- Email: [email protected]
Cryopreservation of the intracellular parasite: Stage V gametocytes of Plasmodium falciparum
Abstracts / Cryobiology 71 (2015) 537e573
millimolar to sub-millimolar range. In vitro studies using human red blood cells (RBCs) and hematopoietic stem cells (HSCs) demonstrate that these ice recrystallization inhibitors are also effective cryoprotectants. In addition, some of these molecules are able to protect cells from cryoinjury associated with transient warming events (TWEs). Funding: NSERC, CBS, CIHR. Conflict of Interests: N/A S18. CRYOPRESERVATION OF THE INTRACELLULAR PARASITE: STAGE V GAMETOCYTES OF PLASMODIUM FALCIPARUM E.R. James, H. Huang, A.O. Awe, T. Li, Y. Abebe, M. Marquette, A. Eappen, M. Li, A.J. Ruben, A. Patil, Y. Getachew, A. Liem, A. Richman, Y. Wen, S. Matheney, J. Overby, K. Nelson, V. Pich, P. Billingsley. Sanaria Inc., Rockville, Maryland, USA E-mail address: [email protected]
Successful cryopreservation of obligate intracellular parasites involves a compromise between methods optimal for the parasite and for the host cell. Stage V gametocytes of Plasmodium, however, may be an exception since exit from their host cell and transformation into gametes is triggered when they transition from a mammalian intravascular environment to the mid gut environment of mosquitoes. We hypothesized, therefore, that survival of the host erythrocyte could be ignored and cryopreservation optimized solely for the gametocytes. In experiments, testing parameters including the type of container, type, concentration, and time of incubation in CPA, cooling rate, warming rate, and method of dilution, we optimized cryopreservation for parasite survival based initially on male gamete activation. All handling before cooling and after thawing maintained the gametocytes at 37 C to prevent parasite transformation. The resulting method yielded good survival of male and female gametocytes postthawing that produced oocysts e the stage in which the human-infective sporozoites develop. Concomitantly, most erythrocytes were destroyed by cryopreservation resulting in a highly enriched population of gametocytes. This enables the goal of banking stable lots of well-characterized cryopreserved gametocytes to initiate mosquito infections on demand and for assays of transmission-blocking antibodies and drugs. Funding: PATH - Malaria Vaccine Initiative. Conflict of Interests: N/A S19. BIOBANKING OF STEM CELLS: IMPROVED METHODS AND PROCEDURES FOR PLURIPOTENT STEM CELL PRESERVATION, STORAGE STABILITY AND VALIDATION Heiko Zimmerman 1, 2. 1 Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280 Sulzbach, Germany; 2 Dept. Molecular & Cellular Biotechnology/Nanotechnology, Saarland University, Campus, 66123 Saarbrücken, Germany E-mail address: [email protected]
The project EBiSC (www.ebisc.org) aims to build up a European biobank for research grade human induced pluripotent stem cells (hIPSCs). The vision of EBiSC leads to the demand for upscaled production methods for these kind of cells leading to the need for automated systems and procedures in stem cell processing and banking. An overview of existing stateof-the-art automation systems is given and the specifications for different applications are compared. Advantages and disadvantages of various bioreactor systems for stem cell engineering are concluded and the application of fully automated workflows using liquid handling systems is shown. Furthermore, modules and concepts for automated cell identification, pluripotency testing, viability and functionality tests are drawn and results are shown. Scalable label-free analysis of pluripotent stem cells using quantitative life cell imaging and on-line image analysis is shown. A specialised system, the automated Hanging Drop technique (DropTech®) is shown. The DropTech system allows fully automated cultivation of hiPSCs on micro carrier using the hanging drop technology and enables applications like the automated Embryonic Stem cell Test (EST) for standardised
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embryotoxicity tests. The last part of the talk deals with the technology of cryopreservation, banking and validation frozen samples. The method of surface-based vitrification of pluripotent stem cells is introduced and the need for a completely closed cool chain is derived from experimental results. Solutions for automated industrial scale biobanking with closed cool chains and with minimal harmful thermal fluctuations are shown and the effect on functionality of cryopreserved cells compared to standard technology is shown. A method for non-invasive monitoring of re-crystalisation and devitrification effects using Raman micro-spectroscopy is presented. Funding: EC-FP7, grant no. 115582-3 (EBiSC), EC-FP7, grant no. 601865 (DropTech) Conflict of Interest: N/A S20. QUALIFICATION OF A CRYOGEN-FREE CONTROLLED RATE FREEZER FOR USE IN THE DELIVERY OF CRYOPRESERVED STEM CELLS BANKS FOR THERAPEUTIC APPLICATIONS J. Man 1, C.J. Hunt 1, L.E. Healy 1, G.J. Morris 2. 