Simulated microgravity impairs respiratory burst activity in human promyelocytic cells

Summary The concept of microgravity (free-fall) influencing cellular functions in nonadherent cells has not been a part of mainstream scientific thought. Utilizing rotating wall vessels (RWVs) to generate simulated microgravity conditions, we found that respiratory burst activity was significantly altered in nonadherent promyelocytic (HL-60) cells. Specifically, HL-60 cells in simulated microgravity for 6, 19, 42, 47, and 49 d had 3.8-fold fewer cells that were able to participate in respiratory burst activity than cells from 1×g cultures (P=0.0011, N=5). The quantity of respiratory burst products from the cells in simulated microgravity was also significantly reduced. The fold increase over controls in mean fluorescence intensities for oxidative products from cells in microgravity was 1.1±0.1 versus 1.8±0.3 for cells at 1 ×g (P=0.013, N=4). Furthermore, the kinetic response for phorbol ester-stimulated burst activity was affected by simulated microgravity. These results demonstrate that simulated microgravity alters an innate cellular function (burst activity). If respiratory burst activity is impaired by true microgravity, then recovery from infections during spaceflight could be delayed. Finally, RWVs provide an excellent model for investigating the mechanisms associated with microgravity-induced changes in nonadherent cells.     

Cultivation of fall armyworm ovary cells in simulated microgravity

Summary A methodology is presented to culture Fall Armyworm Ovary cells in simulated microgravity using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 × 10⁷ viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm² in shaker culture to under 0.5 dyne/cm² in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.     

Three-dimensional macro-scale assessment of regional and temporal wall shear stress characteristics on aortic valve leaflets

The aortic valve (AV) achieves unidirectional blood flow between the left ventricle and the aorta. Although hemodynamic stresses have been shown to regulate valvular biology, the native wall shear stress (WSS) experienced by AV leaflets remains largely unknown. The objective of this study was to quantify computationally the macro-scale leaflet WSS environment using fluid–structure interaction modeling. An arbitrary Lagrangian–Eulerian approach was implemented to predict valvular flow and leaflet dynamics in a three-dimensional AV geometry subjected to physiologic transvalvular pressure. Local WSS characteristics were quantified in terms of temporal shear magnitude (TSM), oscillatory shear index (OSI) and temporal shear gradient (TSG). The dominant radial WSS predicted on the leaflets exhibited high amplitude and unidirectionality on the ventricularis (TSM>7.50 dyn/cm², OSI < 0.17, TSG>325.54 dyn/cm² s) but low amplitude and bidirectionality on the fibrosa (TSM < 2.73 dyn/cm², OSI>0.38, TSG < 191.17 dyn/cm² s). The radial WSS component computed in the leaflet base, belly and tip demonstrated strong regional variability (ventricularis TSM: 7.50–22.32 dyn/cm², fibrosa TSM: 1.26–2.73 dyn/cm²). While the circumferential WSS exhibited similar spatially dependent magnitude (ventricularis TSM: 1.41–3.40 dyn/cm², fibrosa TSM: 0.42–0.76 dyn/cm²) and side-specific amplitude (ventricularis TSG: 101.73–184.43 dyn/cm² s, fibrosa TSG: 41.92–54.10 dyn/cm² s), its temporal variations were consistently bidirectional (OSI>0.25). This study provides new insights into the role played by leaflet–blood flow interactions in valvular function and critical hemodynamic stress data for the assessment of the hemodynamic theory of AV disease.     

流体剪切力对内皮细胞miR-21和miR-199a表达的影响

为探讨流体剪切力对内皮细胞micorRNAs表达的影响。采用旋转锥形圆盘剪切力系统对内皮细胞分别加载低(4dyn/cm2)、中(10 dyn/cm2)和高(15 dyn/cm2)3种不同梯度的剪切力作用24h。对照组未加载剪切力。采用高通量筛选芯片检测microRNAs表达变化,qRT-PCR验证,并进行生物信息学分析。与对照组比较,低剪切力组表达差异的microRNAs有33个(FC1.5或0.5倍,P0.05),其中28个上调,5个下调;中剪切力组表达差异的microRNAs有8个(FC1.5或0.5倍,P0.05),其中6个上调,2个下调;高剪切力组表达差异的microRNAs有31个(FC1.5或0.5倍,P0.05),其中25个上调,6个下调。miR-21在高剪切力组中上调最显著(FC=0.026),在低剪切力组中显著下调(FC=3.531)。miR-199a在低剪切力组中上调最显著(FC=0.075),在高剪切力组中显著下调(FC=3.031)。表达差异的microRNA的靶基因主要与内皮细胞的力学信号转导、细胞跨膜迁移、钙离子信号通路、细胞内吞作用等相关。流体剪切力可诱导内皮细胞miR-21和miR-199a表达发生改变。     

Simulated microgravity affects semicarbazide-sensitive amine oxidase expression in recombinant Escherichia coli by HPLC-ESI-QQQ analysis

