Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Increase of capsular material thickness following in vivo growth of virulent Streptococcus suis serotype 2 strains

Abstract Protein profile and capsular material thickness of Streptococcus suis serotype 2 strains were compared after in vitro and in vivo growth. Three virulent and one avirulent strains were used. These strains were grown in Brain Heart Infusion (BHI) broth, cells were collected by centrifugation, resuspended in a sterile saline solution and injected in diffusion chambers. The devices were then inserted in rat abdomens for 17 h. In vitro grown strains were also inoculated into fresh BHI broth and cultivated for 17 h at 37°C. In vivo as well as in vitro grown bacteria were harvested by centrifugation, processed in a French pressure cell, treated with lysozyme and centrifuged to collect cell proteins for SDS-PAGE analysis. Transmission electron microscopy using polycationic ferritin labeling to stabilize capsular material was also carried out. No significant modification was noted in the protein profile for any strain after in vivo growth except for a 39 kDa protein of one virulent strain. On the other hand, an increase in thickness of capsular material was noted for the three in vivo grown virulent strains while no change was noted for the avirulent strain. This increase in capsular material thickness of virulent strains was accompanied by an increased resistance to killing by pig polymorphonuclear leukocytes. The capacity to produce more capsular material in vivo seems to be an attribute of some virulent S. suis serotype 2 strains.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Effect of growth conditions on heat resistance of Arizona bacteria grown in a chemostat.

The effects of various growth conditions on the heat resistance of Arizona bacteria grown in a continuous-culture device (chemostat) were studied. Using either glucose, NH4Cl, NaH2PO4, or MgCl2 as the rate-limiting nutrient, it was found that the heat resistance, in all cases depended on the dilution rate and, hence, growth rate of the culture. Cells grown at high dilution rates were less heat resistant than those grown at low dilution rates. If, however, the dilution rate was maintained at a constant rate, the higher the growth temperature, the more heat resistant were the cells. Also at any given dilution rate, the cells were most heat resistant when grown at a near neutral pH. Most survival curves were biphasic in shape, indicating the presence in the population of two fractions of cells, one fraction being more resistant than the other. The size of the more heat-resistant fraction varied from almost 100% in very slow-growing cultures to practically 0% in cultures grown at a dilution rate of 0.67 h-1.     

Morphological and biochemical variations of Haemophilus influenzae type b induced by pH and temperature changes

Haemophilus influenzae type b ATCC 10211 was cultured at different temperatures (25 degrees C-49 degrees C) and pH values (5.7-8.7) either in liquid or semisolid medium. Morphological variations of individual cells were noted by optical microscopy depending upon the conditions of growth. At higher temperatures filaments were produced whereby the length of individual cells increased compared to cultures grown at 37 degrees C. Filaments were also observed at lower pH values. Culture conditions also affected colonial morphology. At low pH values colonies had an enhanced lobulated contour and were more wrinkly and rougher than at higher pH. The changes in cellular and colonial morphology were correlated with distinct outer membrane protein profiles. The changes in temperature and pH did not affect identification of the microorganism by the API system.     

Syncytia infectivity of assay of bovine leukemia virus

Bovine embryonic spleen cell cultures were examined to find several factors influencing the specificity, sensitivity and reproducibility of the syncytia infectivity assay of bovine leukemia virus (BLV). The highest sensitivity of the assay were observed when cell sheets of 30 to 50% confluence were inoculated with a stock of BLV, and when cells containing 4 or more nuclei were counted as syncytial cells. Treatment of the cell sheets with a diethylamino-ethyl-dextran solution (25 micrograms/ml) prior to BLV inoculation was found to be essential for the optimal induction of syncytia. Low-passage cultures were found to be more susceptible to the induction of syncytia by BLV than high-passage cultures. Cell-free BLV preparations decreased in syncytia-inducing ability to some extent by the first cycle of freezing (at -70 degrees C) and thawing. No further decrease, however, was caused by repeated cycles of freezing and thawing or by prolonged incuvation at -80 degrees C. The syncytia-inducing activity of BLV was inhibited by all the BLV-precipitating antibody-positive sera originated from both cases of the adult form of bovine leukosis and cases of persistent lymphocytosis. It was not inhibited by the sera of 16 of 17 cattle apparently healthy and negative for BLV-precipitating antibody. These results indicate that the syncytia infectivity assay and syncytia inhibition test are specific for BLV.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system.

