AQUACULTURE
·
2025
·
IF 3.9
Song Wang,Liu Song,Yaru Li,Zhenxin Zhao,Ting Xue,Guoxiang Luan,Huarong Guo
Holothurian cell culture remains unsuccessful due to the lacking of suitable medium and tissues. Here, a reliable in vitro cell culture system has been developed for Apostichopus japonicus by optimizing the basal and growth media as well as the sourced tissues. Blood vessel and longitudinal muscle tissues were first found to be the best two sourced tissues. Subsequently, the composition and content of free amino acids and carbohydrates in holothurian coelomic fluid were analyzed and the obtained data were used to design and obtain an optimal basal medium (SCBM-4). Next, an optimal growth medium (SCGM-24) was further developed by supplementing 1 % FBS, 25 % coelomic fluid and 20 % body wall enzymatic hydrolysate of sea cucumbers into SCBM-4. Primary blood vessel (SCBV) cells could be efficiently isolated by mechanical dissociation in tissue dissociation apparatus and survive healthily for 22 d in SCBM-4, and 58 d in SCGM-24 with a gradually decreased proliferation activity. Primary longitudinal muscle (SCLM) cells could only be efficiently isolated by explant culture and healthily survive for 52 d in SCBM-4 but with no proliferation activity, whereas their viabilities decreased sharply in SCGM-24. The epithelial-shaped SCBV cells could be successfully sub-cultured for 3 times using 4 °C PE solution, whereas the loosely attached round-shaped SCBV and SCLM cells could be passaged for 6 and 4 times by pipetting, respectively. Additionally, shrimp covert mortality nodavirus (CMNV) could cross-species infect and multiply in SCBV cells, and acute heat shock treatment could significantly upregulate the transcriptional expression of heat shock protein genes.
研究领域:
海洋生物学
·
细胞培养
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病毒学
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分子生物学
·
水产养殖
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产品关联
DOI
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2022
·
IF 6.064
Chen Qiaohong,Chen Yi,Xu Quan,Jin Hu,Hu Qiang,Han Danxiang
Chromochloris zofingiensis has obtained particular interest as a promising candidate for natural astaxanthin production. In this study, we established a two-stage heterotrophic cultivation process, by using which both the growth of C. zofingiensis and astaxanthin accumulation are substantially enhanced. Specifically, the ultrahigh biomass concentration of 221.3 g L−1 was achieved under the optimum culture conditions in 7.5 L fermenter during 12 days. When scaled-up in the 500 L fermentor, the biomass yield reached 182.3 g L−1 in 9 days, while the astaxanthin content was 0.068% of DW. To further promote astaxanthin accumulation, gibberellic Acid-3 (GA3) was screened from a variety of phytohormones and was combined with increased C/N ratio and NaCl concentration for induction. When C. zofingiensis was grown with the two-stage cultivation strategy, the astaxanthin yield reached 0.318 g L−1, of which the biomass yield was 235.4 g L−1 and astaxanthin content was 0.144% of DW. The content of the total fatty acids increased from 23 to 42% of DW simultaneously. Such an astaxanthin yield was 5.4-fold higher than the reported highest record and surpassed the level of Haematococcus pluvialis. This study demonstrated that heterotrophic cultivation of C. zofingiensis is competitive for industrial astaxanthin production.
研究领域:
微藻生物学
·
生物技术
·
天然产物开发
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DOI
PubMed
高影响因子
Journal of Oceanology and Limnology
·
2023
·Vol. 41
·期 5
·1918-1939
·
IF 1.6
Guo Liwen,Zhao Yaqi,Guo Huarong
Crab cell line, especially continuous crab cell line, can provide us a useful tool for studies on the virology, immunology, and molecular biology of crabs. However, no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest. In this study, long-term in vitro culture conditions for both two- (2D) and three-dimensions (3D) were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus , designated as PTH cells. In 2D culture, a novel crab basic medium in osmolarity of 990–1 100 mOsm/kg was optimized for the first time, which is different from Leibovitz’s L-15 medium in mainly the components of amino acids, containing double strengths of the contents of free amino acid mixture in the crab serum. Then an optimal crab growth medium was developed by supplementing 5% fetal bovine serum, 50-g/L yeast extract powder, 20-µg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium, and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells. These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen. In 3D culture, using the same crab growth medium, the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60% on Day 103. This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.
