Cells In Liquid Nitrogen Research Articles

Potentiating cancer vaccination by adjuvant-loaded cryo-shocked tumor cells

Cancer vaccine holds vast promise in potentiating tumor immunotherapy. Here, we developed a simple cancer vaccine based on the liquid nitrogen-treated cells (LNT cells) that engineered by one-shot shocking of the live tumor cells in liquid nitrogen. In this vaccine, the obtained LNT cells served as both tumor antigens and delivery vehicles to load the adjuvant imiquimod (R837). This design could achieve efficient co-uptake of antigen/adjuvant by antigen presenting cells (APCs) and prolong in vivo retention of tumor antigens and adjuvants. This adjuvant-loaded LNT cells augmented in vivo antitumor responses and enhanced survival in melanoma-bearing mouse model compared with conventional whole-cell vaccine of the mixture of tumor lysis and adjuvant.

Immunosuppressive dead cell as lung-targeting vehicle and cytokine absorption material for cytokine storm attenuation of pneumonia.

Effectively controlling cytokine storm is important to reduce the mortality of severe pneumonia. In this work a bio-functional dead cell was engineered by one-time quick shock of live immune cells in liquid nitrogen, and the obtained immunosuppressive dead cell could server as both lung-targeting vehicle and cytokine absorption material. After loading the anti-inflammatory drugs of dexamethasone (DEX) and baicalin (BAI), the drug-loaded dead cell (DEX&BAI/Dead cell) could first passively target to the lung after intravenous administration and quickly release the drugs under high shearing stress of pulmonary capillaries, realizing drug enrichment in the lung. Then, the immunosuppressive dead cell acted as the camouflage of normal immune cells with various cytokine receptors exposing on their surface, to "capture" the cytokines and further reduce the state of inflammation. With above formulation design, a synergic anti-inflammatory effect between drugs and carrier could be achieved. In a lipopolysaccharide-induced pneumonia mice model, this system could calm down the cytokine storm with high efficacy and elongate the survival of mice.

Cryo-Shocked Cancer Cells as an Oncolytic Adenovirus Reservoir for Glioblastoma Immunotherapy

Glioblastoma is the most common type of primary brain tumor, which has a high recurrence rate and a high mortality rate. Immunotherapy shows promise in cancer therapy due to its capacity to manipulate the immune system to attack tumor cells with less toxic and durable immune responses. However, the low immunogenicity and limited immune cell infiltration in a glioblastoma lead to a weakened antitumor immune response, resulting in suboptimal therapeutic efficacy. A compelling solution is provided by oncolytic adenovirus (OAs), which can selectively replicate within tumor cells while simultaneously promoting antitumor immunity. Herein, we constructed an oncolytic adenovirus reservoir (OAR) by shocking OA-loaded tumor cells in liquid nitrogen to eliminate proliferation and pathogenicity. OARs showed sustained OAs release and effectively lysed tumor cells in vitro and in vivo. In a mouse intracranial glioblastoma model, OARs could efficiently induce dendritic cells' maturation, facilitate the tumor recruitment, and promote the infiltration of cytotoxic effector T lymphocytes via a single treatment, resulting in specific antitumor immune responses and long-term animal survival. Taken together, these results demonstrated that OAR is a promising synergistic therapeutic strategy for treating glioblastoma.

Biobanking of vegetable genetic resources by in vitro conservation and cryopreservation

Today, application of in vitro culture by means of slow growth storage of shoot cultures and cryopreservation of organs, tissues and cells in liquid nitrogen presents a remarkable strategic tool to support medium- and long-term conservation of plant genetic resources. Over the last 30 years, considerable progresses have been made in the development of both methods that are currently considered as ex situ conservation strategies, complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including strategically-important vegetables, such as garlic, artichoke, asparagus, cassava, Jerusalem artichoke, mint, potato, sweet potato, chicory, taro, thyme and yam. As a consequence, more than 45,000 accessions of vegetable crops are maintained in 22 genetic resources conservation centers (biobanks), located in 16 countries and 6 continents (Europe, Asia, Africa, Oceania, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but cryopreservation is also constantly growing, with almost 8300 vegetable accessions being stored in liquid nitrogen at − 196 °C.

