- Email: [email protected]
66. Experimental investigation of biophysical parameters for MC3T3-E1 murine osteoblasts in absence of cryoprotective agents
324
Abstracts / Cryobiology 63 (2011) 306–342
ing the optimized composition of CPA mixtures is extremely difficult and challenging due to the large number of CPA candidates to be screened for their multiple characteristics. These characteristics include the phase change characteristics, the extent of cryoprotection, toxicity, and osmotic shock during loading/unloading. Systematic and rapid screening and characterization, which can accelerate the optimization process, are highly desired. In the present study, we develop a microfluidic platform to screen and characterize CPA mixtures cell-type specifically and on-chip. This microfluidic platform is based on the ‘‘electro-wetting-on-dielectric” (EWOD) principle and is capable of manipulating reagents droplet-wise using electric field. Droplet-wise liquid motion enables highly complicated multiplexing. Due to this unique capability, it has various advantages for screening complex mixtures of reagents for various properties relevant to cryopreservation. As a proof-of-concept, a mixture of Me2SO-PBS was prepared and characterized for its phase change behavior. Then, the mixture was optimized on-chip for preserving human breast cancer cells (MCF-7) considering the toxicity and cryoprotection. Suspension of MCF-7 cells, solution of viability dyes (mixtures of 40 lM of Hoechst and 30 lM propidium iodide), and stock Me2SO were loaded on the EWOD platform. Droplets of MCF-7 suspension were created and mixed with Me2SO droplets to prepare the suspension droplets with four different concentrations of Me2SO (i.e. 0%, 12.5%, 25%, 50% v/v). For the toxicity assay, the prepared array of cell suspensions was placed in incubator for different times (i.e. 5, 15, 30 min) and the viability was assessed. For the screening for cryoprotection, the cell suspension array were frozen to 50 °C, held for 5 min and thawed to the room temperature. After F/ T, the post-thaw viability was assessed on-chip. Notable toxicity was observed at 25% Me2SO after 30-min exposure (viability = 71.6 ± 4.5%) and at 50% at 15-min exposure (viability = 61.9 ± 10.6%). After F/T, almost all cells are dead at 0% (viability = 4.0 ± 3.2%) and 50% Me2SO (viability = 10.2 ± 6.3%) due to cryoinjury and Me2SO toxicity, respectively. However, the post-thaw survival substantially increased at the 12.5% (viability = 49.6 ± 3.2%) and 25% Me2SO (viability = 61.9 ± 10.6%). These results imply that the optimal Me2SO-PBS concentrations for the cryopreservation of MCF-7 cells are in the range of 12.5% and 25% Me2SO. In summary, the EWOD-based platform is capable of screening and characterizing a variety of CPA mixtures with high accuracy and efficiency for cell-specific cryopreservation. Conflict of interest: None declared. Source of funding: NIH/NIBIB R01 EB008388, Research Enhancement Grant from UT-Arlington.
