- Email: [email protected]
38. A digital microfluidic platform for high throughput optimization of cryoprotective agents
Abstracts / Cryobiology 59 (2009) 370–418 processing time could be reduced from 55 to 20 min. Quite independently of the computational effort reported here, the authors of the published cryopreservation protocol for human oocytes have simplified their protocol based on an empirical approach that has produced a protocol very similar to that predicted using the simplified model developed here [Dr. Giovanni Coticchio, personal communication]. Finally, the model predicts that IIF is very sensitive to both seeding temperature and relaxation time at the seeding temperature, indicating that the coupling between temperature and time during the seeding process must be carefully controlled if reproducibly high recovery is to be expected. (Conflicts of interest: None. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.051
38. A digital microfluidic platform for high throughput optimization of cryoprotective agents. *Bumsoo Han, Hyejin Moon, Department of Mechanical and Aerospace Engineering, University of Texas at Arlington, Arlington, TX 76019, USA Dimethyl sulfoxide Me2SO is the most widely used cryoprotective agent for routine preservation of cells and simple tissues. However, more effective and less toxic CPAs are still highly desired. It has also been reported that mixtures or cocktails of CPAs provide the better protection of tissues from cryoinjury than pure Me2SO . However, determining the optimized composition of CPA cocktails is extremely difficult and sometimes impractical considering the wide variety of CPAs. Thus, a more systematic and mechanistically-driven high throughput (HTP) screening strategy is highly desirable. In this context, microfluidics and micro-manufacturing technologies provide new avenues to achieve HTP optimization of CPA mixtures. In the present study, we propose an HTP platform for CPA mixture preparation and optimization and demonstrate its feasibility. The platform is a microfluidic multiplexing system based on electrowetting-on-dielectric (EWOD) technology. The developed platform is capable of creating, moving, dividing and merging drops with controlled manners. The operation of this platform was demonstrated by creating multiple 100 nL drops of Me2SO, PBS and water mixtures from their reservoirs. The developed EWOD-based multiplexer has many advantages for HTP characterization and optimization of CPA mixtures. First, reagents are well controlled in a small volume (few tens of nL range). Unlike mixing with test tubes and pipettes, there is no dead volume during EWOD mixing and reagent consumption is much less. Second, the number of pipetting operation is dramatically less than that of conventional lab practice. In the present study, just four pipettings to load stock solutions to the reservoirs were necessary. Third, all mixing and aliquoting processes are automated. The sequential operation of the EWOD multiplexer can be programmed prior to the screening process. Therefore, one-click after loading stock solutions, droplets with multiple concentrations are automatically created and positioned in an array format as designed by the user. In summary, this EWOD-based multiplexer technique will facilitate creating various concentrations of CPA mixtures in a small volume with great accuracy. Thus, characterization of CPA mixtures during F/T can be done with multiple droplets simultaneously. This will drastically reduce efforts and the number of experiments for the characterization and optimization of CPA mixtures. (Conflicts of interest: None declared. Source of funding: None declared.)
381
ture measurement rate of 2000 per second is adequate. This exceeds the rate of typical TC readout systems. We chose to use a digital storage oscilloscope as these easily achieve sampling rates 10–1000 times faster than our requirement. The measured voltages are converted to temperatures using the Type T tables and Microsoft Excel. The essential cost-saving step is to use an USB instrument which plugs into an ordinary laptop computer USB port. The laptop provides the power, brains, and display for the instrument. Inexpensive digital USB scopes are available in the price range from 100 to 2000 US$. We settled on the very simple, small, and intuitive to use, StingRay DS1M12 from EasySync Ltd for 220 US$. This unit offers 12 bit resolution, a maximum sensitivity of ±10 mV full scale (perfect for the 7 mV temperature swings between room and LN2 temperatures), and can easily record 10–20 s of data in an Excel readable format. With our thermocouple setup, the system noise was about ±0.2 mV peakto peak which translates to a temperature noise of ±2.5 °C root mean square. For temperature swings of 100 °C this yields a cooling/warming rate accuracy of a few percent; more than adequate for our needs. The final component in the system is a polypropylene CryoTop to which the TC is cemented and on which the 0.1 ll aqueous sample droplet is placed. In practice, this system has proven robust and easy to use. The Excel analysis work sheet is available from the authors, [email protected]. (Conflicts of interest: None declared. Source of funding: US National Institutes of Health Grant R01-RR 18470.) doi:10.1016/j.cryobiol.2009.10.053
