Cryopreservation Of Cell Lines Research Articles

Cryopreservation of Biologically Functional Submandibular Gland Rudiments from Fetal Mice.

Cryopreservation of cell lines has been widely used in the laboratory; however, cryopreservation of organs is still considered to be difficult. The submandibular gland (SMG) of fetal mice is one of the best-characterized organs. We investigated the conditions for cryopreserving SMG rudiments. Embryonic day 13 SMG rudiments were cryopreserved with or without a cryoprotectant. They were thawed and incubated in DMEM/F12 medium. Moreover, the influence of EGF stimulation on the signaling cascade after frozen-thawing the rudiments was analyzed by Western blotting. When SMG rudiments were cryopreserved without a cryoprotectant, all cells in the rudiments died. However, the SMG rudiments that had been preserved in a cryoprotectant showed branching morphogenesis. Additionally, the responsiveness of signaling cascades to EGF did not differ between frozen with a cryoprotectant and non-frozen rudiments. Cryopreservation might be a useful technology for preserving tissues from small organs, such as fetal SMG rudiments.

Abstract 2532: High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation

Abstract Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by H&E-staining. Many human samples provide outstanding quality nuclei suspensions, including cryopreserved cell lines, PBMCs, bone marrow, liver, brain, breast, colon, lung, prostate and melanomas. Fixed and preserved specimens also yield nuclei readily interrogated by targeted DNA sequencing panels. Nuclei suspensions are readily processed with the Tapestri Platform for single-cell DNA analysis. Leveraging droplet microfluidics and barcoding, the workflow enables high uniformity of ~90%, low ADO of ~10%, and typical nuclei recovery rates exceed 10%. Up to 20,000 nuclei can be interrogated in each run with catalog or custom panels for any tumor type. Here, a 59-gene tumor hotspot panel was used to study five melanoma metastases. The single-cell data enabled the unique reconstruction of tumor sample clonal phylogeny unresolved by bulk analysis. Also, low prevalence metastatic subclones masked in bulk NGS data were detected in normal liver samples. In summary, we show that diverse types of archived tumor tissues are readily dissociated with this universal nuclei protocol. Researchers now have a highly sensitive, targeted, customizable solution for revealing genomic variation and clonal propagation in complex archived solid tumor samples. Citation Format: David Ruff, Pedro Mendez, Daniel Mendoza, Nianzhen Li, Adam Sciambi, Kaustubh Gokhale, Dalia Dhingra, Keith Jones, Dennis Eastburn. High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.

Optimization of an in Vitro CYP450 Induction Assay Using Cryopreserved Cell Lines

Cytochrome P450 (CYP450) enzymes are responsible for the majority for Phase I metabolism. Induction occurs when a drug increases the rate of CYP450 enzyme production. As a result, the rates of metabolism of subsequent drugs increase, posing concerns for dosing and safety. To further expand the USAMRICD Absorption, Distribution, Metabolism, Excretion, and Toxicity Center of Excellence (ADMET CoE) profiling capabilities, a CYP450 induction assay is being developed using alternative cell lines to primary human hepatocytes to measure changes in gene expression of CYP1A2 and CYP3A4 isoenzymes.Primary human hepatocytes are the gold standard for evaluating P450 induction in vitro; however, they have inherent limitations such as high costs, fastidious nature, and significant lot‐to‐lot variability. Induction assessments using cryopreserved hepatocytes and two immortalized cell lines, HepaRG and HepatoCells, are being conducted to determine a more robust, cost‐effective alternative model. Each cell line was exposed to the standard inducers rifampicin and omeprazole to assess CYP3A4 and CYP1A2 activity, respectively. Following a 72‐hour incubation period, RNA from the cell cultures was extracted and purified. Fold changes of gene expression were measured using real‐time quantitative PCR (RT‐qPCR). Hepatocytes displayed an average fold change of 92.7 for CYP3A4 and 49.6 for CYP1A2, whereas HepaRG displayed changes of 28.6 and 179.5, respectively. Average values for CYP3A4 and CYP1A2 for HepatoCells were 58.7 and 43.9. Based on the data, HepatoCells most closely mimicked cryopreserved hepatocytes in comparison to HepaRG cells. Additional data on assay parameters and technique optimization will be presented.Support or Funding InformationOpinion disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government. This project was generously supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. Support is also provided by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

