded collagen scaffolds (height of 3 mm, diameter of 15 mm, pore size of 65 lm) were frozen in specially designed, improved tissue freezing containers and thawed using an adapted rewarming equipment. The study was accomplished by a mathematical simulation within the three dimensional samples. The simulation program (Ansys Multiphysics) allows one to visualize the temperature distribution within the sample during cooling with respect to a homogenous temperature distribution. To estimate the reliability of the simulation, the results gained with different freezing protocols were compared with measured data. Therefore, temperature profiles were taken at defined positions within the samples. The simulation allows the determination of cooling rates for the different temperature areas in the TEP. After cryopreservation the thawed, cell-seeded samples were examinated by MTT-assay (cell amount and survival rate measurement) and CalceinAM/EthD-assay (fluorescence live/dead cell detection) to investigate biological effects of the cooling rate, the improved freezing containers with respect to the gradients within the sample. The MTT-Assay showed cell survival rates up to 50% for the improved tissue freezing container at adapted cooling rates. Compared with the results of our previous study, cell survival rate improved by 20% with higher cell activity on the surface of the samples as compared to the inner part. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.136
134. Preservation of engineered bone tissue. Debasree Biswas, Nilay Chakraborty, Amie Sparnell, Ahmed El-Ghannam, Gloria D. Elliott, University of North, Carolina at Charlotte, Charlotte, NC, USA The concept of using porous bioactive ceramic as a vehicle for bone cell delivery to treat large bone defects is very promising and may provide
369
an effective substitute for biological grafts to treat large bone defects in craniomaxillofacial orthopedic surgeries. Many studies have demonstrated that porous bioactive ceramics such as hydroxyapatite enhance bone cell adhesion and function in vitro and in vivo. When cultured bone marrow mesenchymal stem cells are combined with a novel porous resorbable bioactive silica-calcium phosphate nano composite (SCPC), rapid osteoblastic cell differentiation and mineralized bone matrix were formed thereby leading to the creation of cultured artificial bone that is similar to native bone. The enhancement of osteogenic capacity was attributed to the upregulation of osteogenic gene expression by cells attached to the SCPC scaffold. Thus, it is increasingly clear that preservation methods for the cell-ceramic hybrid need to be developed to preserve such living products in good condition to ensure a steady supply for transplantation. In the current work we have cryopreserved rat femur bone marrow cells on SCPC. Cells were pre-seeded onto 10 mm · 2 mm discs of the SCPC scaffold and then incubated at various times to achieve various levels of attachment. Cells were then exposed to increasing concentrations of VM3 vitrification solution (21st Century Medicine, CA) using 1x LM5 solution as a carrier solution. Four separate dilutions of VM3 in LM5 (10%, 25%, 50%, 75% and 100%) were prepared and the cells were exposed for different time intervals for stepwise loading and unloading of VM3. Optimal cooling and warming rates were determined to achieve vitrification of the composite samples and to avoid sample cracking. Vitrification was achieved by suspending the sample (3 mL volume) in a glass vial (20 mL capacity) over liquid nitrogen (LN2) vapor at a known height from the surface. Samples were cooled to a final temperature of 126 C at an optimized cooling rate of 9 C/min. Samples were thawed in 2 steps, warming slowly (10 C/min) to 100 C by raising the vial to a higher height above the LN2, and then quickly to 0 C (100 C/min) by immersion of the vial in a water bath held at 37 C. The cooling and warming rates were recorded by a K type thermocouple inserted into the vial. Two approaches used for the measurement of viability in this work, (1) Trypan Blue exclusion and (2) Calcein-AM/PI fluorescence assays. The highest viability was achieved for samples that were allowed to tether to the scaffold, but were not firmly adhered. This was achieved with a pre-incubation period of 15 minutes. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.137
135. Post-thaw caspase inhibition improves the cryopreservation outcome for bovine corneal endothelial cells. William L. Corwin a,b, John M. Baust a,b, Robert G. Van Buskirk a,b, John G. Baust a, a Binghamton University, Binghamton, NY, USA; b Cell Preservation Services, Inc., Owego, NY, USA Over the past five years, there has been a renewed interest in the development of new technologies designed to improve cryopreservation outcome. This renewed effort and subsequent success is due, in part, to reports detailing the involvement of delayed apoptotic and necrotic events contributing to cryopreservation failure. The efforts to improve cryopreservation outcome have focused primarily on the development of new cryopreservation solutions and protocols which attempt to reduce cellular stress during the preservation process with little attention given to the recovery phase. While this endeavor has been successful in increasing cryopreservation efficacy, there still remains a level of cell death associated with the cryopreservation process that has yet to be overcome through the approach of designing the preservation solution. As such, we set out on a new path to investigate the effect of post-thaw conditioning of cells in an attempt to further enhance cryopreservation outcome. Bovine corneal endothelial (BCE) cells were cryopreserved in DMEM +10% FBS +5% Me2SO (M5) or CryoStor CS2 (CS2), which contains 2% of Me2SO. Samples were cooled at 1 C/min to 80 C and then quenched in liquid nitrogen. A pan-caspase inhibitor VI (Z-VAD-FMK), diluted to a working concentration of 10 lmol/L, was added either to the solution during preservation or to the culture media post-thaw to test the protection it afforded. Post-thaw viability was assessed following 24 hours of culture
