Abstracts: CryoLetters 33 (6), 2012

CryoLetters is a bimonthly, international journal for low temperature science and technology



Volume 33, No. 6 November/December 2012

ISSN 0143-2044



The impact of two different thaw protocols on outcomes of vitrified cleavage-stage embryos transfer
Xiao-jian Zhang, Ye-Zhou Yang, Qun Lv, Yu Wang,
Xue-hua Cao, Xiao-jie Li, Mei-xu Liao and Chun Guo




Cryopreservation of citrus shoot tips using micrografting for recovery
Gayle M. Volk, Remi Bonnart, Robert Krueger and Richard Lee




Cryopreservation of korean oge chicken semen using

Hong Jo Lee, Sung Kyu Kim, Hyun-Jun Jang,
Kyung Soo Kang, Jae-Hwan Kim, Seong-Bok Choi
and Jae Yong Han




The use of fetal bovine serum for cryopreservation of stage III zebrafish (Danio rerio) ovarian follicles
T. Zampolla, DM Rawson and T. Zhang




A comparative study of vitrification and encapsulation-vitrification for cryopreservation of protocorms of Cymbidium eburneum L., a threatened and vulnerable orchid of india
Kiran Gogoi, Suman Kumaria and Pramod Tandon




Long-term, large scale banking of Citrus species embryos: comparisons between cryopreservation and other seed banking temperatures
S. K. Malik, R. Chaudhury and H. W. Pritchard




Conservation of coconut (Cocos nucifera l.) germplasm at sub-zero Temperature
Sisunandar, Peter A Sopade, Yohannes M S Samosir,
Alain Rival and Steve W Adkins




Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation
José M. Álvarez, Millán Cortizo and Ricardo J. Ordás




Compatible solutes improve cryopreservation of human endothelial cells
Huan Sun, Birgit Glasmacher and Nicola Hofmann




Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification
Raphael Adu-Gyamfi and Andy Wetten




Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (nakai) hairy roots using a systematic approach
Haeng-Hoon Kim, Elena V. Popova, Dong-Jin Shin,
Chang-Hyu Bae, Hyung-Jin Baek, Sang-Un Park
and Florent Engelmann




Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device TheraCyte™
Ilya Yakhnenko, Wallace K. Wong, Igor I. Katkov
and Pamela Itkin-Ansari





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CryoLetters 33 (6), 411- 417 (2012)
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Xiao-jian Zhang*, Ye-Zhou Yang, Qun Lv, Yu Wang, Xue-hua Cao,
Xiao-jie Li, Mei-xu Liao and Chun Guo

Department of assisted reproductive medical center ,Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital,32 Yi Huan Lu Xi Er Duan, Chengdu, 610072, Sichuan, People's Republic of China.
*Corresponding author  e-mail:


The objective of this study was to evaluate the gestational results obtained with vitrified-thawed human cleavage-stage embryo by two different thaw protocols. Embryo development was observed to cleavage-stage and embryos were cryopreserved by vitrification on day 3 after oocyte retrieval. 51 cycles were thawed using vitrification warming kit with decreasing concentrations of sucrose in 3 dilutions ( 1.0, 0.5 and 0 mol/L respectively) as group 1, 56 cycles were thawed with decreasing concentrations of sucrose in 5 dilutions ( 0.8, 0.6, 0.33, 0.2 and 0 mol/L respectively) as group 2. Embryo survival (>50% intact blastomeres), complete embryo survival (100% intact blastomeres), pregnancy and implantation rates were compared, and development rates the day after thawing were also compared. Multivariate analysis showed a significant difference in embryo immediate morphological survival rate, complete survival and clinical pregnancies rate between the two groups respectively (87.0 vs. 98.6%, p=0.000; 71.0 vs. 82.0%, p=0.043; 27.5 vs. 46.4%, P=0.048) whereas the embryo subsequent development rates, mean number of transferred embryos was similar between the two groups. (61.4 vs. 61.3%, p=0.502; 2.2±0.5 vs. 2.4±0.6, p=0.113). In addition, no differences in implantation rate were observed between two groups (17.7 vs. 25.6%, P=0.138). No difference in the multiple pregnancy rates was found among the two groups also.

