- Email: [email protected]
Towards dry preservation of mammalian cells
554
Abstracts / Cryobiology 71 (2015) 537e573
than twice, so we suggest that new pores of bigger dimensions were formed. We can conclude that microscopic pores appear during cryopreservation in the membrane of carp spermatozoa, the approximate radius of them is more than 0.78nm. Funding: N/A Conflict of Interests: N/A
experiments were designed to investigate if the cryoprotective effect of these molecules can be attributed to their IRI activity. The results of these experiments will be discussed. Funding: N/A Conflict of Interests: N/A
S70. TOWARDS DRY PRESERVATION OF MAMMALIAN CELLS
S72. FRACTURES, ICE AND ME2SO CONCENTRATION UNDER THE LIGHT OF XRAYS IN A NANOCT
€tte Oldenhof 2, Harald Sieme 2, Willem F. Miao Zhang 1, Harrie Wolkers 1. 1 Institute of Multiphase Processes, Leibniz University, Hannover, Germany; 2 Clinic for Horses e Unit for Reproductive Medicine, University of Veterinary Medicine, Hannover, Germany
Pablo Acosta 1, Ariadna Corral 1, Marcin Balcerzyk 2, Angel n Risco 1, 2. 1 Engineering School, University of Sevilla, Parrando 2, Ramo Sevilla, Spain; 2 Centro Nacional de Aceleradores (Universidad de Sevilla e CSIC e Junta de Andalucía), Seville, Spain E-mail address: [email protected]
The major challenge in dry preservation of mammalian cells is to introduce membrane-impermeable lyoprotectants such as trehalose into the cells for intracellular protection. Here it was tested if freezinginduced membrane phase transitions can be used to incorporate trehalose into fibroblasts and if this increases cryosurvival. It was found that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas the cells display highly cooperative fluid-to-gel membrane phase transitions during freezing. Fibroblasts that were frozen in the presence of the membrane-impermeable fluorescent dye lucifer yellow, exhibited intracellular lucifer yellow after slow, as well as rapid freezing with the fluorescent dye equally distributed within the cell. A plot of cryosurvival versus the cooling rate when using trehalose as cryoprotective agent showed a narrow inverted-‘U’ curve, whereas cells cryopreserved with dimethyl sulfoxide showed a much broader inverted-‘U’ curve. Cells cryopreserved with trehalose displayed good proliferation using the optimal cooling rate, indicating trehalose can be used for cryopreservation of fibroblasts. In summary, cell membranes display an increased permeability during passage through freezing-induced membrane phase transitions resulting in uptake of membrane-impermeable molecules, which we suggest can be used to incorporate trehalose into cells stabilizing them during freezing and freeze-drying. Funding: This work is supported by funding from Cook General BioTechnology (Indianapolis, Indiana, USA), as well as the German Research Foundation (DFG) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grant WO1735/61, SI1462/4-1. Conflict of Interests: N/A S71. CARBOHYDRATE-BASED SMALL MOLECULE ICE RECRYSTALLIZATION INHIBITORS AS CRYOPRESERVATIVES FOR RED BLOOD CELLS Jennie Briard, Jessica Poisson, Tracey Turner, Priya Chandran, Jason Acker, David Allan, Robert Ben. University of Ottawa, Ottawa, Ontario, Canada E-mail address: [email protected]
The ability to inhibit ice recrystallization (IRI activity) is a very desirable property for a cryoprotectant. Studies have demonstrated that ice recrystallization during the freezing and thawing stages of cryopreservation is a significant contributor to cellular damage and death. Our laboratory has designed a number of small carbohydrate-based molecules that inhibit ice recrystallization and prevent cryoinjury. In addition, these compounds permit the use of reduced amounts of glycerol (15% v/v) in conjunction with slow freezing rates for the cryopreservation of red blood cells (RBCs). Interestingly, the molecules that possessed the most IRI activity were able to reduce the hemolysis of cryopreserved RBCs compared to the control, while less active IRIs were not. Additionally, several of these small-molecule IRIs are able to improve the functionality of hematopoietic stem cells (HSCs) compared to standard 10% dimethyl sulphoxide (MeSO2) cryosolution post-thaw. It was again noticed that compounds which possess potent IRI activity improved functionality when compared to compounds that did not possess IRI activity. Several