1 UK Stem Cell Bank, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, United Kingdom; 2 Asymptote Ltd. St. John’s Innovation Centre, Cambridge, United Kingdom Production of cells for direct clinical application as well as seed stocks intended as starting materials for cellular therapies require adherence to strict regulatory requirements generally based on Good Pharmaceutical Manufacturing Practice (GMP). This requires a consistent and robust cryopreservation step currently served either by cryogen-fed controlled rate freezers (CRF) or passive cooling devices (PCD). Both present issues of regulatory compliance if used in a GMP cleanroom environment. One approach to overcoming the issues arising from these devices is to use cryogen-free, Stirling-cycle cooling technology as the basis for a CRF. We have compared the recovery and biological status of hESC and hiPSC lines frozen down in a prototype of the Asymptote Via-Freeze with a PCD (Mr Frosty™) using a standardised cell bank protocol. Cells were tested for “cell health” (propidium iodide/acridine orange, intracellular glutathione, mitochondrial membrane potential), stem cell characteristics (Tra-1-60, SSEA1, 3 and 4), karyology (G-banding) and markers for self-renewal, attachment and recovery immediately upon thawing and 5 passages postthaw. Cryopreservation using the prototype Via-Freeze did not significantly alter cell adhesion or other cell health markers, nor were there any changes in expression of self-renewal genes and phenotypic characteristics when compared to pre-freeze controls. In summary the Via Freeze provided comparable results to the passive cooling device and unlike cryogen-fed CRFs is suitable for use in a cleanroom environment. Funding: Innovate UK (Technology Strategy Board) Conflict of Interests: N/A S21. EFFICIENT AUTOMATABLE AND ASEPTIC VITRIFICATION OF HUMAN PLURIPOTENT STEM CELLS USING BIO-SAFE AND CHEMICALLY DEFINED MEDIA D. Connan 1, F.J. Ectors 2, J. Piret 3, N. Antoine 3, L. Grobet 4. 1 First Spin-off Project Leader VITRICELL, Embryology Unit, FARAH & GIGA, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium; 2 Mammalian Transgenic Platforms, FARAH & GIGA, University of Liege, Liege, Belgium; 3 Laboratory of Animal Histology, FARAH, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium; 4 Embryology Unit, FARAH & GIGA, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium E-mail address: [email protected]
Human pluripotent stem cells (hPSCs) are considered as the novel reference bio-material for biomedical research and cell-based therapies. However, their use in clinics requires efficient and bio-safe handling. Cryopreservation is an obligate key step of storage and transportation, during which the cells undergo extreme physical and chemical conditions prone to alter their viability as well as their biological properties. Moreover, commonly used slow freezing and vitrification protocols cannot
millimolar to sub-millimolar range. In vitro studies using human red blood cells (RBCs) and hematopoietic stem cells (HSCs) demonstrate that these ice recrystallization inhibitors are also effective cryoprotectants. In addition, some of these molecules are able to protect cells from cryoinjury associated with transient warming events (TWEs). Funding: NSERC, CBS, CIHR. Conflict of Interests: N/A S18. CRYOPRESERVATION OF THE INTRACELLULAR PARASITE: STAGE V GAMETOCYTES OF PLASMODIUM FALCIPARUM E.R. James, H. Huang, A.O. Awe, T. Li, Y. Abebe, M. Marquette, A. Eappen, M. Li, A.J. Ruben, A. Patil, Y. Getachew, A. Liem, A. Richman, Y. Wen, S. Matheney, J. Overby, K. Nelson, V. Pich, P. Billingsley. Sanaria Inc., Rockville, Maryland, USA E-mail address: [email protected]
Successful cryopreservation of obligate intracellular parasites involves a compromise between methods optimal for the parasite and for the host cell. Stage V gametocytes of Plasmodium, however, may be an exception since exit from their host cell and transformation into gametes is triggered when they transition from a mammalian intravascular environment to the mid gut environment of mosquitoes. We hypothesized, therefore, that survival of the host erythrocyte could be ignored and cryopreservation optimized solely for the gametocytes. In experiments, testing parameters including the type of container, type, concentration, and time of incubation in CPA, cooling rate, warming rate, and method of dilution, we optimized cryopreservation for parasite survival based initially on male gamete activation. All handling before cooling and after thawing maintained the gametocytes at 37 C to prevent parasite transformation. The resulting method yielded good survival of male and female gametocytes postthawing that produced oocysts e the stage in which the human-infective sporozoites develop. Concomitantly, most erythrocytes were destroyed by cryopreservation resulting in a highly enriched population of gametocytes. This enables the goal of banking stable lots of well-characterized cryopreserved gametocytes to initiate mosquito infections on demand and for assays of transmission-blocking antibodies and drugs. Funding: PATH - Malaria Vaccine Initiative. Conflict of Interests: N/A S19. BIOBANKING OF STEM CELLS: IMPROVED METHODS AND PROCEDURES FOR PLURIPOTENT STEM CELL PRESERVATION, STORAGE STABILITY AND VALIDATION Heiko Zimmerman 1, 2. 1 Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280 Sulzbach, Germany; 2 Dept. Molecular & Cellular Biotechnology/Nanotechnology, Saarland University, Campus, 66123 Saarbrücken, Germany E-mail address: [email protected]