Keywords: Cleavage-stage embryos, Vitrified, Thawed method, Outcomes



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CryoLetters 33 (6), 418 - 426 (2012)
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Cryopreservation of Citrus Shoot tips using micrografting for recovery

Gayle M. Volk1*, Remi Bonnart1, Robert Krueger2 and Richard Lee2

1USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA
2USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, 1060 Martin Luther King Blvd., Riverside, CA 92507, USA
*Corresponding author


The USDA-ARS National Plant Germplasm System (NPGS) and the University of California Citrus Variety Collection maintain more than 888 unique accessions representing 132 taxa of Citrus, Fortunella, and citrus wild species within field, screenhouse, and greenhouse collections. We have identified a cryopreservation method by which Citrus genetic resources that are not maintained in vitro can be successfully conserved. Shoot tips were excised from actively growing vegetative flushes of protected trees. Surface-disinfected shoot tips were precultured overnight in 0.3 M sucrose, loaded with a loading solution for 20 min and treated with PVS2 for 30 or 60 min at 0oC, prior to direct immersion in liquid nitrogen. Rewarmed shoot tips post-cultured overnight on survival medium were then micrografted on ‘Carrizo’ seedling rootstocks to produce whole plants. Micrografted shoot tips recovered quickly and rooted plants could be transferred to the greenhouse within months. Regrowth of whole plants after micrografting averaged 53% for cryopreserved shoot tips of cultivars representing eight Citrus and Fortunella species. This method has several advantages: it uses screenhouse or greenhouse plants as source materials, it is not dependent upon cultivar-specific recovery media, and it avoids seedling juvenility.

Keywords: citrus, cryopreservation, micrografting, shoot tip



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CryoLetters 33 (6), 427 - 434 (2012)
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cryopreservation of korean oge chicken semen using n-methylacetamide

Hong Jo Lee1, Sung Kyu Kim1, Hyun-Jun Jang1, Kyung Soo Kang1,
Jae-Hwan Kim2, Seong-Bok Choi2 and Jae Yong Han1*

1WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Korea
2National Institute of Animal Science, R.D.A., Namwon 590-830, Korea
*Corresponding author  email:


The importance of genetic resource preservation has been highlighted in the literature as a means of maintaining genetic diversity. Among the various methods of preserving such resources, semen cryopreservation can be advantageous because it reduces the time of restoring genetic resources and is less technique-dependent. The Korean Oge (KO) chicken is a Natural Monument and is recognized as an important genetic resource in Korea. However, successful cryopreservation methods for KO chickens have yet to be reported. Therefore, we completed cryopreservation methods in KO chickens using N-methylacetamide (MA) as a cryoprotectant. Also we performed additional experiments to identify whether fertility and hatchability are affected by long-term storage. Finally, we examined sperm viability in the cryopreserved semen. Our results suggest that the cryopreservation method using MA can be applied to KO chickens regardless of storage period and could be a useful tool for the preservation the endangered avian species.

Keywords: Cryopreservation, N-methylacetamide, Korean Oge



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CryoLetters 33 (6), 435 - 442 (2012)
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T. Zampolla1, DM Rawson1 and T. Zhang2*

1iBEST Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK
2School of Applied Sciences, Bournemouth University, Poole House, Talbot Campus, Fern Barrow, Poole, Dorset, BH12 5BB, UK
*Corresponding author  email:


