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Abstracts: CryoLetters 27 (1), 2006

CryoLetters 27 (1), 17-28 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CHARACTERISATION OF CRYOPRESERVED CELLS FRESHLY ISOLATED FROM HUMAN BONE MARROW

Philippa A. Rusta*, Costas Tingeridesa, Stephen R. Cannonb,
Timothy W.R. Briggsb and Gordon W. Blunna

*Department of Orthopaedics, Bonney House, St Mary's Hospital, Praed Street, London W2 1NY, UK.
Tel.: +44 (0) 20 8747 8982, Email:
philipparust@hotmail.com
aBiomedical Engineering, Institute of Orthopaedics and Musculo-Skeletal Science, University College London, Brockley Hill, Stanmore HA7 4LP, UK.
bDepartment of Orthopaedic Surgery, Royal National Orthopaedic Hospital Trust, Stanmore, HA7 4LP, UK

Abstract

Osteoblast progenitor cells (OBPCs) isolated from bone marrow have the ability to differentiate into osteoblasts and thus potential therapeutic use to tissue-engineer bone. In order for OBPCs to be available for clinical use a means of storing viable cells is necessary.  The aim of this study was to determine whether a simple method of cryopreservation had an effect on osteogenic differentiation or growth of OBPCs isolated from fresh human bone marrow. Stro-1 was used to identify the isolated OBPCs. The osteoblastic potential of the marrow cells was confirmed as culture with osteogenic supplements (OS) significantly increased osteoblastic protein production (alkaline phosphatase (ALP), osteopontin and osteocalcin) compared with standard conditions (P < 0.05). Ten further marrow aspirates were harvested; each was halved for either cryopreservation or control culture. Primary cultures from both populations formed colonies with recognised OBPC morphology. OS stimulated both cryopreserved and control populations to produce significantly more osteoblastic proteins (P < 0.05) and there was no significant difference between the increase in osteogenic proteins when cultured with OS (P > 0.2). The proliferation rate after 5 days in culture was not significantly affected by cryopreservation (P > 0.05).  It has been suggested that OBPCs are immuno-privileged; so allogenic cells could be implanted into patients for tissue engineering bone without causing a hypersensitivity reaction. Our study demonstrates a method of storage, which allows OBPCs to be available for use without affecting osteoblastic potential or viability.

Keywords: Mesenchymal stem cells, osteoblast progenitor cells, cryopreservation, phenotype characterisation.

 

 

CryoLetters 27 (1), 29-42 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF SHOOT TIPS OF BLACKBERRY AND RASPBERRY BY ENCAPSULATION-DEHYDRATION AND VITRIFICATION

Sandhya Gupta* and Barbara M. Reed

U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, Oregon 97333, USA
Email:
corbr@ars-grin.gov
*Corresponding author, Present address: Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110012, India. Email:
sandhya_gupta87@yahoo.com.

Abstract

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm.  Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to ~20% moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5% dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25C, followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60-100% using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71% average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.

Keywords: cold acclimation, germplasm, liquid nitrogen, Rubus

 

 

CryoLetters 27 (1), 43-50 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

fREEZE-DRYING OF HUMAN PLATELETS: INFLUENCE OF INTRACELLULAR TREHALOSE AND EXTRACELLULAR PROTECTANTS

Xin-Li Zhou1, Hong Zhu2, Shao-Zhi Zhang 1, Fa-Ming Zhu2,
Guang-Ming Chen1* and Li-Xing Yan2

1Cryobiology laboratory, Institute of Refrigeration and Cryogenic Engineering, Zhejiang University, Hangzhou 310027, China.
*E-mail: gmchen@zju.edu.cn 
2 Blood Center of Zhejiang Province, Hangzhou 310006, China.

Abstract

Freeze-drying is an ideal alternative for long-term preservation of platelets in blood banks. Intracellular trehalose is believed to be an effective lyoprotectant for preserving cells during freeze-drying. In this study, 13 mM intracellular trehalose was loaded into human platelets through fluid-phase endocytosis pathway. Bovine serum albumin and trehalose were used as extracellular protectants. The effects of intracellular trehalose and extracellular protectants on freeze-dried platelets were studied respectively. The results showed 13 mM intracellular trehalose was beneficial to freeze-dried human platelets, but only slightly enhanced the protection afforded by extracellular protectants. Loaded with 13 mM intracellular trehalose, platelets were freeze-dried in a formulation of 1% bovine serum albumin and 1% trehalose, 40 days later, the survival rate of rehydrated platelets was about 85%, the morphology of rehydrated platelets was intact and the aggregation percentage with thrombin (1 U/ml) was 97.3%.

