Construction methods and application of assembloids / 中国组织工程研究

BACKGROUND:In recent years,many studies have confirmed that assembloids can make up for the shortcomings of organoids,which cannot fully reproduce the interaction between cell and cell and between cell and matrix.Since the assembloids construction methods are in the early stage of development,there is no unified standard. OBJECTIVE:To review the current construction methods,applications,advantages,and disadvantages of assembloids,guide the development and improvement of vitro cell models. METHODS:PubMed,CNKI,and WanFang databases were searched with English search terms"assembloids,organoids,tumor microenvironment,organoids AND assemble,organoids AND microenvironment"and Chinese search terms"assembloids,organoids,tumor microenvironment,organoid reorganization,multicellular model".Totally 94 articles were screened out for review after excluding irrelevant articles and deduplication. RESULTS AND CONCLUSION:(1)According to the different sources of cells,the construction of assembloids can be divided into three methods:self-assembly,direct-assembly,and mixed-assembly.According to the differences of cell culture methods,it can be divided into suspension culture method,matrix culture method,organ chip culture method,and 3D bio-printing.(2)The process of self-assembly covers early stages of cell and tissue development,so it has broad prospects in the fields of organ development and developmental disorders.The function of differentiated mature cells is relatively perfect,and the assembloids directly assembled by them have more potential in the study of functional disorders and cell-damaging diseases.Self-assembly may be better in organ transplantation,and direct-assembly will be more suitable for the repair of tissue damage.Mixed-assembly combines the advantages of the former two and is mostly used to explore the physiological and pathological mechanisms of cells in the microenvironment,as well as drug screening.(3)Although different assembloids have their own advantages,they all face the problem of imperfect vasculature system,then,each method has its own limitations,for example,the degree of cell differentiation in self-assembly assembloids may still be different from that in vivo,and the fixed cell types in direct-assembly models cannot simulate complex microenvironments in vivo.These are urgent problems to be solved.(4)In the future,with the continuous improvement of assembloids culture technology,scientists can assemble biomimetic organoids with more complex tissues in vitro,providing infinitely realistic models for the study of physiological and pathological processes of human tissue and organ.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production

Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.

Suspension culture of Vero cells for the production of adenovirus type 5

Elicitation effect on the production of asiaticoside and asiatic acid in shoot, callus, and cell suspension culture of Centella asiatica

Centella asiatica is an important medicinal plant which contains various phytocompounds. Asiatic acid and asiaticosideare two major compounds which are responsible for its various pharmaceutical activities. The present study analyzesthe effect of elicitor, i.e., methyl jasmonate on the synthesis of asiaticoside and asiatic acid (ATA) in shoot, callus, andcell suspension cultures of C. asiatica. A high-performance liquid chromatography analysis showed that the elicitationwith 100 µM concentration of methyl jasmonate enhanced asiaticoside content by 69-fold in callus culture, 39-fold inshoot cultures, and ATA by 1.9-fold in cell suspension culture. Thus, elicitation with methyl jasmonate is an effectivemethod of increasing the rate of biosynthesis of asiaticoside and ATA in plant cell cultures of C. asiatica

Effect of drought stress on accumulation of two respective phenylethanoid glycosides in tissue culture of Cistanche tubulosa / 中草药

Objective: To establish the plant tissue culture system of Cistanche tubulosa, and determine the effect of drought stress on accumulation of two respective phenylethanoid glycosides in it. Methods The major chemical constituents of C. tubulosa by tissue culture were analyzed by HPLC-UV and HR-MS. The cell growth curves were also determined. In addition, the effects of drought stress on the phenylethanoid glycosides (echinacoside and acteoside) content in the tissue culture system of C. tubulosa were also studied by using NaCl, mannitol and PEG6000 as osmotic regulators, respectively. Results:Chemical constituents analyses revealed that callus and suspension cultures of C. tubulosa could produce the respective phenylethanoid glycosides of echinacoside and acteoside as in wild plant; Cell growth curves indicated that 30 d were the optimum culture period of callus culture; The cell growth rate and the accumulation of echinacoside and acteoside were mostly inhibited when the callus cells were under drought stress induced by NaCl or mannitol. Meanwhile, the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa could be effectively enhanced by treatment with PEG6000. The maximum biomass of echinacoside and acteoside could reach to (1.07 ± 0.10) g/L and (0.12 ± 0.01) g/L 15 d after induction, respectively. And their contents were 20.94% and 2.27% separately based on the cell dry weight (DW) after 15 d of treatment with 6% PEG6000, which were 1.29 and 1.19 fold higher than the control group. Conclusion:Drought stress induced by PEG6000 could effectively enhance the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa.

