Calcium phosphate transfection optimization for serum-free suspension culture

Aim of this study was to identify optimal conditions for suspension transfection in the absence of serum. Transfection parameters for suspension culture can be very different to ones in adherent cells. Most transfection protocols have been developed and optimizedfor adherent culture. Using green fluorescent protein (GFP) as reporter, FCS was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. A reduction of the calcium concentration from 250 mM to 100 mM, yielded a 25-fold increase in the expression of the recombinant protein compared to the serum-free standard conditions. Altering the phosphate concentration, 1.4 mM in the transfection buffer, did not improve the protein expression. Interestingly, reduction of DNA quantity by half to a concentration of 0.5 μg per milliliter of culture volume resulted in a two-fold increase of protein production. Addition of albumin to serum-free medium protected the cells against the toxicity of the calcium phosphate transfection particles (CaPi) yielding higher protein expression. All the experiments were executed in a shaken multi-well system, allowing high multiplicity parameter screening to speed up optimizations. The culture system is inexpensive, simple and efficient, minimizing costs for labor and consumables. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tapping culture-an improved method for cell suspension culture

Summary A new culture vessel was designed for cell suspension culture. A silicone-convered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of “stirring” in cultures the bar moves vertically with a “tapping” motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures than in stirring cultures. Serial cultivation of cells in tapping cultures was also successful. This work was supported in part by the grants for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Cell aggregate suspension culture for large-scale production of biomolecules

Summary A method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm² flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1×10¹⁰ cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.     

Plant cell suspension culture rheology

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. (c) 1993 John Wiley & Sons, Inc.     

Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.     

Reactor design for large scale suspension animal cell culture

The scale of operation of freely suspended animal cell culture has been increasing and in order to meet the demand for recombinant therapeutic products, this increase is likely to continue. The most common reactor types used are stirred tanks. Air lift fermenters are also used, albeit less commonly. No specific guidelines have been published for large scale (≥10 000 L) animal cell culture and reactor designs are often based on those used for microbial systems. However, due to the large difference in energy inputs used for microbial and animal cell systems such designs may be far from optimal. In this review the importance of achieving a balance between mixing, mass transfer and shear effects is emphasised. The implications that meeting this balance has on design of vessels and operation, particularly in terms of strategies to ensure adequate mixing to achieve homogeneity in pH and dissolved gas concentrations are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Handling culture medium composition for optimizing plant cell suspension culture in shake flasks

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L^?1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g^?1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g^?1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g^?1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg^?1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg^?1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.     

Microwave sterilization of plastic tissue culture vessels for reuse.

A simple protocol has been developed for recycling plastic tissue culture vessels. The killing properties of microwaves were used to decontaminate plastic tissue culture vessels for reuse. Nine bacterial cultures, four gram-negative and five gram-positive genera, including two Bacillus species, were used to artificially contaminate tissue culture vessels. The microwaves produced by a "home-type" microwave oven (2.45 gHz) were able to decontaminate the vessels with a 3-min exposure. The same exposure time was also used to completely inactivate the following three test viruses: polio type 1, parainfluenza type 1 (Sendai), and bacteriophage T4. The recycling procedure did not reduce the attachment and proliferation of the following cell types: primary chicken and turkey embryo, HEp-2, Vero, BGMK, and MK-2.     

Atmospheric stability in cell culture vessels

Summary We have investigated the atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O₂ and CO₂ in the media of both seeded and unseeded culture vessels incubated at 37°C. There was no diffusion of either O₂ or CO₂ through glass vessels. At low partial pressures of oxygen (P_{O 2}), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mm Hg per 24 hr, and at high P_{O 2}, oxygen diffused slowly out of polystyrene flasks. CO₂ diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the P_{O 2} ⩽ room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO₂ and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO₂ out of the vessels. Presented, in part, by Dr. Arthur Balin in partial fulfillment of the requirements for the Ph.D. This work was supported by USPHS grants AG-00378 from the National Institute of Aging and CA-14345 from the National Cancer Institute and NR 202-005 from the Office of Naval Research. A.K.B. is a trainee of the Medical Scientist Training Program, National Institutes of Health (GM 02046).     

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l^–1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.     

Bench-scale calorimetry of hybridomas in suspension culture

Heat produced by hybridoma suspension cultures was continuously monitored in a two-liter heat-flux calorimeter. Heat measurements reflected changes in metabolic state of the culture under different aeration conditions. A highly anaerobic metabolism was indicated by the low ratios of heat produced to glucose consumed.     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.     

Device for holding a variety of tissue culture vessels during microscopy

Summary A device for holding tissue culture tubes, plates and flasks has been designed for use in microscopy of cell cultures, which eliminates changing holders when using varied types or sizes of vessel.     

Biomass segregation in sage cell suspension culture

The biomass of sage (Salvia officinalis L.) cell suspension culture was composed of single cells and cell aggregates. The development of aggregated cell culture from a single-cell suspension was monitored by particle size distribution for four particle size classes. Particle size distribution was compared between the biomass grown in bioreactor and shake flasks. The size of the particles had a strong influence on content of secondary metabolite, ursolic acid (UA). The single cell biomass fraction accumulated up to 7.7 mg UA g^–1 DW which was up to 50 times higher compared to aggregated biomass fractions.