研究领域:
海洋生物学
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细胞生物学
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免疫学
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病毒学
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分子生物学
✓ 已验证
DOI
高影响因子
STAR Protocols
·
2024
·
IF 1.3
Zitian Wang,Xiaowei Tang,Davit Khutsishvili,Gan Sang,Edgar A. Galan,Jie Wang,Shaohua Ma
Summary In vitro organoids, including cerebral organoids, are usually developed without mechanical compression, which may contribute to a delay in maturation. Here, we present a protocol for encapsulating cerebral organoids with a thin shell of low-concentration alginate hydrogel. We describe steps for organoid generation, microfluidic chip culture, Matrigel coating, expansion culture, and alginate encapsulation. We then detail procedures for maturation culture and organoid characterization. The moderate compressive stimulation that the shell provides promotes cell proliferation and neuronal maturation. For complete details on the use and execution of this protocol, please refer to Tang et al. 1
CAS / MDL:
研究领域:
神经科学
·
组织工程
·
生物医学工程
·
细胞生物学
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 3.8
Chuanfu Huo,Yuanyuan Zhao,Meng Song,Yuzhe Guo,Shihao Li,Wensong Tan,Yan Zhou
Exosomes are nanoscale vesicles containing a variety of bioactive factors, showing significant potential as cell-free therapeutic agents. However, the large clinical doses required for exosome administration highlight the need for a scalable serum-free production system to increase exosome yield. In this study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs) were cultured in three-dimensional (3D) environments using polyethylene terephthalate (PET) fiber membranes. Two key pathways promoting exosome secretion and biogenesis through 3D culture were identified. Specifically, 3D culture reduced the expression of Integrin β1, leading to the decondensation of cortical actin, and increased the expression of RAC1, which promoted actin aggregation on the membranes of multivesicular bodies (MVBs). Building on this mechanistic understanding, we simulated the depolymerization/aggregation of the cytoskeleton in 3D culture and designed a serum-free medium optimized for exosome production. For large-scale exosome production, hUCMSCs were cultured in a fixed-bed bioreactor using PET fiber rolls as the fixed-bed layer with the developed serum-free medium. This setup increased exosome production per cell by 16-fold compared to 2D culture, yielding 2.6 × 10^14 exosome particles. The exosome products demonstrated enhanced in vitro angiogenesis, immunomodulation, and in vivo wound healing capabilities. This study provides valuable insights and strategic guidance for the serum-free, large-scale production of exosomes using bioreactors, with significant implications for therapeutic applications.
研究领域:
细胞生物学
·
再生医学
·
生物工程
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产品关联
DOI
高影响因子
AQUACULTURE
·
2024
·
IF 3.9
Yaqi Zhao,Ting Xue,Jinwu Wang,Huarong Guo
Long-term in vitro cultured crayfish cells can serve as a valuable tool for investigating various aspects of crayfish biology, encompassing cell physiology and viral mechanisms. Regrettably, they are difficult to maintain a highly active state and survive during long-term and continuous culture in vitro . In order to generate highly viable crayfish cell cultures, it is crucial to optimize dependable culture conditions and utilize a suitable cell medium. In this study, we have successfully developed reliable in vitro long-term culture systems for hematopoietic (HPT) cells of crayfish Cherax quadricarinatus by formulating an optimal crayfish cell growth medium (OCCM), including under two-dimensional (2D) and three-dimensional (3D) conditions. The novel medium was optimized by a mix of exogenous nutrients, crayfish plasma (CP) and shrimp serum-derived amino acids and saccharides within the Leibovitz's L-15 (L-15) medium. Our newly developed crayfish cell growth medium (CCM-28) enabled the culturing of HPT cells for over 142 days under 2D and over 60 days under 3D conditions, with consistent active cell proliferation observed over a 30-day incubation period. This proliferation was comparable to in vivo conditions, with a cell division rate exceeding 20%, suggesting that a high mitotic rate has been successfully achieved during long-term in vitro culture. Flow cytometry analysis further elucidated the mitosis-arrest of HPT cells cultured in vitro was predominantly observed in the G 0 /G 1 phase. Furthermore, the cell subculture techniques have been established using mechanical dissociation method (in 2D) and substrate exchange method (in 3D). Additionally, the susceptibility of these cell cultures to covert mortality nodavirus (CMNV) and white spot syndrome virus (WSSV) was confirmed by capturing cytopathic effects (CPE) and analyzing viral copy numbers. Species identification using the cytochrome oxidase subunit I ( COI ) gene established the origin of the cell cultures from C. quadricarinatus . In conclusion, the successful development of highly viable crayfish cells in long-term in vitro culture systems, as demonstrated in this study, will significantly advance the research on immortalization in crayfish cells and the study of mechanisms related to viruses.
