Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20–25 days duration at cell concentrations up to 50 × 10⁶ cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.     

Separation of CHO cells using hydrocyclones

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.     

A preliminary investigation into the potential use of a hydrocyclone to concentrate entomopathogenic nematodes from suspension

A 32-mm diameter hydrocyclone was used to investigate whether the entomopathogenic nematode Steinernema carpocapsae could be recovered and concentrated from a dilute suspension. By altering the sizes of the underflow and overflow apertures of the hydrocyclone, it was possible to recover high percentages of the nematodes from the initial suspension in reduced volumes of fluids. All underflow and overflow configurations tested gave an increased concentration of nematodes in the underflow of between 1.26 and 4.55 times the initial feed concentration. The highest nematode concentrations were achieved with a 4-mm underflow and an 8-mm overflow. It is proposed that through further refinements of hydrocyclone performance, it may be possible to utilise hydrocyclones in the recovery and harvesting processes of in vitro nematode production systems.     

Application of hydrocyclone for separation of Pichia pastoris produced r-HBsAg from fermentation culture: impact of concentration and pressure on hydrocyclone performance

Abstract

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated.

Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R² = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3?bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19?mm and 21?mm were used for separating the yeast cells.     

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.     

Optimization of an acoustic cell filter with a novel air-backflush system

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1). The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.     

Cell separator operation within temperature ranges to minimize effects on Chinese hamster ovary cell perfusion culture

A cell retention device that provides reliable high-separation efficiency with minimal negative effects on the cell culture is essential for robust perfusion culture processes. External separation devices generally expose cells to periodic variations in temperature, most commonly temperatures below 37 degrees C, while the cells are outside the bioreactor. To examine this phenomenon, aliquots of approximately 5% of a CHO cell culture were exposed to 60 s cyclic variations of temperature simulating an acoustic separator environment. It was found that, for average exposure temperatures between 31.5 and 38.5 degrees C, there were no significant impacts on the rates of growth, glucose consumption, or t-PA production, defining an acceptable range of operating temperatures. These results were subsequently confirmed in perfusion culture experiments for average exposure temperatures between 31.6 and 38.1 degrees C. A 2(5-1) central composite factorial design experiment was then performed to systematically evaluate the effects of different operating variables on the inlet and outlet temperatures of a 10L acoustic separator. The power input, ambient temperature, as well as the perfusion and recycle flow rates significantly influenced the temperature, while the cell concentration did not. An empirical model was developed that predicted the temperature changes between the inlet and the outlet of the acoustic separator within +/-0.5 degrees C. A series of perfusion experiments determined the ranges of the significant operational settings that maintained the acoustic separator inlet and outlet temperatures within the acceptable range. For example, these objectives were always met by using the manufacturer-recommended operational settings as long as the recirculation flow rate was maintained above 15 L day(-1) and the ambient temperature was near 22 degrees C.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Propagation of MM Virus in Continuous Cell Lines

Baby hamster kidney (BHK), McCoy, and L cell lines were found to be suitable for isolation of MM virus from infected mouse brain tissue. The virus was recovered in high titer in the first passage in BHK and McCoy cells, with concomitant cytopathic effect (CPE). In L cells, virus yield was lower than in the other two cell lines and CPE was incomplete. Adaptation of the virus to BHK and McCoy cells by serial passages was evidenced by accelerated development of the CPE and increase in the virus titer. Plaques were obtained in all three cell lines when inoculated with infected mouse brain or with the tissue culture-propagated virus. In the BHK cells, the virus release preceded the appearance of CPE and maximal yield of virus was obtained after 1 to 3 days of incubation, depending on the size of inoculum. The BHK-propagated virus had the same lethality for mice as did the mouse brain-propagated stock, and there was no difference in the course of the disease caused by the two preparations.     

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells.

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.     

Effect of the water extracts of propolis on stimulation and inhibition of different cells

The water extracts of propolis (WEP) could inhibit growth of different cell lines namely McCoy, HeLa, SP2/0, HEp-2, and BHK21 and stimulate growth of normal cell named human lymphocyte, rat kidney, rat liver, and rat spleen. In these experiments 1 and 2 mg of WEP were added to 1 ml RPMI media with 5% FCS. Cell counts and cell viability of propolis-treated and propolis-free cells were assessed by Trypan blue dye exclusion test and MTT assay. The results showed that in case of McCoy, HeLa, SP20, HEp-2, and BHK21 cell lines, the water extracts of propolis could inhibit cell growth as well as reduction on size of the cells. In contrast the same amount of WEP could stimulate growth of normal cells up to 60% with the same concentration used for cell lines. Thus our study indicates that although WEP consists only of the soluble part of propolis, it enables to inhibit different cell lines and increase growth of normal cells. This indicates also that WEP contains the specific compounds with bioactivity against cell lines. Although propolis contain different number of compounds it is clear that WEP has enough biological compounds useful for the treatment of some diseases, medical and related applications.     

