Death of Lactobacillus bulgaricus Resulting from Liquid Nitrogen Freezing

Concentrated cell suspensions of Lactobacillus bulgaricus prepared from cells grown in semisynthetic media were frozen in liquid nitrogen. After storage for 24 hr, the cell suspensions were found to have decreased colony counts and acid-producing capacity in milk. The amount of loss varied among the different strains tested. The addition of known cryoprotective agents to cell suspensions of the most labile strain before freezing provided little or no protection to the cells. However, storage stability of all strains investigated was improved by supplementing the growth medium with Tween 80 (polyoxyethylene sorbitan monooleate). The concentration of Tween 80 necessary for maximal storage stability varied among strains.     

The use of freeze-preserved treponemes in the Treponema pallidum immobilization test

Treponema pallidum extracted from rabbit testes and quick frozen in liquid nitrogen were vigorously motile upon thawing, when cryoprotected with dimethyl sulfoxide (DMSO). However, this chemical was toxic to unfrozen treponemes unless normal serum, 10–20% final concentration, was added to suspensions before freezing, or immediately after thawing. Such organisms survived the in vitro incubation of the T. pallidum immobilization (TPI) test, but DMSO partially inhibited immune immobilization. This inhibition was concentration dependent and was not observed in treponeme preparations diluted fourfold with fresh medium after 10% DMSO storage. Frozen treponeme suspensions used in this fashion yielded TPI test results comparable to those of fresh suspensions, and were still satisfactory after 12 months storage.     

Differences in the in vitro growth pattern of fresh and cryopreserved granulo-monopoietic precursors

A study of the in vitro growth model of human granulo-monopoietic precursors (CFU-GM) before and after cryopreservation using both leukocyte feeder layers and GCT conditioned medium as the source of colony stimulating activity (CSA) is reported. The number of colonies produced with fresh cells was linearly related to the amount of marrow seeded with both CSA sources, whereas after cryopreservation this was true with feeder layers, and with GCT only at relatively high cell concentrations. This might indicate the production of granulopoietic stimulators on the part of a second population that is at least partly resistant to freezing. It seems more likely, however, that these results depend mainly on a sublethal damage to CFU-GM induced by freezing, thus making the cells hyporesponsive to some forms of CSA, such as those contained in GCT conditioned medium.     

Recovery of CFU-GM after freezing of normal human bone marrow: effect of three dilution techniques after thawing

In the cryopreservation procedures intended for autotransplantation of human bone marrow a controversial point is represented by the methods of reconstruction of the cellular suspension after thawing and before infusion into the patient. To evaluate how the dilution rate after thawing affects bone marrow viability, we cryopreserved the bone marrow from 16 hematologically normal patients in DMSO at a concentration of 10%. After thawing, the cells were diluted according to three different techniques and their viability was tested by the growth of CFU-GM in methylcellulose. The average recovery of CFU-GM, in comparison with that of fresh cells, was satisfactory and not affected by the type of dilution. In conclusion, if we accept that the resistance to osmotic stress due to the cryoprotectant is similar for stem cells and CFU-GM, we can maintain that a slow, gradual dilution is not a necessary condition to assume the staminality of bone marrow designed for autotransplantation.     

Does storage affect epifluorescence microscopic counts of total bacteria in freshwater samples?

Preservation with formalin and storage at 4 degrees C of freshwater samples, associated with sonication before counting, proved to be efficient for a few days of storage (no differences with the initial cell count within 7 d of storage); after 8 months, 95% of the initial count was preserved. This procedure always gave higher counts than those obtained from the freezing technique of filter storage at -18 degrees C after sample filtration.     

Freeze Preservation of Somatic Embryos and Clonal Plantlets of Carrot (Daucus carota L)

Cell suspensions of carrot (Daucus carota L.) can be cryopreserved by slow freezing (about 2 C per minute) in medium containing dimethylsulfoxide as a cryoprotectant. After storage in liquid nitrogen and thawing they demonstrate a high viability and are able to resume growth. Such a method entirely fails to preserve clonal plantlets; somatic embryos cease organized development at the time of freezing and recover growth only by secondary embryogenesis. Modification of the procedure, involving the removal of superficial moisture from cryoprotectant-treated embryos and plantlets and enclosing them in a foil envelope before freezing, greatly improves their survival potential. The use of dimethylsulfoxide at levels between 2.5 and 20% (v/v) and freezing at rates between 1 and 5 C per minute yielded viable preparations under appropriate thawing conditions. In general, treatments which increased tissue dehydration before or during freezing were most successful when followed by relatively slow thawing. Conversely where dehydration to a lesser degree was achieved, more rapid thawing was advantageous. Postthawing washing or inoculation into liquid media was inhibitory to recovery. On semisolid regrowth medium, somatic embryos resumed normal development, whereas in plantlets the root and shoot meristem regions gave rise to new growth. In both cases, inclusion of activated charcoal in the medium promoted organized growth.     

