Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.
Animals ; Batch Cell Culture Techniques ; Bioreactors ; standards ; CHO Cells ; Cell Count ; Computer Simulation ; Cricetinae ; Cricetulus ; Industrial Microbiology ; instrumentation ; methods

OBJECTIVETo establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.

METHODSThree 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.

RESULTSCompared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).

CONCLUSION4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Chloral Hydrate ; pharmacology ; Cilia ; drug effects ; enzymology ; physiology ; Male ; Osteoblasts ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley

Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.
Biomass ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Culture Media ; chemistry ; metabolism ; Eriobotrya ; chemistry ; growth & development ; metabolism ; Triterpenes ; analysis ; metabolism

Suspension cultures cell of Sorbus aucuparia (SASC) was used as materials, the changes of physiological and biochemical indexes of SASC after treatment with yeast extract (YE) were detected, and the synthetic mechanism of secondary metabolites in SASC treated with YE was preliminarily explored. The results were as follows: under the assay conditions, SASC was induced to synthesize five biphenyl compounds, and these compounds content changed differently with induction time prolonging; YE treatment inhibited cell growth, the culture medium pH was gradually reduced after treatment; water-soluble protein content showed a trend of slow decline, which was significantly increased in YE treatment group (YE group) compared with the control group (CK group), the maximum relative content was 147.76% in contrast with CK group; both YE group and CK group were extracellular Ca2+ flow influx, but the YE group flow was significantly slow than CK group. The results indicate that YE induced the cells in a stress state, which was not conducive to the growth of cells and forced the cells to synthesize biphenyl compounds against external stress; water-soluble protein may serve as intracellular enzymes involved in the synthesis of compounds regulation; Ca2+ may as signal molecule mediate cell signal transduction respond to YE stress.
Cell Culture Techniques ; instrumentation ; methods ; Culture Media ; chemistry ; metabolism ; Saccharomyces cerevisiae ; chemistry ; Secondary Metabolism ; Sorbus ; growth & development ; metabolism

To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.
Agrobacterium ; physiology ; Benzofurans ; analysis ; metabolism ; Cell Culture Techniques ; instrumentation ; methods ; Cinnamates ; analysis ; metabolism ; Culture Media ; chemistry ; metabolism ; Depsides ; analysis ; metabolism ; Drugs, Chinese Herbal ; analysis ; metabolism ; Plant Roots ; chemistry ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; chemistry ; growth & development ; metabolism ; microbiology

AIM: To investigate the effects of feeding phenylalanine (Phe) and tyrosine (Tyr) on the accumulation of total phenolic compounds and four phenylethanoid glycosides (PeGs) to a cell suspension culture of the parasitic plant Cistanche deserticola. METHOD: A cell suspension culture of C. deserticola was established and precursors of different concentrations were fed. In each group, the cell was sampled at the 24(th) day after inoculation. The content of total phenolic compounds and four PeGs compounds were determined using the Folin-Ciocalteu method and an HPLC method, respectively. RESULTS: In the Phe fed cells, the maximum PeGs yield was achieved when Phe was fed at 1.5 mmol·L(-1) and the yield reached 1.13 times the control cell concentration. In the Tyr fed cells, the maximum yield of PeGs was 1.60 times of control when 0.75 mmol·L(-1) Tyr was fed to the cells. Furthermore, it was found that the salidroside yield was 4.01 times of control group when 5 mmol·L(-1) Tyr was fed. CONCLUSION Tyr is a better precursor for PeGs accumulation compared with Phe, and the rate limiting enzymes might be involved in the Tyr branch.
Cell Culture Techniques ; instrumentation ; methods ; Cistanche ; chemistry ; growth & development ; metabolism ; Culture Media ; chemistry ; metabolism ; Glycosides ; analysis ; metabolism ; Phenylalanine ; metabolism ; Tyrosine ; metabolism