doi:10.1016/j.cryobiol.2011.09.068
66. Experimental investigation of biophysical parameters for MC3T3-E1 murine osteoblasts in absence of cryoprotective agents. Ateeq J. Suria 1, Baolin Liu 1,2, Sanket A. Unhale 1, John J. McGrath * 1, 1 Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA, 2 Institute of Cryo-Medicine and Food Refrigeration, University of Shanghai for Science and Technology, China Understanding key biophysical parameters of biological cells is essential for development of optimal cryopreservation protocols. Here we present the experimental estimates of some of these parameters for MC3T3-E1 murine osteoblast cells. These cells are important in the development of tissue engineered cortical bone substitutes i.e. osteoblast seeded hydroxyapatite (OB:HA) bone implants [B.L. Liu, J.J. McGrath, L. McCabe, M. Baumann, Response of murine osteoblasts and porous hydroxyapatite scaffolds to two-step, slow freezing and vitrification processes, Cell Preservation Technology 1.1 (2002) 33–44]. Murine osteoblasts were cultured at 37 °C and 5% CO2 environment for 4–5 days in the presence of – aMEM culture medium supplemented with fetal bovine serum (FBS). The isotonic diameter of osteoblasts was measured to be 15.8 ± 2.4 lm. Boyle van’t Hoff analysis of osteoblasts yielded an osmotically-inactive volume fraction of 26% of isotonic cell volume. Membrane hydraulic permeability, Lp, was determined at room temperature using a microdiffusion chamber and a micro-fluidic device. Regression of the shrinkage dynamics yielded an estimate of 0.51 (±0.13) lm/atm-min for cells exposed to 1000 mOsm in the micro-diffusion chamber. Regression of the shrinkage dynamics yielded estimates of 0.81 (±0.18), 0.48 (±0.04) and 0.31 (±0.09) lm/atm-min for cells exposed to 500, 700 and 1000 mOsm in the micro-fluidic device. The concentration dependence of the permeability is consistent with previous reports for various cells. A conduction cryomicroscope system was used to study the response of osteoblasts suspended in isotonic culture medium to cooling at various constant cooling rates. The cellular response to freezing was observed and recorded using a cryomicroscope, while logging the sample temperatures. Cell diameters at subzero temperatures were measured and plotted in the form of normalized cell volume as a function of temperature. Nonlinear regression of the data using a least squared curve fit algorithm (‘lsqcurvefit’ in Matlab, utilizing the Levenberg–Marquardt scheme) yielded the membrane permeability parameters. The hydraulic permeability (Lp,0) at the reference temperature of 0 °C and the activation energy (Ea) were estimated to be 4.7e 13 m/Pa s (2.9 lm/min-atm) and 48.9 kJ/mol (11.7 kcal/mol), respectively. Formation of intracellular ice was recorded by observing ‘‘flashing” or darkening of cells upon cooling. Cumulative incidence of intracellular ice was plotted as a function of temperature and non-linear regression was used to estimate the heterogeneous nucleation parameters. The kinetic nucleation parameter, X0, was estimated to be
1.6e+10 m 2 s 1 and the thermodynamic nucleation parameter, j0, was estimated to be 1.5e+09 K[5]. The estimated values of all measured parameters were within the range of known parameters for other cell types. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.069
67. The effects of synthetic ice blockers on thermal expansion of vitrifying DP6 and their implications on large-scale cryopreservation. Yoed Rabin *, David P. Eisenberg, Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, PA, USA Developing reliable techniques for large-scale cryopreservation represents an unmet need. The process of vitrification as an alternative for large-scale cryopreservation combines two competing requirements on the cryoprotective agent (CPA) concentration: decreasing the CPA concentration in order to decrease toxicity effects, and increasing the CPA concentration in order to enable vitrification at the typical low cooling rates achievable at the center of a large specimen. The structural integrity of the cryopreserved specimen post thawing is essentially dominated by two categories of physical effects: biotransport and thermo-mechanical stress with fracture formation as its most dramatic outcome. Decreasing the cooling rates during vitrification is key to decreasing thermo-mechanical stresses and preserving the structure of the specimen. A possible alternative to decreasing the CPA concentration during vitrification is mixing it with the so-called synthetic ice blockers (SIB). Unlike common CPAs, which promote vitrification by the elevation of the viscosity of the material, the SIBs inhibit crystallization by strongly interacting with the water molecules. Either way, the outcome of vitrification is path and rate dependent. The combination of CPAs with SIBs can potentially decrease the overall concentration of the vitrification-promoting cocktail, thereby reconciling the competing needs to decrease toxicity and to preserve structural integrity. This study is aimed at providing insight on how SIBs can potentially affect the structural integrity of the specimen. The current study focuses on measuring the thermal expansion of the CPA cocktail DP6 when mixed with the following SIBs: 12% polyethylene glycol (PEG 400), 6% 1,3-cyclohexadiene (1,3-CHD), and 6% 2,3-butanediol. Thermal expansion is the driving mechanism of thermomechanical stress and can serve as