The project EBiSC (www.ebisc.org) aims to build up a European biobank for research grade human induced pluripotent stem cells (hIPSCs). The vision of EBiSC leads to the demand for upscaled production methods for these kind of cells leading to the need for automated systems and procedures in stem cell processing and banking. An overview of existing stateof-the-art automation systems is given and the specifications for different applications are compared. Advantages and disadvantages of various bioreactor systems for stem cell engineering are concluded and the application of fully automated workflows using liquid handling systems is shown. Furthermore, modules and concepts for automated cell identification, pluripotency testing, viability and functionality tests are drawn and results are shown. Scalable label-free analysis of pluripotent stem cells using quantitative life cell imaging and on-line image analysis is shown. A specialised system, the automated Hanging Drop technique (DropTech®) is shown. The DropTech system allows fully automated cultivation of hiPSCs on micro carrier using the hanging drop technology and enables applications like the automated Embryonic Stem cell Test (EST) for standardised
541
embryotoxicity tests. The last part of the talk deals with the technology of cryopreservation, banking and validation frozen samples. The method of surface-based vitrification of pluripotent stem cells is introduced and the need for a completely closed cool chain is derived from experimental results. Solutions for automated industrial scale biobanking with closed cool chains and with minimal harmful thermal fluctuations are shown and the effect on functionality of cryopreserved cells compared to standard technology is shown. A method for non-invasive monitoring of re-crystalisation and devitrification effects using Raman micro-spectroscopy is presented. Funding: EC-FP7, grant no. 115582-3 (EBiSC), EC-FP7, grant no. 601865 (DropTech) Conflict of Interest: N/A S20. QUALIFICATION OF A CRYOGEN-FREE CONTROLLED RATE FREEZER FOR USE IN THE DELIVERY OF CRYOPRESERVED STEM CELLS BANKS FOR THERAPEUTIC APPLICATIONS J. Man 1, C.J. Hunt 1, L.E. Healy 1, G.J. Morris 2. 1 UK Stem Cell Bank, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, United Kingdom; 2 Asymptote Ltd. St. John’s Innovation Centre, Cambridge, United Kingdom Production of cells for direct clinical application as well as seed stocks intended as starting materials for cellular therapies require adherence to strict regulatory requirements generally based on Good Pharmaceutical Manufacturing Practice (GMP). This requires a consistent and robust cryopreservation step currently served either by cryogen-fed controlled rate freezers (CRF) or passive cooling devices (PCD). Both present issues of regulatory compliance if used in a GMP cleanroom environment. One approach to overcoming the issues arising from these devices is to use cryogen-free, Stirling-cycle cooling technology as the basis for a CRF. We have compared the recovery and biological status of hESC and hiPSC lines frozen down in a prototype of the Asymptote Via-Freeze with a PCD (Mr Frosty™) using a standardised cell bank protocol. Cells were tested for “cell health” (propidium iodide/acridine orange, intracellular glutathione, mitochondrial membrane potential), stem cell characteristics (Tra-1-60, SSEA1, 3 and 4), karyology (G-banding) and markers for self-renewal, attachment and recovery immediately upon thawing and 5 passages postthaw. Cryopreservation using the prototype Via-Freeze did not significantly alter cell adhesion or other cell health markers, nor were there any changes in expression of self-renewal genes and phenotypic characteristics when compared to pre-freeze controls. In summary the Via Freeze provided comparable results to the passive cooling device and unlike cryogen-fed CRFs is suitable for use in a cleanroom environment. Funding: Innovate UK (Technology Strategy Board) Conflict of Interests: N/A S21. EFFICIENT AUTOMATABLE AND ASEPTIC VITRIFICATION OF HUMAN PLURIPOTENT STEM CELLS USING BIO-SAFE AND CHEMICALLY DEFINED MEDIA D. Connan 1, F.J. Ectors 2, J. Piret 3, N. Antoine 3, L. Grobet 4. 1 First Spin-off Project Leader VITRICELL, Embryology Unit, FARAH & GIGA, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium; 2 Mammalian Transgenic Platforms, FARAH & GIGA, University of Liege, Liege, Belgium; 3 Laboratory of Animal Histology, FARAH, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium; 4 Embryology Unit, FARAH & GIGA, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium E-mail address: [email protected]
Human pluripotent stem cells (hPSCs) are considered as the novel reference bio-material for biomedical research and cell-based therapies. However, their use in clinics requires efficient and bio-safe handling. Cryopreservation is an obligate key step of storage and transportation, during which the cells undergo extreme physical and chemical conditions prone to alter their viability as well as their biological properties. Moreover, commonly used slow freezing and vitrification protocols cannot