Cryopreservation of feline red blood cells in liquid nitrogen using glycerol and hydroxyethyl starch.

The objective of this study was to evaluate the techniques and short-term effects of cryopreservation of feline red blood cells (RBCs) in liquid nitrogen using glycerol or hydroxyethyl starch (HES) as a cryoprotectant. Feline RBCs were manually mixed with either 20% glycerol or 12.5% HES and frozen for 24 h in liquid nitrogen. The samples were thawed and glycerolized samples were manually washed. Success of the freeze/thaw process was determined by recovery rate of RBCs and evaluation of morphological changes using scanning electron microscopy (SEM). A unit of canine packed RBCs was also subjected to the same methodology to evaluate the cryopreservation handling technique. Feline RBCs preserved with 20% glycerol had a high recovery rate (94.23 ± 1.25%) immediately after thawing. However, the majority of the cells were lost during the washing process, with a final packed cell volume of <1%. A recovery rate was unable to be assessed for samples preserved with HES owing to the high viscosity of the mixture. SEM revealed significant morphological changes after glycerol was added to the feline RBCs. Although these morphological changes were partially reversed after thawing, the majority of the RBCs were lost during the washing process. Minimal morphological changes were noted in the HES sample. Similar results were noted with the canine RBCs. The described manual technique for cryopreservation using glycerol was not able to successfully preserve feline or canine RBCs. In the present study, it was difficult to make conclusions about the efficacy of HES. Further studies evaluating HES as a cryoprotectant are warranted.

Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness.

This review describes 120years history of technology for cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It is presented an explanation why cryoprotectant-free vitrification for some human ejaculates is better than conventional freezing and vitrification with the presence of cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria and micoplasmas after cryoprotectant-free cryopreservation in culture medium and even in distilled water. This fact increases the potential risk of disease transmission through liquid nitrogen. It is concretized the concept "asepticity" as obvious parameter for any medical assisted reproduction technology which includes the cooling of cells in liquid nitrogen. It is described the role of nonpermeable compounds of mediums for cryoprotectant-free vitrification: carbohydrates, proteins, lipoproteins, antioxidants. This review summarizes concerned data regarding two groups of different current technologies for cryoprotectant-free vitrification of human spermatozoa: with direct contact of spermatozoa with liquid nitrogen as well as with full isolation of these cells from liquid nitrogen (aseptic technologies).

Mammalian Cell Culture

AbstractMammalian cell culture is the process of growing animal cells in vitro in a flask or dish. This unit describes the methods, equipment, supplies, and reagents used in a cell culture laboratory. Because preventing contamination and maintaining purity of cell cultures is arguably the greatest challenge in animal cell culture, the principles of aseptic technique in mammalian cell culture are presented in detail. Cell culture media and additives are discussed. A method of counting cells and assessing their viability with a hemacytometer is illustrated. The steps needed to maintain an adherent cell line by feeding and passing cells from one culture vessel to another are presented, and variations for passing suspension‐grown cells are also outlined. Long‐term cryogenic storage of cells in liquid nitrogen is discussed and a protocol for freezing cells is provided, as are details for thawing and recovering viable cells from frozen stocks. © 2015 by John Wiley &amp; Sons, Inc.

High temperature is essential for preserved human sperm function during the devitrification process.

Sperm vitrification is a cryopreservation method based on high-speed freezing by direct exposure of cells in liquid nitrogen (N2L), thereby avoiding the traditional cooling curves of freezing. The objective of this work was to determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilisation potential. Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures for 5 and 10 s at 38, 40 and 42 °C. Sperm motility was evaluated by the CASA system and the sperm membrane function by HOST test. It was detected that progressive motility of sperm warmed at 38, 40 and 42 °C was 26.4 ± 8.4%; 56.6 ± 16.3% and 65.4 ± 15%, respectively, with statistically significant differences between the temperatures of 38 and 40 °C and 38 and 42 °C (P < 0.05). The plasma membrane function evaluated by HOST test was better preserved at 42 °C (76.3 ± 2.0%) compared to 40 °C (43 ± 2%) and 38 °C (65.6 ± 1.5%). The temperature in the thawing process can affect the motility and plasma membrane integrity and function. The warming at 42 °C for thawed vitrified sperm is the optimum temperature to preserve the sperm physiological parameters.