Although several studies on fish ovarian follicles cryopreservation have been carried out, their cryopreservation still remains unsuccessful. In this study, for the first time the effect of Fetal Bovine Serum (FBS) in combination with several concentrations of methanol (1, 2 or 4M) as cryoprotectant on stage III ovarian follicles viability was investigated and cryopreservation using controlled slow cooling was undertaken. Membrane integrity assessed by trypan blue (TB) staining and ATP level of stage III ovarian follicles were evaluated following cryoprotectant treatment and cryopreservation. The results showed that the survival of ovarian follicles after cryopreservation, assessed by TB staining, was significantly decreased using all three concentrations of CPAs solutions when compared to room temperature controls. The results showed a further decrease of viability when the TB test was performed 2h post-thaw. 2M methanol+10% FBS resulted in higher survival rate compared with the other methanol concentrations in combination with 10% FBS, viability results obtained 10 min and 2 hours post-thaw were 70.2 ± 4.2% and 46.2 ± 10.4% respectively. Whilst results obtained with 1M methanol supplemented with 10% FBS 10min and 2 hours post-thaw were 51.8 ± 2.4 and 3.4 ± 1.5 and those obtained with 4M supplemented with 10% FBS were 36.0 ± 5.3 and 6.4 ± 3.3 respectively.  However, a decrease of ATP content was observed with all three cryoprotectant solutions indicating a compromised metabolic status. The addition of 10% fetal bovine serum to media used for cryopreservation zebrafish ovarian follicles did not allow the achievement of successful cryopreservation.

Keywords: zebrafish; ovarian fragments; methanol; ATP; fetal bovine serum



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CryoLetters 33 (6), 443 - 452 (2012)
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Kiran Gogoi, Suman Kumaria* and Pramod Tandon

Plant Biotechnology Laboratory, Department of Botany, North-Eastern Hill University, Shillong 793 022, India.
*Corresponding author  e mail:


Cryopreservation of the threatened orchid Cymbidium eburneum L. was successfully achieved using encapsulation-vitrification and vitrification. Comparing the two methods tested, it was observed that regeneration of protocorms cryopreserved using encapsulation-vitrification was higher than with vitrification. To achieve optimal regrowth after cryopreservation, protocorms were precultured for 24 h with 0.2 M sucrose for vitrification and with 0.7 M sucrose for encapsulation-vitrification, reaching 60% and 70% regeneration, respectively. With both techniques employed, a 20 min exposure duration to Plant Vitrification Solution 2 (PVS2) led to optimal regeneration after cryopreservation. A maximum of 66% regeneration was achieved after cryopreservation using encapsulation-vitrification, whereas it was only 50% after cryopreservation using vitrification. The same regeneration pattern was observed with protocorms cryopreserved using both techniques employed. This is the first report of long-term conservation by cryopreservation of C. eburneum protocorms.

Keywords: Cryopreservation, encapsulation-vitrification, protocorms, PVS2, vitrification



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CryoLetters 33 (6), 453 - 464 (2012)
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S. K. Malik1*, R. Chaudhury1 and H. W. Pritchard2

1Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110012, INDIA.
2Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK.
*Corresponding author  e-mail:


The long-term, large scale application of embryo cryopreservation has been assessed rarely and comparisons of viability loss for partially dried material with conventional seed bank storage conditions infrequently made. Five citrus species were cryopreserved following air drying of embryos (seed minus the testa) and embryonic axes: rough lemon (Citrus jambhiri), pommelo (C. grandis), mandarin (C. reticulata), citron (C. medica) and kagzi lime (C. aurantifolia). Although drying rates to c. 10% moisture content (MC) were approximately 10-times faster for isolated axes compared to embryos, the optimum MCs for cryopreservation were generally similar within a species, varying from c. 10% (C. jambhiri) to c. 20% (C. medica). Nonetheless, the hydration window for cryopreservation of the axis was usually wider than for the embryo. For all species, embryo or axis survival after cryopreservation ranged from 65% to 96% (C. medica axes), producing normal healthy seedlings from embryos and plantlets from axes without intervening callus growth in vitro. Whilst partially dried embryos of all five species survived fully liquid nitrogen vapour storage for 120 days, viability loss was rapid at -20°C, 5°C and ambient temperature, with a maximum interpolated half-life across these temperatures of c. 80 days for C. grandis at 5°C. The developed cryopreservation protocols were applied routinely to cryobank 377 accessions of Citrus germplasm from field genebanks, farmer’s orchards, semi-wild and wild sources. After an average of 6.3 to 8.4 years cryo-storage, between 69 and 81% of accessions per species retained ≥ 70% of the viability after desiccation. The results provide irrevocable evidence for the importance of cryopreservation for the banking of seeds of higher plants. 

