Culture of amebocytes in a nutrient mist bioreactor

Summary We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells. The nutrient mist approximated the amebocyte stem tissue’s natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crabLimulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of theLimulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay. Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium. However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.     

<Emphasis Type="Italic">Ex vivo</Emphasis> expansion of hematopoietic cells from CD34<Superscript>+</Superscript> cord blood cells in various culture conditions

This study compared the cell expansion and colony-forming ability of human cord blood stem cells cultured ex vivo with 2 types of cytokine combinations, and 2 types of media characterized by the presence or absence of fetal bovine serum (FBS) in 2 or 3 dimensional (2D or 3D) culture environments. Purified CD34⁺ cells derived from different donors were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) and Ultraculture serum-free medium (SFM) containing the cytokine cocktail-I (coc-I) (EPO, GM-CSF, SCF, and IL-3) or cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher CFU-GM values were observed in the IMDM compared to the SFM. In the coc-I conditions, the ‘IMDM + coc-I’ and ‘IMDM + coc-I + FBS’ conditions gave the greatest cell (1,667 ± 274 and 1,600 ± 140-fold, respectively) and colony-forming units (CFU) expansions (BFU-E: 21 ± 3, 36 ± 5; CFU-GM: 95 ± 19, 81 ± 17; and CFU-GEMM: 2 ± 1, 3 ± 1-fold, respectively) in 26 day culture, respectively. In the coc-II conditions, the ‘SFM + coc-II’ condition gave the greatest cell expansion (2,143 ± 134-fold), but the ‘IMDM + coc-II’ condition gave the best CFU-GM expansion (924 ± 110-fold) in 26 day culture. In conclusion, ‘IMDM + coc-I’ and ‘IMDM + coc-II’ were the most accessible conditions for CFU expansion for all culture cases. The 2D stationary culture had affirmative effect on CFU expansion compared to the 3D culture using semisolid Methocult™. These results are believed to be significant in the ex vivo expansion of hematopoietic stem cells.     

In vitro biosynthesis of juvenile hormone in larval honey bees: Comparison of six media

Summary We have formulated a tissue culture medium based on the components of larval honey bee hemolymph. Using an in vitro radiochemical assay to measure juvenile hormone biosynthesis, we compared our larval-based medium to four commercially available media (Grace’s, Medium-199; Shields and Sang M3, and Minimum Essential Medium), and a medium based on adult honey bee hemolymph. All media were formulated without methionine. There was no significant difference in the amounts of juvenile hormone produced by the larval medium and Grace’s; both of these media, however, were more suitable than the remaining four. Our larval-based tissue culture medium should prove useful in studies aimed at elucidating the underlying hormonal mechanism(s) of caste development in honey bees.     

Embryonic development of an endoparasitoid,Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media

Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400. The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.A preliminary report of this work has been presented elsewhere (Liu and Liang, 1984).     

Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.     

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (β,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Insect cell culture media for cultivation of new world Leishmania

Monophasic insect cell culture media were evaluated for suitability in the propagation of strains of Leishmania brasiliensis, L. mexicana and L. enriettii. Amastigotes were inoculated into Grace's, Schneider's and Mitsuhashi-Maramorosch's Media containing antibiotics and varying concentrations of fetal calf serum. After 4–7 days incubation at 27°C, promastigote yields exceeded 1 × 10⁸/rr.l in some instances in contrast to yields of approximately 2 × 10⁷/ml in blood-based media. Optimal concentrations of fetal calf serum were determined to be 15% or more for most of the strains tested.     

Inactivation of endotoxin by Limulus amoebocyte lysate.

The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed.     

Primary culture and characteristic morphologies of neurons from the cerebral ganglion of the mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in modified Medium 199 (M199) medium than that in Liebowitz’s L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics were identified in the neuron outgrowth. They were “small cells”, “veilers”, “branchers”, “multipolar cells”, “super-large cell”, and “bipolar cells”. Among all of the cells, bipolar cells were identified for the first time in crustacean neurons culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual degradation.     