40. Levitating vitrified droplets. *Young S. Song a, Douglas Adler a, Hasan O. Keles a, Emre Kayaalp b, Aida Nureddin c, Raymond M. Anchan d, Richard Maas d, Utkan Demirci a,e, a Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Bioengineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, b Montreal Medical School, QC, Canada, c Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, d Center for Infertility and Reproductive Surgery, Obstetrics Gynecology & Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, e Harvard-Massachusetts Institute of Technology Health Sciences and Technology, Cambridge, MA, USA The vitrification phenomenon is observed usually when the time for ice crystal formation during cooling is limited. The vitrification process is critical for applications in cell cryopreservation, especially using cell-encapsulating droplets. Such an ultrarapid vitrification in general accompanies the Leidenfrost phenomenon, which diminishes the cooling rate of droplets. Thus, it is important to understand the correlation between phase transition from liquid to vitrified solid and Leidenfrost effect of droplets. In this study, we demonstrate a droplet that levitates on liquid nitrogen and vitrifies during quenching. For the cryopreservation application, a cryoprotectant agent (CPA) is employed as a droplet liquid. The phase transition and levitation of CPA droplets are analyzed by using a theoretical model based on three dimensionless numbers (Stefan, Biot, and Fourier numbers). Unlike typical vitrification methods with 100 °C/ min cooling rate, our droplet-based vitrification approach can achieve more than 10,000 °C/min cooling rate of droplets. The model allows us to predict a threshold radius to vitrify a CPA droplet. The threshold radius is found to be around 100 lm assuming that the cut-off degree of crystallization is 0.5 for 3 M cryoprotectant. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2009.10.052 doi:10.1016/j.cryobiol.2009.10.054
39. Simple, inexpensive measurement of very rapid cooling and warming rates. *F.W. Kleinhans a,b, Shinsuke Seki b, Peter Mazur b, a Department of Physics, IUPUI, Indianapolis, IN 46202, USA, b Fundamental and Applied Cryobiology Group, Department of Biochemistry & Cellular & Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA
41. Numerical study of the dependence on temperature and duration of loading for cryoprotectant and toxic injury. Zhang Shaozhi, Xu Mengjie, Chen Guangming, Cryobiology Laboratory, Refrigeration & Cryogenic Engineering Institute, Zhejiang University, China
Currently our group is investigating some effects and interactions of very rapid cooling and warming rates in cryobiology. The reason is that there is increasing emphasis on using vitrification procedures for the cryopreservation of mammalian oocytes using devices like the CryotopÒ. One problem is that the cooling and warming rates are so high, that it becomes difficult to measure them. Consequently, we have developed a simple, inexpensive (<300 US$) temperature measurement system. The design goal was to measure the temperature of a 0.1 ll aqueous sample at rates up to 1 million °C/min using a temperature sensing element which would not perturb the system. To achieve the latter, a simple, fine gauge thermocouple (TC) proved suitable. We used Type T (copper–constantan) which is available in pre-made wire sizes as small as 25 lm diameter (Omega). We settled on 50 lm diameter wire as a compromise between ease of handling (under a stereo microscope) versus minimum system perturbation. We estimate the heat capacity of the 50 lm diameter wire TC tip at 1/5 the heat capacity of 0.1 ll of water; sufficiently small to yield only a modest thermal perturbation of the system. Secondly, it is necessary to record the TC temperature at a sufficiently high rate. For a simple thermal ramp, e.g. a plunge from room temperature to LN2, a tempera-
Cryoprotectants are necessary for almost all protocols of cryopreservation of biomaterials. The loading of cryoprotectant into cells needs to consider the following factors: deformation limit, toxicity and duration. Temperature has an impact on the membrane permeability for water and cryoprotectant, thus on the loading duration and toxic injury. The temperature dependence of the loading duration is numerically studied here using a two-parameter model. The loading duration tL is defined as the time needed when the intracellular cryoprotectant concentration reaches 95% of the extracellular cryoprotectant concentration. Typical ranges for parameters in the model are adopted through literature survey. They include: (1) membrane hydraulic conductivity Lp, 4 10 16–2.0 10 12 m s/Pa; (2) cryoprotectant permeability P, 1.0 10 19–2.0 10 6 m/s; (3) isotonic osmolality Mis, 0.25–0.35 M; (4) cell diameter D, 5–25 lm; (5) osmotically inactive fraction Vb, 0.15–0.55; (6) crp, a dimensionless parameter defined as P/(LpRTMiso), 0.1–10; (7) cell surface area ratio, a dimensionless parameter defined as A/(pD2), 1–2; (8) activation energy for Lp, Ea, 10–250 kJ/mol; (9) activation energy for P, Ep, 40–120 kJ/mol. More than 20,000 random cases are calculated. It is found that the loading duration can be fitted as an exponential function of temperature, tL = a eb/T with R2 > 0.95. The constant b is more
