Efficient Cryopreservation Protocol Research Articles

Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis

The successful use of human induced pluripotent stem cells (iPSCs) for research or clinical applications requires the development of robust, efficient, and reproducible cryopreservation protocols. After cryopreservation, the survival rate of iPSCs is suboptimal and cell line-dependent. We assessed the use of ice recrystallization inhibitors (IRIs) for cryopreservation of human iPSCs. A toxicity screening study was performed to assess specific small-molecule carbohydrate-based IRIs and concentrations for further evaluation. Then, a cryopreservation study compared the cryoprotective efficiency of 15 mM IRIs in 5 % or 10 % DMSO-containing solutions and with CryoStor® CS10. Three iPSC lines were cryopreserved as single-cell suspensions in the cryopreservation solutions and post-thaw characteristics, including pluripotency and differential gene expression were assessed. We demonstrate the fitness-for-purpose of 15 mM IRI in 5 % DMSO as an efficient cryoprotective solution for iPSCs in terms of post-thaw recovery, viability, pluripotency, and transcriptomic changes. This mRNA sequencing dataset has the potential to be used for molecular mechanism analysis relating to cryopreservation. Use of IRIs can reduce DMSO concentrations and its associated toxicities, thereby improving the utility, effectiveness, and efficiency of cryopreservation.

Micropropagation and Shoot Tip Cryopreservation of 'Sunny Gold' Freesia.

Cryopreservation is a promising method for the long-term preservation of plant germplasm, especially for vegetatively propagated species like freesias. In this study, we investigate streamlining the cryopreservation process for 'Sunny Gold' Freesia, starting from effective in vitro initiation and proliferation using various plant growth regulator combinations. We also assess the impact of subculture on regrowth rates after cryopreservation. The shoot tips were successfully initiated in vitro after sterilization. The shoots were multiplied an average of three times in media containing N6-benzyladenine and kinetin. The regrowth rates of non-cryopreserved shoot tips excised from different subculture cycles did not differ significantly, with rates of 44% observed for plants from more than five subcultures and 47% for those from three subcultures. However, only the shoot tips excised from cultures subjected to three subculture cycles were able to recover after cryopreservation, with a regrowth rate of 31%. Our findings lay the groundwork for the development of an efficient cryopreservation protocol for freesias in the future.

Application of D Cryo-Plate Technique for The Cryopreservation of In Vitro-grown Shoot Tips of Tarenaya Spinosa (Cleomaceae)

BACKGROUND: Tarenaya spinosa is a medicinal species used for treating respiratory and inflammatory diseases. Various biotechnological studies have been developed for in vitro establishment of plants and long-term conservation of this species. Objective: This study aimed to establish a new cryopreservation protocol using the D cryo-plate technique for in vitro-grown shoot tips of T. spinosa. MATERIALS AND METHODS: Different steps of the cryopreservation procedure were evaluated in this work: preculture; sucrose concentration of calcium alginate gel; concentration and time of exposure to osmoprotective loading solution; time of exposure to silica gel; and regrowth on recovery medium. RESULTS: The optimal procedure included preculture with increasing sucrose concentration (from 0.25 to 0.50 M), encapsulation and dehydration over silica-gel for 60 min. Increasing sucrose concentration in the loading solution or exposure duration had a negative effect on recovery of cryopreserved shoot tips. However, the association of calcium alginate gel enriched with 0.6 M sucrose with post-rewarming culture with BAP for 2 weeks resulted in the most efficient cryopreservation protocol (76% survival and 70% shoot recovery). CONCLUSION: The plants developed after cryopreservation maintained their in vitro multiplication capacity and demonstrated the efficiency of long-term conservation by D cryo-plate technique for T. spinosa.

Development of efficient and sustainable droplet-vitrification cryoconservation protocol for shoot tips for long-term conservation of Dahlia germplasm

Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005–06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient droplet-vitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm.

Freezing for the Future: Obtaining Fibroblast Samples from Deceased Wild Mammals for the Brazilian Cerrado Germplasm Bank.

