Perfusion Cell Culture Research Articles

Development of a shake tube-based scale-down model for perfusion cultures.

The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption.

Real-time measurement of cholesterol secreted by human hepatocytes using a novel microfluidic assay.

The use of microfluidics has become widespread in recent years because of the use of lesser resources such as small size, low volume of reagents, and physiological representation of mammalian cells. One of the advantages of microfluidic-based cell culture is the ability to perfuse culture media which tends to improve cellular health and function. Although measurement of cellular function conventionally is carried out using well-plates and plate readers, these approaches are insufficient to carry out in-line analysis of perfused cell cultures because of mismatch between volumes and sensitivity. We report the development of a novel microfluidic device and assay that is carried out under perfusion, in-line to measure the cholesterol secreted from a human hepatocyte tissue-chip. The heart of the assay is the unique implementation of enzymatic chemistry that is carried out on a polystyrene bead. Using this approach, we successfully measured cholesterol secreted by the perfused human hepatocytes.

Improved Performance in Mammalian Cell Perfusion Cultures by Growth Inhibition.

Mammalian cell perfusion cultures represent a promising alternative to the current fed-batch technology for the production of various biopharmaceuticals. Long-term operation at a fixed viable cell density (VCD) requires a viable culture and a constant removal of excessive cells. Product loss in the cell removing bleed stream deteriorates the process yield. In this study, the authors investigate the use of chemical and environmental growth inhibition on culture performance by either adding valeric acid (VA) to the production media or by reducing the culture temperature (33.0 °C) with respect to control conditions (36.5 °C, no VA). Low temperature significantly reduces cellular growth, thus, resulting in lower bleed rates accompanied by a reduced product loss of 11% compared to 26% under control conditions. Additionally, the cell specific productivity of the target protein improves and maintained stable leading to media savings per mass of product. VA shows initially an inhibitory effect on cellular growth. However, cells seemed to adapt to the presence of the inhibitor resulting in a recovery of the cellular growth. Cell cycle and Western blot analyses support the observed results. This work underlines the role of temperature as a key operating variable for the optimization of perfusion cultures.

Development of a microfluidic perfusion 3D cell culture system

Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

Manipulation of the sodium-potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures.

Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed.

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics.

Removing nonviable cells from a cell suspension is crucial in biotechnology and biomanufacturing. Label-free microfluidic cell separation devices based on dielectrophoresis, acoustophoresis, and deterministic lateral displacement are used to remove nonviable cells. However, their volumetric throughputs and test cell concentrations are generally too low to be useful in typical bioreactors in biomanufacturing. In this study, we demonstrate the efficient removal of small (<10 μm) nonviable cells from bioreactors while maintaining viable cells using inertial microfluidic cell sorting devices and characterize their performance. Despite the size overlap between viable and nonviable cell populations, the devices demonstrated 3.5-28.0% dead cell removal efficiency with 88.3-83.6% removal purity as well as 97.8-99.8% live cell retention efficiency at 4 million cells per mL with 80% viability. Cascaded and parallel configurations increased the cell concentration capacity (10 million cells per mL) and volumetric throughput (6-8 mL min-1). The system can be used for the removal of small nonviable cells from a cell suspension during continuous perfusion cell culture operations.

Synchrony changes on different time-scales during in vitro neuronal network development

Synchrony changes on different time-scales during in vitro neuronal network development

Microfluidic system for synthesis of nanofibrous conductive hydrogel and muscle differentiation.

Microscale hydrogels can be synthesized within microfluidic systems and subsequently assembled to make tissues composed of units such as myofibers in muscle tissue. Importantly, the nanofibrous surface of hydrogels is essential for tissue engineering aims due to inducing beneficial cell-surface interactions. In this study, a new microfluidic platform, embedded with a hydrogel, was introduced that allowed for performing multiple non-parallel steps for the synthetic approaches. Satellite cells, isolated from skeletal tissues of 10-day Naval Medical Research Institute-murine were cultured on the prepared hydrogel within the microfluidic system. The normal proliferation of satellite cells occurred after the employment of continuous perfusion cell culture. Interestingly, the positive results of the immuno-staining assay along with the cellular bridge formation between hydrogel fragments confirmed the muscle differentiation of seeded satellite cells. Further on, COMSOl simulations anticipated that the thermodynamic conditions of the microfluidic system during hydrogel synthesis had to be kept steady while a shear stress value of 15 × 10-6 Pa was calculated, exhibiting a cell culture condition free of environmental stress.