133. Improvement of cell survival in TE products after cryopreservation. Inga Bernemann, Birgit Glasmacher, Leibniz Universitaet Hannover, Hannover, Germany Effective cooling and thawing of tissue and tissue engineered products (TEP’s) pose a specific challenge in cryopreservation for the purpose of medical application. 293T-cell seeded collagen scaffolds (height of 3 mm, diameter of 15 mm, pore size of 65 lm) were frozen in specially designed, improved tissue freezing containers and thawed using an adapted rewarming equipment. The study was accomplished by a mathematical simulation within the three dimensional samples. The simulation program (Ansys Multiphysics) allows one to visualize the temperature distribution within the sample during cooling with respect to a homogenous temperature distribution. To estimate the reliability of the simulation, the results gained with different freezing protocols were compared with measured data. Therefore, temperature profiles were taken at defined positions within the samples. The simulation allows the determination of cooling rates for the different temperature areas in the TEP. After cryopreservation the thawed, cell-seeded samples were examinated by MTT-assay (cell amount and survival rate measurement) and CalceinAM/EthD-assay (fluorescence live/dead cell detection) to investigate biological effects of the cooling rate, the improved freezing containers with respect to the gradients within the sample. The MTT-Assay showed cell survival rates up to 50% for the improved tissue freezing container at adapted cooling rates. Compared with the results of our previous study, cell survival rate improved by 20% with higher cell activity on the surface of the samples as compared to the inner part. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.136
134. Preservation of engineered bone tissue. Debasree Biswas, Nilay Chakraborty, Amie Sparnell, Ahmed El-Ghannam, Gloria D. Elliott, University of North, Carolina at Charlotte, Charlotte, NC, USA The concept of using porous bioactive ceramic as a vehicle for bone cell delivery to treat large bone defects is very promising and may provide
369
an effective substitute for biological grafts to treat large bone defects in craniomaxillofacial orthopedic surgeries. Many studies have demonstrated that porous bioactive ceramics such as hydroxyapatite enhance bone cell adhesion and function in vitro and in vivo. When cultured bone marrow mesenchymal stem cells are combined with a novel porous resorbable bioactive silica-calcium phosphate nano composite (SCPC), rapid osteoblastic cell differentiation and mineralized bone matrix were formed thereby leading to the creation of cultured artificial bone that is similar to native bone. The enhancement of osteogenic capacity was attributed to the upregulation of osteogenic gene expression by cells attached to the SCPC scaffold. Thus, it is increasingly clear that preservation methods for the cell-ceramic hybrid need to be developed to preserve such living products in good condition to ensure a steady supply for transplantation. In the current work we have cryopreserved rat femur bone marrow cells on SCPC. Cells were pre-seeded onto 10 mm · 2 mm discs of the SCPC scaffold and then incubated at various times to achieve various levels of attachment. Cells were then exposed to increasing concentrations of VM3 vitrification solution (21st Century Medicine, CA) using 1x LM5 solution as a carrier solution. Four separate dilutions of VM3 in LM5 (10%, 25%, 50%, 75% and 100%) were prepared and the cells were exposed for different time intervals for stepwise loading and unloading of VM3. Optimal cooling and warming rates were determined to achieve vitrification of the composite samples and to avoid sample cracking. Vitrification was achieved by suspending the sample (3 mL volume) in a glass vial (20 mL capacity) over liquid nitrogen (LN2) vapor at a known height from the surface. Samples were cooled to a final temperature of 126 C at an optimized cooling rate of 9 C/min. Samples were thawed in 2 steps, warming slowly (10 C/min) to 100 C by raising the vial to a higher height above the LN2, and then quickly to 0 C (100 C/min) by immersion of the vial in a water bath held at 37 C. The cooling and warming rates were recorded by a K type thermocouple inserted into the vial. Two approaches used for the measurement of viability in this work, (1) Trypan Blue exclusion and (2) Calcein-AM/PI fluorescence assays. The highest viability was achieved for samples that were allowed to tether to the scaffold, but were not firmly adhered. This was achieved with a pre-incubation period of 15 minutes. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.137
135. Post-thaw caspase inhibition improves the cryopreservation outcome for bovine corneal endothelial cells. William L. Corwin a,b, John M. Baust a,b, Robert G. Van Buskirk a,b, John G. Baust a, a Binghamton University, Binghamton, NY, USA; b Cell Preservation Services, Inc., Owego, NY, USA Over the past five years, there has been a renewed interest in the development of new technologies designed to improve cryopreservation outcome. This renewed effort and subsequent success is due, in part, to reports detailing the involvement of delayed apoptotic and necrotic events contributing to cryopreservation failure. The efforts to improve cryopreservation outcome have focused primarily on the development of new cryopreservation solutions and protocols which attempt to reduce cellular stress during the preservation process with little attention given to the recovery phase. While this endeavor has been successful in increasing cryopreservation efficacy, there still remains a level of cell death associated with the cryopreservation process that has yet to be overcome through the approach of designing the preservation solution. As such, we set out on a new path to investigate the effect of post-thaw conditioning of cells in an attempt to further enhance cryopreservation outcome. Bovine corneal endothelial (BCE) cells were cryopreserved in DMEM +10% FBS +5% Me2SO (M5) or CryoStor CS2 (CS2), which contains 2% of Me2SO. Samples were cooled at 1 C/min to 80 C and then quenched in liquid nitrogen. A pan-caspase inhibitor VI (Z-VAD-FMK), diluted to a working concentration of 10 lmol/L, was added either to the solution during preservation or to the culture media post-thaw to test the protection it afforded. Post-thaw viability was assessed following 24 hours of culture