We isolated and further characterized fibroblasts obtained from postmortem skin biopsies of three different Brazilian wild species (Chrysocyon brachyurus-maned wolf, Cerdocyon thous-crab-eating fox, Mazama gouazoubira-brown brocket deer). The effects of two cryoprotectants, 10% dimethyl sulfoxide (DMSO) and 5% dimethylformamide (DMF), were assessed to determine the most efficient cryopreservation protocol. Such an investigation promotes the creation of germplasm banks, using samples that would otherwise be rejected and permanently lost following the death of the animals. We utilized animal corpses that were involved in highway accidents, found dead in the natural environment, or referred to us from the veterinary hospital at the Brasília Zoo. Fibroblasts from C. brachyurus specimens presented a delay in cell growth in Dulbecco's modified Eagle's medium in relation to other species. This observation is a limiting factor for the future storage of cells from this species. Differences in cellular morphology were observed between C. brachyurus, C. thous, and M. gouazoubira, presenting branched, fusiform, and spherical forms, respectively. The cryoprotective solution containing 10% DMSO was more efficient than 5% DMF medium in preserving the viability of fibroblasts of the three species (p < 0.05). After defining the best cryopreservation solution, a germplasm bank was successfully formed. This biological reservoir is configured as the first germplasm bank containing somatic cells and gametes of wild mammals of the Cerrado biome of Brazil. This material will be used for future characterization of the species and multiplication by means of nuclear transfer cloning.

Optimization of a sperm cryopreservation protocol for giant grouper (Epinephelus lanceolatus)

Characterized by the extremely fast growth performance, giant grouper (Epinephelus lanceolatus) is one of the most promising marine culture species in South East Asia. Motivated by the rapid expansion in grouper aquaculture, giant grouper semen remains in increasing demand for massive seed production, but which is often constrained by the shortage of broodstock and asynchronous availability of gametes for the bulk of farmers. Sperm cryopreservation has a great potential for increased flexibility and efficiency for artificial seed production in aquaculture. To develop an efficient and standardized cryopreservation protocol for giant grouper semen, some key factors were investigated in this study, including the effects of different extenders (MPRS, E2, ELRS3), freezing heights (3, 5, 7 and 9 cm) above the liquid nitrogen surface corresponding to different freezing rates, final sperm concentration in the straw (1–10 × 109 spermatozoa/mL), equilibration (0–180 min) and pre-freezing storage time (0–60 h). Moreover, the fertilizing ability of cryopreserved semen was tested at sperm:egg ratios ranging from 100:1 to 1× 106:1. With 10% DMSO as cryoprotectant, the glucose-based extender ELRS3 at freezing height 5–7 cm produced higher post-thaw sperm motility. The optimal sperm concentrations in straws ranged from 2 to 8 × 109 spermatozoa/mL. Besides, sperm motility after thawing was not affected by a 120-min equilibration, but a significant decrease was detected after 180 min of equilibration. Concerning pre-freezing storage, semen stored within 24 h after collection could be successfully cryopreserved, while extended storage time would compromise the cryopreserved sperm motility. Similar fertilization results were obtained at the range of 1000 to 1 × 106 spermatozoa per egg for cryopreserved semen. Compared with fresh semen, the fertilization capacity of thawed sperm was not affected by 120 min of post-thaw storage, although with reduced motility parameters. This standardized procedure will be useful for the implementation of cryopreserved giant grouper semen in hatchery practice.

Influence of ethylene glycol on Eucalyptus grandis cryopreservation using the V cryo-plate technique

The conservation of plant genetic resources is fundamental for the development of agriculture. The development of efficient cryopreservation protocols has become an effective tool to preserve cells, tissues and organs of different plant species. We sought here to develop an efficient method of cryopreservation of Eucalyptus grandis employing the V cryo-plate technique. This experiment examined the exposure of shoot tips to three different cryoprotectants (PVS2, PVS3, and PVS4). The cryoprotectants PVS2 and PVS4 proved to be more efficient among the cryoprotectants tested, as 44.4% of the shoot tips immersed in those solutions survived, and 31.1% generated new shoots. Ethylene glycol was found to be a key compound for the successful cryopreservation of shoot tips, in light of its low toxicity as compared to other cryoprotective compounds. Thus, the methodology developed here represents an effective method for the conservation of E. grandis germplasm through cryopreservation and the use of the V cryo-plate technique.

Determining the effect of pretreatments on freeze resistance and survival of cryopreserved temperate fruit tree dormant buds