研究领域:
细胞生物学
·
病毒学
·
水产养殖
·
海洋生物学
✓ 已验证
产品关联
DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2025
·
IF 9
Yuntong Xu,Guiyuan Yu,Zhitong Wang,Wen Cao,Min Feng,Wenwen Wei,Liejin Guo
Microalgae offer high lipid content and significant carbon–neutral potential, yet inefficient bioprocess and carbon source management fundamentally hinder their scalability. Addressing these challenges, this study developed an innovative integrated two-stage continuous cultivation system for Synechococcus sp., combining pH-triggered CO 2 delivery with wastewater nutrient recycling. Elucidating the relationships among growth, pH, and inorganic carbon, the precise CO 2 -based pH strategy eliminated salt stress, enhancing biomass productivity by 25 % over conventional chemical methods. The optimized nitrogen-deficiency two-stage mode achieved a 2.3-fold increase in lipid productivity and enabled high-efficiency nutrient removal (NH 4 + >70 %, PO 4 3− near-complete). A comprehensive techno-economic analysis confirmed superior cost-effectiveness, demonstrating a ∼45 % reduction in minimum biodiesel production cost compared to single-stage operations. These findings establish a highly feasible, scalable, and sustainable wastewater-to-bioenergy solution.
研究领域:
环境生物技术
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可再生能源
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微生物生理学
·
废水处理
·
生物能源工程
·
碳循环管理
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工业生物技术
·
系统生物工程
✓ 已验证
DOI
PubMed
高影响因子
Biology-Basel
·
2020
·Vol. 9
·期 7
·171
·
IF 3.796
Jianming Zhang,Yushan Bu,Chengcheng Zhang,Huaxi Yi,Daqun Liu,Jingkai Jiao
At present, De Man, Rogosa and Sharpe (MRS) broth is the medium of choice for promoting bacteriocin production. However, this medium is expensive and not applicable for large-scale production. Therefore, a low-cost and high-efficiency culture medium for bacteriocin Lac-B23 production byLactobacillus plantarumJ23 was developed. First, the effects of the composition of MRS broth on bacteriocin Lac-B23 production and bacterial growth were researched by a one variable at a time approach. Then, a Plackett-Burman design was used to screen significant components for production. Finally, the steepest ascent and central composite designs were used to obtain an optimum medium. The final composition of the modified MRS was much simpler than MRS broth, and the modified MRS contained only glucose, yeast extract, dipotassium phosphate, manganese sulfate monohydrate, Tween 80 and sodium acetate anhydrous. The highest bacteriocin Lac-B23 production reached 2560 activity units (AU)/mL in the modified MRS, which is nine times higher than that in MRS broth (280 AU/mL). Meanwhile, the cost per liter of the modified MRS (8.56 Ren Min Bi (RMB)/L) is 34.70% the cost of MRS broth (13.11 RMB/L), and the cost per arbitrary units of bacteriocin Lac-B23 in the modified MRS is approximately fourteen times more convenient (3.34 RMB/106AU) than in the MRS broth (46.82 RMB/106AU).Keywords:bacteriocin Lac-B23;low-cost;high-efficiency;culture medium
研究领域:
微生物学
·
食品科学
✓ 已验证
DOI
PubMed
高影响因子
EUROPEAN FOOD RESEARCH AND TECHNOLOGY
·
2021
·Vol. 247
·期 11
·2765-2776
·
IF 2.998
Ni Zhenghang,Ye Pian,Liu Jian,Huang Jun,Zhou Rongqing