Improved process robustness, product quality and biological efficacy of an anti-CD52 monoclonal antibody upon pH shift in Chinese hamster ovary cell perfusion culture

pH is an important and commonly researched parameter that affects the production and quality of monoclonal antibodies (mAbs). Our previous study showed that culture at high pH (7.15 ± 0.05) yielded a high cell density, at a low pH (6.85 ± 0.05) yielded a high titer and low byproduct. However, the effects of a pH shift on the production process, quality, and biological potency of mAbs are seldom reported in perfusion culture. In this study, we performed a pH shift perfusion culture (from 7.15 ± 0.05 to 6.85 ± 0.05 at day 9) and a control group (constant at 7.15 ± 0.05) in a 15-L bioreactor and compared the production process, critical attributes and biological efficacy (complement-dependent cytotoxicity, CDC; antibody-dependent cell-mediated cytotoxicity, ADCC) of an anti-CD52 mAb. The cells maintained a higher density and viability, and productivity was significantly enhanced with the pH shift. The charge and size heterogeneity of the mAb were comparable to the control group. However, the glycosylation content, especially that of galactosylation and afucosylation, was significantly higher with the pH shift, which lead to increased biological efficacy. Thus, a pH shift in perfusion culture had a noticeably positive effect on process robustness: it prolonged the culture duration, enhanced productivity, improved product quality and increased biological efficacy.

Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells

In vitro recapitulation of an organotypic stromal environment, enabling efficient angiogenesis, is crucial to investigate and possibly improve vascularization in regenerative medicine. Our study aims at engineering the complexity of a vascular milieu including multiple cell-types, a stromal extracellular matrix (ECM), and molecular signals. For this purpose, the human adipose stromal vascular fraction (SVF), composed of a heterogeneous mix of pericytes, endothelial/stromal progenitor cells, was cultured under direct perfusion flow on three-dimensional (3D) collagen scaffolds. Perfusion culture of SVF-cells reproducibly promoted in vitro the early formation of a capillary-like network, embedded within an ECM backbone, and the release of numerous pro-angiogenic factors. Compared to static cultures, perfusion-based engineered constructs were more rapidly vascularized and supported a superior survival of delivered cells upon in vivo ectopic implantation. This was likely mediated by pericytes, whose number was significantly higher (4.5-fold) under perfusion and whose targeted depletion resulted in lower efficiency of vascularization, with an increased host foreign body reaction. 3D-perfusion culture of SVF-cells leads to the engineering of a specialized milieu, here defined as an angiogenic niche. This system could serve as a model to investigate multi-cellular interactions in angiogenesis, and as a module supporting increased grafted cell survival in regenerative medicine.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs invitro to boost their bone forming capacity invivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks invivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days invitro improves the quantity and quality of bone formation invivo. This study highlights the need for optimizing invitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.

Microfluidic Cell Retention Device for Perfusion of Mammalian Suspension Culture

Continuous production of biologics, a growing trend in the biopharmaceutical industry, requires a reliable and efficient cell retention device that also maintains cell viability. Current filtration methods, such as tangential flow filtration using hollow-fiber membranes, suffer from membrane fouling, leading to significant reliability and productivity issues such as low cell viability, product retention, and an increased contamination risk associated with filter replacement. We introduce a novel cell retention device based on inertial sorting for perfusion culture of suspended mammalian cells. The device was characterized in terms of cell retention capacity, biocompatibility, scalability, and long-term reliability. This technology was demonstrated using a high concentration (>20 million cells/mL) perfusion culture of an IgG1-producing Chinese hamster ovary (CHO) cell line for 18–25 days. The device demonstrated reliable and clog-free cell retention, high IgG1 recovery (>99%) and cell viability (>97%). Lab-scale perfusion cultures (350 mL) were used to demonstrate the technology, which can be scaled-out with parallel devices to enable larger scale operation. The new cell retention device is thus ideal for rapid perfusion process development in a biomanufacturing workflow.