A banking strategy toward customized therapy in breast cancer

In breast cancer, various clinical parameters are assessed to define clinical stage and thus obtain a more accurate prognosis. However, banks of tumor tissues are an important source of material for studies of risk of recurrence and of features governing clinical outcome in breast cancer. Although the heterogeneous characteristics of individual tumors, subtle phenotypes and stem cells can only be identified in viable cells, tissue banks often give low priority to the preservation of living cells because it is labor-intensive and expensive. The present study was designed to evaluate the feasibility of introducing, within the routine procedures of tissue preservation, a cryopreservation protocol that allows the recovery of living cells after storage. We analyzed the effect of storage time on cell viability, growth rates, and protein expression of ten human breast cancer specimens subjected to various cryopreservation techniques. Cryopreservation of cancer tissue specimens for 12 months allowed protein characterization but not the recovery of living cells. Here we show that enzymatic digestion immediately before slow freezing, and storage in liquid nitrogen permits the recovery and expansion of living cells that can be tailored to specific requirements and projects.     

Long-term preservation of canine bone marrow: in vitro studies.

In vitro studies were performed on canine bone marrow frozen with DMSO and stored in liquid nitrogen for 2 to 6 months. The results are compared with previously reported parallel in vivo experiments that demonstrated no loss of stem cells. When studies were performed immediately after thawing, there was no substantial drop in the count of nucleated cells and, except for megakaryocytes, there was no alteration of the bone marrow morphology. After two washes, and removal of DMSO, the nucleated cell count dropped to 50% of its previous value. Optic and electron microscopy showed severe damage in mature myeloid elements. In some instances, the cells had a condensed nucleus similar to the red-purple inclusion body of LE cells (as observed in systemic lupus erythematosus), and electron microscopy showed heavy chromatin clumping. On the other hand, both optic and electron microscopy showed a good preservation of lymphocytes, plasmocytes, and erythroid precursors. Two-hour DNA synthesis slightly dropped after storage, and this drop appeared more consistent when related to a constant volume of bone marrow (50 microliters) rather than to a constant number of nucleated cells (10(6)). In five instances frozen and thawed bone marrow was grown in short-term cultures, and analysis of 98 metaphases showed no major aberrations of the chromosomes and only 2% of minor aberrations, such as breakages and fragments. These data, compared with the results of previous in vivo experiments that showed no loss of stem cells after 5 months storage, suggest that stem cells are less sensitive to freezing and thawing injury than myeloid elements and/or that it might be safer for the thawed bone marrow not to be manipulated before infusion.     

Germination and Food Reserves in Bambarra Groundnut Seeds (Voandzeia subterranea Thouars) after Different Periods of Storage

Studies were made on bambarranut seeds (Voandzeia subterraneaThouars) after 0, 6, 12, 18 and 24 months of storage in gunnybags under laboratory conditions (25–35 °C). Seeddeterioration during storage was indicated by delayed germination,reduced germinability, reduced growth of seedlings and increasednumber of stunted seedlings culminating in a total failure ofgermination after two years. Slight depletion of food reserves occurred during seed storage.The loss in fat was higher than starch or protein. Total solublesugars decreased while the content of total fatty acids andamino acids and soluble protein increased. Total nitrogen (N)remained unaffected while soluble-N and amino-N increased. Allthese components showed a rapid change (increase or decrease)from 12 months to 18 months of storage which was associatedwith commencement of rapid decline in germinability of the seedsand growth of the seedlings. Initial rapid imbibition of water was observed in viable aswell as non-viable seeds, though at a higher rate in the latterand followed by a lag period in both. At the end of 24 h ofimbibition, water content in non-viable seeds was less thanthat in viable ones. Key words: Voandzeia subterranea, Seed germination, Seed storage     

Batch cultivation of Xanthobacter Py2 on 1-pentene

Summary The propene isolated strain Xanthobacter Py2 was able to grow on 1-pentene. The biomass yield for growth under 1-pentene limiting conditions was 0.48 (Ceq/Ceq). Upon storage at both +4°C and -20°C no loss of enzymatic epoxide degrading activity in resting cell suspensions was observed after a month. However, activity decay was pronounced during the stationary phase of growth as well as under reaction conditions.     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

Liquid Nitrogen Freezing in Microbiological Assay Systems: I. Preservation of Lactobacillus leichmannii for Direct Use in the Vitamin B12 Assay

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B₁₂ assays. Complete recovery of viable cells was obtained when the suspensions in basal B₁₂ medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B₁₂ assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.     