It was aimed at investigating the effect of co-cultivating Torulaspora delbrueckii (hereinafter abbreviated as T . delbrueckii ) CCTCC M2019523 with Saccharomyces cerevisiae (hereinafter abbreviated as S. cerevisiae ) on the quality of greengage wine and the diversity of the microbial community in the mash, including the ratio and sequence of cultivation. Among samples, it shows high quality of the fresh wine when sequential inoculated at the ratio of 1.2:1 and co-cultured. The content of the sugar and succinic acid decreased, while that of alcohol increased. Of them, the content of phenylethyl alcohol and capric aldehyde enhanced, while decanoic acid decreased, so that the intensity of rose and plant aroma was enhanced, the rare delicacy of spoilage and sourness was reduced. The acidity of the fresh wine was also significantly reduced by fed-batch culture and its plant aroma was also enhanced. It provides a new avenue to improve the flavor and quality of wine brewed by a featuring characteristic fruit.
研究领域:
食品科学
·
微生物学
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2021
·
IF 3.307
Yuanyuan Miao,Yanyun Hu,Shanyong Yi,Xuejia Zhang,Ninghua Tan
Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes -mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rol B and rol C genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g −1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g −1 DW for shoots and roots respectively) and calli (1.082 μg g −1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g −1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g −1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g −1 DW). Hairy root culture of R. yunnanensis , with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.
研究领域:
植物生物技术
·
生药学
·
分子生物学
·
药物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
·
2022
·
IF 3.141
Dong Zhang,Yingling Bao,Zhi Ma,Jiawei Zhou,Hanchi Chen,Yuele Lu,Linjiang Zhu,Xiaolong Chen
Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett–Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.
研究领域:
微生物学
·
生物技术
·
药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Biomass Conversion and Biorefinery
·
2022
·Vol. 13
·期 18
·16807-16818
·
IF 4.05
Wang Qing,Jin Wenbiao,Han Wei,Song Kang,Chen Yidi,Chen Chuan,Jiang Guangming,Zhou Xu
In order to achieve strain improvement and reduction of fermentation medium cost, atmospheric and room temperature plasma (ARTP) mutagenesis and microbial microdroplet culture (MMC) high-throughput screening were used to obtain high-yield Aurantiochytrium sp. strain. Meanwhile, low-cost food waste liquid was added into culture medium to reduce cost. The results showed that the optimal treatment time of ARTP mutagenesis for Aurantiochytrium sp. was 25 s, and 20 mutant strains were screened out by MMC to investigate their performance. The most significant increase in DHA yield was mutant Aurantiochytrium sp. MA20, whose biomass, lipid, and DHA yield were 29.0 ± 0.24, 15.8 ± 0.11, and 9.22 ± 0.37 g/L and 5.77, 16.9, and 83.2% higher than those of original strain, respectively. Compared with the original strain, the DHA proportion in total fatty acids of Aurantiochytrium sp. MA20 increased from 37.2 to 58.3%, while the DHA proportion in biomass increased from 18.4 to 31.7%. The genetic stability for Aurantiochytrium sp. MA20 showed that the yield of biomass, lipid, and DHA of the 50th generation were 29.1 ± 0.13, 16.2 ± 0.31, and 9.47 ± 0.26 g/L, respectively, which was no significant difference between original strain, indicating that the mutant MA20 could be inherited stably. Using food waste liquid to replace 100% yeast extract and 50% sea water of the fermentation medium, the DHA yield reached 9.54 g/L, which was equivalent to that of traditional medium, while the cost of medium was reduced by 45% that achieved the recycle of food waste resource. Graphical abstract
研究领域:
微生物学
·
生物技术
·
环境科学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）