Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Precision-cut luteal slices: A promising approach for studying luteal function in pigs

The study was aimed to validate the precision-cut luteal slices to investigate porcine luteal function. Corpora lutea (CLs) were cut into 180-μm thick slices using Krumdick Tissue Slicer. The viability, tissue structure and steroidogenic acute regulatory protein (STAR) expression in the luteal slices did not differ between the beginning and the end of the 24-h incubation period. The luteal progesterone secretion showed a time- and dose-dependent response to porcine luteinizing hormone. The effects of prostaglandin F_2α and 17β-estradiol on progesterone secretion by porcine luteal slices were comparable to the previously reported in vivo results of the CL microdialysis system in the pig.     

Pharmacological Modulation of Cisplatin Toxicity Rhythms with Buthionine Sulfoximine in Mice Bearing Pancreatic Adenocarcinoma (PO3)

In a previous report, we showed that the circadian rhythm of cisplatin (cis-diamminedichloroplatinum, CDDP) toxicity in healthy mice was modified by buthionine sulfoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. In the present study, the effects of BSO on the rhythms of CDDP toxicity and antitumor efficacy were investigated in mice bearing a transplantable pancreatic adenocarcinoma (PO3). B₆D₂F₁ mice were inoculated widi two 4 mm³ tumor fragments, one in each flank, then were synchronized with an alternation of 12h of light (L) and 12h of darkness (D) (LD 12: 12). Three weeks later, a single dose of CDDP (12 mg/kg iv) was injected at 3h, 7h, 11h, 15h, 19h, or 23h after light onset (HALO) with or without prior BSO (450 mg/kg ip 4h earlier). The antitumor activity of CDDP as assessed by tumor weight change and tumor growth delay was weak in this tumor model irrespective of prior BSO administration or CDDP dosing time. Nevertheless, toxic effects of CDDP as gauged by body weight loss or survival varied significantly according to CDDP dosing time. Body weight loss was least in mice receiving CDDP alone at the mid-to-late active span. Survival rate was 97% in mice treated with CDDP alone and 47% in those receiving prior BSO (χ² = 23.6, p <. 0001). BSO pretreatment further shifted the period of survival or body weight change from 24h to (10 + 24)h, an effect similar to that earlier reported in healthy mice. Thus, PO3 tumor at a measurable stage altered neither the circadian rhythm in CDDP toxicity nor the ultradian rhythm in the toxicity of BSO-CDDP combination. The results suggest that rhythms in target tissues for drug actions can be manipulated with biochemical modulators, thus partly escaping central clock control.     

Effects of cadmium-metallothionein on renal organic ion transport and lipid peroxidation in rats

The effects of cadmium-metallothionein (Cd-MT) on organic ion uptake in renal cortical slices and lipid peroxidation in the kidney were studied in rats. For in vitro studies, slices were prepared from kidneys of control animals and incubated in buffer containing either cadmium chloride (CdCl₂) or Cd-MT in equimolar Cd concentrations ranging from 5 × 10^?6 to 2 × 10^?4 M. Uptake into the slices of the organic anion p-aminohippuric acid (PAH) was found to be inhibited by both forms of Cd in a dose-dependent manner. Although this inhibition was slightly greater in the presence of Cd-MT, accumulation of Cd into the slices was approximately 12 times greater with CdCl₂ than Cd-MT. Tetraethylammonium (TEA) uptake was less sensitive to the inhibitory effects of both CdCl₂ and Cd-MT, although a dose-dependent inhibition did occur with higher Cd concentrations. To study the in vivo effects of Cd-MT on transport function and lipid peroxidation in the kidney, rats were injected with Cd-MT (0.3 mg Cd per kilogram body weight [bw]) and sacrificed at specific time intervals. Similar to the in vitro studies, PAH uptake into the renal cortical slices was markedly inhibited within 12 hours after Cd-MT injection whereas inhibition of TEA uptake was less and not observed until 48 hours after injection. Only a small increase (1.4-fold) in lipid peroxidation, as measured by generation of malondialdehyde (MDA), in the kidney was detected at four hours postinjection, and no further increase was observed at later time periods. The results suggest that Cd-MT affects the transport of organic anions and cations during its renal uptake but that lipid peroxidation may play only a minor role in Cd-MT-induced renal toxicity.     

Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: Effects of oxygen tension

We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37°C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO₂–95% air (WPCS/air or ROCS/air) or 5% CO₂–70% O₂–25% N₂ (WPCS/ O₂ or ROCS/ O₂). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/ O₂ were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/ O₂ for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/ O₂ than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/ O₂. ROCS/ O₂ emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.     

Reversal of cisplatin sensitization and abrogation of cisplatin-enriched cancer stem cells in 5-8F nasopharyngeal carcinoma cell line through a suppression of Wnt/β-catenin-signaling pathway

Nasopharyngeal carcinoma (NPC) is one of the rare cancers in western countries but predominant in Southeast Asian countries including Thailand. One major cause for failure of NPC chemotherapeutic treatments is reportedly correlated with the elevation of cancer stem cell (CSC) fractions. Thus, this present study aims to investigate the effect of cisplatin (CDDP) treatment on the enrichment of cancer stem-like cells (CSCs) and its associated signaling pathway in EBV-negative NPC cells. Cisplatin-pretreated 5-8F NPC cells (5-8F CDDP) were first generated by treating the cells with 0.5 μM cisplatin for 48 h. After the instant treatment, 5-8F CDDP showed increased IC₅₀ values, demonstrating a decrease in CDDP sensitization. Besides, the proportion of NPC cells with cancer stem-like phenotypes comprising side population (SP), key stemness-related gene expressions including SOX2, ALDH1, CD24 was significantly enhanced. Additionally, 5-8F CDDP displayed the upregulation of β-catenin gene, suggesting its association with the CSC-initiating mechanism. Furthermore, a tankyrase inhibitor for Wnt/β-catenin pathway, XAV939, substantially reduced CSCs and retrieved the cisplatin sensitivity in 5-8F CDDP. This confirms that the Wnt/β-catenin signaling is accountable for rising of the CSC population in EBV-negative NPC. Finally, the combined treatment of CDDP and XAV939 exhibited lower 5-8F CDDP cell viability compared to the treatment of CDDP alone, suggesting the reversal of cisplatin sensitization. In conclusion, the enhancement of CSCs in 5-8F NPC cells caused by the instant cisplatin treatment is initially mediated through the upregulation of β-catenin and activation of Wnt/β-catenin signaling pathway. As a result, a primary chemotherapeutic treatment with closely monitoring the targeted Wnt/β-catenin signaling pathway could potentially prevent the development of CSCs and improve the treatment efficiency in NPC.

     

Hippocampal slice (K+)e: depth profiles and changes induced by stimulation or anoxia

In an attempt to develop a technique which would allow early assessment of the functional state of explanted brain tissue, (K+)e was measured in the CA1 region of rat hippocampal slices using K+-selective microelectrodes. In slices (450 micron) maintained at the boundary between the incubation medium and 95% O2/5% CO2 atmosphere, (K+)e was highest (up to 25-30 mmol/l) immediately below the exposed surface and gradually decreased with depth to (K+) of the bathing fluid (5 mmol/l). (K+)e below the exposed surface remained high throughout the 2 h of incubation. In submersed slices, (K+)e was the highest in the center of the slice (200 micron, 10 mmol/l) and decreased towards both surfaces. During 2 h incubation, (K+)e decreased in the center of the slice to 6 mmol/l in viable preparations remaining high in the deteriorating ones. Electrical stimulation of Schaffer's collaterals (15 V; 0.2 ms; 10 Hz) increased (K+)e of viable slices 200 micron below the surface by 2-3 mmol/l. Similar but slower (K+)e changes were elicited by brief (3 min) anoxic episodes (perfusion with incubation medium equilibrated with 95% N2/5% CO2). It is concluded that submersed slices have a more uniform (K+)e profile as compared to the exposed ones and that low (K+)e in the early phase of incubation is a good predictor of slice viability.     

