Design of a large-scale mammalian cell,suspension culture facility

The design of a suspension culture facility capable of producing approximately 10¹² cells per week has been developed on a small-scale system which has evolved from various architectural, engineering, biological, and biohazard considerations. The smaller system is composed of spinner flasks (50 ml to 8 liters) modified for semicontinuous culture conditions, metal reservoirs, a continuous flow centrifuge, and supportive equipment. The large system which is under construction is composed of metallic vessels of up to 500 liter working volume with hard plumbing, monitors, controllers, recorders, continuous flow centrifuge and other ancillary equipment. This system begins with medium preparation and ends with harvesting of cells and disposition of supernatant. The design of this turn-key operation was developed over a two and one-half year period through the cooperation of private industry, the federal government, and the academic community.     

Large-scale mammalian cell culture: Design and use of an economical batch suspension system

Large-scale mammalian cell culture in the absence of antibiotics requires stringent conditions of sterility for all vessels, procedure, and systems used. Application of existing fermentation technology suffers from the differences between mammalian and bacterial cultures. Relatively simple and inexpensive 100-L vessels have been designed specifically for medium storage and antibiotic-free mammalian cell culture. These vessels are portable and sterilized in a 2 x 3 x 5 ft conventional or VACUMATIC autoclave. They consist of 30-gal 316 stainless-steel sanitary process drums whose heads have been modified to meet the rapid pressure changes that occur during autoclaving. The vessels incorporate systems for aseptic introduction and removal of both liquids and gases required for inoculation, growth, and harvesting of cell suspensions. A two-disk vibromixer is used for agitation with inoculation at a laminar flow hood and incubation in a warm room. These vessels have been used for culture of one rat and eight human tumor lines for over 2 x 10(5) L of suspension.     

Development of a hollow-fiber system for large-scale culture of mammalian cells

A flat-bed hollow-fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge-type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless-steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm² of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Development of a single-pass ceramic matrix bioreactor for large-scale mammalian cell culture

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via sllicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up. (c) 1992 John Wiley & Sons, Inc.     

A simple system for plant cell microinjection and culture

Microinjection of plant protoplasts and cells has been recently reported, however a system that combines simplicity of design, harmless immobilization, high resolution visibility and ability to monitor individual target cells is lacking. This report describes a system which combines these features. It consists of a microinjection-microculture dish containing immobilized protoplasts and a simple chamber that maintains sterility and humidity during injection. Highly purified protoplast preparations are plated at low population density as a thin monolayer of widely separated cells embedded in agarose layered over a thicker (0.2 mm at center to 1 mm at edge) layer of agarose-solidified medium. This physical arrangement allows for rapid location, mapping and injection of the immobilized protoplasts and also their subsequent location for growth monitoring. The double layers of agarose provide adequate nutrition for culturing injected cells to the microcalli stage. In addition to protoplast injection, this system was also used to inject 3- to 4-day old nonspherical cells derived from protoplasts. Colony formation rates from injected protoplasts and cells with regenerated walls were equivalent to those of uninjected controls. Furthermore, tobacco protoplasts stored at 4°C in liquid medium for up to two weeks remained fully competent for plating and injection. These cold-stored protoplasts, when injected, formed colonies at rates similar to those from fresh preparations. The ability to store protoplasts without loss of viability considerably increases the ease and convenience of cell injection experiments.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of the other products that may also be suitable.     

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.     

哺乳动物细胞灌流培养工艺开发与优化

近年来生物药市场需求量激增,高产量、高质量、低成本的哺乳动物细胞灌流培养工艺顺势成为工业界和学术界普遍关注的热点。文中围绕灌流培养工艺特有的操作环节及工艺优化应着重关注的细节展开论述,综述了近年来在灌流培养工艺开发和优化上取得的进步和提出的策略,以期为哺乳动物细胞灌流培养技术的开发提供参考。     

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.     

New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells

We present a new approach for biomass assessment in cell culture using a disposable microcentrifuge tube. The specially designed tube is fitted with an upper chamber for sample loading and a lower 5 microL capillary for cell collection during centrifugation. The resulting packed cell volume (PCV) can be quantitatively expressed as the percentage of the total volume of the sample. The present study focused on the validation of the method with mammalian cell lines that are widely used in bioprocessing. Using several examples, the PCV method was shown to be more precise, rapid, and reproducible than manual cell counting.     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Computational fluid dynamics modeling of an inverted frustoconical shaking bioreactor for mammalian cell suspension culture

We previously developed an inverted frustoconical shaking bioreactor (IFSB) which had high mammalian cell culture performance when compared with a mechanically stirred tank reactor (STR) or a flat-bottom shaking bioreactor (FBSB). Here, we determined the mixing time (t) and volumetric oxygen transfer coefficient (k _La) of this IFSB at various speeds, and simulated the fluid hydrodynamics, including the shear stress and specific surface area, by computational fluid dynamics. The shortest mixing time was observed in a STR. The maximum kLa value of 12/h was achieved in the IFSB at an aeration rate of 4 L/h, demonstrating that our IFSB has enhanced oxygen transfer capabilities needed to meet the demands of mammalian cells. Simulation studies revealed a 3% greater specific surface area and a 21% lower shear strain in the IFSB compared to an FBSB under the same conditions. Additionally, the conical angle of the vessel, which significantly affected cell growth and recombinant protein production, was tested here. We conclude that, compared to the STR and FBSB, the IFSB has an increased liquid surface area for oxygen uptake and exhaust CO₂ stripping, an enhanced k _La for cell robust growth to a high cell density, and a lower shear stress to alleviate cell damage.     

一次性生物反应器悬浮培养HEK293细胞生产Ad-IFNγ的工艺

腺病毒载体是极具发展前景的基因治疗载体之一,为获得一条新型腺病毒规模化生产工艺,研究采用5 L振荡激流式一次性生物反应器悬浮培养HEK293细胞来生产重组腺病毒载体。细胞经种子链逐步扩增后,接种至AP10生物反应器,采用CD293无血清培养基流加培养悬浮HEK293细胞,细胞密度达约2.0×106个/m L时,以30 MOI(Multiplicity of infection)感染重组Ad-IFNγ(Recombinant adenovirus-interferon gamma),48 h后收获细胞,3次冻融裂解离心收获上清病毒粗产品。采用壳蛋白免疫法快速测定粗产品滴度。结果表明,采用振荡激流式一次性生物反应器,流加HEK293细胞悬浮培养6 d,密度可达2.0×106个/m L,Ad-IFNγ粗产量达1.49×1013 IFU(Infectious unit),单细胞包装量达3 800 IFU/cell。采用阴离子交换层析法纯化重组腺病毒,回收率35.9%。建立了利用5 L激流式一次性生物反应器悬浮培养HEK293细胞生产重组腺病毒载体Ad-IFNγ的初步工艺。     

A simple,disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

Summary Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and ligual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.     

A simple method for hemoglobin measurement in cell culture system.

A simple method of hemoglobin analysis in a cell culture system is described. Hemoglobins synthesized in cell cultures are labeled with radioactive amino acids. The cell extract containing radiolabeled hemoglobin is mixed with A, F, S, C, hemoglobin markers and separated by cellulose acetate electrophoresis. Individual bands of hemoglobin are cut from the gel and analyzed for radioactivity. This method is especially useful for determination of newly synthesized minute amount of hemoglobin in cell extracts that are difficult to visualize by staining procedure.