Optimization of noni callus induction and establishment of callus suspension system / 生物工程学报

The aim of the study was to obtain the secondary metabolites in the stem segment of noni and to establish genetic transformation system. The stem segments (no axillary buds) of noni were used as explants to induce the callus, and then to establish the cell suspension system. The factors affecting callus induction and cell suspension were studied. The results showed that the optimal culture medium for induction was MS with 1.0 mg/L 6-Benzylaminopurine (6-BA) and 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and the optimum culture medium for suspension was MS with 1.0 mg/L 6-BA and 0.1 mg/L 2,4-D, 3% sucrose and the pH of 5.85, with the initial inoculation amount of 37.5 g/L, and the speed of 110 r/min and 25±2 °C applying darkness culture. The suspension cells grew well and showed the maximum growth rate. The growth curve of the suspension cells from the stem segment of noni was in "S-typed" trend, and it should be transformed to the fresh medium between 12 and 20 d. During the culture, the pH of the culture medium decreased and then slowly increased, and the optimum pH for the suspension cells culture of callus from noni's stem segments was 4.5-5.0. In this study, the stable cell suspension system of the stem segment of noni was successfully established.

Experience in the extraction and culture of neural stem cells / 天津医药

@#There are many ways to extract and culture neural stem cells in vitro, but the viability and stability of neural stem cells obtained by different methods are different. By thinking about the process of extracting and culturing neural stem cells in vitro from the cerebral cortex of SD fetal rats, we summarized extraction steps, the main points of extraction, the selection basis of culture medium, selection of inoculation density, cultural method, methods of solution changing, passage time and passage methods. At last a large number of neural stem cells with high vitality and stability have been obtained and applied to the basic research of neural stem cells.

Comparative study on extraction and culture method of neural stem cells / 中国药理学通报

Aim To explore the best method of neural stem cellextraction and culture, and provide a technical reference for thebasic research of neural stem cells. Methods Different extractionand culture methods of neural stem cells were compared.The rate of ball of neural stem cells and the stability of neuralstem cells in undifferentiated state were observed by extraction offetal and neonatal rats cortex, using different types of medium,different inoculation density, different culture methods, differentmethods of changing liquid and different passage methods. Neuralstem cells' activities were detected by Varioskan LUX MultimodeMicroplate Reader. Results ① The brain cortex of fetalrats of 14 d had higher proportion of neural stem cells, less othercells and more neurospheres than newborn rats of 24 h. ② Neuralstem cells could be stabilized in undifferentiated state by usingserum-free medium, while most of the neural stem cellswere differentiated into neurons and glial cells by using serummedium. ③ Neural stem cells, seeding at 1. 0 ×109 ·L-1 , hada large number of neurosperes and were in good condition. ④Suspension culture was beneficial to form a stable neurosphereand keep the neural stem cells in an undifferentiated state thanadherent culture. ⑤ The state of neurosphere by changing halfof the medium and adding medium without discarding was betterthan that by replacing all medium. ⑥ The neurospheres couldbe separated into single cells by mechanical blow in primary generationand the second generation of neurospheres cultured in serum-free medium. While the percentage of viable cells in neuralstem cells was the highest digested with stem cell lysates afterthree generations and the neurospheres re-formed were better. ⑦Neural stem cells' activity of 14 d fetal rat in Accutase digestiongroup was significantly higher than that of the other threegroups, and the difference was significant (P <0. 05). ConclusionsNeural stem cells proliferate and divide well, with highrate of ball formation and good neurosphere condition, which canmaintain a stable undifferentiated state by extracting the cerebralcortex of 14 d fetal rats, using serum-free medium, inoculatinginto a 25 cm2 flask at a density of 1. 0 × 109 ·L-1 , and takingthe suspension culture (adding the medium 1 ~ 1. 5 mL every 2~3 d and passage every 6 ~8 d ).