Renal function and organic anion and cation transport during dehydration and/or food restriction in chickens

The effect of dehydration in the presence or absence of continued food intake on renal function was evaluated in chickens. In addition, renal transport of organic anions and cations under these conditions was assessed in vitro by uptake of ¹⁴C-para-aminohippuric acid and ¹⁴C-tetraethylammonium bromide by renal slices. Water restriction with continued food intake resulted in increases in serum osmolality and serum concentrations of sodium, uric acid, calcium and total protein. If food was restricted in addition to water, only serum osmolality and sodium concentration were significantly increased after 48 hours. Dehydration with continued access to food resulted in marked decreases in extracellular fluid volume, glomerular filtration rate and effective renal plasma flow. If food was restricted during dehydration, the decrease in effective renal plasma flow was attenuated despite reductions in glomerular filtration rate and extracellular fluid comparable to that seen in dehydrated birds allowed free access to food. Transport of organic anions was significantly increased after 24 and 48 hours of water restriction, regardless of whether food was withheld. Enhanced transport of organic anions in the presence of decreased glomerular filtration rate and effective renal plasma flow during dehydration may promote precipitation of urates and nephrosis in chickens.Abbreviations cpm counts per minute - dpm disintegrations per minute - ECF extracellular fluid - ERPF effective renal plasma flow - GFR glomerular filtration rate - PAH para-aminohippuric acid - SEM standard error of the mean - TEA tetraethylammonium bromide     

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCl₂, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl₂ demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells

Resistance to cisplatin [cis-diamminedichloroplatinum(II), CDDP] chemotherapy is a major problem in the clinic. Understanding the molecular basis of the intracellular accumulation of CDDP and other platinum-based anticancer drugs is of importance in delineating the mechanism of resistance to these clinically important therapies. Different molecular mechanisms may coexist, but defective uptake of CDDP is one of the most consistently identified characteristics of cells selected for CDDP resistance. We have studied the impact of intracellular chloride concentration on platinum-based compound accumulation in the human GLC4, GLC4/CDDP, and K562 tumor cell lines. We show that (1) a decrease of intracellular chloride concentration yielded an increase of CDDP accumulation and vice versa and (2) the intracellular chloride concentration in GLC4/CDDP cells is higher than in sensitive cells, whereas CDDP accumulation shows the opposite behavior. The identification of chloride as a critical determinant of CDDP intracellular accumulation and the molecular mechanisms by which CDDP-resistant cells modulate chloride concentration may allow alternative therapeutic approaches. Our findings indicate that increase of intracellular chloride concentration may be a major determinant of CDDP resistance.     

In vitro culture of precision-cut testicular tissue as a novel tool for the study of responses to LH

In vitro culture systems are valuable tools for investigating reproductive mechanisms in the testis. Here, we report the use of the precision-cut in vitro system using equine testicular slices. Testes were collected from immature light breed stallions (n = 3) and cut into slices (mean slice weight = 13.85 ± 0.20 mg; mean slice thickness = 515.00 ± 2.33 μm) using the precision-cut tissue-slicing method. Four tissue slices were placed on a grid floating on medium in individual vials. After a 1-h preincubation, they were exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50, and 500 ng/ml for 6 h at 32°C. Viability of the tissue was maintained based on histological integrity and lack of appreciable lactate dehydrogenase in the medium. The production and release of testosterone (T) and estradiol-17β (E2) into the medium was measured following in vitro culture. The addition of oLH increased T and E2 at least 400% and 120%, respectively, over the 0-ng oLH control cultures. Testicular gene expression was assessed with in situ hybridization methodology for steroidogenic acute regulatory protein (StAR protein), phosphodiesterase 3B (PDE3B), and outer dense fiber of sperm tails 2 (ODF2) mRNAs. In situ hybridization revealed an oLH concentration-dependent increase in the concentration of StAR protein mRNA in Leydig cells. No differences were observed for the expression of PDE3B or ODF2 genes in seminiferous tubules among treatment groups as expected. These results demonstrate the value of in vitro culture of the precision-cut tissue slices for studies of the regulation of steroidogenesis and gene expression in the stallion testes.     