39 Embryo Development of Kazakh Argali (Ovis ammon collium) by Handmade Cloning

Wildlife conservation requires innovative preservation methods in order to preserve gene and species biodiversity. Nuclear transfer has the potential to preserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where cryopreserved cell lines have been conserved in cryobanks. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of cryopreserved somatic cells from the Kazakh argali (Ovis ammon collium) to enucleated domestic sheep (Ovies aries) oocytes. Frozen-thawed fibroblasts were diluted with DMEM (1:5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL−1. Fibroblasts were placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and incubated at 5% CO2, 95% relative humidity, and 37°C. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s PBS + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300 × g for 10 min. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries. Subsequently, the cumulus cells were removed by pipetting in 1 mg mL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2 mg mL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1 min. Bisection was performed by hand under a stereomicroscope using a microblade in 5 μg mL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Fusions were performed 24 to 28 h after the start of maturation. One cytoplast was attached to one fibroblast in 500 μg mL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, and 0.01% polyvinyl alcohol), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a single DC pulse of 100V, each pulse for 9 μs. Successfully fused embryos were activated 1 h after the end of fusion by incubation in 2 μM calcium ionophore (Sigma, St. Louis, MO, USA) in T20 for 5 min followed by 3-h incubation in microdrops of culture medium containing 2 mM 6-DMAP. After successful reconstruction, 79 nuclear transferred and activated embryos were cultured in well-of-the-wells in trigas (5% O2, 5% CO2, 90% N2) in Submarine incubation system for 7 days. All except 15 embryos cleaved; 35 (44.3%) developed to compacted morula, and 15 (18.9%) to the blastocyst stage. In conclusion, argali embryos developed from reconstruction using their frozen–thawed fibroblasts combined with domestic sheep cytoplasts; however, in vitro developmental ability to the blastocyst stage was limited. Additional research that establishes the early embryo development with optimising nuclear transfer techniques may have a potential role in the conservation of critically endangered wildlife species.

Erratum to: In vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P < 0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l−1 abscisic acid (ABA), 50 g l−1 maltose, and 100 g l−1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P = 0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.

Cryopreservation of canine cardiosphere-derived cells: Implications for clinical application.

The clinical application of cardiosphere-derived cells (CDCs) to treat cardiac disease has gained increasing interest over the past decade. Recent clinical trials confirm their regenerative capabilities, although much remains to be elucidated about their basic biology. To develop this new treatment modality, in a cost effective and standardized workflow, necessitates the creation of cryopreserved cell lines to facilitate access for cardiac patients requiring urgent therapy. Cryopreservation may however lead to alterations in cell behavior and potency. The aim of this study was to investigate the effect of cryopreservation on canine CDCs. CDCs and mesenchymal stem cells (MSCs) isolated from five dogs were characterized. CDCs demonstrated a population doubling time that was unchanged by cryopreservation (fresh vs. cryopreserved; 57.13 ± 5.27 h vs. 48.94 ± 9.55 h, P = 0.71). This was slower than for MSCs (30.46 h, P < 0.05). The ability to form clones, self-renew, and commit to multiple lineages was unaffected by cryopreservation. Cryopreserved CDCs formed larger cardiospheres compared to fresh cells (P < 0.0001). Fresh CDCs showed a high proportion of CD105+ (89.0% ± 4.98) and CD44+ (99.68% ± 0.13) cells with varying proportions of CD90+ (23.36% ± 9.78), CD34+ (7.18% ± 4.03) and c-Kit+ (13.17% ± 8.67) cells. CD45+ (0.015% ± 0.005) and CD29+ (2.92% ± 2.46) populations were negligible. Increasing passage number of fresh CDCs correlated with an increase in the proportion of CD34+ and a decrease in CD90+ cells (P = 0.003 and 0.03, respectively). Cryopreserved CDCs displayed increased CD34+ (P < 0.001) and decreased CD90+ cells (P = 0.042) when compared to fresh cells. Overall, our study shows that cryopreservation of canine CDCs is feasible without altering their stem characteristics, thereby facilitating their utilization for clinical trials. © 2017 International Society for Advancement of Cytometry.

In Vitro Cryopreservation of Date Palm Caulogenic Meristems.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulation-vitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.