CARDIAC GLYCOSIDES, PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY FROM PLANT CELL SUSPENSION CULTURES OF Thevetia peruviana / PRODUCCIÓN DE GLÍCOSIDOS CARDIOTÓNICOS, COMPUESTOS FENÓLICOS Y ACTIVIDAD ANTIOXIDANTE EN CULTIVOS DE CÉLULAS VEGETALES EN SUSPENSIÓN DE Thevetia peruviana

Cell suspension cultures of Thevetia peruviana were established under dark for 19 days to investigate kinetic behavior related to biomass, substrate, cardiac glycoside, polyphenols, reactive oxygen species and anti-oxidant activity. The results showed high biomass production (18.80gDW/L) as well as sucrose consumption in 7 days. Preferential glucose over fructose consumption was observed. Intracellular production of cardiac glycosides reached 2.58mg DE/gDW at day 19. Highest extracellular production was reached between day 2 and 7 (6.19mg DE/L). Highest extracellular phenolic content was 80.61 ± 5.16mg GAE/L at day 7. Intracellular phenolic content increased to 2.76 ± 0.14mg GAE/gFW at day 7 and remained constant until day 19. ROS production at day 7 could be related to sucrose and glucose total consumption. Pearson Product-Moment Correlation Coefficient (ρ) showed that the phenolic compounds in cell suspension cultures of T. peruviana were responsible for the observed anti-oxidant activity. All together, these results give the first steps in metabolic behavior in cell suspension cultures of T. peruviana.

Se establecieron cultivos en suspensión de la especie vegetal Thevetia peruviana en oscuridad, durante 19 días, para estudiar el comportamiento cinético de producción de biomasa, consumo de sustrato, producción de glicósidos cardiotónicos, polifenoles, especies reactivas de oxígeno y actividad antioxidante. Los resultados mostraron una alta producción de biomasa (18,80g PS/L), al igual que consumo total de sacarosa, a los 7 días de cultivo. Se observó un consumo preferencial de glucosa sobre fructosa durante todo el cultivo. La producción de glicósidos cardiotónicos intracelulares alcanzó valores de 2,58mg ED/g PS, al día 19. La mayor producción extracelular (6,19mg ED/L), se alcanzó entre los días 2 y 7. El mayor contenido de compuestos fenólicos extracelular fue de 80,61 ± 5,16mg GAE/L, en el día 7. El contenido de compuestos fenólicos intracelulares incrementó a 2,76 ± 0,14mg AGE/gPF, al día 7 y se mantuvo constante, hasta el día 19. La producción de EROs, al día 7, puede estar relacionada con el consumo total de sacarosa y glucosa. El coeficiente de correlación producto-momento de Pearson (ρ) indicó que los compuestos fenólicos en cultivos celulares en suspensión de T. peruviana eran los responsables de la actividad antioxidante observada. En conjunto, estos resultados brindan las primeras bases relacionadas al comportamiento metabólico de cultivos celulares en suspensión, de T. peruviana.

Expression and antiviral activity of a chimeric porcinized monoclonal antibody (cHQ06) against E2 protein of classical swine fever virus / 生物工程学报

Classical swine fever (CSF), one of OIE-listed diseases, is a highly contagious and economically important disease of pigs. Classical swine fever virus (CSFV) is the causative agent of CSF. The capsid (C) protein and the glycoproteins Erns, E1 and E2, are structural components of the virus. E2 is the most immunogenic protein of the CSFV glycoproteins, inducing neutralizing antibodies that provide protection against lethal CSFV challenge. In a previous study, we developed a murine MAb HQ06 against the E2 protein of CSFV. In this study, the variable region genes from HQ06 and constant regions gene of swine antibody are fused and cloned into the eukaryotic expression vectors to establish a cell line which can stably express a chimeric porcinized MAb (cHQ06) against E2 in CHO cell. The purified cHQ06 antibody protein was determined to be successfully generated, which exhibited high reactivity between cHQ06 and the E2 protein of CSFV by enzyme-linked immunosorbent assay (ELISA) and Western blotting. More importantly, we investigated the neutralizing activity of cHQ06 against CSFV. In conclusion, this study generated cHQ06 for efficient and stable production which can be used against to develop novel diagnostic assays, investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives

Objective To evaluate the impact of plant growth regulators including kinetin (KN), benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis (P. vietnamensis) in cell suspension culture. Methods Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate. Results All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d, (57.0 ± 0.9) and (3.1 ± 0.1) mg/mL fresh and dry weight, respectively, whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold. Conclusions The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives

OBJECTIVE@#To evaluate the impact of plant growth regulators including kinetin (KN), benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis (P. vietnamensis) in cell suspension culture.@*METHODS@#Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.@*RESULTS@#All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d, (57.0 ± 0.9) and (3.1 ± 0.1) mg/mL fresh and dry weight, respectively, whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4-2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8-2.6 fold.@*CONCLUSIONS@#The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.