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

Plant root is an organ composed of multiple cell types with different functions. This multicellular complexity limits our understanding of root biology because -omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e., uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes), the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous treatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000~mg of plant cells from 60 root systems. Using soybean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soybean root hair specific between preparations of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach.

22. Long-termed cryopreservation of bone marrow stem cells, peripheral lymphocytes, and umbilical cord cells in liquid nitrogen since 1972: Part-2

This study was performed to confirm viability and differentiation in culture of bone marrow cells cryopreserved since 1972. Materials and Methods were presented previously as part 1. Bone marrow cells from 293 advanced cancer patients were collected and frozen in 1972 for allogeneic and after 1982 for autologous transplantation. Containers with bone marrow which were not re-infused into patients were continuously preserved in a liquid nitrogen stock.

164 - Isolation, Proliferation, and Phenotyping of Cancer Stem Cells From Primary Breast Cancers: A Model for Evaluating the Response to Anti-Tumor Drugs

ABSTRACT Background Recent years have witnessed an increasing interest in the role of cancer stem cells (CSCs) because of selfrenewal, promoting metastasis, and resisting radio- and chemotherapies. In addition, the high levels of CSCs in tumor mass were linked to a poor prognosis, relapse and metastasis. For the former reasons SCS is currently a main focus of cancer research. Objective This study aims at establishing a CSC cellular model, which might enable the testing of new chemotherapeutics and/or combinatorial therapies for currently used agents. Material and methods We culture tumor cells obtained from primary breast tumors of Syrian patients. This was followed by isolating stem cell population existing in the primary tumor by means of serial dilution of cells, and the characterization of these cells by flow cytometry based on their morphology, surface antigen profile (CD44 + /high/CD24-/low), and their growth and enrichment in both adherent and non-adherent conditions. Finally, the viability of CSCs was tested after freezing cells in liquid nitrogen. Results and discussion In this study, we were able to isolate CSCs from primary breast tumor with high purity exceeding 90%, and we showed their CSC characteristics, which included: i) CD44high/CD24-/low profile, ii) their ability to form tumorspheres in non-adherent/suspension conditions, iii) and their ability of resisting chemotherapeutic agents in comparison with non-purified/non-stem breast tumor cells. Conclusions Our results confirm the establishment of a cellular model representing the characteristics of cancer stem cell population. This breast CSC model could be useful in conducting research enabling the identification of responsible mechanisms behind breast cancer resistance to chemotherapeutics. In addition, this model might enable the examining of new combinatorial therapeutics by evaluating the effects of drug combinations targeting the different and heterogeneous cell populations inside breast tumors, both tumor non-stem as well as tumor stem cells, thus, achieving the desirable therapy outcomes. Disclosure All authors have declared no conflicts of interest.

Breeding or Assisted Reproduction? Relevance of the Horse Model Applied to the Conservation of Endangered Equids

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

BackgroundStudies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.FindingsHere we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.ConclusionsWe have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

Mammalian Cell Culture

AbstractMammalian cell culture is the process of growing animal cells in vitro in a flask or dish. This unit describes the methods, equipment, supplies, and reagents used in a cell culture laboratory. Because preventing contamination and maintaining purity of cell cultures is arguably the greatest challenge in animal cell culture, the principles of aseptic technique in mammalian cell culture are presented in detail. Cell culture media and additives are discussed. A method of counting cells and assessing their viability with a hemacytometer is illustrated. The steps needed to maintain an adherent cell line by feeding and passing cells from one culture vessel to another are presented and variations for passing suspension‐grown cells are also outlined. Long‐term cryogenic storage of cells in liquid nitrogen is discussed and a protocol for freezing cells is provided, as are details for thawing and recovering viable cells from frozen stocks. Curr. Protoc. Essential Lab. Tech. 5:4.3.1‐4.3.32. © 2011 by John Wiley &amp; Sons, Inc.

