Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015     

Large‐scale image‐based profiling of single‐cell phenotypes in arrayed CRISPR‐Cas9 gene perturbation screens

High‐content imaging using automated microscopy and computer vision allows multivariate profiling of single‐cell phenotypes. Here, we present methods for the application of the CISPR‐Cas9 system in large‐scale, image‐based, gene perturbation experiments. We show that CRISPR‐Cas9‐mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image‐based phenotyping. We developed a pipeline to construct a large‐scale arrayed library of 2,281 sequence‐verified CRISPR‐Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine‐learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in‐depth characterization of gene perturbation effects. This approach enables genome‐scale image‐based multivariate gene perturbation profiling using CRISPR‐Cas9.     

Using single‐cell genomics to understand developmental processes and cell fate decisions

High‐throughput ‐omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision‐making is inherently a unicellular process to which “bulk” ‐omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single‐cell methods bridge this gap, allowing high‐throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single‐cell gene expression data and highlight areas of developmental biology where single‐cell techniques have made important contributions. These include understanding of cell‐to‐cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.     

A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid‐handling units with disposable plastic bags. Such single‐use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell‐growth‐based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held‐media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:332–337, 2014     

Verification of a new biocompatible single‐use film formulation with optimized additive content for multiple bioprocess applications

Single‐use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single‐use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein‐free cell culture medium that had been used to extract leachables from freshly gamma‐irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma‐irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma‐irradiation, have been established to ensure lot‐to‐lot consistency. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:1171–1176, 2014     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

Evaluating the toxicity of bDtBPP on CHO‐K1 cells for testing of single‐use bioprocessing systems considering media selection,cell culture volume,mixing, and exposure duration

Single‐use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale‐up. Bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4‐di‐tert‐butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single‐use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO‐K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO‐K1 cells. While CHO‐K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry‐standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO‐K1 cells. It was found that the sensitivity of cell culture to leachates from single‐use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318–1323, 2016     

A fluid dynamics approach to bioreactor design for cell and tissue culture

The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three-dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Fluid flow through a high cell density fluidized‐bed during centrifugal bioreactor culture

An increasing demand for products such as tissues, proteins, and antibodies from mammalian cell suspension cultures is driving interest in increasing production through high‐cell density bioreactors. The centrifugal bioreactor (CCBR) retains cells by balancing settling forces with surface drag forces due to medium throughput and is capable of maintaining cell densities above 10⁸ cells/mL. This article builds on a previous study where the fluid mechanics of an empty CCBR were investigated showing fluid flow is nonuniform and dominated by Coriolis forces, raising concerns about nutrient and cell distribution. In this article, we demonstrate that the previously reported Coriolis forces are still present in the CCBR, but masked by the presence of cells. Experimental dye injection observations during culture of 15 μm hybridoma cells show a continual uniform darkening of the cell bed, indicating the region of the reactor containing cells is well mixed. Simulation results also indicate the cell bed is well mixed during culture of mammalian cells ranging in size from 10 to 20 μm. However, simulations also allow for a slight concentration gradient to be identified and attributed to Coriolis forces. Experimental results show cell density increases from 0.16 to 0.26 when centrifugal force is doubled by increasing RPM from 650 to 920 at a constant inlet velocity of 6.5 cm/s; an effect also observed in the simulation. Results presented in this article indicate cells maintained in the CCBR behave as a high‐density fluidized bed of cells providing a homogeneous environment to ensure optimal growth conditions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A simple eccentric stirred tank mini‐bioreactor: Mixing characterization and mammalian cell culture experiments

In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5–100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple—easy to assemble, easy to use, easy to clean—cell culture mini‐bioreactors for lab‐scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini‐bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini‐bioreactor were comparable to those observed for 6‐well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini‐bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini‐bioreactor. Biotechnol. Bioeng. 2013; 110: 1106–1118. © 2012 Wiley Periodicals, Inc.     

Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development

Increasing the throughput and efficiency of cell culture process development has become increasingly important to rapidly screen and optimize cell culture media and process parameters. This study describes the application of a miniaturized bioreactor system as a scaled-down model for cell culture process development using a CHO cell line expressing a recombinant protein. The microbioreactor system (M24) provides non-invasive online monitoring and control capability for process parameters such as pH, dissolved oxygen (DO), and temperature at the individual well level. A systematic evaluation of the M24 for cell culture process applications was successfully completed. Several challenges were initially identified. These included uneven gas distribution in the wells due to system design and lot to lot variability, foaming issues caused by sparging required for active DO control, and pH control limitation under conditions of minimal dissolved CO2. A high degree of variability was found which was addressed by changes in the system design. The foaming issue was resolved by addition of anti-foam, reduction of sparge rate, and elimination of DO control. The pH control limitation was overcome by a single manual liquid base addition. Intra-well reproducibility, as indicated by measurements of process parameters, cell growth, metabolite profiles, protein titer, protein quality, and scale-equivalency between the M24 and 2 L bioreactor cultures were very good. This evaluation has shown feasibility of utilizing the M24 as a scale-down tool for cell culture application development under industrially relevant process conditions.     

Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single‐cell precision to define cell identities in a large and heterogeneous population of cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi‐ATOM, based upon ultrafast phase‐gradient encoding, outperforms state‐of‐the‐art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi‐ATOM for large‐scale, label‐free, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi‐ATOM could empower new strategies in large‐scale biophysical single‐cell analysis with applications in biology and enriching disease diagnostics.      

Quantification of heparin's antimetastatic effect by single‐cell force spectroscopy

In circulation, cancer cells induce platelet activation, leading to the formation of a cancer cell‐encircling platelet cloak which facilitates each step of the metastatic cascade. Since cancer patients treated with the anticoagulant heparin showed reduced metastasis rates and improved survival, it is supposed that heparin suppresses the cloak's formation by inhibiting the interaction between platelet's adhesion molecule P‐selectin with its ligands on cancer cells. To quantify this heparin effect, we developed a single‐cell force spectroscopy approach and quantified the adhesion (maximum adhesion force [F_A] and detachment work [W_D]) between platelets and human non‐small cell lung cancer cells (A549). A configuration was used in which A549 cells were glued to tipless cantilevers and force‐distance (F‐D) curves were recorded on a layer of activated platelets. The concentration‐response relationship was determined for heparin at concentrations between 1 and 100 U/mL. Sigmoid dose‐response fit revealed half‐maximal inhibitory concentration (IC₅₀) values of 8.01 U/mL (F_A) and 6.46 U/mL (W_D) and a maximum decrease of the adhesion by 37.5% (F_A) and 38.42% (W_D). The effect of heparin on P‐selectin was tested using anti‐P‐selectin antibodies alone and in combination with heparin. Adding heparin after antibody treatment resulted in an additional reduction of 9.52% (F_A) and 7.12% (W_D). Together, we quantified heparin's antimetastatic effect and proved that it predominantly is related to the blockage of P‐selectin. Our approach represents a valuable method to investigate the adhesion of platelets to cancer cells and the efficiency of substances to block this interaction.     

Optofluidic rotation of living cells for single‐cell tomography

Flow cytometry provides a high throughput, multi‐dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z‐resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.