Freeze resistance is critical to successful dormant bud (DB) cryopreservation, and is affected by genotype, environmental conditions, dormancy phase and processing techniques. Pretreatment induced freeze resistance may contribute to more successful and efficient protocols for cryopreserving DB. Differential thermal analysis (DTA) was used to quantify the effects of cryopreservation pretreatments on freeze resistance of dormant budwood. Low temperature exotherm (LTE) profiles created by DTA could rapidly identify pretreatments that are contributing to increased freeze resistance in tree fruit species. In this study, DTA was used to help elucidate the effects of varying pretreatments (sucrose, desiccation and their combination) on peach, a model crop in tree fruit physiology that has shown little cryosurvival using the DB method in the past. Post cryopreservation recovery trials using an antimicrobial forced bud development (AFBD) protocol evaluated the ability of selected pretreatments, that improved freeze resistance based on DTA, to improve recovery of dormant budwood of various deciduous tree fruit and nut species. Precryogenic exposure to sucrose solution (5.0 M, 96 h), desiccation to 30% moisture content (MC) and their combination tested for their efficacy on improving postcryogenic viability in peach, apricot, sweet cherry, little walnut, black walnut, English walnut, apple, and pear. Among the different pretreatments tested, desiccation to 30% MC had the greatest impact on increasing freeze resistance and cryosurvival across most fruit species tested and little walnut. Gradual reduction of MC (from 40 to 25%) levels increased freeze resistance in peach (R2=0.95) and increased some recovery outcomes (leaf, shoot and bud swell), however, this was not correlated with equal cryorecovery outcomes as severe bud cracking was observed. Overall, our approach linking freeze resistance and preconditioning treatments could help establish efficient species-specific cryopreservation protocols for a number of important temperate woody crops which could be recovered as complete plants by coupling AFBD and plant tissue culture.

Production of common carp donor-derived offspring from goldfish surrogate broodstock

Common carp (Cyprinus carpio) is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining a carp gene bank in situ can be demanding due to the large body size of the fish. Moreover, ex situ gene banking in common carp is as in other fish species limited to sperm cryopreservation. Recent progress in reproductive biotechnology in fishes has allowed the transfer of germ stem cells (gamete precursors) between individuals or even species. After maturation, the recipients are producing gametes of the donor. Surrogacy can serve as a valuable tool in germplasm banking to recover cryopreserved germ stem cells. Some unfavourable characteristics of the donor species such as long maturation time or large body size hampering its reproduction can be overcome by choosing a surrogate with more convenient characteristics. Efficient protocols for cryopreservation of common carp male and female germ stem cells have been recently developed in our laboratory. The next step has been to assess the potential of smaller goldfish (Carassius auratus) surrogates to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells.High transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp at the age of 3 years giving rise to viable progeny. Donor-derived identity of the offspring was confirmed by genotyping and the production of a typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control common carp and goldfish eggs. Interestingly, flagellum length in donor-derived spermatozoa was comparable to the common carp flagellum and significantly shorter than goldfish flagellum. Genotyping of the Y chromosome locus in chimeric goldfish provided a novel insight on the fate of transplanted cells, where only genotypic females were capable of producing eggs, while both genotypic males and females were capable of producing sperm. The present technology can be combined with cryopreservation procedures to ease needs for common carp breeds preservation and their recovery using many times smaller goldfish surrogates but also serve as a convenient model to study interspecific surrogacy in cyprinids.

Optimization of UC-MSCs cold-chain storage by minimizing temperature fluctuations using an automatic cryopreservation system

The effective long-term cryopreservation of human mesenchymal stem cells is an essential prerequisite step and represents a critical approach for their sustained supply in basic research, regenerative medicine, and tissue engineering applications. Off-the-shelf availability of human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. In the long-term low-temperature storage process of cells, traditional manual storage has a great impact on cell activity, recovery, and function due to repeated exposure of cells to room temperature. To minimize the effect of fluctuation in ambient temperature on stored cells, we designed an automatic cryopreservation system that handles cells under controlled temperatures. In this work, UC-MSCs were utilized to investigate and compare the influence of manual and automatic cryopreservation approaches. To simulate the manual process, the UC-MSCs were transferred back and forth repeatedly (up to 400 times) between the liquid nitrogen (LN2) tank (−150 °C) and room temperature by a robotic arm. Similarly, the UC-MSCs from the same batch were collected and transferred repeatedly between two storage units by the automatic cryopreservation system, where the cells were maintained below-150 °C throughout the cold chain process. Viability, percent recovery, adherence capability, cell proliferation, and multilineage differentiation ability were assessed for UC-MSCs. Compared to the manual approach, UC-MSCs handled by the automatic system demonstrated higher viability, percent recovery, and cell proliferation, as well as improved adherence to culture plate with greater potential in multilineage differentiation after 400 temperature cycles. The described entire cold chain system may provide a powerful tool to develop safe, reliable and efficient protocols for manufacturing and banking of UC-MSCs, improving their off-the-shelf availability for regenerative medicine applications.