Use of cyanobacterial gas vesicles as oxygen carriers in cell culture

The gas vesicles isolated from the cells of filamentous cyanobacterium Anabaena flos-aquae were treated and sterilized with glutaraldehyde and then evaluated for their effectiveness as gas carriers in cell culture. Anchorage-dependent Vero cells were grown in a packed bed of microcarrier beads under the perfusion of Dulbecco’s Modified Eagle’s Medium with 1% serum. The culture medium supplemented with 1.8% (v/v) gas vesicles was found to support a 30% higher maximum glucose utilization rate than the same medium without gas vesicles. The gas vesicle suspension was confirmed to have no apparent effects on cell metabolism in T-flask cultures. The study results indicated that the gas vesicles, with high oxygen carrying capacity, can be used to increase the oxygen supply in cell culture systems.     

Insect protein digestion improves purity of Steinernema carpocapsae in vitro culture and reduces culture period

Insect protein, used for in vitro culture media for entomopathogenic nematode, produces nematodes of high quality. However, the time-consuming culture and poor purity of nematodes hinder the commercial application of insect protein media. We show that hydrolyzed insect protein improves nematode purity in in vitro culture. The results revealed that nematode purity was increased by more than 90 %, and the culture period was reduced by 6 days. Estimated economic efficiency of using hydrolyzed insect protein medium was increased by 44.25 % over that obtained with non-hydrolyzed insect medium.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Observations of endotoxin-like activity associated with the Legionnaires' disease bacterium

Suspensions of Legionnaires' disease bacterium stored in sterilized tap water 279–287 days produced gelation ofLimulus amebocyte lysate. A 1-ml suspension of washed cells containing 10⁹ viable organisms had aLimulus amebocyte lysate activity equivalent to 4 mg of endotoxin. This activity remained stable in samples that had been autoclaved at 121°C for 15 min. Both the autoclaved cells and filtrate of autoclaved cells were pyrogenic in inoculated rabbits. The Legionnaires' disease bacterium produces a substance or substances that have biological properties associated with endotoxin of more typical Gram-negative bacteria.     

Temporal and spatial patterns of organic carbon are linked to egg deposition by beach spawning horseshoe crabs (<Emphasis Type="Italic">Limulus polyphemus</Emphasis>)

The spring spawning by the American horseshoe crab (Limulus polyphemus L.) results in temporally and spatially discrete inputs of eggs onto sandy beaches in Delaware Bay, USA. We tested the hypothesis that seasonal patterns of sediment organic carbon on Delaware Bay beaches is linked to this pulsed input of horseshoe crab eggs. At a location with minimal horseshoe crab spawning activity (Higbee Beach), there was little seasonal variation in sediment organic carbon, no distinction between organic carbon levels as a function of shoreline position or sediment depth, and no significant correlation between the abundance of crab eggs and percent organic carbon. Conversely, at a prime horseshoe crab spawning habitat (North Beach), organic carbon levels were seasonally pulsed and were correlated with egg abundance. Moreover, the strongest evidence of seasonality was seen at the middle foreshore location at the 15–20 cm depth, consistent with the highest input of horseshoe crab eggs. Although some of the organic carbon contributed by horseshoe crab eggs in May–June leaves the beach in the form of hatched larvae later in the year, there is a net input of organic carbon to the system in the form of unfertilized and/or dead eggs, egg membranes, and embryonic molts. We suggest that the inputs of eggs from horseshoe crabs and other beach spawning animals, such as grunion and capelin, make significant contributions to the energy budget of sandy beaches.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Cardioinhibitory peptides from Limulus polyphemus: modulation of the neurogenic heart

Four neuropeptides have been isolated and sequenced from acetone extracts of brains of the horseshoe crab Limulus polyphemus. They belong to a newly discovered peptide family in invertebrates. A possible role of the four peptides from Limulus as cardioregulatory neurotransmitters has been tested on the isolated Limulus heart. Three of the peptides (DEGHKMLYFamide, GHSLLHFamide, and PDHHMMYFamide) produce dose-dependent decreases in both amplitude and rate of the heart contractions, whereas DHGNMLYFamide reduces only the amplitude of the heartbeat. All four peptides differ in threshold, potency and duration of their effects.Abbreviations DIC diisopropylcarbodiimide - DTT dithiotreitol - Fmoc 9-fluorenylmethyloxycarbonyl - GABA gamma amino butyric acid - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - IBMX 3-isobutyl 1-methyl-xanthine - Lip-HP Limulus head peptide - LP Limulus peptide - TBTU 2-(1H-benzotriazole-1yl)-1,1,3,3,tetramethyluronium tetrafluoroborate - TFA trifluoroacetic acid - TLC thin-layer chromatography