The concentration of vitrification solutions, the presence of ice and the existence of fractures can be quantified by a novel technique based in the detection of the sulfur atom in the Me2SO molecule. The high number of electrons in this atom makes it visible at energies around 75KeV. Water, Me2SO and air/solution (fractures) have very different CT values. Bishop et al. also used the CT to detect changes of phase glycerol solutions, although they based the attenuation in density differences, and therefore, with a lower contrast. Results: X-ray attenuation was proportional to the Me2SO concentration at room temperature. The proportionality of the attenuation with the Me2SO concentration was also proved at cryogenic temperature (-140 C), and in volumes from 200mm to 20ml. Results also showed that Me2SO absolute concentration can be measured inside ovarian tissue and rabbit kidneys. This technique has also been used to avoid macrofractures (>0.1mm wide) of 7.05 molar solutions of Me2SO. However, microfractures (<0.1mm wide) were visible in all samples. To get rid of the macrofractures, an accurate control of the temperature between -110 and -135 C was critical. A vertical position of the (tubular) container was the best way for avoiding contractions in unwanted places. Funding: N/A Conflict of Interests: N/A S73. CRYOPRESERVATION OF HUMAN SYNOVIAL TISSUE FOR BIOBANKING PURPOSES M. De Vries, M. Broeren, M. Bennink, P. van Lent, R. Thurlings, P. van der Kraan, M. Koenders, F. van de Loo. Experimental Rheumatology, Dept of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands E-mail address: [email protected]
Human synovial tissue derived from joint surgery of arthritis patients is valuable material for studying fundamental research questions. However, due to improved treatment options for these patients, less surgery is needed and less articular material becomes available for research. Additionally, performing a standardized comparative experiment with fresh tissue is difficult as material is scarce and infrequently available. It would be ideal to store and collect all this valuable tissue for later use. The aim of this study is to develop a cryopreservation method for human synovium by which the cryopreserved tissue performs as freshly obtained material. Human synovial tissue is derived, with informed consent, from joint surgery and frozen as 3mm biopsies. Cryosections are made to prove synovial origin. Biopsies are frozen by slow freezing using several CPA solutions. Thawing has been performed at 37 C, followed by quick removal of the CPA solution. To determine preservation of the inflammatory tissue phenotype, cytokine secretion was measured by Luminex analysis. Results indicate that the tissue is able to respond to inflammatory stimuli. Preliminary results show similar RNA integrity numbers for control and frozen tissue. Gene expression analysis is ongoing to investigate preservation of a panel of disease related genes after cryopreservation. Funding: ZonMW. Conflict of Interests: N/A
Abstracts / Cryobiology 71 (2015) 537e573
than twice, so we suggest that new pores of bigger dimensions were formed. We can conclude that microscopic pores appear during cryopreservation in the membrane of carp spermatozoa, the approximate radius of them is more than 0.78nm. Funding: N/A Conflict of Interests: N/A
experiments were designed to investigate if the cryoprotective effect of these molecules can be attributed to their IRI activity. The results of these experiments will be discussed. Funding: N/A Conflict of Interests: N/A
S70. TOWARDS DRY PRESERVATION OF MAMMALIAN CELLS
S72. FRACTURES, ICE AND ME2SO CONCENTRATION UNDER THE LIGHT OF XRAYS IN A NANOCT
€tte Oldenhof 2, Harald Sieme 2, Willem F. Miao Zhang 1, Harrie Wolkers 1. 1 Institute of Multiphase Processes, Leibniz University, Hannover, Germany; 2 Clinic for Horses e Unit for Reproductive Medicine, University of Veterinary Medicine, Hannover, Germany
Pablo Acosta 1, Ariadna Corral 1, Marcin Balcerzyk 2, Angel n Risco 1, 2. 1 Engineering School, University of Sevilla, Parrando 2, Ramo Sevilla, Spain; 2 Centro Nacional de Aceleradores (Universidad de Sevilla e CSIC e Junta de Andalucía), Seville, Spain E-mail address: [email protected]
The major challenge in dry preservation of mammalian cells is to introduce membrane-impermeable lyoprotectants such as trehalose into the cells for intracellular protection. Here it was tested if freezinginduced membrane phase transitions can be used to incorporate trehalose into fibroblasts and if this increases cryosurvival. It was found that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas the cells display highly cooperative fluid-to-gel membrane phase transitions during freezing. Fibroblasts that were frozen in the presence of the membrane-impermeable fluorescent dye lucifer yellow, exhibited intracellular lucifer yellow after slow, as well as rapid freezing with the fluorescent dye equally distributed within the cell. A plot of cryosurvival versus the cooling rate when using trehalose as cryoprotective agent showed a narrow inverted-‘U’ curve, whereas cells cryopreserved with dimethyl sulfoxide showed a much broader inverted-‘U’ curve. Cells cryopreserved with trehalose displayed good proliferation using the optimal cooling rate, indicating trehalose can be used for cryopreservation of fibroblasts. In summary, cell membranes display an increased permeability during passage through