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.     

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.     

Growth and cell wall composition of glucose-limited bean cells (Phaseolus vulgaris L.)

Glucose-limited bean cells (Phaseolus vulgaris L.) were grown in a modified bacterial fermentor at a constant pH of 4.8. The cultures were kept in steady state at different specific growth rates varying from 0.00216 h^–1 to 0.0106 h^–1. Culture conditions are described that are needed to start a continuous culture. First, it was essential to use log-phase cells as starting material. Second, it was important to increase the dilution rate gradually, otherwise cells in the culture aggregated. Cells grown at the highest dilution rate employed contained twice as much protein per gram dry weight as cells grown at the lowest dilution rate. The composition of the cell walls also varied with the dilution rate in contrast to their relatively constant composition when grown in batch culture.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila.

The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48 degrees-65 degrees C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7 degrees C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30 degrees C) were twice as resistant as those in the early stage (5 h at 30 degrees C), and the maximum D-value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z-values significantly. The heat resistance of cells incubated for 48 h at 30 degrees C increased (twice) after holding at 7 degrees C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30 degrees C) did not influence their D-values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 to 4.0 reduced D-values by a factor of 5. Although the strain studied was heat-sensitive (D55 degrees C = 0.17 min; z = 5.11 degrees C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in tower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.     

Growth Characteristics, Bile Sensitivity, and Freeze Damage in Colonial Variants of Lactobacillus acidophilus

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneous culture of Lactobacillus acidophilus RL8K. R and S types were stable upon repeated transfer on agar, but revertant colonies did appear after broth transfers. When propagated in commercial MRS broth, R and S cultures showed similar growth characteristics, and both cell types were insensitive to freezing and frozen storage at −20°C. Alternatively, during growth in scratch MRS broth, R cultures shifted to a reduced rate of growth during the late logarithmic phase. R cells grown under these conditions were susceptible to death by freezing and injury at −20°C. Microscopically, R cells were observed as long gram-positive rods with small nonstainable blebs protruding from the cell wall. In bile sensitivity studies of R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1% bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bile resistance and freeze damage of R and S cells suggest that colonial and cellular morphologies are important considerations for the selection of Lactobacillus strains as dietary adjuncts and for the development of growth conditions for preparing frozen concentrated cultures from either cell type.     

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40 degrees C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP-Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43 degrees C (at least 25 times lower than those observed when the growth temperature was 37 degrees C). The synthesis of these enzymes increased rapidly when bacteria grown at 43 degrees C were transferred to 37 degrees C and decreased dramatically when cells grown at 37 degrees C were transferred to 43 degrees C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.     

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+-ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore-resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+-ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.0. In the cells grown at pH 7.5, they were moderately observed. The Na+-stimulated ATPase activity of the cells grown at pH 9.5 was about 4-fold higher than that of the cells grown at pH 6.0. Thus, amplification of the Na+-ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila

S. CONDON, M.L. GARCIA, A. OTERO AND F.J. SALA. 1992. The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48°-65°C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7°C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30°C) were twice as resistant as those in the early stage (5 h at 30°C), and the maximum D -value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z -values significantly. The heat resistance of cells incubated for 48 h at 30°C increased (twice) after holding at 7°C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30°C) did not influence their D -values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 4.0 reduced D -values by a factor of 5. Although the strain studied was heat-sensitive ( D _55°C= 0.17 min; z = 5.11°C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.