an indicator of (partial) crystallization during vitrification. Results of this study indicate that the addition of the SIBs under investigation moderately affects the thermal expansion coefficient of DP6, ranging up to 16% for the cocktail DP6 + PEG 400 when compared with the baseline. Those cocktails led to a high degree of vitrification at relatively low cooling rates of about 3 °C/ min, although complete tissue vitrification was more difficult to achieve when compared with vitrification of the CPA cocktail alone. Measurements were conducted down to about 90 °C, a range in which the CPA cocktail behaves like a liquid in any practical time scale. Measurements at lower temperatures, where the CPA cocktail behaves like a solid, are currently underway using a different measurement technique. The low achievable cooling rates and the magnitude of thermal expansion variation in the presence of SIBs warrant further investigation on the resulting thermo-mechanical stress in typical scenarios relevant to cryopreservation. Acknowledgments: This project has been supported by Award Number R21EB011751 from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Conflict of interest: The authors declare no actual or potential conflicts of interest. doi:10.1016/j.cryobiol.2011.09.070
Keynote session 5 Mathematical modeling of ice formation
68. From needle crystals to spherulites: Phase-field modeling of complex solidification morphologies. L. Gránásy, Research Institute for Solid State Physics and Optics, H-1525 Budapest, P.O. Box 49, Hungary, BCAST, Brunel University, Uxbridge UB8 3PH, UK Results of a recently proposed phase-field theory of polycrystalline solidification will be reviewed. The phase-field theory can be regarded as a multi-field generalization of the Cahn–Hilliard/Ginzburg–Landau type field theoretic models, in which the structural change during solidification is monitored by a structural order parameter, the phase field, whose time evolution is assumed to follow relaxation dynamics, and is coupled to the evolution of other conserved and/or non-conserved fields, such as temperature, concentration, or density. The phase-field model to be presented incorporates the phase-field, a concentration field, while differences in the crystallographic orientations are captured by an appropriate orientational field (scalar field in 2D and quaternion field in 3D). Langevin noise is added to the equations of motion to represent the thermal fluctuations. Accordingly, the model includes homogeneous and heterogeneous nucleation of growth centers [L. Gránásy et al., Phys. Rev. Lett. 88 (2002)
Abstracts / Cryobiology 63 (2011) 306–342
ing the optimized composition of CPA mixtures is extremely difficult and challenging due to the large number of CPA candidates to be screened for their multiple characteristics. These characteristics include the phase change characteristics, the extent of cryoprotection, toxicity, and osmotic shock during loading/unloading. Systematic and rapid screening and characterization, which can accelerate the optimization process, are highly desired. In the present study, we develop a microfluidic platform to screen and characterize CPA mixtures cell-type specifically and on-chip. This microfluidic platform is based on the ‘‘electro-wetting-on-dielectric” (EWOD) principle and is capable of manipulating reagents droplet-wise using electric field. Droplet-wise liquid motion enables highly complicated multiplexing. Due to this unique capability, it has various advantages for screening complex mixtures of reagents for various properties relevant to cryopreservation. As a proof-of-concept, a mixture of Me2SO-PBS was prepared and characterized for its phase change behavior. Then, the mixture was optimized on-chip for preserving human breast cancer cells (MCF-7) considering the toxicity and cryoprotection. Suspension of MCF-7 cells, solution of viability dyes (mixtures of 40 lM of Hoechst and 30 lM propidium iodide), and stock Me2SO were loaded on the EWOD platform. Droplets of MCF-7 suspension were created and mixed with Me2SO droplets to prepare the suspension droplets with four different concentrations of Me2SO (i.e. 0%, 12.5%, 25%, 50% v/v). For the toxicity assay, the prepared array of cell suspensions was placed in incubator for different times (i.e. 5, 15, 30 min) and the viability was assessed. For the screening for cryoprotection, the cell suspension array were frozen to 50 °C, held for 5 min and thawed to the room temperature. After F/ T, the post-thaw viability was assessed on-chip. Notable toxicity was observed at 25% Me2SO after 30-min exposure (viability = 71.6 ± 4.5%) and at 50% at 15-min exposure (viability = 61.9 ± 10.6%). After F/T, almost all cells are dead at 0% (viability = 4.0 ± 3.2%) and 50% Me2SO (viability = 10.2 ± 6.3%) due to cryoinjury and Me2SO toxicity, respectively. However, the post-thaw survival substantially increased at the 12.5% (viability = 49.6 ± 3.2%) and 25% Me2SO (viability = 61.9 ± 10.6%). These results imply that the optimal Me2SO-PBS concentrations for the cryopreservation of MCF-7 cells are in the range of 12.5% and 25% Me2SO. In summary, the EWOD-based platform is capable of screening and characterizing a variety of CPA mixtures with high accuracy and efficiency for cell-specific cryopreservation. Conflict of interest: None declared. Source of funding: NIH/NIBIB R01 EB008388, Research Enhancement Grant from UT-Arlington.