freezing-induced membrane phase transitions resulting in uptake of membrane-impermeable molecules, which we suggest can be used to incorporate trehalose into cells stabilizing them during freezing and freeze-drying. Funding: This work is supported by funding from Cook General BioTechnology (Indianapolis, Indiana, USA), as well as the German Research Foundation (DFG) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grant WO1735/61, SI1462/4-1. Conflict of Interests: N/A S71. CARBOHYDRATE-BASED SMALL MOLECULE ICE RECRYSTALLIZATION INHIBITORS AS CRYOPRESERVATIVES FOR RED BLOOD CELLS Jennie Briard, Jessica Poisson, Tracey Turner, Priya Chandran, Jason Acker, David Allan, Robert Ben. University of Ottawa, Ottawa, Ontario, Canada E-mail address: [email protected]
The ability to inhibit ice recrystallization (IRI activity) is a very desirable property for a cryoprotectant. Studies have demonstrated that ice recrystallization during the freezing and thawing stages of cryopreservation is a significant contributor to cellular damage and death. Our laboratory has designed a number of small carbohydrate-based molecules that inhibit ice recrystallization and prevent cryoinjury. In addition, these compounds permit the use of reduced amounts of glycerol (15% v/v) in conjunction with slow freezing rates for the cryopreservation of red blood cells (RBCs). Interestingly, the molecules that possessed the most IRI activity were able to reduce the hemolysis of cryopreserved RBCs compared to the control, while less active IRIs were not. Additionally, several of these small-molecule IRIs are able to improve the functionality of hematopoietic stem cells (HSCs) compared to standard 10% dimethyl sulphoxide (MeSO2) cryosolution post-thaw. It was again noticed that compounds which possess potent IRI activity improved functionality when compared to compounds that did not possess IRI activity. Several
The concentration of vitrification solutions, the presence of ice and the existence of fractures can be quantified by a novel technique based in the detection of the sulfur atom in the Me2SO molecule. The high number of electrons in this atom makes it visible at energies around 75KeV. Water, Me2SO and air/solution (fractures) have very different CT values. Bishop et al. also used the CT to detect changes of phase glycerol solutions, although they based the attenuation in density differences, and therefore, with a lower contrast. Results: X-ray attenuation was proportional to the Me2SO concentration at room temperature. The proportionality of the attenuation with the Me2SO concentration was also proved at cryogenic temperature (-140 C), and in volumes from 200mm to 20ml. Results also showed that Me2SO absolute concentration can be measured inside ovarian tissue and rabbit kidneys. This technique has also been used to avoid macrofractures (>0.1mm wide) of 7.05 molar solutions of Me2SO. However, microfractures (<0.1mm wide) were visible in all samples. To get rid of the macrofractures, an accurate control of the temperature between -110 and -135 C was critical. A vertical position of the (tubular) container was the best way for avoiding contractions in unwanted places. Funding: N/A Conflict of Interests: N/A S73. CRYOPRESERVATION OF HUMAN SYNOVIAL TISSUE FOR BIOBANKING PURPOSES M. De Vries, M. Broeren, M. Bennink, P. van Lent, R. Thurlings, P. van der Kraan, M. Koenders, F. van de Loo. Experimental Rheumatology, Dept of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands E-mail address: [email protected]
Human synovial tissue derived from joint surgery of arthritis patients is valuable material for studying fundamental research questions. However, due to improved treatment options for these patients, less surgery is needed and less articular material becomes available for research. Additionally, performing a standardized comparative experiment with fresh tissue is difficult as material is scarce and infrequently available. It would be ideal to store and collect all this valuable tissue for later use. The aim of this study is to develop a cryopreservation method for human synovium by which the cryopreserved tissue performs as freshly obtained material. Human synovial tissue is derived, with informed consent, from joint surgery and frozen as 3mm biopsies. Cryosections are made to prove synovial origin. Biopsies are frozen by slow freezing using several CPA solutions. Thawing has been performed at 37 C, followed by quick removal of the CPA solution. To determine preservation of the inflammatory tissue phenotype, cytokine secretion was measured by Luminex analysis. Results indicate that the tissue is able to respond to inflammatory stimuli. Preliminary results show similar RNA integrity numbers for control and frozen tissue. Gene expression analysis is ongoing to investigate preservation of a panel of disease related genes after cryopreservation. Funding: ZonMW. Conflict of Interests: N/A