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34⁺ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34⁺ cell viabilities before freezing were 99.8 ± 0.5% and after thawing were 99.8 ± 0.4% in the large bag compartments and 99.7 ± 0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34⁺ cells, CFU-GM, and total CFU were 94.8 ± 16.0%, 99.3 ± 18.6%, 103.9 ± 20.6%, and 94.3 ± 12.5%, respectively in the large compartments, and 95.8 ± 25.9%, 106.8 ± 23.9%, 101.3 ± 23.3%, and 93.8 ± 19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.     

Recovery of sublethally heat-injured Salmonella typhimurium on supplemented plating media.

The efficacy of 32 additives to Levine eosin-methylene blue-salts agar medium (EMBS) for the recovery of sublethally heat-injured Salmonella typhimurium was evaluated. In order of decreasing effectiveness, lactate, mannitol, and alpha-glycerophosphate mediated 90% or more recovery of injured cells; similar levels of recovery were obtained on EMBS supplemented with 1% (wt/vol) tryptic soy broth, protease peptone, or plate count agar. Other additives showed little or no capacity for repair or strongly inhibited heated and nonheated cell suspensions. Conditions of growth and storage before heat treatment were also found to markedly affect susceptibility to heat injury.     

Recovery of sublethally heat-injured Salmonella typhimurium on supplemented plating media.

The efficacy of 32 additives to Levine eosin-methylene blue-salts agar medium (EMBS) for the recovery of sublethally heat-injured Salmonella typhimurium was evaluated. In order of decreasing effectiveness, lactate, mannitol, and alpha-glycerophosphate mediated 90% or more recovery of injured cells; similar levels of recovery were obtained on EMBS supplemented with 1% (wt/vol) tryptic soy broth, protease peptone, or plate count agar. Other additives showed little or no capacity for repair or strongly inhibited heated and nonheated cell suspensions. Conditions of growth and storage before heat treatment were also found to markedly affect susceptibility to heat injury.     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

Seleno-DL-methionine and the protection of human platelets against freezing injury

The effect of seleno-DL-methionine, which has antioxidative properties, on the recovery of human platelets after freezing with 0.5 mol/liter glycerol or 0.7 mol/liter (5% v/v) dimethyl sulfoxide was investigated. Incubation of platelets with 2 mumol/liter seleno-DL-methionine for 30 min at room temperature before equilibration with the protective additives improved the post-thaw uptake of 5-hydroxytryptamine and the percentage reversal in the hypotonic stress test. The effect was small, but in view of the ability of seleno-DL-methionine to inhibit lipid peroxidation in membranes, the results suggest that oxidative damage is implicated in freezing injury. The dimethyl sulfoxide protocol apparently afforded greater protection to the platelets than the glycerol protocol. But, if the platelets were incubated for 24 hr at 37 degrees C after thawing, there was a marked improvement in the response of cells in the hypotonic stress test, particularly in the samples frozen with glycerol, and there was no longer any difference between the two additives. There was, however, a concomitant loss of almost half the number of cells in the thawed suspensions during the prolonged incubation at 37 degrees C.     

Long-term storage of nuclear suspensions prepared from paraffin-embedded material for flow cytometric DNA analysis

Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.     

Stable Mycoplasma Antigen Preparations for Indirect Hemagglutination Tests

In immunological studies of mycoplasmas, the use of glutaraldehyde for the fixative makes it possible to use erythrocytes from commercially available defibrinated sheep blood. It eliminates the necessity of having to screen blood from individual sheep to obtain a suitable source of erythrocytes, as when employing tannic acid for fixation and sensitization. The chemical bonding of soluble mycoplasma proteins to glutaraladehyde-fixed sheep erythrocytes by bis-diazotized 3,3'dimethoxy derivative, benzidine, yields preparations that are satisfactory antigens for performing the indirect hemagglutination test by the microtiter technique. The antigenic preparations are satisfactory for use after storage at 4 or -10 C for many months. Incorporation of 5% glycerine in the final suspending milieu makes it possible to obtain uniform suspensions of the fixed and sensitized sheep erythrocytes after freezing and after repeated freezing and thawing. Proteins from Mycoplasma arthritidis and M. hominis have been coupled to glutaraldehyde-fixed erythrocytes by diazotization. The last mentioned preparation detected the presence of antibodies in titers greater than 1:10 in 37% of 237 pregnant women whose ages ranged between 20 and 30 years. There was no correlation between the presence of specific antibodies in the blood and the isolation of M. hominis from the cervical canal.