Sequestration at the cell wall and plasma membrane facilitates zinc tolerance in the moss Pohlia drummondii

Bryophytes are, evolutionarily, between the green algae and flowering plants that lack proper vascular and root systems as well as a protecting cuticle on the gametophyte. Bryophytes are sensitive to environmental stress factors and are widely used as bioindicators. However, bryophytes also show a high physiological flexibility and are able to inhabit ecological niches that range from highly irradiated peat bogs to salt lakes and metal-contaminated sites. The moss Pohlia drummondii lives on metal-contaminated habitats in Austria that contain high amounts of copper, iron and zinc. Zinc and copper are essential micronutrients for plants but are harmful if they are applied at high doses. Our experiments showed that P. drummondii prospered in vitro on solid media spiked with 1 mM ZnEDTA and 10 mM ZnEDTA. Using energy dispersive X-ray microanalysis (EDX), we detected the uptake and accumulation of zinc from the medium into the gametophyte, but the cellular location of the metal remained unclear. After breaking open the cells in liquid nitrogen, we tried to differentiate between the cell wall and the inside of the vacuole, which were both putative sites for heavy metal sequestration. However, we could not detect a significant difference between the two fractions. The zinc-specific fluorescent dye FluoZin™-3, which was applied to plant cells for the first time, produced more satisfying results. The cell walls of P. drummondii grown on both zinc concentrations showed bright fluorescent labelling, whereas the control cells showed no staining. Plasmolysis of the zinc-treated cells indicated that the dye entered the preplasmolytic space but did not pass the plasma membrane of the leaf cells. From these results, we propose that the cell walls of P. drummondii together with the plasma membrane formed a functional barrier to protect the cytoplasm from harmful doses of zinc, thereby providing an effective mechanism for P. drummondii to tolerate metal contamination and survive on zinc-loaded soils.

Fabrication of 3‐Dimensional Tissue Models to Study Head and Neck Squamous Cell Carcinoma

Methods: Primary HNSCC tissue was obtained from patients at biopsy or resection, transported to the lab in culture medium (Fig. 1) on ice, then rinsed in 70% alcohol (Fig. 2) and three washes of PBS (Fig. 3). Each specimen was then cut in less than 5mm3 segments (Fig. 4), placing half the specimen in 2D monolayer culture (Fig. 5) and banking some of the cells in liquid nitrogen while the other half was formalin fixed for routine pathologic analysis. After reaching confluence (Fig. 7), the 2D tumor associated fibroblasts were trypsinized (Fig. 8) and then transferred to a contracted collagen matrix in order to generate 3D tissues by growing cells at an air-liquid interface. Once grown, the tissues are being tested for cytokines/biomarkers that are believed to play a role in the pathogenesis of HNSCC.

ES Cell Extract‐Induced Expression of Pluripotent Factors in Somatic Cells

Reprogramming of somatic cells was induced by ES cell-free extract. The system relied on the transient uptake of regulatory components from a nuclear and cytoplasmic extract derived from ES cells by the nucleus of a reversibly permeabilized NIH3T3 cell. NIH3T3 cells were permeabilized by streptolysin O (SLO). Reprogramming cell-free extracts were prepared by repeatedly freezing and thawing ES cells in liquid nitrogen. After incubation in the extract for 1 hr, permeabilized NIH3T3 cells were resealed by CaCl(2) and continually cultured for weeks to assess expression of ES cell specific markers. As we observed using FACS and fluorescence microscope, the optimal SLO concentration for permeabilizing NIH3T3 cells was 25 U. After 2 weeks of culture, the treated NIH3T3 cells began to express Nanog, c-Myc, Klf4, and 6 weeks later Oct4 was detectable. However, Sox2 was detected only after 8 weeks of culture. Differentiated somatic cells could be reprogrammed in ES extract in vitro, which provides a new approach to decreasing differentiation levels in somatic cells without disturbing the DNA sequences.