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.     

Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals

Human liver and kidney organ slices were used to investigate the biotransformation competence of the slices in combination with several markers of cell viability and function. The immunosuppressant cyclosporin A (CSA) is extensively metabolized in liver slices to the three known primary metabolites and many secondary metabolites. In kidney cortex slices the biotransformation of CSA is far more pronounced in humans than in rats. In human liver slices, levels of CYP3A, the proteins metabolizing CSA, are depressed about 25% by 1 and 10 mol/L CSA within 24 h, indicating that high blood or tissue concentrations will inhibit CSA clearance.A clinical marker for liver damage is the release of cellular -glutathione-S-transferases (GST). In this study the GST levels were used to assess donor organ quality, organ slice incubation conditions, and compound exposure. A marker for cell death in human cells is the solubilization and release of nuclear matrix proteins (Numa). Increases were apparent only after 48 h of culture.A side-effect of CSA is that it induces hypertension and perturbs the lipid profile of transplant recipients. A potential marker for lipid disturbances is levels of serum lipoprotein (a) (Lp(a)), which is synthesized in the liver and found only in humans, apes, and nonhuman primates. CSA increases Lp(a) levels in the human liver slice cultures about 2-fold.This study has demonstrated that the biotransformation capability of the organ slices contributes to the optimization of thein vitro system and to the evaluation of markers for drug induced side-effects or toxicity. Assays were identified that could be used clinically to monitor CSA-induced organ damage or rejection (GST), hypertension (Lp(a)), and toxicity (Numa).Abbreviations GST -glutathione-S-transferases - CSA cyclosporin A - CYP3A cytochrome P4503A - CSX undefined cyclosporin - Lp(a) lipoprotein (a) - IMM SDZ IMM 125 - Numa nuclear matrix proteins     

Kidney structure and function of obligate and facultative hibernators: the white-tailed prairie dog (Cynomys leucurus) and the black-tailed prairie dog (Cynomys ludovicianus)

The white-tailed prairie dog is an obligate hibernator that enters a heterothermic phase when maintained in the cold with low intensity light and ad libitum food and water. The black-tailed prairie dog (a facultative hibernator) will not hibernate under similar conditions. It has been suggested that the black tailed prairie dog remains active during the winter because it can conserve water more effectively due to a more efficient kidney. The present study revealed no significant differences between the species in renal morphology: relative medullary thickness, nephron heterogeneity, renal vasculature, or fornix dimensions, all of which are structures associated with the urinary concentrating mechanism. In addition, there was no difference in number of nephrons between the two species. The black-tailed prairie dog does produce a more concentrated urine when food and water deprived. However, this difference was not observed when the animals were salt loaded. The water-deprivation and salt-loading experiments suggest that the higher urine osmolality produced by the back-tailed prairie dog during fasting is a result of a higher urea load due to a greater protein catabolism and not because of a differential capacity to concentrate urine.Abbreviations C cortex - GFR glomerular filtration rate - H height - IS inner stripe - IZ inner zone of medulla - L length - OS outer stripe - PE polythylene - RMT relative medullary thickness - T _a ambient temperature - W width     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease.Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (Δ +480%; p < 0.001) compared to sham-operated controls. , indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (Δ −34%; p < 0.05) and 90 min (Δ −47%; p < 0.05). renin release from renal cortical slices of ADX rats was also significantly greater (Δ +130%; p < 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 μg/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values.Prostaglandin E₂ (PGE₂) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE₂ synthesis in glomeruli microdissected from ADX rats was significantly increased (Δ +110%; p < 0.001) compared to controls. PGE₂ synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10⁻⁶ M) decreased PGE₂ synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices.These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