Establishment and characteristics of Iranian Sistani cattle fibroblast bank: a way to genetic conservation

Cryopreservation of cell lines from endangered animals is an important approach to conserve genetic resources. In this study, ear fibroblasts from 51 Iranian Sistani cattle (Bos indicus) were cultured and cryopreserved in vitro, and their biological characteristics, proliferation and chromosomal features were analyzed. The results indicated that Sistani cattle fibroblasts could be successfully obtained by explant culture. The cultured cells were typical fibroblasts, with normal morphology and population doubling time of approximately 24 ± 0.5 h. Karyotyping showed that frequency of cells with chromosome number 2n = 60 was >95%. Bacterial, fungal, yeast and mycoplasma contaminations were negative. The efficiencies of VSV-G (pMDG) and lentiviral pCSGW vectors encoding fluorescent proteins were around 60%. Multiplex PCR for identification of species confirmed that Sistani cattle cell lines had no cross-contamination. The samples identification information was prepared and is available in this research. These established cell lines are valuable genetic pools from endangered Iranian Sistani cattle breed, and would be a source of many biological studies such as genomic, postgenomic and somatic cell cloning researches.

CRYOBANKING OF SOMATIC CELLS IN CONSERVATION OF ANIMAL GENETIC RESOURCES: PROSPECTS AND SUCCESSES (review)

Extinction of many species is irreversible and is a part of the natural evolution, but human activities have influenced this process, making it much faster comparing to speciation. According to FAO, approximately 20 % of the breeds of cattle, goats, pigs, horses and poultry in the world are currently at risk of disappearance, many have died in the past few years, as a result their genetic characteristics lost forever. The role of banks in the management of genetic resources and the conservation of endangered species is particularly noticeable in the last decade. Most cryobanks focus on the cryopreservation of gametes (primarily sperm) and embryos. Their main goal is to produce offspring using reproductive technologies, which include artificial insemination, in vitro fertilization and embryo transfer. The discovery of the phenomenon of reprogramming somatic cell nuclear allowed expanding the range of forms of biological material in programs for cryopreservation. Creating cryobanks of somatic cells as donors of nuclei for cloning considered an auxiliary instrument for the preservation and improvement of the gene pool of farm animals and poultry. To obtain viable cryopreserved cell lines very small amount of biopsy material, including that of dead animals, is sufficient, but such lines contain the complete genome and proteome. In contrast to germ cells, embryos and generative tissues, the cryopreserved somatic cells after repeated thawing are capable to regenerate, i.e. almost infinitely may serve as a source of biomaterial for use in assisted reproductive technologies and biological research, including retrospective reconstruction. Furthermore, due to the small size the somatic cells are more resistant to cryopreservation. This review also provides a brief description of the principles and history of cloning. The advantages of the use of different cell types as karyoplasts are discussed. In particular, almost all types of cells (e.g. embryonic cells, mammary cells, cumulus, granulosa, oviduct, liver, fibroblasts, white blood cells and embryonic stem cells) can be used for the production of cloned animals, but the cloning efficiency depends significantly on the type of cells. Aiming embryo development and birth of live offspring, the fetal fibroblasts as donors of nuclear material for cloning are most effective. Alternatively, the stem cells may be a source of the nuclei. Stem or progenitor cells (i.e., stem, determined to differentiate in specific type cells) are easier reprogrammed than terminally differentiated cells. Also when stem cells nuclei are used as karyoplasts the number of cloned embryos significantly increased. The advances in interspecific cloning as a strategy for restoration of rare and endangered species are discussed. Numerous examples show that somatic cells can be considered the most promising material for the recovery of animal genetic resources of different types. Particularly from 1997 to 2012 using the differentiated somatic cells domestic and wild animals of different species were obtained such as sheep, mice, cows, goats, pigs, guar, mouflon, domestic cat, rabbits, mule, horse, rat, wildcat, dog, banteng, ferret, wolves, buffalo, deer, mountain goat, camel, coyote. Cattle are still the leader in the production of cloned offspring with the efficacy 10 % on average, and in some cases up to 25 %, while for most other animals it does not exceed 1 %. Under controlled conditions in farms with good management, the productivity of clones should vary only within the remaining natural variability and mitochondrial genetic variability due to cloning technology.

КРИОБАНКИ СОМАТИЧЕСКИХ КЛЕТОК КАК ПЕРСПЕКТИВНЫЙ СПОСОБ СОХРАНЕНИЯ ГЕНЕТИЧЕСКИХ РЕСУРСОВ ЖИВОТНЫХ (обзор)

Extinction of many species is irreversible and is a part of the natural evolution, but human activities have influenced this process, making it much faster comparing to speciation. According to FAO, approximately 20 % of the breeds of cattle, goats, pigs, horses and poultry in the world are currently at risk of disappearance, many have died in the past few years, as a result their genetic characteristics lost forever. The role of banks in the management of genetic resources and the conservation of endangered species is particularly noticeable in the last decade. Most cryobanks focus on the cryopreservation of gametes (primarily sperm) and embryos. Their main goal is to produce offspring using reproductive technologies, which include artificial insemination, in vitro fertilization and embryo transfer. The discovery of the phenomenon of reprogramming somatic cell nuclear allowed expanding the range of forms of biological material in programs for cryopreservation. Creating cryobanks of somatic cells as donors of nuclei for cloning considered an auxiliary instrument for the preservation and improvement of the gene pool of farm animals and poultry. To obtain viable cryopreserved cell lines very small amount of biopsy material, including that of dead animals, is sufficient, but such lines contain the complete genome and proteome. In contrast to germ cells, embryos and generative tissues, the cryopreserved somatic cells after repeated thawing are capable to regenerate, i.e. almost infinitely may serve as a source of biomaterial for use in assisted reproductive technologies and biological research, including retrospective reconstruction. Furthermore, due to the small size the somatic cells are more resistant to cryopreservation. This review also provides a brief description of the principles and history of cloning. The advantages of the use of different cell types as karyoplasts are discussed. In particular, almost all types of cells (e.g. embryonic cells, mammary cells, cumulus, granulosa, oviduct, liver, fibroblasts, white blood cells and embryonic stem cells) can be used for the production of cloned animals, but the cloning efficiency depends significantly on the type of cells. Aiming embryo development and birth of live offspring, the fetal fibroblasts as donors of nuclear material for cloning are most effective. Alternatively, the stem cells may be a source of the nuclei. Stem or progenitor cells (i.e., stem, determined to differentiate in specific type cells) are easier reprogrammed than terminally differentiated cells. Also when stem cells nuclei are used as karyoplasts the number of cloned embryos significantly increased. The advances in interspecific cloning as a strategy for restoration of rare and endangered species are discussed. Numerous examples show that somatic cells can be considered the most promising material for the recovery of animal genetic resources of different types. Particularly from 1997 to 2012 using the differentiated somatic cells the domestic and wild animals of different species such as sheep, mice, cows, goats, pigs, guar, mouflon, domestic cat, rabbits, mule, horse, rat, wildcat, dog, banteng, ferret, wolves, buffalo, deer, mountain goat, camel, coyote were obtained. Cattle are still the leader in the production of cloned offspring with the efficacy 10 % on average, and in some cases up to 25 %, while for most other animals it does not exceed 1 %. Under controlled conditions in farms with good management, the productivity of clones should vary only within the remaining natural variability and mitochondrial genetic variability due to cloning technology.

Magnetic resonance diffusion and relaxation characterization of water in the unfrozen vein network in polycrystalline ice and its response to microbial metabolic products

Polycrystalline ice, as found in glaciers and the ice sheets of Antarctica, is a low porosity porous media consisting of a complicated and dynamic pore structure of liquid-filled intercrystalline veins within a solid ice matrix. In this work, Nuclear Magnetic Resonance measurements of relaxation rates and molecular diffusion, useful for probing pore structure and transport dynamics in porous systems, were used to physically characterize the unfrozen vein network structure in ice and its response to the presence of metabolic products produced by V3519-10, a cold tolerant microorganism isolated from the Vostok ice core. Recent research has found microorganisms that can remain viable and even metabolically active within icy environments at sub-zero temperatures. One potential mechanism of survival for V3519-10 is secretion of an extracellular ice binding protein that binds to the prism face of ice crystals and inhibits ice recrystallization, a coarsening process resulting in crystal growth with ice aging. Understanding the impact of ice binding activity on the bulk vein network structure in ice is important to modeling of frozen geophysical systems and in development of ice interacting proteins for biotechnology applications, such as cryopreservation of cell lines, and manufacturing processes in food sciences. Here, we present the first observations of recrystallization inhibition in low porosity ice containing V3519-10 extracellular protein extract as measured with Nuclear Magnetic Resonance and Magnetic Resonance Imaging.