Mechanistic modeling of the loss of protein sieving due to internal and external fouling of microfilters.

Fed-batch and perfusion cell culture processes used to produce therapeutic proteins can use microfilters for product harvest. In this study, new explicit mathematical models of sieving loss due to internal membrane fouling, external membrane fouling, or a combination of the two were generated. The models accounted for membrane and cake structures and hindered solute transport. Internal membrane fouling was assumed to occur due to the accumulation of foulant on either membrane pore walls (pore-retention model) or membrane fibers (fiber-retention model). External cake fouling was assumed to occur either by the growth of a single incompressible cake layer (cake-growth) or by the accumulation of a number of independent cake layers (cake-series). The pore-retention model was combined with either the cake-series or cake-growth models to obtain models that describe internal and external fouling occurring either simultaneously or sequentially. The models were tested using well-documented sieving decline data available in the literature. The sequential pore-retention followed by cake-growth model provided a good fit of sieving decline data during beer microfiltration. The cake-series and cake-growth models provided good fits of sieving decline data during the microfiltration of a perfusion cell culture. The new models provide insights into the mechanisms of fouling that result in the loss of product sieving. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1323-1333, 2017.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017.

Abstract 4080: Cytochrome P450 enzyme activity is enhanced in hepatocytes grown using a perfused 3D cell culture drug screening system

Abstract A critical requirement for assessing drug toxicity in vitro is cytochrome P450 (CYP) function that is physiologically representative. Due to low levels of CYP enzyme activity, cell lines such as HepG2 hepatocytes have largely been supplanted with primary cells derived from human donor tissue. While these cells retain CYP activity ex vivo, drug screening is limited to short-term assessments due to de-differentiation. This limits their efficacy in testing slow-clearing, large-molecule compounds such as antibody-based therapies and low-turnover drugs. Cell lines in monolayer cultures have been effective in assessing the safety of small molecule drugs, but the disparity between cell lines and in vivo tissue biology proves too substantial for current potential therapies. Three-dimensional (3D) cultures have been shown to be more physiologically relevant models of in vivo tissue. However, even they fail to fully mimic living tissue and suffer from the lack of active oxygen and nutrient transport. Lena Biosciences has developed a groundbreaking in vitro system that facilitates long-term survival of 3D cultures. Cells seeded in 3D hydrogels supported by a rigid scaffold are maintained in statistically independent wells in their own medium which floats atop a high-density blood-substitute in which medium is immiscible. Infusion and withdrawal of this blood-substitute introduces perfusion into a medium-throughput multiwell system without the need for separate pumps for each culture condition. Internal validation experiments using hepatocytes cultured in a 12-well system showed a two-fold improvement in cellular respiration (measured by alamarBlue assay) over cultures maintained in a standard multiwell plate when the blood-substitute was introduced, and a three-fold improvement when the cultures were perfused. Cell viability (measured by LDH release) was significantly improved when perfusion was introduced. CYP activity (assessed using both fluorescent and luminescent substrate-based assays) was also significantly higher in the perfused hepatocyte cultures. These data demonstrate the substantial advantages to growing cells in this novel perfused cell culture model. Scaling the system to include multiple cell types (primary or cell lines), would allow for simultaneous testing of target and off-target effects of potential small or large molecule therapies. Thus, this system serves as a major improvement over the current organ-on-a-chip solutions in the area of drug safety testing. Citation Format: James T. Shoemaker, Jelena Vukasinovic. Cytochrome P450 enzyme activity is enhanced in hepatocytes grown using a perfused 3D cell culture drug screening system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4080. doi:10.1158/1538-7445.AM2017-4080

At-line Monitoring von Zellkulturprozessen mittels Massenspektrometrie

Perfusion becomes a reasonable alternative to traditional batch or fed-batch processes, partly due to the increasing availability of suitable re-usable or single-use equipment. High cell densities and long duration of perfusion processes require reliable process control. Amongst others, monitoring of cell stress becomes important. Intact cell MALDI-TOF mass spectrometry (ICM-MS) biotyping is described here as a robust at-line monitoring method of cell stress in perfusion cell culture.

