High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

苏云金杆菌补料高密度培养的研究

研究了苏云金杆菌的补米高密度培养。系统探讨了补料方式、补料成分和补料时间对发酵水平的影响。结果表明,利用同样物料,补料高密度培养比分批培养的晶体含量提高４２．２％,效价提高３６．４％。     

High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10⁸ cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20–30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.     

A multiparallel bioreactor for the cultivation of mammalian cells in a 3D‐ceramic matrix

For adherently growing cells, cultivation is limited by the provided growth surface. Excellent surface‐to‐volume ratios are found in highly porous matrices, which have to face the challenge of nutrient supply inside the matrices' caverns. Therefore, perfusion strategies are recommended which often have to deal with the need of developing an encompassing bioreactor periphery. We present a modular bioreactor system based on a porous ceramic matrix that enables the supply of cells with oxygen and nutrients by perfusion. The present version of the reactor system focuses on simple testing of various inoculation and operation modes. Moreover, it can be used to efficiently test different foam structures. Protocols are given to set‐up the system together with handling procedures for long‐time cultivation of a CHO cell line. Experimental results confirm vital growth of cells inside the matrices' caverns. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

酿酒酵母S.cerevisiae高密度培养条件优化研究

考察了培养基组成和培养条件对酿酒酵母Saccharomyces cerevisiae发酵的影响。以TB培养基为初始培养基,通过正交实验设计优化培养基组成,确定了影响酵母细胞产量最主要的因素是葡萄糖,最适培养基组成为:酪蛋白胨15 g/L,酵母粉25 g/L,葡萄糖30 g/L,KH2PO42.4g/L,K2HPO4.3H2O 16.34 g/L。并确定了最佳培养条件:温度30℃,转速150 r/min。采用优化培养基及培养条件下进行发酵,菌液最高OD600值和细胞密度分别达15.82和2.03×108/mL,比优化前分别提高24.2%和22.0%。     

High density cell culture of a marine photosynthetic bacterium Rhodovulum sp. with self-flocculated cells

High density cell cultivation of a marine photosynthetic bacterium, Rhodovulum sp. PS88 with self-flocculated cells was established by using a single-tower fermenter. High density cell culture with continuous cultivation was yielded 43 g dry matter l–1 with acetate as a substrate consumed at 22.5 g/l day. © Rapid Science Ltd. 1998     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.     

酿酒酵母工程菌高密度培养生产香紫苏醇

为提高酿酒酵母工程菌S7香紫苏醇产量,采用摇瓶培养,研究了其生长和代谢特点,发现产物合成与菌体生长密切关联。在3 L发酵罐中通过补料-溶氧联动控制的方式,以葡萄糖、乙醇和葡萄糖/乙醇混合物为碳源进行高密度培养,香紫苏醇产量分别达到253 mg/L、386 mg/L和408 mg/L,最高产量是摇瓶培养的27倍。说明添加乙醇作为碳源有助于香紫苏醇合成。研究结果对优化酿酒酵母细胞工厂,高效生产萜类化合物具有重要参考价值。     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×10⁸ cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×10⁷ cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×10⁷ cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM Eagle's minimal essential medium - Dulbecco's modified Eagle MEM Dulbecco's modified Eagle minimal essential medium     

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h⁻¹ dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.     

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.     

High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.