In recent years, Chinese hamster ovary (CHO) production vessel volume has reached more than 1 000 L in Chinese biopharms, and 10 000 L in foreign big biopharms, such as Lonza and Genetech. In general, there are some steps seed bioreactor for seed expansion, which decreases the efficiency of production process. In this work, a perfusion-based process was developed to drastically increase the split ratio during the scale-up of CHO cell cultures. Fed-batch cultures were inoculated with cells propagated in either batch or perfusion cultures that grown in disposable Cellbags using the WAVE Bioreactor system. The higher cell concentration of 2 x 10(7) cells/mL with 95% viability allowed to increase the split ratio to about 1:50-1:100 for inoculum propagated in perfusion culture. The method described here could reduce the number of required expansion steps and eliminate two or three bioreactors. Disposable perfusion bioreactor with only a few liters working volume have the potential to directly inoculate volumes of up to 1 000 liters. This would allow to shorten process time in these bioreactors, which often are the bottleneck in plant throughput.
Animals ; Bioreactors ; CHO Cells ; cytology ; Cell Culture Techniques ; instrumentation ; methods ; Cricetinae ; Cricetulus

OBJECTIVETo study orientation remodeling without stress in bone harvest chamber.

METHODSThe bone harvest chamber (BHC) methodology is adopted in this study. Five female Japanese white rabbits were allowed unrestricted activity. The bone harvest chamber was a cylindrical Ti implant body with a transverse 1 mm wide canal for bone ingrowths. Retrieval of the contents of the canal was allowed with minimal disturbance to the surrounding bone or outer cylinder. After bone harvest chambers were implanted into the tibia of rabbits for 8 weeks, the chambers were considered to be osseointegrated with the bone. After harvested, the tissue were fixed and decalcified, then embedded in paraffin. Each rabbit was put into surgical operation 4 times for 4 stages: vacant for the first time; the tissue were cut into longitudinal sections at the second and third stages; harvesting tissues were cut into transverse sections at the fourth stage. Directional analysis: the standard deviation of the orientation of cell nucleus in each section was used as statistics, the difference between longitudinal section and transverse section were analyzed.

RESULTSOf the tissue into bone harvest chamber, directionality of cells arranged was more significantly on longitudinal section than on transverse section and there was statistical ignificecne.

CONCLUSIONUnder the no-stress circumstance of BHC bone remodeling showed directivity. Stress is not the direct leading signal about bone reconstitution. The structure of BHC might be related to orientation remodeling, which suggests that the relationship between orientation and stress is mediated by blood vessel. The effect of stress may be to affect vessel distributing in some orietation.

Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Female ; Osteocytes ; chemistry ; cytology ; Rabbits ; Tibia ; chemistry ; cytology

China consumes and exports traditional Chinese medicinal resources the most in the world. However, we cannot anchor our hope on field production of traditional Chinese medicinal materials and their active ingredients, due to limited land resources. Therefore, the development of biotechnology is of great importance for China to solve the problem of traditional Chinese medicinal resources. Plant cell culture is an important approach for the sustainable development of precious medicinal resources. This essary summarizes the optimization of conditions for medicinal plant cell culture, the regulation of secondary metabolic pathways and cell bioreactor culture, and realizes that the authentic commercial production of more medicinal plants requires efforts from all aspects.
Bioreactors ; Biotechnology ; methods ; trends ; Cell Culture Techniques ; instrumentation ; methods ; China ; Drugs, Chinese Herbal ; metabolism ; Plants, Medicinal ; cytology ; growth & development ; metabolism ; Research ; trends

Based on single-chip microcomputer, we have established a mechanical strain loading system with multi-channel to study the biological behavior of cultured cells in vitro under mechanical strain. We developed a multi-channel cell strain loading device controlled by single-chip microcomputer. We controlled the vacuum pump with vacuum chamber to make negative pressure changing periodically in the vacuum chamber. The tested cells were seeded on the surface of an elastic membrane mounted on the vacuum chamber, and could be strained or relaxed by cyclic pressure. Since the cells are attached to the surface of the membrane, they presumably experience the same deformation as that was applied to the membrane. The system was easy to carry and to operate, with deformation rate (1%-21%) and frequency (0-0. 5Hz) which could be adjusted correctly according to experimental requirement, and could compare different deformation rate of three channels at the same time. The system ran stably and completely achieved design aims, and provided a method to study the biological behavior of cultured cells attached to the surface of the elastic membrane under mechanical strain in vitro.
Cell Culture Techniques ; instrumentation ; methods ; Computer Simulation ; Equipment Design ; Mechanotransduction, Cellular ; physiology ; Microcomputers ; Stress, Mechanical ; Tensile Strength