硝酸盐对牛粪梭菌甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响

【背景】异于同型产乙酸菌通常利用Wood-Ljungdahl途径将2分子CO₂还原为1分子乙酰辅酶A,Clostridium bovifaecis缺失Wood-Ljungdahl途径甲基支路第1步将CO₂还原为甲酸的甲酸脱氢酶,需甲酸存在时将1分子甲酸和1分子CO₂还原为乙酰辅酶A发生葡萄糖的同型产乙酸型发酵。已有报道显示,硝酸盐也可作为同型产乙酸菌的电子受体,而且对不同同型产乙酸菌的代谢影响有所不同,然而硝酸盐对这种独特的甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响尚不清楚。【目的】探究硝酸盐对C.bovifaecis甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响。【方法】硝酸盐浓度分别为10 mmol/L和30 mmol/L时,以未添加硝酸盐为对照实验,研究C.bovifaecis在葡萄糖+甲酸+CO₂为基质条件下的细菌生长、底物消耗和产物生成情况。【结果】10 mmol/L和30 mmol/L硝酸盐存在时,主要产物乙醇浓度分别为5.80 mmol/L和1.66 mmo...     

不同磷、硫及二氧化碳浓度对标志链带藻生长和碳水化合物积累的影响

【目的】为了研究不同磷、硫及二氧化碳浓度对标志链带藻(Desmodesmus insignis)生长与碳水化合物积累的影响,本实验以改良BG11培养基为基础,设计了8种不同初始K_2HPO_4浓度、8种不同初始MgSO_4浓度及4种二氧化碳浓度培养标志链带藻。【方法】采用干重法和苯酚-硫酸法分别测定其生物质浓度与总碳水化合物的含量。【结果】实验结果显示,在高磷浓度(0.460 mmol/L)下生物量达到最高为6.37 g/L,磷浓度为0.230 mmol/L (对照组)时总碳水化合物含量及单位体积产率达到最高,分别为45.40%(%干重)和0.20 g/(L·d)。不同初始MgSO_4浓度实验结果显示,高硫浓度有利于标志链带藻生长及碳水化合物的积累,生物量、总碳水化合物含量及单位体积产率分别在硫浓度为1.217 mmol/L、0.609 mmol/L和1.824 mmol/L时达到最高,分别为7.02 g/L、51.6%(%干重)及0.26 g/(L·d)。当二氧化碳浓度为3%(V/V)时,标志链带藻生物量、总碳水化合物含量及单位体积产率均达到最高,分别为6.81 g/L、44.03%和0.20 g/(L·d)。【结论】因此,磷浓度为0.230 mmol/L、硫浓度为1.824 mmol/L和二氧化碳浓度为3%时最有利于标志链带藻生长及碳水化合物的积累。     

Effect of bioreactor configuration on substrate uptake by cell suspension cultures of the plant Eschscholtzia californica

Summary The uptake of carbohydrates and oxygen by cell suspension cultures of the plant Eschscholtzia californica (California poppy) was studied in relation to biomass production in shake flasks, a 1-1 stirred-tank bioreactor and a 1-1 pneumatically agitated bioreactor. The sequence of carbohydrate uptake was similar in all cases, with sucrose hydrolysis occurring followed by the preferential uptake of glucose. The uptake of fructose was found to be affected by the oxygen supply rate. Carbohydrate utilization occurred at a slower rate in the bioreactors. Apparent biomass yields, Y _X/S, ranged from 0.42 to 0.50 g biomass/g carbohydrate, while true biomass yields, Y _X/S, were about 0.69 g/g. The maintenance coefficient for carbohydrate, m _S, ranged between 0.002 and 0.008 g/dry weight (DW) per hour. The maximum measured specific oxygen uptake rate was 0.56 mmol O₂/g DW per hour and occurred early in the growth stage. The decline in specific uptake rate coincided with a decline in cell viability. The oxygen uptake rate was faster in shake flasks, corresponding to the higher growth rate obtained. The true growth yield on oxygen, YX/O₂, was calculated to range from 0.83 to 1.23 g biomass/g O₂, while the maintenance coefficient, mO₂, ranged from 0.15 to 0.25 mmol O₂/g DW per hour. The growth yields for oxygen determined from the stoichiometry of an elemental balance were within 10% of those calculated from experimental data. Offprint requests to: Raymond L. Legge