doi:10.1016/j.cryobiol.2011.09.068
66. Experimental investigation of biophysical parameters for MC3T3-E1 murine osteoblasts in absence of cryoprotective agents. Ateeq J. Suria 1, Baolin Liu 1,2, Sanket A. Unhale 1, John J. McGrath * 1, 1 Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA, 2 Institute of Cryo-Medicine and Food Refrigeration, University of Shanghai for Science and Technology, China Understanding key biophysical parameters of biological cells is essential for development of optimal cryopreservation protocols. Here we present the experimental estimates of some of these parameters for MC3T3-E1 murine osteoblast cells. These cells are important in the development of tissue engineered cortical bone substitutes i.e. osteoblast seeded hydroxyapatite (OB:HA) bone implants [B.L. Liu, J.J. McGrath, L. McCabe, M. Baumann, Response of murine osteoblasts and porous hydroxyapatite scaffolds to two-step, slow freezing and vitrification processes, Cell Preservation Technology 1.1 (2002) 33–44]. Murine osteoblasts were cultured at 37 °C and 5% CO2 environment for 4–5 days in the presence of – aMEM culture medium supplemented with fetal bovine serum (FBS). The isotonic diameter of osteoblasts was measured to be 15.8 ± 2.4 lm. Boyle van’t Hoff analysis of osteoblasts yielded an osmotically-inactive volume fraction of 26% of isotonic cell volume. Membrane hydraulic permeability, Lp, was determined at room temperature using a microdiffusion chamber and a micro-fluidic device. Regression of the shrinkage dynamics yielded an estimate of 0.51 (±0.13) lm/atm-min for cells exposed to 1000 mOsm in the micro-diffusion chamber. Regression of the shrinkage dynamics yielded estimates of 0.81 (±0.18), 0.48 (±0.04) and 0.31 (±0.09) lm/atm-min for cells exposed to 500, 700 and 1000 mOsm in the micro-fluidic device. The concentration dependence of the permeability is consistent with previous reports for various cells. A conduction cryomicroscope system was used to study the response of osteoblasts suspended in isotonic culture medium to cooling at various constant cooling rates. The cellular response to freezing was observed and recorded using a cryomicroscope, while logging the sample temperatures. Cell diameters at subzero temperatures were measured and plotted in the form of normalized cell volume as a function of temperature. Nonlinear regression of the data using a least squared curve fit algorithm (‘lsqcurvefit’ in Matlab, utilizing the Levenberg–Marquardt scheme) yielded the membrane permeability parameters. The hydraulic permeability (Lp,0) at the reference temperature of 0 °C and the activation energy (Ea) were estimated to be 4.7e 13 m/Pa s (2.9 lm/min-atm) and 48.9 kJ/mol (11.7 kcal/mol), respectively. Formation of intracellular ice was recorded by observing ‘‘flashing” or darkening of cells upon cooling. Cumulative incidence of intracellular ice was plotted as a function of temperature and non-linear regression was used to estimate the heterogeneous nucleation parameters. The kinetic nucleation parameter, X0, was estimated to be
1.6e+10 m 2 s 1 and the thermodynamic nucleation parameter, j0, was estimated to be 1.5e+09 K[5]. The estimated values of all measured parameters were within the range of known parameters for other cell types. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.069
67. The effects of synthetic ice blockers on thermal expansion of vitrifying DP6 and their implications on large-scale cryopreservation. Yoed Rabin *, David P. Eisenberg, Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, PA, USA Developing reliable techniques for large-scale cryopreservation represents an unmet need. The process of vitrification as an alternative for large-scale cryopreservation combines two competing requirements on the cryoprotective agent (CPA) concentration: decreasing the CPA concentration in order to decrease toxicity effects, and increasing the CPA concentration in order to enable vitrification at the typical low cooling rates achievable at the center of a