Isolation and Identification of Human Ovarian Cancer Stem-like Cells Induced by Short-term Treatment with Carboplatin / 中国医科大学学报

Objective To isolate carboplatin-resistant human ovarian cancer stem-like cells and provide a cellular model for the development and screening of second-line drugs for carboplatin-resistant ovarian cancer.Methods Primary ovarian cancer cells were isolated and cultured from ovarian cancer tissues and ascites,and ovarian cancer stem-like cell spheres were isolated from serum-free suspension culture directly or after carboplatin induction.The expression of stem cell markers was evaluated by immunofluorescent assay or flow cytometry.Results The ovarian cancer stem-like cell spheres were isolated successfully from both ovarian cancer tissues and ascites and expressed high levels of stem cell markers CD133,ALDH1,ABCG2,and CD24.The abundance of CD24+ cells in the ovarian cancer stem cells was significantly increased upon short-term induction with carboplatin.Condusion Ovarian cancer stem-like cells can be enriched and purified by short-term induction with carboplatin.This cell model can be used in research on chemoresistant ovarian cancer in the future.

Isolation and Adherent Culture of Neural Stem Cells from Mouse Embryo / 华中科技大学学报(医学版)

Objective To develop a modified method of adherent culturing NSCs to replace the traditional suspension culturing method.Methods Neural stem cells(NSCs)were isolated from brain of fetal mouse at 15.5-day fetal age.In culture bottle which had been pre-coated with poly-L-ornithine hydrochloride/fibronectin(PO/FN),the cells were adherently cultured and passaged.The cell morphology was observed under inverted microscope.NSCs and their differentiated cells were identified by immunofluorescence.Results NSCs obtained in this study were successfully adherently cultured and expressed specific markers.Conclusion NSCs are successfully adherently cultured in this study.As compared with the traditional suspension culturing method,adherent culturing is simpler and has lower wastage of passage.It is easy to observe cell morphology and differentiation by this method.It is also helpful to culturing and storage of NSCs.

Isolation of breast cancer stem cell from MDA-MB231 cell line using vincristine / Aislamiento de células madre de cáncer de mama de la línea celular MDA-MB231 utilizando vincristina

Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs' viability (P> 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P>0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.

El cáncer ha sido considerado como una enfermedad de células madre. Recientemente se ha propuesto cultivo en suspensión en combinación con medicamentos contra el cáncer para aislamiento de las células madre del cáncer (CMC). En este estudio se utilizó la vincristina como fármaco anticanceroso combinado con cultivo en suspensión para el aislamiento y purificación de las células madre cancerosas, de la línea celular de cáncer de mama humano (MDA-MB231). Las células se trataron con diferentes concentraciones de vincristina (0, 2, 4, 6 y 8 ng/ml). Las células madre se identificaron mediante la expresión de los genes OCT4, Nanog, SOX2 y nucleostemin por RT-PCR. La unidad de formación mammosphere se midió a través de cultivo en suspensión que contenía EGF, bFGF, LIF, B27, insulina y BSA. Los mammospheres aislados fueron estudiados para la expresión de CD44. Los resultados mostraron que 4 ng/ml de vincristina durante 72 horas podrían ser utilizados como la mejor y más fiable dosis que permite eliminar alrededor del 80 % de las células madre no cancerosas, sin causar un efecto destructivo sobre la viabilidad de las CMC (P> 0,05). La RT-PCR mostró que en las células tratadas con él fármaco hubo expresión de los genes OCT4, Nanog, SOX2 y nucleostemin. La unidad de formación de las células pretratadas con vincristina fue significativamente más alta que las unidades sin tratamiento previo (P>0,05). La inmunofluorescencia para CD44 muestró una alta expresión del marcador de CMC entre mammospheres aisladas. La vincristina en combinación con el cultivo en suspensión puede ser considerado como un método apropiado para aislar CMC.