A new method for yeast recovery in batch ethanol fermentations: Filter aid filtration followed by separation of yeast from filter aid using hydrocyclones

In the Melle-Boinot process for alcohol production, centrifuges are normally used for yeast recovery at the end of a batch fermentation. Centrifuges are expensive equipment and represent an impressive part of the equipment costs in alcohol industries. In the present work, an alternative method for yeast recovery using less expensive equipment was studied. Instead of using centrifuges, yeast was separated from the fermented broth by filter aid filtration, followed by separation of yeast from the filter aid using hydrocyclones. A stainless steel plate-and-frame filter of filtration area 1.14 m² and two 30 mm hydrocyclones, which followed the Bradley and Rietema recommended proportions, were used in this work. The filter aid was perlite. Tests of direct separation of yeast from the fermented broth using the Bradley hydrocyclone proved to be completely unfeasible, since the maximal reduced total efficiency obtained was only 1%. When the hydrocyclones were used to separate perlite from the resuspended filtration cake, the perlite total separation efficiency obtained in the underflow was as high as 95% when using the Bradley hydrocyclone with an underflow diameter of 3 mm. To show the feasibility of the proposed new method of yeast recovery, a complete cycle of experiments, which included fermentation, yeast separation, and new fermentation using the recycled cells, was performed with good results.     

麻疹病毒沪191对HeLa肿瘤细胞抑制作用的体内外实验研究

研究麻疹病毒减毒疫苗沪191株(MV沪191)在组织培养中和裸鼠体内对HeLa肿瘤细胞的抑制作用.用空斑实验测定MV沪191感染HeLa细胞后细胞裂解液中病毒量;用MTF试验测定MV沪191感染对细胞活性的影响;用流式细胞仪分析测定MV沪191感染引起的细胞凋亡和对细胞周期的影响;HeLa肿瘤细胞背部皮下接种BALB/C裸鼠引起的肿瘤,评估MV沪191体内抑瘤作用.MV沪191感染HeLa细胞后可引起广泛的CPE,感染的HeLa细胞与对照组相比细胞活性明显降低.MV沪191感染HeLa细胞后随着时间延长,G1/G0细胞率明显增多,S期率明显减少,细胞凋亡率明显增加(P＜0.01).给药第60天时瘤内治疗组、静脉治疗组和对照组肿瘤体积平均分别为15.5、64.6、156.4 mm3.瘤内治疗组与对照组相比有显著差异(P＜0.01);静脉治疗组与对照组相比有明显差异(P＜0.05).MV沪191减毒株在组织培养中和裸鼠体内对HeLa肿瘤有明显的杀伤作用.     

Tyrosine phosphorylations specific to mitosis in human and hamster cells

Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and ～ 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.     

CHO细胞无血清结团灌流培养：超声-沉降柱二合一灌流系统

利用CHO细胞能在培养过程中自然结团的特性,采用超声—沉降柱二合一灌流系统能促进细胞结团和加强截留的特性,我们用无血清培养基连续灌流培养基因重组CHO细胞MK3-A2株,分泌表达rhTNK-tPA获得了成功。培养周期为77-110天,细胞结团率为90%左右,直径在285～570μm之间,细胞截留率保持在95%左右,成活率为85%以上,细胞密度达到2×107/ml左右,rhTNK-tPA生产率平均为89 mg/L/d,最高时达216mg/L/d。 结果表明,使用该灌流系统进行细胞结团培养可以取代微载体培养用于动物细胞制药的规模化生产。     

Controlled shear filtration: A novel technique for animal cell separation.

A novel rotary microfiltration technique specifically suited for the separation of animal cells has been developed. The concept allows the independent adjustment of wall shear stress, transmembrane pressure, and residence time, allowing straightforward optimization of the microfiltration process. By using a smooth, conically shaped rotor, it is possible to establish a controlled shear field in which animal cells experience a significant hydrodynamic lift away from the membrane surface. It is shown in preliminary experiments that shear-induced cell-rupture speeds up membrane clogging and that cell debris poses the most significant problem in harvesting of BHK cell cultures by dynamic microfiltration. However, a threshold value of shear stability exists which depends on the frequency of passing the shear field, the residence time in the shear field, as well as on cell status. By operating close to this threshold value, cell viability can be maintained while concentration polarization is efficiently minimized. By applying this concept, it is possible to attain flux rates several times higher compared to conventional crossflow filtration. Controlled shear filtration (CSF) can be used for batch harvesting as well as for cell retention in high cell density systems. In batch harvesting of hIL-2 from rBHK cell culture, a constant flux rate of 290 L h-1 m-2 has been adjusted without indication of membrane clogging or fouling.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

Characterization and optimization of acoustic filter performance by experimental design methodology

Acoustic cell filters operate at high separation efficiencies with minimal fouling and have provided a practical alternative for up to 200 L/d perfusion cultures. However, the operation of cell retention systems depends on several settings that should be adjusted depending on the cell concentration and perfusion rate. The impact of operating variables on the separation efficiency performance of a 10-L acoustic separator was characterized using a factorial design of experiments. For the recirculation mode of separator operation, bioreactor cell concentration, perfusion rate, power input, stop time and recirculation ratio were studied using a fractional factorial 2(5-1) design, augmented with axial and center point runs. One complete replicate of the experiment was carried out, consisting of 32 more runs, at 8 runs per day. Separation efficiency was the primary response and it was fitted by a second-order model using restricted maximum likelihood estimation. By backward elimination, the model equation for both experiments was reduced to 14 significant terms. The response surface model for the separation efficiency was tested using additional independent data to check the accuracy of its predictions, to explore robust operation ranges and to optimize separator performance. A recirculation ratio of 1.5 and a stop time of 2 s improved the separator performance over a wide range of separator operation. At power input of 5 W the broad range of robust high SE performance (95% or higher) was raised to over 8 L/d. The reproducible model testing results over a total period of 3 months illustrate both the stable separator performance and the applicability of the model developed to long-term perfusion cultures.