large specimen. The structural integrity of the cryopreserved specimen post thawing is essentially dominated by two categories of physical effects: biotransport and thermo-mechanical stress with fracture formation as its most dramatic outcome. Decreasing the cooling rates during vitrification is key to decreasing thermo-mechanical stresses and preserving the structure of the specimen. A possible alternative to decreasing the CPA concentration during vitrification is mixing it with the so-called synthetic ice blockers (SIB). Unlike common CPAs, which promote vitrification by the elevation of the viscosity of the material, the SIBs inhibit crystallization by strongly interacting with the water molecules. Either way, the outcome of vitrification is path and rate dependent. The combination of CPAs with SIBs can potentially decrease the overall concentration of the vitrification-promoting cocktail, thereby reconciling the competing needs to decrease toxicity and to preserve structural integrity. This study is aimed at providing insight on how SIBs can potentially affect the structural integrity of the specimen. The current study focuses on measuring the thermal expansion of the CPA cocktail DP6 when mixed with the following SIBs: 12% polyethylene glycol (PEG 400), 6% 1,3-cyclohexadiene (1,3-CHD), and 6% 2,3-butanediol. Thermal expansion is the driving mechanism of thermomechanical stress and can serve as an indicator of (partial) crystallization during vitrification. Results of this study indicate that the addition of the SIBs under investigation moderately affects the thermal expansion coefficient of DP6, ranging up to 16% for the cocktail DP6 + PEG 400 when compared with the baseline. Those cocktails led to a high degree of vitrification at relatively low cooling rates of about 3 °C/ min, although complete tissue vitrification was more difficult to achieve when compared with vitrification of the CPA cocktail alone. Measurements were conducted down to about 90 °C, a range in which the CPA cocktail behaves like a liquid in any practical time scale. Measurements at lower temperatures, where the CPA cocktail behaves like a solid, are currently underway using a different measurement technique. The low achievable cooling rates and the magnitude of thermal expansion variation in the presence of SIBs warrant further investigation on the resulting thermo-mechanical stress in typical scenarios relevant to cryopreservation. Acknowledgments: This project has been supported by Award Number R21EB011751 from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Conflict of interest: The authors declare no actual or potential conflicts of interest. doi:10.1016/j.cryobiol.2011.09.070
Keynote session 5 Mathematical modeling of ice formation
68. From needle crystals to spherulites: Phase-field modeling of complex solidification morphologies. L. Gránásy, Research Institute for Solid State Physics and Optics, H-1525 Budapest, P.O. Box 49, Hungary, BCAST, Brunel University, Uxbridge UB8 3PH, UK Results of a recently proposed phase-field theory of polycrystalline solidification will be reviewed. The phase-field theory can be regarded as a multi-field generalization of the Cahn–Hilliard/Ginzburg–Landau type field theoretic models, in which the structural change during solidification is monitored by a structural order parameter, the phase field, whose time evolution is assumed to follow relaxation dynamics, and is coupled to the evolution of other conserved and/or non-conserved fields, such as temperature, concentration, or density. The phase-field model to be presented incorporates the phase-field, a concentration field, while differences in the crystallographic orientations are captured by an appropriate orientational field (scalar field in 2D and quaternion field in 3D). Langevin noise is added to the equations of motion to represent the thermal fluctuations. Accordingly, the model includes homogeneous and heterogeneous nucleation of growth centers [L. Gránásy et al., Phys. Rev. Lett. 88 (2002)