Comparison of cryopreservation methods for the long term storage of the marine diatom Haslea ostrearia (simonsen)

Long term maintenance of microalgal strains by serial subculturing is often expensive and time-consuming. Alternative methods, such as cryopreservation, present several benefits and thus seem more relevant. Our study aimed at comparing two cryopreservation procedures applied to the marine diatom Haslea ostrearia (Simonsen): (1) a two-step freezing method in liquid media using 5%, 10% and 20% MeOH, Me₂SO or Glycerol, and (2) an immobilization-dehydration method consisting in an algal cell entrapped in 0.7 M sucrose dehydrated and air-flow desiccated calcium alginate beads before “direct” or “two-step” freezing. Our results showed that the cryopreservation of H. ostrearia was feasible. With the two-step freezing protocol only Me₂SO maintained cell viability without contamination but the low percentage of viability (<10%) prevents its use. Conversely, the immobilization-dehydration methods tested in this study were effective. Average viability of 57% and 77% were obtained with the “direct” and the “two step” cooling assays respectively, ensuring preservation of the genetic traits of H. ostrearia.     

Alligator mandibular development during long term organ culture

Summary The development of the first branchial (mandibular) arch of the American alligator (Alligator mississippiensis) is studied in organ culture for the first time. There is extensive mandibular morphogenesis in vitro and the pattern of the differentiated elements, bones, muscles, and cartilage, is comparable to that found during normal development in ovo. In addition, we observe the development of paired lingual swellings and the formation of a small tongue consisting of fibrous, lipid containing, and muscular tissues, as found in the normal tongue. Several culture variables are examined: (a) although alligator embryos normally develop at 30°C, we successfully culture the mandibular rudiments with good short term (3 wk) results at 37°CC; (b) at 30° C, we are able to culture arches for long term periods of 6 wk; (c) the mandibular arches develop well in both serum containing medium and in a chemically defined medium free from added hormones. The reptilian mandibular arches exhibit excellent, patterned, development and may have less stringent culture requirements than their avian and mammalian counterparts. Collection and specimen export was performed under U.S. Fish and Wildlife Permit PRT2-2511 and Northern Ireland Permit B/WL2/80 issued to M.W.J.F. Collaborative study was made possible by the 1981 award of a Research Travelling Scholarship to M.W.J.F. from The Wellcome Trust, for which we are grateful. This work is supported by Grant 8113610 CB from the MRC of Great Britain, Grant EP109/74/75 from the Northern Ireland Eastern Health & Social Services Board, and Grants DE-02848 and DE-03569 from the National Institutes of Health, Bethesda, MD.     

Radiosensitivity of human T-lymphocytes proliferating in long term culture

A method is described for determining the radiation sensitivity of dividing human T-lymphocytes in long-term culture. The results are fitted to a single-hit multi-target model. For cobalt-60 gamma radiation Do values of cells from five normal individuals range from 0.99 to 1.37 Gy with an overall Do of 1.09 Gy, and the extrapolation numbers range from 1.20 to 1.71 with an overall extrapolation number of 1.42.     

Ploidy variation of pronamide-treated maize calli during long term culture

Summary Anther-derived calli of corn were treated with 10 M pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r²=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.     

A procedure for Poitou jackass sperm cryopreservation

We have tried to establish sperm banking for the endangered Poitou donkeys. No successful cryopreservation technique had been described for spermatozoa of this species; our preliminary work indicated that a particular medium and procedure may be effective for cryopreservation of Poitou jackass spermatozoa as evaluated by sperm motility, membrane integrity and pregnancy rate after AI with frozen-thawed semen. We found that glutamine at 80 mM and 10% (v/v) quail egg yolk in a basal medium containing 4% (v/v) glycerol (T2-94 medium) improved the post-thaw total and progressive motility and velocity assessed with the automated analyzer ATS-M. The T2-94 medium also preserved the sperm nuclear, acrosom, and plasma membrane integrity as assessed with the acridine orange method, fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) lectin procedure, and hypo-osmotic swelling test, respectively. Semen frozen-thawed in T2-94 medium as used to artificially inseminate. 13 Poitou jennies from the beginning of estrus to ovulation during 4 cycles at a rate of one AI per day. Heigh pregnancies and 3 foals were obtained, but only when the glycerol was removed from sperm before AI. We conclude that the cryopreservation of Poitou jackass semen for sperm banking may succeed by using the T2-94 medium and removing the glycerol post-thaw, but before AI.     

Significance of freeze-drying in long term storage of date palm pollen

The authors attempt to determine the optimal conditions for maintaining long term viability of date palm pollen using a special method of freeze-drying. They have undertaken a systematic study of the viability over time of a sample of the same pollen stored and conserved under different conditions (freeze-drying, temperature, gaseous atmosphere and storage time).

The results are thoroughly analysed by means of different approaches of multiparametric modelisation. These numerical treatments permit us to:

Les auteurs ont déterminé, par une méthode particulière de lyophilisation, les conditions optimales permettant le maintien de la viabilité du pollen du palmier dattier (Phoenix dactylifera) à long terme. Une étude systématique a été réalisée sur des lots provenant d'un même échantillon de pollen conservé et stocké selon différentes conditions: (lyophilisation, température, atmosphère gazeuse et en fonction du temps de stockage).

Ils ont cherché à analyser de manière approfondie les résultats obtenus au moyen de plusieurs approches de modélisation multiparamétrique.

Ces traitements numériques ont permis:

1. demonstrate that such a phenomenon submits to the usual laws and can be modelled

2. propose a general approach for the study of the ageing of pollen with time and with respect to different conservation and storage conditions.