The Evolution of the Cryopreservation Techniques in Reproductive Medicine—Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Nowadays, cryopreservation of gametes and embryos is a fundamental, integral, and indispensable part of infertility treatment or fertility preservation. Cryopreservation is not only needed for the policy of single embryo transfer and cryopreservation of surplus embryos, but for deferring embryo transfer in the case of ovarian hyperstimulation syndrome, uterine pathologies, and suboptimal endometrium built-up or when preimplantation genetic testing is needed. Several current strategies in assisted reproduction technology (ART) would be inconceivable without highly efficient cryopreservation protocols. Nevertheless, cryopreservation hampered for a long time, especially in terms of low survival rates after freezing and thawing. Only the technical progress during the last decades, namely, in regard to the implementation and advancement of vitrification, leveraged its application, and thus, even allows the cryopreservation of human oocytes—a process that is far from being easy. This review aims to provide a deeper insight into the physical processes of cryopreservation and to explore the character of the vitrified state in the extra and intracellular milieu in order to demonstrate that the common denominator to all cryopreservation procedures is the establishment of an intracellular amorphous condition that hinders the likelihood of crystallization.

Criopreservación de semen canino (Canis familiaris) en pajilla francesa en el municipio de Medellín, Antioquia

El objetivo de este trabajo fue aplicar la metodología de criopreservación de semen canino en pajilla francesa en el municipio de Medellín- Antioquia. Se realizó una colecta seminal a ocho caninos adultos, por método de mano enguantada, cada eyaculado fue evaluado macroscópica y microscópicamente y posteriormente diluido con Triladyl® a una concentración final de 80 x 106 espermatozoides/ml. Se empacaron en pajillas de 0,5 ml y fueron sometidas a criopreservación con un descenso de temperatura rápido, sometiendo las pajillas a vapores de nitrógeno durante 25 minutos y luego transferidas directamente a nitrógeno líquido (método de pajilla francesa). En el semen fresco, se observó una apariencia blanquecina lechosa, un volumen de 2.27 ml (±1.40) una concentración de 388.5 x106 espermatozoides/ mL (±228.069), una motilidad individual de 79% (± 4%) y un vigor de 3.96 (±0.327). Se obtuvieron 123 pajillas, de las cuales se tomaron 27 aleatoriamente y se les evaluó la motilidad individual (51 ± 19%) y vigor (2.89 ± 1.02). En conclusión, la aplicación de la metodología de criopreservación rápida por pajilla francesa, usando Triladyl® como crioprotector, se sugiere como protocolo eficiente para la criopreservación de semen canino en el municipio de Medellín.

Criopreservação: uma ferramenta para conservação de recursos genéticos de videira

Abstract – Plant genetic resources conservation is essential for the development of agriculture. The development of efficient cryopreservation protocols has become an effective tool to conserve vegetatively propagated plant species. Traditionally, genetic resources have been maintained primarily in field collections. As field collections, Vitis accessions are vulnerable to abiotic stresses and biotic threats, such as pathogens and pests. Thus, the availability of robust cryopreservation methods is an opportunity to preserve collections for long periods, safely and with low maintenance costs. Several cryopreservation methods have been described for grapevines and, so far, droplet-vitrification has been the most effective for multiple Vitis species. This method seems to be promising to overcome species- and genotype-specific responses to a determined protocol, factors that have been bottlenecks for the widespread use of Vitis cryopreservation. This review article presents updated and relevant information on the use of cryopreservation as a complementary tool for the conservation of Vitis genetic resources.

An Easy Method for Cryopreservation of Zebrafish (Danio rerio) Sperm.

We developed an easy, efficient, and cheap protocol for zebrafish sperm cryopreservation carried out on dry ice (20 min) using simple composition solution (200 mM glucose, 40 mM KCl, 30 mM Tris, pH = 8.0). The average efficiency of the present cryopreserve method was between 10% and 20% (expressed as fertilization rate). The experiments were conducted and repeated at two different locations, in different countries, yielding very similar results, showing the reproducibility and applicability of the method.

Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Cryopreservation of somatic embryos from Petiveria alliacea L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.

DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival.

Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.

Evaluation of the Efficiency of Two Different Freezing Media and Two Different Protocols to Preserve Human Spermatozoa from Cryoinjury.

It is universally recognized that cryopreservation impairs sperm quality. In order to improve postthawing sperm survival and motility, media of different composition and different protocols have been proposed. However, no clear evidence is available to understand which are the most efficient protocol and medium for sperm cryopreservation. The present study evaluates the efficiency of two different cryopreservation protocols and two common freezing media (FM) containing different cryoprotectants (CPs), TEST Yolk Buffer (TYB) and Sperm Freeze (SF), to preserve human sperm quality. Our data suggest that TYB is better than SF both in terms of postthaw viability and in terms of progressive motility, while the direct addition of FM to the sperm sample resulted in the most efficient protocol in terms of postthaw viability but not in terms of progressive motility.

Comparative studies of mesenchymal stem cells derived from different cord tissue compartments – The influence of cryopreservation and growth media

IntroductionWe have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. MethodsUsing an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). ResultsExpression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. DiscussionOur efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200mM trehalose is an efficient cryopreservation protocol for bovine SSCs.