Effect of L-tryptophan and its derivatives on cephalotaxine production in Cephalotaxu mannii suspension cells / 中草药

Objective: To investigate the effects of L-tryptophan and its derivative 5-methyl-DL-tryptophan (5-MT) on suspension cells growth and cephalotaxine production of Cephalotaxu mannii. Methods: The cell suspension cultures were treated with L-tryptophan and 5-MT on day 15, respectively. Then the cell growth, cephalotaxine production, and activity of key enzymes in the metabolism pathway were determined. Results: L-tryptophan and 5-MT could enhance cephalotaxine production. The cultures treated with 5 mg/L L-tryptophan showed the highest cephalotaxine yield (2.944 mg/L). While treated with 10 mg/L L-tryptophan and 5 mg/L 5-MT, the cephalotaxine yields were 1.947 and 2.192 times of the control culture (1.343 mg/L); The biomass of suspension cultures were decreased by 9.35% and 18.04%, respectively, less than those in the control group (2.406 g/L); The activity of 3-deoxy-D-arabino- heptulosonate-7-phosphate synthase was decreased by 12.4% and 5.57% compared with the control (0.67 U/mg); The activity of phenylalanine ammonium-lyase activity increased by 37.72% and 28.02% and the total phenolics content increased by 19.14% and 9.61% compared with the control (78.21 U/mg and 0.36 mg/mL). Conclusion: L-tryptophan and its derivative 5-MT can inhibit the L-tryptophan biosynthetic pathway and promote the accumulation of cephalotaxus alkaloids.

Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures

ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.

The expression and significance of Nanog and CD44 in spheroid body-forming cells of gastric cancer cell line MKN-45 / 天津医药

Objective To detect the expression of stem-cell related factors Nanog and CD44 in spheroid body-form?ing cells of gastric cancer cell line MKN-45. Methods The gastric cancer cell line MKN-45 was used to culture spheroid bodies in non-adherent condition in a serum-free medium supplemented with epidermal growth factor (EGF) and basic fibro?blast growth factor (bFGF). Using Western blot analysis, immunofluorescence staining and quantitative real time polymerase chain reaction(qRT-PCR), the expression levels of stem cell-related genes Nanog and CD44 were studied. Results In this study, we observed that MKN45 cells formed spheroid bodies in non-adherent condition in a serum-free medium, and the levels of Nanog and CD44 mRNA expression in spheroid body-forming cells were 2.34 ± 0.22 and 1.18 ± 0.04,respectively, which were higher than those in parental cells (1.00±0.00 and 1.00±0.05). The levels of Nanog and CD44 protein expression in spheroid body-forming cells were 0.18±0.02 and 0.24±0.04, respectively, which were significantly higher than those in pa?rental cells (0.07±0.02 and 0.18±0.01, P<0.05). Nanog protein was positively stained within the perinuclear and cytoplasm of the spheroid body-forming cells, and CD44 was positively stained mainly in the membrane. Dual staining of Nanog/CD44 indicated that the embryonal protein Nanog was co-localized with CD44 in the spheroid body-forming cells. Conclusion Spheroid body-forming cells developed from human gastric cancer cell line MKN-45 in serum-free medium supplemented with EGF and bFGF show characteristics of cancer stem cell (CSC). The cells co-expressed of CD44 and Nanog maybe a phe?notype of gastric CSCs.

Culture process for fully human anti-VEGF165 monoclonal antibody / 中国药科大学学报

@#This study was focus on the optimizing cell culture process of recombinant DG44 cells which expressing novel fully human anti-VEGF165 monoclonal antibody(HVAB). Feed-batch culture and two-phase temperature control were studied for optimizing the HVAB productivity in shaken flasks. The influences of pH changes were determined to study the growth of DG44 cell and expression of HVAB in bioreactors. The HVAB productivity was rose from 101 mg/L to 654 mg/L after feed-batch culture in shaken flask, and cell viability maintained above 80% after reduce the temperature in middle growth phase. It is also suggested that pH range at 6. 4-7. 4 is beneficial to DG44 cells growth and HVAB expression. The productivity in bioreactor is 526 mg/L decreased 11% compare with in shaken flask, which laid an foundation for further pilot scale production.