1. de démontrer qu'un tel phénomène obéit globalement à des lois statistiques simples, et qu'il est de ce fait modélisable

2. de proposer une approche plus générale du vieillissement des grains de pollen en fonction du temps et des conditions de conservation et de stockage.     

Preservation of functional integrity during long term storage of a biological membrane

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca²⁺-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca²⁺ transport, and coupling between Ca²⁺ transport and ATP utilization are maintained essentially intact for at least 110 days.     

Preservation of functional integrity during long term storage of a biological membrane

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca2+-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca2+ transport, and coupling between Ca2+ transport and ATP utilization are maintained essentially intact for at least 110 days.     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Rapid and stable propagation ofOrnithogalum umbellatum L. in long term culture

Callus cultures were raised from bulb scale segments ofOrinthogalum umbellatum L. (Liliaceae), on a Murashige and Skoog (1962) medium (MS) with 8 mg/l naphthaleneacetic acid (NAA). Bulbous shoots developed from calli after 2 months using MS medium with 2 mg/l NAA and 0.5 mg/l N⁶ - benzyladenine (BA). Shoots were also induced directly from scales of regenerated bulb used as secondary explants on MS medium supplemented with 0.5 mg/l BA. Shoots developed roots in 1/2 - strength MS medium. Regenerants multiplied rapidly in 1/2-MS liquid medium. Chromosome instability was reduced in callus grown on 2 mg/l NAA compared to callus grown on 8 mg/l NAA. Callus retained regeneration potential for 5 years in this modified MS medium. The chromosome analysis of regenerants dervied from callus, even from long term culture of 5 years, revealed only diploid cells with normal karyotype comprising 2n=46 chromosomes. Stable nature of callus and regenerants were further confirmed by cytophotometry. This procedure can be applied for securing stable regenerants on a mass scale inO. umbellatum.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Increased expression of keratin polypeptides in long term culture of adult rat hepatocytes.

Adult rat hepatocyte cultures showed the presence of albumin after 1 week of seeding. As the cultures aged, the cells commenced expressing alpha-foetoprotein and later keratin polypeptides 55 and 52 kD but ceased expressing albumin and alpha-foetoprotein. Enhanced expression of keratin polypeptides was confirmed by Western blot analysis in long term cultures. A possible mechanism for dedifferentiation of hepatocytes in culture is discussed.     

A high throughput system for long term application of intermittent cyclic hydrostatic pressure on cells in culture

The process of bone remodeling is governed by mechanical stresses and strains. Studies on the effects of mechanical stimulation on cell response are often difficult to compare as the nature of the stimuli and differences in parameters applied vary greatly. Experimental systems for the investigation of mechanical stimuli are mostly limited in throughput or flexibility and often the sum of several stimuli is applied. In this work, a flexible system that allows the investigation of cell response to isolated intermittent cyclic hydrostatic pressure (icHP) on a high throughput level is shown. Human bone derived cells were cultivated with or without mechanical stimulus in the presence or absence of chemical cues triggering osteogenesis for 7-10 days. Cell proliferation and osteogenic differentiation were evaluated by cell counting and immunohistochemical staining for bone alkaline phosphatase as well as collagen 1, respectively. In either medium, both cell proliferation and level of differentiation were increased when the cultures were mechanically stimulated. These initial results therefore qualify the present system for studies on the effects of isolated icHP on cell fate and encourage further investigations on the details behind the observed effects.     

长期施肥对红壤稻田氮储量的影响

稻田背景氮高是我国氮肥利用率低的主要原因之一,减少氮肥施用量对提高氮肥的农学利用率和缓解环境压力具有重要的意义。研究采用长期定位试验(19902006年)土壤全氮、稻谷产量等数据,分析施肥模式对稻田耕层土壤氮储量、氮肥农学利用率的影响,探讨在降低常规施用氮量的33.3%而不明显减产措施的可行性。结果表明:长期有机物质循环利用能显著提高耕层土壤全氮含量,氮储量与试验前相比平均提高了18.8%,仅施用化肥对土壤全氮含量没有显著影响,减量施肥处理(JS)对耕层土壤氮的积累效应一直优于仅施化肥的处理(NP、NPK)。17a的JS处理并没有降低稻谷产量,与常量NPK储量相比年际产量相对误差仅为3.2%,而输入N的农学利用率提高了12%。在半量稻草还田的条件下减少氮肥的施用量到180kg·hm-2是可行的,红壤稻田产量可维持在10t·hm-2左右。     

Simple procedure for sperm cryopreservation in the larvacean tunicate Oikopleura dioica

The larvacean tunicate Oikopleura dioica is an attractive organism for studies of the development, evolution, and physiology of chordates, showing considerable promise for genetic approaches given its short life cycle of five days. To facilitate future genetic studies, the development of protocols for the maintenance of individual strains is essential. Here we report a simple and practical protocol for the cryopreservation of sperm using liquid nitrogen (-196°C) and dimethyl sulfoxide (DMSO) as a protective agent. The quality of the frozen-thawed sperm was evaluated in terms of fertilizing ability and subsequent development of the fertilized eggs. We examined several parameters to optimize the efficiency of cryopreservation, such as the concentration of DMSO, the method for acclimation of sperm to DMSO before freezing, and for placing sperm in liquid nitrogen, as well as the pH of the seawater used in resuspending the thawed sperm. We confirmed that viable sperm were recovered after preservation for more than one year. In addition, mature animals, and even a subsequent generation, were obtained from eggs fertilized by the cryopreserved sperm. The present procedure seems to be simple and sufficiently practical for maintenance of future established lines of O. dioica using frozen sperm.