Keywords: Embryo axis, cryo-base collection, commodity species, crop wild relative, tropical fruit.



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CryoLetters 33 (6), 465 - 475 (2012)
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Sisunandar1,2*, Peter A Sopade3, Yohannes M S Samosir1,4,
Alain Rival5 and Steve W Adkins1

1The University of Queensland, Integrated Seed Research Unit, School of Agriculture and Food Sciences, St Lucia, Queensland, Australia 4072
2 The Muhammadiyah University of Purwokerto, Biology Education Department, Kampus Dukuhwaluh, Purwokerto, Indonesia, 53182.
3The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Queensland, Australia 4072
4Bakrie Agriculture Research Institute (BARI), Bakrie Sumatera Plantations, Jl. Ir.Juanda 1, Kisaran 21202, North Sumatera, Indonesia
5Cirad, UMR DIADE, Palm Developmental Biology Group, F-34398 Montpellier, France
*Corresponding author email:
Ph. +62281630463, Fax. +62281637239.


Protocols are proposed for the low (-20°C) and ultra-low (-80°C) temperature storage of coconut (Cocos nucifera L.) embryos. A tissue dehydration step prior to storage, and a rapid warming step upon recovery optimized the protocol. The thermal properties of water located within the embryos were monitored using differential scanning calorimetry (DSC). In the most efficient version of the protocol, embryos were dehydrated under a sterile air flow in a dehydration solution containing glucose (3.33 M) and glycerol (15%) for16 hours. This protocol decreased the embryo water content from 77 to 29% FW and at the same time reduced the amount of freezable water down to 0.03%. The dehydrated embryos could be stored for up to 3 weeks at -20°C (12% producing normal plants upon recovery) or 26 weeks at -80°C (28% producing normal plants upon recovery). These results indicate that it is possible to store coconut germplasm on a medium term basis using an ultra-deep freezer unit. However for more efficient, long term storage, cryopreservation remains the preferred option.

Keywords: dehydration, cryopreservation, differential scanning calorimetry, embryo culture, medium-term conservation, recalcitrant seeds.



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CryoLetters 33 (6), 476 - 484 (2012)
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José M. Álvarez1, Millán Cortizo1,2 and Ricardo J. Ordás1*

1Laboratorio de Biotecnología Agroforestal, Escuela Politécnica de Mieres, Universidad de Oviedo, C/ Gonzalo Gutiérrez Quirós, ES-33600 Mieres, Spain
2Present address: INRA, UMR1318, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France
*Corresponding author email:   Fax: +34 985458001


Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150ºC freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100% of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

Keywords: cryopreservation, Pinus pinaster, somatic embryogenesis, plant regeneration.



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CryoLetters 33 (6), 485 - 493 (2012)
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Compatible Solutes improve cryopreservation of human endothelial cells

Huan Sun, Birgit Glasmacher and Nicola Hofmann*

Institute for Multiphase Processes, Leibniz Universität Hannnover, Callinstr. 36, 30167 Hannover, Germany.
*Corresponding author e-mail:


Dimethyl sulfoxide (DMSO) is till now a widely used cryoprotective agent for cryopreservation of cells. Since high concentrations of DMSO have detrimental effects on cell functioning the aim of this study is to reduce the DMSO concentration by using compatible solutes (CS). These are small organic osmolytes that microorganisms synthesize and accumulate to counteract stress factors. Three CS, hydroxyectoine, ectoine and L-proline were investigated as cryoprotective agents for the cryopreservation of the human endothelial cell line HPMEC-ST1.6R. They were either supplemented to freezing or to cell culture medium. L-proline was the most effective CS, the efficiency of recultivation was improved by more than 100%. A combination of L-proline and ectoine in the cell culture medium resulted in an improvement by 63%. Our results show that L-proline and ectoine could be used as additional CPAs to improve the cryopreservation of human endothelial cells in the presence of low DMSO concentration.

