A novel dielectrophoresis-based device for the selective retention of viable cells in cell culture media

Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 239-250, 1997.     

High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler

A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.     

外循环气升式反应器培养新疆紫草细胞

采用两步培养法进行新疆紫草细胞悬浮培养及5L外循环气升式反应器扩大培养,探讨了培养过程中细胞生长、紫草色素合成与培养液的电导率、可溶性糖含量变化之间的关系。第一步培养时细胞生长迅速,但也有一部分色素合成,电导率及可溶性糖含量迅速下降;第二步培养初期电导率也开始下降,但当色素合成达到高峰并有一部分外泌到培养基后,电导率又开始回升。可溶性糖捎耗很快,到后期巳测不出其存在。因此通过监测培养液中电导率及可溶性糖的变化情况,可以为新疆紫草细胞大规模培养与色素合成提供有用的参数指标。     

表达内皮抑素的rCHO细胞的微囊化培养

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。种子细胞是生物微胶囊治疗作用的执行者, 是构建微囊微反应器的基本元素。如何获得大量高活性的种子细胞已经成为规模化制备生物微胶囊所面临的最关键的限制因素。本实验考察了搅拌式生物反应器内扩增的重组CHO细胞进行包囊及微囊化细胞在生物反应器内规模化培养的可行性。实验结果显示:重组CHO细胞在生物反应器内可以快速生长,并且对数期细胞包囊,微囊化细胞活性良好。制备的微囊化细胞可以在生物反应器内培养,与培养板培养比较细胞生长较快、内皮抑素表达量较高。应用生物反应器培养技术能够在体外快速、大量扩增重组CHO细胞,满足微囊化细胞制备对种子细胞量与质的要求,微囊化细胞可以在生物反应器内培养。     

Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks

Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Novel,linked bioreactor system for continuous production of biologics

A novel, alternative intensified cell culture process comprised of a linked bioreactor system is presented. An N-1 perfusion bioreactor maintained cells in a highly proliferative state and provided a continuous inoculum source to a second bioreactor operating as a continuous-flow stirred-tank reactor (CSTR). An initial study evaluated multiple system steady-states by varying N-1 steady-state viable cell densities, N-1 to CSTR working volume ratios, and CSTR dilution rates. After identifying near optimum system steady-state parameters yielding a relatively high volumetric productivity while efficiently consuming media, a subsequent lab-scale experiment demonstrated the startup and long-term operation of the envisioned manufacturing process for 83 days. Additionally, to compensate for the cell-specific productivity loss over time due to cell line instability, the N-1 culture was also replaced with younger generation cells, without disturbing the steady-state of the system. Using the model cell line, the system demonstrated a two-fold volumetric productivity increase over the commercial-ready, optimized fed-batch process.     

Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES 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.     

Improving performance of mammalian cells in fed-batch processes through "bioreactor evolution"

The amount of recombinant product obtained from mammalian cells grown in a bioreactor is in part limited by achievable cell densities and the ability of cells to remain viable over extended periods of time. In an attempt to generate cell lines capable of better bioreactor performance, we subjected the DG44 Chinese Hamster Ovary (CHO) host cell line and a recombinant production cell line to an iterative process whereby cells capable of surviving the harsh conditions in the bioreactor were selected. This selective process was termed "bioreactor evolution". Following the selective process, the "evolved" host cells attained a 2-fold increase in peak cell density and a 72% increase in integral cell area. Transient transfection experiments demonstrate that the evolved cells have the same transfection efficiency and the same secretory potential as the initial cells. The "evolved" host was also found to contain a large subpopulation of cells that did not require insulin for growth. From this, a new population of growth-factor-independent cells was obtained. These improvements in host properties should prove beneficial in the expression of recombinant proteins in fed-batch processes. The selective process was also applied to a recombinant production cell line. The evolved cells from this selection exhibited a 38% increase in peak cell density, a 30% increase in integral cell area, and a 36% increase in product titer. These increases were obtained without any appreciable impact on product quality, demonstrating the usefulness of this simple approach to improve the performance of recombinant cell lines.     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.     

Variable functions of bcl-2 in mediating bioreactor stress- induced apoptosis in hybridoma cells

It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modified CelliGen-packed bed bioreactors for hybridoma cell cultures

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG₁ monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen^™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10⁸ cells per cm³ of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.     

Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell- surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.     

Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor.

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 x 10(7) cells/mL was achieved, as was an antibody productivity of over 50 micrograms/(mL.day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.     

A cell-detaching reactor for inoculation of anchorage-dependent CHD and Vero cells between stepwise-expanded bioreactors

A cell-detaching reactor was developed to collect cells growing on microcarriers for inoculation between stepwise-expanded bioreactors. It consisted of a trypsinization zone and a separation zone, which were separated by a 200-mesh stainless steel screen. The screen allowed the cells only to pass through to the next bioreactor, after the cells have been trypsinized and detached from microcarriers. The operating feasibility of the cell-detaching reactor was tested with anchorage-dependent recombinant Chinese hamster ovary (rCHO) and African green monkey kidney (Vero) cells. rCHO and Vero cells were first cultured in a small microcarrier bioreactor, and then inoculated via the cell-detaching reactor into either a packed-bed bioreactor (for rCHO cells) or a larger microcarrier bioreactor (for Vero cells). For rCHO cells, the cell density reached 1.3 × 10⁷ cells/ml in the perfusion culture, and Vero cells reached 1.3 × 10⁶ cells/ml in the batch culture.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.     

Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution

Abstract Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.