Detection of bone marrow involved by nonhematopoietic neoplasms using flow cytometry

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Key words: Neoplasms, glandular and epithelial; Bone marrow neoplasms; Neoplasm invasiveness; Keratins; Flow cytometry

Ganglioside signatures of primary and nodal metastatic melanoma cell lines from the same patient

The primary cutaneous melanoma initially migrates to the regional lymph nodes (LNs). Human melanoma overexpresses gangliosides, the sialylglycosphingolipids. The ganglioside signatures may differ between primary and LN melanomas owing to the differences in the tumor microenvironments. The melanoma cells obtained from the primary and LN of the same patient might be useful to evaluate the above hypothesis. For this purpose, the cryopreserved cell lines from a primary cutaneous melanoma (IGR-39) and its nodal metastasis (IGR-37) from the same patient were used. We have also compared the ganglioside signatures of freshly obtained melanoma cells from primary, LN and organ metastases from different patients. Gangliosides were extracted, purified and identified by resorcinol and specific murine monoclonal antibodies. Comparison of the primary cell line with the nodal metastatic line obtained from the same patient distinctly showed the following features: (i) an increased production of gangliosides, (ii) O-acetylation of GM2 and GD3, (iii) an increased and altered O-acetylation of GD2 and (iv) possibly de-N-acetylation of GD3. These findings suggest that the nodal microenvironment might favor activation of O-acetyl-transferases capable of O-acetylating both alpha2, 3 and alpha2, 8 sialic acids of gangliosides. Supporting this, the primary melanoma cells obtained from different patients, showed no O-acetylation of GD3 or GD2. The cell line from groin LN showed the presence of O-acetyl (O-Ac)GD3. The cell lines from thyroid, spleen and jejunum expressed O-AcGD2. In all metastatic melanoma cell lines GD1a is more prevalent than GD3, suggesting that GD1a may be a major melanoma-ganglioside.

Brief history of the tissue bank, charles university hospital, hradec králové, czech republic.

A brief history of the Tissue Bank (TB) of the University Hospital Hradec Králové, Czech Republic, established by Dr. R. Klen in 1952 is presented. In Dr. Klen's original concept the TB was defined as a department specialised in the harvesting, processing, preservation, storage and distribution of various kinds of tissue for clinical and experimental practice. The first kinds of tissue collected in cadaveric donors were corneas, bone and skin. Xenogeneic cartilage and bone grafts were prepared at the same time. Later, preparation of soft connective tissues and chorion-amnion was introduced. During the first 15 years of activity a total of 11,443 grafts preserved by hypothermy at +4 degrees C or freezing in absence of cryoprotectants (-20 degrees C) were prepared. In the 60's freeze-drying of tissue grafts was introduced and the bank of cryopreserved cell lines was established. In the 80's cryopreservation of haematopoietic progenitor cells for clinical transplantations was started and the spectrum of tissue grafts was enlarged (xenogeneic pericardium and allogeneic specially treated dura mater for neurosurgical operations, pigskin for burn treatment, demineralised bone for parodontology and implantology). In the 90's human keratinocyte culture for treatment of burns and chronic skin defects was started. The human milk bank and organ bank co-operating with the Regional Transplantation Centre are component parts of the TB as well. The TB is an institutional member of the European Association of Tissue Banks and annually delivers approximately 1000 grafts that are used in University and county hospitals as well as in surgeons' private practices. Health insurance companies reimburse all grafts on a non-profit and tax-free basis.

Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica

The aim of the present study was to apply cryopreservation for the embryogenic cultures of open-pollinated Abies cephalonica, as well as to test the genetic fidelity of the cryopreserved cell lines using random amplified polymorphic DNAs, i.e. RAPD markers. Dimethylsulfoxide (DMSO) and two mixtures of polyethylene glycol, glucose and DMSO (PGD I and PGD II), were used as cryoprotectants and the cryoprotectant treatments were performed with and without storage in liquid nitrogen. All the tested cryopreservation methods, i.e. DMSO, PGD I or PDG II used as cryoprotectant combined with slow cooling and fast thawing, proved to be applicable for A. cephalonica SE cultures. The best results, measured as survival percentage and mean growth ratio, were achieved using the PGD I treatment. The RAPD assays performed revealed considerable genetic variation in the DMSO treated but non-frozen samples, i.e. 16.8% of the produced RAPD profiles showed intraclonal variation while background variation was seen in 1.7% of the control amplifications. The present results show that cryoprotectants may cause a risk for genetic fidelity of plant material and thus, monitoring it is important. This is particularly true in the breeding of the long-living conifers, since the effects of occasional mutations or genetic rearrangements may not be readily observed in young plants but expressed substantially later on in mature trees.

Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.)

A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15-21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.