38. A digital microfluidic platform for high throughput optimization of cryoprotective agents. *Bumsoo Han, Hyejin Moon, Department of Mechanical and Aerospace Engineering, University of Texas at Arlington, Arlington, TX 76019, USA Dimethyl sulfoxide Me2SO is the most widely used cryoprotective agent for routine preservation of cells and simple tissues. However, more effective and less toxic CPAs are still highly desired. It has also been reported that mixtures or cocktails of CPAs provide the better protection of tissues from cryoinjury than pure Me2SO . However, determining the optimized composition of CPA cocktails is extremely difficult and sometimes impractical considering the wide variety of CPAs. Thus, a more systematic and mechanistically-driven high throughput (HTP) screening strategy is highly desirable. In this context, microfluidics and micro-manufacturing technologies provide new avenues to achieve HTP optimization of CPA mixtures. In the present study, we propose an HTP platform for CPA mixture preparation and optimization and demonstrate its feasibility. The platform is a microfluidic multiplexing system based on electrowetting-on-dielectric (EWOD) technology. The developed platform is capable of creating, moving, dividing and merging drops with controlled manners. The operation of this platform was demonstrated by creating multiple 100 nL drops of Me2SO, PBS and water mixtures from their reservoirs. The developed EWOD-based multiplexer has many advantages for HTP characterization and optimization of CPA mixtures. First, reagents are well controlled in a small volume (few tens of nL range). Unlike mixing with test tubes and pipettes, there is no dead volume during EWOD mixing and reagent consumption is much less. Second, the number of pipetting operation is dramatically less than that of conventional lab practice. In the present study, just four pipettings to load stock solutions to the reservoirs were necessary. Third, all mixing and aliquoting processes are automated. The sequential operation of the EWOD multiplexer can be programmed prior to the screening process. Therefore, one-click after loading stock solutions, droplets with multiple concentrations are automatically created and positioned in an array format as designed by the user. In summary, this EWOD-based multiplexer technique will facilitate creating various concentrations of CPA mixtures in a small volume with great accuracy. Thus, characterization of CPA mixtures during F/T can be done with multiple droplets simultaneously. This will drastically reduce efforts and the number of experiments for the characterization and optimization of CPA mixtures. (Conflicts of interest: None declared. Source of funding: None declared.)
381
ture measurement rate of 2000 per second is adequate. This exceeds the rate of typical TC readout systems. We chose to use a digital storage oscilloscope as these easily achieve sampling rates 10–1000 times faster than our requirement. The measured voltages are converted to temperatures using the Type T tables and Microsoft Excel. The essential cost-saving step is to use an USB instrument which plugs into an ordinary laptop computer USB port. The laptop provides the power, brains, and display for the instrument. Inexpensive digital USB scopes are available in the price range from 100 to 2000 US$. We settled on the very simple, small, and intuitive to use, StingRay DS1M12 from EasySync Ltd for 220 US$. This unit offers 12 bit resolution, a maximum sensitivity of ±10 mV full scale (perfect for the 7 mV temperature swings between room and LN2 temperatures), and can easily record 10–20 s of data in an Excel readable format. With our thermocouple setup, the system noise was about ±0.2 mV peakto peak which translates to a temperature noise of ±2.5 °C root mean square. For temperature swings of 100 °C this yields a cooling/warming rate accuracy of a few percent; more than adequate for our needs. The final component in the system is a polypropylene CryoTop to which the TC is cemented and on which the 0.1 ll aqueous sample droplet is placed. In practice, this system has proven robust and easy to use. The Excel analysis work sheet is available from the authors, [email protected]. (Conflicts of interest: None declared. Source of funding: US National Institutes of Health Grant R01-RR 18470.) doi:10.1016/j.cryobiol.2009.10.053
40. Levitating vitrified droplets. *Young S. Song a, Douglas Adler a, Hasan O. Keles a, Emre Kayaalp b, Aida Nureddin c, Raymond M. Anchan d, Richard Maas d, Utkan Demirci a,e, a Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Bioengineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, b Montreal Medical School, QC, Canada, c Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, d Center for Infertility and Reproductive Surgery, Obstetrics Gynecology & Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, e Harvard-Massachusetts Institute of Technology Health Sciences and Technology, Cambridge, MA, USA The vitrification phenomenon is observed usually when the time for ice crystal formation during cooling is limited. The vitrification process is critical for applications in cell cryopreservation, especially using cell-encapsulating droplets. Such an ultrarapid vitrification in general accompanies the Leidenfrost phenomenon, which diminishes the cooling rate of droplets. Thus, it is important to understand the correlation between phase transition from liquid to vitrified solid and Leidenfrost effect of droplets. In this study, we demonstrate a droplet that levitates on liquid nitrogen and vitrifies during quenching. For the cryopreservation application, a cryoprotectant agent (CPA) is employed as a droplet liquid. The phase transition and levitation of CPA droplets are analyzed by using a theoretical model based on three dimensionless numbers (Stefan, Biot, and Fourier numbers). Unlike typical vitrification methods with 100 °C/ min cooling rate, our droplet-based vitrification approach can achieve more than 10,000 °C/min cooling rate of droplets. The model allows us to predict a threshold radius to vitrify a CPA droplet. The threshold radius is found to be around 100 lm assuming that the cut-off degree of crystallization is 0.5 for 3 M cryoprotectant. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2009.10.052 doi:10.1016/j.cryobiol.2009.10.054
39. Simple, inexpensive measurement of very rapid cooling and warming rates. *F.W. Kleinhans a,b, Shinsuke Seki b, Peter Mazur b, a Department of Physics, IUPUI, Indianapolis, IN 46202, USA, b Fundamental and Applied Cryobiology Group, Department of Biochemistry & Cellular & Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA
41. Numerical study of the dependence on temperature and duration of loading for cryoprotectant and toxic injury. Zhang Shaozhi, Xu Mengjie, Chen Guangming, Cryobiology Laboratory, Refrigeration & Cryogenic Engineering Institute, Zhejiang University, China
Currently our group is investigating some effects and interactions of very rapid cooling and warming rates in cryobiology. The reason is that there is increasing emphasis on using vitrification procedures for the cryopreservation of mammalian oocytes using devices like the CryotopÒ. One problem is that the cooling and warming rates are so high, that it becomes difficult to measure them. Consequently, we have developed a simple, inexpensive (<300 US$) temperature measurement system. The design goal was to measure the temperature of a 0.1 ll aqueous sample at rates up to 1 million °C/min using a temperature sensing element which would not perturb the system. To achieve the latter, a simple, fine gauge thermocouple (TC) proved suitable. We used Type T (copper–constantan) which is available in pre-made wire sizes as small as 25 lm diameter (Omega). We settled on 50 lm diameter wire as a compromise between ease of handling (under a stereo microscope) versus minimum system perturbation. We estimate the heat capacity of the 50 lm diameter wire TC tip at 1/5 the heat capacity of 0.1 ll of water; sufficiently small to yield only a modest thermal perturbation of the system. Secondly, it is necessary to record the TC temperature at a sufficiently high rate. For a simple thermal ramp, e.g. a plunge from room temperature to LN2, a tempera-
Cryoprotectants are necessary for almost all protocols of cryopreservation of biomaterials. The loading of cryoprotectant into cells needs to consider the following factors: deformation limit, toxicity and duration. Temperature has an impact on the membrane permeability for water and cryoprotectant, thus on the loading duration and toxic injury. The temperature dependence of the loading duration is numerically studied here using a two-parameter model. The loading duration tL is defined as the time needed when the intracellular cryoprotectant concentration reaches 95% of the extracellular cryoprotectant concentration. Typical ranges for parameters in the model are adopted through literature survey. They include: (1) membrane hydraulic conductivity Lp, 4 10 16–2.0 10 12 m s/Pa; (2) cryoprotectant permeability P, 1.0 10 19–2.0 10 6 m/s; (3) isotonic osmolality Mis, 0.25–0.35 M; (4) cell diameter D, 5–25 lm; (5) osmotically inactive fraction Vb, 0.15–0.55; (6) crp, a dimensionless parameter defined as P/(LpRTMiso), 0.1–10; (7) cell surface area ratio, a dimensionless parameter defined as A/(pD2), 1–2; (8) activation energy for Lp, Ea, 10–250 kJ/mol; (9) activation energy for P, Ep, 40–120 kJ/mol. More than 20,000 random cases are calculated. It is found that the loading duration can be fitted as an exponential function of temperature, tL = a eb/T with R2 > 0.95. The constant b is more