Simulated microgravity has been reported to affect the gene, protein expression, and its function in the cells. Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in vascular cells, smooth muscle cells, and adipocytes. It is noteworthy whether the expression of SSAO is affected under simulated microgravity or not. In this study, an SSAO-transformed Escherichia coli BL21 was constructed firstly. Then, a sensitive, selective, and accurate method based on high-performance liquid chromatography electrospray ionization triple quadrupole (HPLC-ESI-QQQ) was developed to determine the amount of SSAO in the E. coli BL21. The limit of detection and limit of quantification were 5.0 and 10 fmol, respectively. Finally, SSAO expression in the recombinant E. coli BL21 was evaluated with various gravity and temperature conditions by HPLC-ESI-QQQ analysis. It is interesting that the tendency in the alteration of SSAO under simulated microgravity showed temperature difference. At 18 °C, the amount of SSAO in the inclusion bodies and soluble fractions under the simulated microgravity increased by 83% and 116%, respectively, compared with normal gravity. However, the decrease by 38% and 49% in the inclusion bodies and soluble fractions under the simulated microgravity was observed at 37 °C. Results obtained here indicate that the SSAO expression under simulated microgravity is dramatically sensitive to the temperature. On the other hand, a novel bioreactor from this study may also be useful for the recombinant protein expression in the field of gene engineering.     

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth''s 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47^phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47^phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47^phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47^phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.     

Tissue Factor-Expressing Tumor Cells Can Bind to Immobilized Recombinant Tissue Factor Pathway Inhibitor under Static and Shear Conditions In Vitro

Mammary tumors and malignant breast cancer cell lines over-express the coagulation factor, tissue factor (TF). High expression of TF is associated with a poor prognosis in breast cancer. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF, is constitutively expressed on the endothelium. We hypothesized that TF-expressing tumor cells can bind to immobilized recombinant TFPI, leading to arrest of the tumor cells under shear in vitro. We evaluated the adhesion of breast cancer cells to immobilized TFPI under static and shear conditions (0.35 – 1.3 dyn/cm²). We found that high-TF-expressing breast cancer cells, MDA-MB-231 (with a TF density of 460,000/cell), but not low TF-expressing MCF-7 (with a TF density of 1,400/cell), adhered to recombinant TFPI, under static and shear conditions. Adhesion of MDA-MB-231 cells to TFPI required activated factor VII (FVIIa), but not FX, and was inhibited by a factor VIIa-blocking anti-TF antibody. Under shear, adhesion to TFPI was dependent on the TFPI-coating concentration, FVIIa concentration and shear stress, with no observed adhesion at shear stresses greater than 1.0 dyn/cm². This is the first study showing that TF-expressing tumor cells can be captured by immobilized TFPI, a ligand constitutively expressed on the endothelium, under low shear in vitro. Based on our results, we hypothesize that TFPI could be a novel ligand mediating the arrest of TF-expressing tumor cells in high TFPI-expressing vessels under conditions of low shear during metastasis.     

Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells

The key mechanism responsible formaintaining cell volume homeostasis is activation ofvolume-regulated anion current (VRAC). The role of hemodynamicshear stress in the regulation of VRAC in bovine aortic endothelialcells was investigated. We showed that acute changes in shear stresshave a biphasic effect on the development of VRAC. A shear stress stepfrom a background flow (0.1 dyn/cm²) to 1 dyn/cm² enhanced VRAC activation induced by an osmoticchallenge. Flow alone, in the absence of osmotic stress, did not induceVRAC activation. Increasing the shear stress to 3 dyn/cm²,however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5-10dyn/cm²), the current was immediately strongly suppressed.Suppression of VRAC was observed both in cells challenged osmoticallyand in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor inregulating the ability of vascular endothelial cells to maintain volume homeostasis.

     

Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm² to 6, 10, or 20 dyn/cm² elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.     

Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

Summary Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein—Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4–7 d. The cells were then transferred to 50-cm³ tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated withn-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2–3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2–3 or 48 h with TPA. TPA reactivation of EBV following a 2–3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3–4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.     

Constant surface tension molecular dynamics simulations of lipid bilayers with trehalose

Surface areas and fluctuations evaluated from 50 ns molecular dynamics simulations of fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers in a 1:2 trehalose:lipid ratio carried out at surface tensions 10, 17 and 25 dyn/cm/leaflet are compared with those of pure bilayers under the same conditions. Trehalose increases the surface area, as consistent with the surface tension lowering observed in simulations at constant area. The system bulk elastic modulus K _b  = 1.5 ± 0.3 × 10¹⁰ dyn/cm². It is independent of bilayer surface area and trehalose content within statistical error. In contrast, the area elastic modulus K _a shows a strong area dependence. At 64 Å²/lipid (the experimental surface area), K _a  = 138 ± 26 dyn/cm for a pure DPPC bilayer and 82 ± 10 dyn/cm for one with trehalose; i.e. trehalose increases fluidity of the bilayer surface at this area per lipid.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Electrically measuring viscoelastic parameters of adherent cell layers under controlled magnetic forces