Keywords: ectoine, hydroxyectoine, L-proline, compatible solutes, cryopreservation.



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CryoLetters 33 (6), 494 - 505 (2012)
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Raphael Adu-Gyamfi1, 2* and Andy Wetten1

1School of Biological Sciences, University of Reading, Whiteknights, Reading, RG6 6AS,  UK.
2Agronomy Department, University for Development Studies, P. O. Box TL 1882, Tamale, Ghana
*Corresponding author e-mail:


Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5%) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

Keywords: cocoa, Theobroma cacao, somatic embryo, conservation, cryopreservation, vitrification, PVS2



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CryoLetters 33 (6), 506 - 517 (2012)
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Haeng-Hoon Kim1,2, Elena V. Popova3, Dong-Jin Shin4,
Chang-Hyu Bae2, Hyung-Jin Baek1, Sang-Un Park5*
and Florent Engelmann 6,7

1National Agrobiodiversity Center, RDA-NAAS, Suwon 441-707, Korea.
2Department of Well-being Resources, Sunchon National University, Suncheon 540-742, Korea (current address).
3Korea Forest Research Institute, Korea Forest Service, Suwon 441-847, Korea.
4Species Restoration Center, Korea National Park Service, Gurye 542-853, Korea.
5Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea.        
6Institut de recherche pour le développement (IRD), UMR DIADE, BP 64501, 34394 Montpellier cedex 5, France.
7Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Correspondence author  email:


A systematic approach using a set of 13 treatments was applied to develop a droplet-vitrification protocol for Rubia akane hairy roots, based on their responses to preculture, loading, dehydration and cooling/rewarming steps. The roots were very sensitive to osmotic stress induced by both preculture in liquid sucrose-enriched medium (up to 0.5 M sucrose) and by dehydration with highly concentrated vitrification solutions (VSs). Loading was necessary before dehydration of explants with VS, and the composition of the loading solution (LS) significantly affected their post-cryopreservation regeneration. Due to high sensitivity of roots to both chemical cytotoxicity and osmotic stress produced by VSs, cryoprotection with alternative VSs, i.e. B5-80% (40% glycerol + 40% sucrose, w/v) at room temperature for 15 min or with A3-70% (29.2% glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0°C for 20 min ensured the highest post-cryopreservation regeneration. However, when using these solutions, endothermic peaks with -2.9 and -5.8 J g-1 FW enthalpies, respectively, were recorded by differential scanning calorimetry (DSC) during the rewarming phase. Droplet-vitrification using foil strips showed higher post-cryopreservation regeneration (86%) compared with vitrification in cryovials (59%), possibly due to the higher cooling and rewarming rates achieved with droplet-vitrification. The developed protocol was applied to hairy roots of five other species with minor modifications in explant type, the duration of the last subculture before explant excision, and the dehydration duration with VS B5-80%.

Keywords: droplet-vitrification, hairy roots, loading, preculture, Rubia akane, thermal analysis, vitrification solution.



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CryoLetters 33 (6), 518 - 531 (2012)
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Ilya Yakhnenko1,3, Wallace K. Wong2, Igor I. Katkov1,3
and Pamela Itkin-Ansari2,4,*

1Cryocore of the Stem Cell Center and 2Development and Aging Program, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA
3CELLTRONIX, San Diego, California 92126, USA
4Dept. of Pediatrics, University of California, San Diego, California, La Jolla 92093-0816, USA
*Corresponding author  email:


Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (>106 cells) device (TheraCyte™). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10%-DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

Keywords: diabetes; insulin producing cells; immunoisolation; macro-encapsulation; TheraCyte; cryopreservation; DMSO toxicity.

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