Rotating wall vessels (RWVS), an ingenious apparatus for three-dimensional suspension culture, is widely used to build a simulated microgravity-effect on cell. Independent researchers have proposed hypotheses to illustrate why RWVS can simulate certain aspects of microgravity. Many of the hypotheses stated that the culture condition in RWVS is determined by the cellular mechanical environment which is a result of low fluid shear and microcarrier's motion. The microcarrier's motions consist of primary and secondary motions. In the light of the analysis of forces loaded by the microcarriers, some conclusions are drawn from the data on microcarriers' primary and secondary motions about which many simulations and observations have already been conducted.
Cell Culture Techniques ; instrumentation ; methods ; Gravitation ; Models, Theoretical ; Motion ; Rotation ; Stress, Mechanical ; Weightlessness Simulation

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

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

Three-dimensionally controlled cell-assembly technique makes fabricating tissues and organs in vitro to be possible. However, for real tissues and organs with complex structure and various cells, fabricating tissues and organs in vitro need a technique that could assemble and locate multi cells and materials precisely in the space. Facing the needs of multi-cell assembly, we designed a mixer nozzle and the matching pulse switching circuit which based on the single-nozzle cell assembly system, and developed a multi-cell-assembly system. We also carried out some assembly experiments with this system using materials that were similar to the multi-component extracellular matrix materials. The results demonstrated that the system could assemble various cells and materials into three-dimensional inhomogeneous structures precisely.
Bioartificial Organs ; Cell Culture Techniques ; instrumentation ; Cell Physiological Phenomena ; Equipment Design ; methods ; Extracellular Matrix ; chemistry ; Humans ; Tissue Engineering ; methods

Transplantation of the microencapsulated recombinant cells is a novel alternative approach to gene therapy of tumors. The semi-permeable membrane of microcapsule protects cells from host's immune rejection, increases the efficiency of gene transfer and reduces the need for frequent injection. Optimization of the preparation and culture is needed to acquire biological microcapsule with high cell viability and protein production. In this work, we studied the effect of different preparation and culture condition on the microencapsulated recombinant CHO cells growth and endostatin production. The result showed that the inoculum cells growth phase and seeding density potently affected the growth and endostatin production of the recombinant CHO cells in the microcapsule. The exponential growth phase recombinant CHO cells with a seeding density of 1 x 10(6) - 2 x 10(6) cells/ mL microcapsules benefited to the cells growth and endostatin production. The time of preparation was another important effect factor of cells viability, the cells viability decreased with the increase of preparation time and the time of preparation should be under 5h for maintaining the cell viability and endostain production. The highest viable cell density and endostatin production was acquired when the microcapsule percentage was 5% in the culture of the microencapsulated cells, the cell growth and endostatin production decreased with the increase of the microcapsule percentage.
Animals ; CHO Cells ; Capsules ; Cell Culture Techniques ; Cell Proliferation ; Cricetinae ; Cricetulus ; Endostatins ; metabolism ; Technology, Pharmaceutical ; instrumentation ; methods ; Time Factors

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 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

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

A hydrostatic compression device has been designed to study the effects of intermittent or continuous compressive forces on cells by trying to mimic in vivo forces. The model system is composed of two pistons that deliver and release compressed air under control of a motor to form an enclosed tissue culture chamber housing numerous tissues culture dishes. The continuous compressive forces and intermittent compressive forces are within 100-300 KPa and 0-30 KPa respectively. The compressive force is uniform in the system and can also result in the generation of shear forces along the interface between the cells and culture substratum, so the hydrostatic compression device can be used to study the characteristics of in vitro cells loading models.
Biomechanical Phenomena ; Cell Culture Techniques ; instrumentation ; methods ; Compressive Strength ; Computer-Aided Design ; Equipment Design ; Humans ; Shear Strength ; Stress, Mechanical