Keywords: platelets, freeze-drying, intracellular trehalose, extracellular protectants

 

 

CryoLetters 27 (1), 51-58 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CHARACTERISTICS OF FRESH GANDER SEMEN AND ITS SUSCEPTIBILITY TO CRYOPRESERVATION IN SIX GENERATIONS DERIVED FROM GEESE INSEMINATED WITH FROZEN-THAWED SEMEN

Ewa Łukaszewicz

Institute of Animal Science, Department of Poultry Breeding, Agricultural University of Wrocław, 50-631 Wrocław, Chełmońskiego 38c, Poland,
Phone: +48 71 3205-774; fax: +48 71 3205-776; e-mail:
ewa@gen.ar.wroc.pl

Abstract

The paper summarises seven years experiments designed to determine the effect of continuous insemination with frozen-thawed semen on fresh semen quality and sperm susceptibility to freezing stress in succeeding generations. During course of experiments, semen was collected from 10-12 White Kołuda® ganders at the age of 8-9 months, then subjected to freezing and used after thawing for insemination of 10 geese in order to obtain the subsequent generation of males. Semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4C, mixed with 6% (v/v) of dimethyl-formamide (DMF), frozen to temp. -1400C at a rate 60C/min and then transferred into LN2 container. Semen samples were thawed prior to insemination in a 60C water-bath. It is difficult to conclude whether freezing stress affected the fresh semen quality, since average volume of SQF (index comprising ejaculate volume, sperm concentration and percentage of live normal cells) varied between generations from 19.3 to 56.2. Continuous goose reproduction by insemination with frozen-thawed semen resulted in significant increase (P 0.01) in spermatozoa resistance to cryoinjury in every subsequent generation. In the relation to adequate fresh semen the percentage of live morphologically intact spermatozoa which withstood freezing procedure increased from 27.2 in 1st generation to 74.4 in 6th generation.

Keywords: gander semen, cryopreservation, spermatozoa morphology

 

 

CryoLetters 27 (1), 59-64 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ULTRA RAPID FREEZING AND VITRIFICATION OF HUMAN EMBRYOS DERIVED FROM ABNORMALLY FERTILISED ZYGOTES

Molina I.1*, Duque C.C.1, Alfonso J.2, Cervera R.P3 and Romeu A.1

1Human Assisted Reproduction Unit. University Hospital la Fe of Valencia, 46009, Valencia, Spain.
2Reproductive Medicine Institute (IMER), 46009, Valencia, Spain.
3Laboratory of Animal Reproduction and Biotechnology. Polytechnic University of Valencia, Spain.
*To whom correspondence should be addressed at: Human Assisted Reproduction Unit. University Hospital la Fe of Valencia, 46009, Valencia, Spain.
E-mail:
molina_inmbot@gva.es.

Abstract

The present study was undertaken to compare the developmental capacity of human embryos derived from abnormally fertilised zygotes (1PN, ≥3 PN; 16-18 hours after ICSI) cryopreserved using two techniques: ultra rapid freezing and vitrification. At 2-4 cell stage, (48 hours after ICSI), these abnormally fertilised embryos were then distributed in three groups: a) embryos that were cryopreserved by ultra rapid freezing (URF Group), b) embryos cryopreserved by vitrification (V Group) and c) embryos that were not cryopreserved (Control group). Survival rates and embryo development after 24 hours of in vitro culture (72 hours after ICSI) were compared.  42 embryos were cryopreserved by ultra rapid freezing in 0.5mL straws, using a mixture of dimethyl sulphoxide (3M) and sucrose (0.25M) in a base solution consisting of IVF medium plus 20% (v/v) of Human Serum Albumin (HSA), and 24 embryos were vitrified in 0.25 ml straws, using a two step protocol with an equilibration solution consisting of 10% ethylene glycol (1.79M) and 10% dimethyl sulphoxide (1.41M) in a base solution of modified phosphate buffered saline (PBS) with 20% of HSA and a vitrification solution consisting of 20% ethylene glycol (3.58M), 20% dimethyl sulphoxide (2.82M) and 0.5M sucrose in base solution.  The recovery rate after thawing/warming was lower for the vitrification group (75% V; 83% URF).  The number of embryos with less than 50% of intact blastomeres after cryopreservation was significantly higher for the URF group (0% V; 34% URF). After in vitro culture, the rate of embryos not cryopreserved (Control group) that developed in vitro (72 hours after ICSI) was the highest (86%), followed by group V (50%), while group URF was the lowest (13%). These differences were statistically significant. This straw method of vitrification is successful and safe.

Keywords: cryopreservation, embryo, vitrification, freezing

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