Intercellular adhesion molecule-1 and gelatinase expression in human peritoneal mesothelial cells during propagation in culture

Mesothelial cells are involved in a variety of biological processes, which include the formation of peritoneal adhesions. The cultures of human peritoneal mesothelial cells comprise an important tool to investigate the behavior and functions of mesothelial cells. Very little is known about the differences among mesothelial cells isolated from different sources and about the changes in specific functions as caused by cell propagation in vitro or that result from storage of cells at low temperatures. This study aims to characterize 2 particular cellular activities relevant for tissue repair, which include the expression of intercellular adhesion molecule-1 (ICAM-1) and the gelatinase activity; in addition, this study will assess the effect of hyaluronan, which is an antiadhesive agent, on these cellular activities. Viable cell lines were established from both omentum and peritoneal lavage fluid from 7 patients. Both ICAM-1 expression, which was measured by enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinase (MMP) bioactivity, which was measured by zymography, were measured in the 2nd and 4th passage; the latter also was measured after freezing and storing of cells in liquid nitrogen. The effects of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), phorbol myristate acetate (PMA), and hyaluronan were analyzed. ICAM-1 was constitutively expressed and stimulated by IL-1beta, TNF-alpha, and PMA. All cell lines produced both MMP-2 and MMP-9. Only the latter activity was affected by TNF-alpha and, especially so, PMA. Differences were found between the 2nd and 4th passage, as well as between cells of different lineage, mostly so if the relative stimulation by the various agents was compared. The addition of sodium hyaluronate either to control cultures or to cultures together with any of the 3 stimuli examined did not significantly change either ICAM-1 expression or gelatinase activity. The freezing and storage of cells did not affect their functions. Both the human omentum and peritoneal lavage fluid are good sources to establish mesothelial cell lines, which can be propagated also after freezing without qualitative changes in their ability to express ICAM-1 and produce the gelatinases. For omental cells, a differential effect of stimulation occurs depending on whether the cells have been passaged 2 or 4 times. The presence of hyaluronan did not affect the expression of ICAM-1 or the gelatinases.

Autologous transplant of hematopoietic cells: Results from cryopreservation in Sanatorio Allende from Cordoba

17530 Background: Preservation of hematopoietic cells (HC) is usually achieved by storing cells in liquid nitrogen at - 1960C (Cryopreservation) (CP). We aim to: observe how many leukapheresis (LK) were necessary in order to obtain the minimal number (n°) of HC (CD34+/kg) for a proper engraftment (EG); correlate the n° of cryopreservated and infused cells with the time of EG; determine if the time of CP at -800C influences the time of EG; determine if the pre-transplant (PT) condition (partial or complete remission) plays a role in the time of EG and/or the time of hospitalization of the patients (pts). Methods: This was a retrospective study. We included all pts that received autologous transplant which HC after CP in our institution between September 2004 and August 2006. Qualitative results were analyzed by Fischer exact test and relation between qualitative and quantitative results by Mann-Whitney/Wilcoxon two- sample test (Kruskal-Wallis test for two groups). Results: 27 pts were included (N=27); the average age was 48.3± 16.6 years with a range of 64 (4–68) years. Diagnosis included: Multiple Myeloma 40%, NHL 22%, Hodgkin disease 15%, AML 15%, Amyloidosis 4%, and Ependymoma 4%. In 66.7% of the pts only one round of LK was needed for recollection. The CP time at -800C had a range of 113 (8–122) days. The median time for EG was 11 days for neutrophils (nt) and 17 days for platelets (pl). There was not significant correlation between PT condition and: n° of nt before or after day 11 (p=0.515); n° of pl before or after day 17 (p=0.972); hospitalization time (p=0.55); time for EG of nt (p-0.553) and pl (p=0.936). There was a significant difference between receiving =5x106 CD34+/Kg and pl EG time before day 17 (p=0.01) but not for nt EG time (p=3.83). CP time had no significant difference in relation to the pl EG before or after day 17 (p=0.08) or nt EG before or after day 11 (p=0.49). Conclusions: The majority of pts had a proper CD34+/Kg count with one LK round. Doses =5x106 CD34+/Kg were associated with less time for pl EG. The storage of HC at -800C showed to be safe for periods between one week and 4 months. The time of storage at -800C did not seem to affect the time of EG but showed a trend towards slowing pl recovery. The PT condition did not correlate with the time for EG or the hospitalization time. No significant financial relationships to disclose.