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.

Differentiation of primary pre‐adipocytes into adipose tissue in a microfluidic chip

The growing problem of obesity has reached epidemic proportion worldwide and with over 1.5 billion adults aged 20 years and older characterized as being overweight [1]. Being overweight or obese remarkably increases the risks of developing other metabolic diseases such as diabetes. Adipose tissue is a key player in metabolic disorders including obesity and diabetes. Recent studies highlight the critical role adipose plays in maintaining metabolic homeostasis [2, 3]. Development of in vitro models of adipose tissue is highly desirable to improve our understanding of disease biology of obesity and metabolic diseases. Microfluidic on‐chip in vitro models offer advantages compared to traditional static culture system in terms of mimicking the complex physiological environment for culturing, differentiating, and stimulating adipocytes in a small‐volume perfused cell culture system [4, 5]. Here, we aim to establish a model for differentiation of pre‐adipocytes into mature adipose depots on a microfluidic chip. Mouse primary pre‐adipocytes isolated from subcutaneous white adipose tissue (WAT) were differentiated into mature white adipocytes in a microfluidic chip under perfused flow of 5μL/h. Our unique microfluidic chip enables for long‐term culture of cells and allows stimulation of cells through perfusion – we can culture, differentiate, and maintain the differentiated adipose tissue for over 21 days in the device. Upon differentiation, the cells increasingly accumulated triglycerides, which was quantified using fluorescence microscopy. Further confirmation of the differentiation was assessed by measuring the gene expression of markers including leptin and adiponectin. In addition, we collected and analyzed the perfusate for adipose secretions including adiponectin. Our results indicate an improved understanding of adipose differentiation under dynamic conditions and can serve as disease models for obesity and other metabolic disorders.Support or Funding InformationThis work was supported by NIH grant DK095984. [1] Ahima RS. Digging deeper into obesity. Journal of Clinical Investigation 2011;121:2076–9.[2] Clark AM, Sousa KM, Chisolm CN, MacDougald OA, Kennedy RT. Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on‐line chemical analysis of cultured adipocyte secretions. Analytical and Bioanalytical Chemistry 2010;397:2939–47.[3] Lee PL, Tang Y, Li H, Guertin DA. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease. Molecular Metabolism 2016;5:422–32.[4] Engler AJ, Hemmingsen M, Vedel S, Skafte‐Pedersen P, Sabourin D, Collas P, et al. The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose‐derived Stem Cells. PLoS ONE 2013;8:e63638.[5] Godwin LA, Brooks JC, Hoepfner LD, Wanders D, Judd RL, Easley CJ. A microfluidic interface for the culture and sampling of adiponectin from primary adipocytes. The Analyst 2015;140:1019–25.

Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.

Using resonant ultrasound field-incorporated dynamic photobioreactor system to enhance medium replacement process for concentrated microalgae cultivation in continuous mode

Holding cells in the cultivation broth for subsequent medium replacement is often considered as one of the most difficult procedures during perfusion cell culture. Although a number of harvest approaches have been reported in the past decades, their drawbacks such as high cost, increased contamination, and/or labor consuming remain obstacles for practical application. To overcome these challenges, a novel resonant ultrasound field-incorporated dynamic photobioreactor system (RUF-DPBS) was developed in which the cell retention and medium replacement are carried out by acoustic radiation forces and gravity, respectively. Based on the collection efficiency of microalgae, the RUF operation was optimized by 1MHz and output intensity of 8W/cm2 with circulating velocity of 2mL/min whereby 93% of Nannochloropsis oculata in 30mL can be collected within 2-h operation. Moreover, the cells cultured with RUF-DPBS in which the medium was changed every three days exhibited increased volumetric productivity that the yields of biomass, total lipid, and eicosapentaenoic acid of the N. oculata after 12-days cultivation significantly enhanced 2.6, 2.1, and 2.5 folds (P<0.05 for each), respectively, as compared to the group without medium replacement. In summary, the semiautomatic RUF-DPBS offers a non-fouling, labor-efficient, and cost-effective means for high-density microalgae culture in continuous mode.