Electrical cell-substrate impedance sensing (ECIS) was used to measure the time-dependence and frequency-dependence of impedance for current flowing underneath and between cells. Osteosarcoma cells with a topology similar to a short cylinder (coin-like) surmounted by a dome were used in this study. Application of a small step increase in net vertical stress to the cells (4 and 7 dyn/cm²), via magnetic beads bound to the dorsal (upper) surface, causes an increase in cell body height and an increase in cell-cell separation, as well as stretching of the cell-substrate adhesion bonds. This results in a fast drop in measured resistance (less than 2 s), followed by a slower change with a time constant of 60–150 s. This time constant is about 1.5 times longer at 22 °C than that at 37 °C; it also increases with applied stress. Our frequency scan data, as well as our data for the time course of resistance and capacitance, show that the fast change is associated with both the under-the-cells and between-the-cells resistance. The slower change in resistance mainly reflects the between-the-cells resistance. To obtain viscoelastic parameters from our data we use a simple viscoelastic model comprising viscous and elastic elements (i.e., a dashpot and two springs) for the cell body, and an elastic element (a spring) for the cell-substrate adhesion system. Our results show that the spring constants and the viscosity of the cell body components of this viscoelastic model decrease as the temperature increases, whereas the elastic modulus of cell-substrate adhesion increases with temperature. At 37 °C, for the cell body we obtain a value of about 10⁵ P for the viscous element of the viscoelastic model, and a spring constant expressed in units of an elastic modulus of about 10⁴ dyn/cm² for the spring in series with the viscous element, with another spring with a modulus of about 2×10³ dyn/cm² in parallel with these. In comparable units, we have a modulus for the cell-substrate adhesion system of about 3×10³ dyn/cm². Received: 23 March 1998 / Revised version: 23 June 1998 / Accepted: 1 July 1998     

Cultivation of cell-polymer tissue constructs in simulated microgravity

Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm(2)). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. (c) 1995 John Wiley & Sons, Inc.     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

Fluctuations and mechanical strength of α-helices of polyglycine and poly(L-alanine)

By using the static correlations of fluctuations in the dihedral angles of the α-helices of polyglycine and poly(L -alanine) calculated previously, geometrical fluctuations of a section (consisting of up to 18 peptide units) of the α-helices of infinite length are calculated. These fluctuations are found to differ in some respects (e.g., the dependence of amplitudes on the length of section) from those of a circular rod made of homogeneous continuous material. However, the moduli of the mechanical strengths (tensile Young's modulus, bending Young's modulus, and the shear modulus) of a circular rod are calculated, whose geometrical fluctuations are approximately equal to the fluctuations of a section consisting of 18 peptide units. They are of the order of 10¹¹ dyn/cm². The tensile rigidity, flexural rigidity, and torsional rigidity are calculated to be 1.20 × 10^?3 dyn, 2.46 × 10^?19 dyn·cm² and 1.79 × 10^?19 dyn·cm² for polyglycine, and 1.96 × 10^?3 dyn, 4.05 × 10^?19 dyn·cm² and 3.28 × 10^?19 dyn·cm² for poly(L -alanine), respectively.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

CRITICAL LEVELS OF LIGHT AND TEMPERATURE REGULATING THE GAMETOGENESIS OF THREE LAMINARIA SPECIES (PHAEOPHYCEAE)1

Gametophytes of three Laminaria species occurring near Helgoland, North Sea, were cultivated 4 wk in a 12:12 LD regime at different temperatures in artificial light fields, and in the sea at different water depths. In the artificial light fields underwater spectral distribution was simulated according to Jerlov water Types 5, 7, 9. Blue light in the simulated light fields amounted to 17, 12 or 4% of total quanta. The rate of vegetative growth did not depend on spectral distribution, was light-saturated at 4–6 W · m^?2, and increased with temperature up to 15 C. L. saccharina (L.) Lamour. exhibited the highest tolerance towards temperature, light and UV. Gametophytes survived 1 wk at 21 C ± 0.1, but not 22 C ± 0.1. Gametophytes of L. hyperborea (Gunn.) Fosl. and L. digitata (Huds.) Lamour. survived 1 wk at 20 C ± 0.1, but not at 21 C ± 0.1. In sunlight, and in the light field of a xenon lamp, 50% of L. saccharina gametophytes were killed by a quantum dose of 50 μEin · cm^?2, and 100% of the plants by 90 μEin · cm^?2. Approximately half of these quantum doses killed the corresponding percent of the other species gametophytes. Appreciably higher quantum doses were survived in visible light, with red being the most detrimental. Fertility depended on a critical quantum dose of blue light which decreased almost exponentially with decreasing temperature. The quantum dose (400–512 nm) required for induction of fertilization of 50% of the female gametophytes (males react similarly) was 90 μEin · cm^?2 at 5 C, 110 μEin · cm^?2 at 10 C, 230 (560 in L. digitata)μEin · cm^?2 at 15 C, and 560 (L. hyperborea) or about 850 (other 2 species) μEin · cm^?2 at 18 C. In the sea the gametophytes survived the dark winter months in the unicellular stage, with almost no vegetative growth of the primary cell, due to lack of light. In early spring the female gametophytes matured in the unicellular, and the males in a few-celled stage at the depth of 2 m, as did the laboratory cultures under conditions inducing maximal fertility.