Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single‐use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large‐scale production and feasibility of meeting clinical‐grade standards. The current work evaluated the capacity of a single‐use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k_La) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h^?1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7‐fold with a total cell number of 1.2 ± 0.2 × 10⁹ cells L^?1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single‐use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362–369, 2018     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

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。     

Bioreactors for 3-dimensional high-density culture of human cells

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.     

Ex vivo expansion of bone marrow mesenchymal stem cells using microcarrier beads in a stirred bioreactor

Bone marrow mesenchymal stem cells (bmMSCs) have recently gained attention as a useful resource in the fields of regenerative medicine and tissue engineering. However, the number of bmMSCs obtained from available donors is very low. Here we developed a culture strategy for in vitro expansion of bmMSCs in a 1.5 L stirred bioreactor with microcarrier beads. First, the microcarriers (Cytodex 3) were equilibrated in culture medium containing 3% fetal bovine serum (FBS) for at least 30 min prior to cell addition. After inoculation, the FBS concentration of the medium was maintained at 3% (v/v) in the first 24 h and thereafter maintained at 1% (v/v) and a developed feeding regimen was applied over 5 days. The maximum cell density of 2.6 × 10⁶ cells/mL was achieved at day 5, corresponding to a 10.4 ± 0.8 fold increases in total cell number. Among the harvested cells, 98.95% expressed CD29 and 84.48% expressed CD90, suggesting that the majority of expanded bmMSCs still retained their differentiation potential. Therefore, the developed microcarrier-based stirred bioreactor culture system is an effective method to generate significant numbers of bmMSCs for potential applications and research studies.     

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters. In this work, power input, mixing time, impeller tip speed, and Reynolds number have been compared systematically for the cultivation of the human cell line AGE1.HN within three partner laboratories using five different bioreactor systems. Proper operation ranges for the bioreactors were identified using the maximal cell‐specific growth rate (μ_max) as indicator. Common optimum values for process transfer criteria were found in these geometrically different bioreactors, in which deviations of μ_max between cultivation systems can be importantly reduced. The data obtained in this work are used for process standardization and comparability of results obtained in different bioreactor systems, i.e. to guarantee lab‐to‐lab consistency for systems biology approaches using mammalian cells.     

搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

目的：摸索搅拌式生物反应器培养小鼠胚胎干细胞（mESC）的最佳条件,建立一种批量制备拟胚体（EB）的方法。方法：研究mESC不同接种密度及生物反应器初始搅拌速度对EB形成的数量和质量的影响,以细菌培养皿中形成的EB为对照,用抗坏血酸诱导其向心肌细胞分化,比较两种培养体系对EB心肌细胞分化潜能的影响,通过免疫荧光染色及RT PCR对ESC来源的心肌细胞进行鉴定。结果：当mESC接种密度为1×105~3×105个/ml,搅拌速度设定为15~30r/min时,搅拌式生物反应器能高效制备出大量相对均一的EB,EB中几乎没有坏死细胞。与细菌培养皿制备的EB相比,生物反应器培养的EB向心肌细胞分化的效率更高,并表达心肌特异性基因。结论：搅拌式生物反应器培养促进EB的形成及其向心肌细胞分化,是一种更为理想的EB培养系统。     

Aujeszky’s disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 10⁶ cells ml^-1, the reactor was inoculated with 9 ml of gE^- Bartha K-61 strain ADV suspension (10^5.9 TCID₅₀) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10^7.0 TCID₅₀ ml⁻¹, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.     

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.     

Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post‐translational modification. In this study, bioreactor production of recombinant human alpha‐1‐antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25‐fold increase in extracellular functional rAAT (603 µg/L) and a higher ratio of functional rAAT to total rAAT (85–90%). Surprisingly, sustained rAAT production and steady state, long‐term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010; 106: 408–421. © 2010 Wiley Periodicals, Inc.     

Production of Superoxide Dismutase from Streptococcus lactis Using a Bioreactor with a Microfiltration Module

The performance of a bioreactor with a microfiltration module for the production of an intracellular enzyme, superoxide dismutase (SOD), by Streptococcus lactis is described. The fermentation system involving the bioreactor enables the continuous removal of metabolites inhibitory for cell growth and the complete recycling of the cells to the bioreactor. In a fed-batch (FB) culture with filtration, in which the main metabolite, lactic acid, in the culture broth was maintained at a low concentration, S. lactis was cultivated to the high concentration of 15.5 g-dry cells/1. The SOD content of the cells remained at almost a constant level throughout the cultivation and the productivity of SOD as well as cells per unit time was 4.3-fold as high as that in the case of a conventional batch culture without filtration. Repeating the FB culture with filtration enhanced the productivities of SOD and cells further, as compared with those in the case of the FB culture with filtration.     

Expansion of mesenchymal stem cells under atmospheric carbon dioxide

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO₂ environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO₂ levels negatively affecting cell growth, but more importantly, the need to sparge CO₂ into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO₂ environment in static cultures. MSC expanded under atmospheric CO₂ with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self‐renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1298–1306, 2013     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo. (c) 1994 John Wiley & Sons, Inc.     

The medium cycle bioreactor (MCB): Monoclonal antibody production in a new economic production system

The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system. Eighty percent of the spent medium was re-used twice. The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances. Therefore, only 20% of the actual medium was necessary to run the recycling process. During seven days culture time in a two liter scale 5 grams of IgG₁ type monoclonal antibody was produced. During that period the cell specific productivity was constant. Renewal of proteins was omitted because the protein content in the system persisted at a high level. Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function). Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step. Only cheap metabolites that are consumed by the cells had to be supplemented. Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium.