Cell Culture Volume Research Articles

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15-18%, without any detrimental effect on cell viability. Despite utilizing 10-50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 μM and 25.4 ± 8.3 μM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

Platforms for Optogenetic Stimulation and Feedback Control

Harnessing the potential of optogenetics in biology requires methodologies from different disciplines ranging from biology, to mechatronics engineering, to control engineering. Light stimulation of a synthetic optogenetic construct in a given biological species can only be achieved via a suitable light stimulation platform. Emerging optogenetic applications entail a consistent, reproducible, and regulated delivery of light adapted to the application requirement. In this review, we explore the evolution of light-induction hardware-software platforms from simple illumination set-ups to sophisticated microscopy, microtiter plate and bioreactor designs, and discuss their respective advantages and disadvantages. Here, we examine design approaches followed in performing optogenetic experiments spanning different cell types and culture volumes, with induction capabilities ranging from single cell stimulation to entire cell culture illumination. The development of automated measurement and stimulation schemes on these platforms has enabled researchers to implement various in silico feedback control strategies to achieve computer-controlled living systems—a theme we briefly discuss in the last part of this review.

Measuring Glucose Consumption and Gluconeogenesis in 3D Human Tissue Cultures with Nanoliter Input Volumes.

Organotypic and microphysiological culture of primary human tissues and cancers has emerged as a powerful set of technologies that allow to faithfully mimic cellular metabolism and functions ex vivo. The predominant 3D culture methods include spheroids and microfluidic chips. These cultures use low cell numbers and culture volumes, which, however, poses important limitations for the available amounts of sample for downstream analyses. Here, we describe a detailed method for the measurement of glucose consumption dynamics in organotypic culture using a bienzymatic colorimetric assay that accurately quantifies glucose levels using nanoliter input volumes. As an example we utilize spheroids consisting of primary human hepatocytes. The assay has been carefully optimized and benchmarked and is compatible with both longitudinal and high-throughput screening in both static and perfused conditions. The method is straightforward and only requires a microplate reader capable of running absorbance kinetic measurements.

New insights on molecular internalization and drug delivery following plasma jet exposures.

In this study, we evaluated cold atmospheric plasmas as a physical drug delivery tool for human cervical cancer HeLa cells and murine breast carcinoma 4T1 cells. Different cell exposure protocols - plasma jet, plasma treated medium, and combinations of plasma-induced electric field and plasma treated medium- have been proposed and assessed to provide new insight on plasma-induced uptake mechanism. Cell culture medium composition and volume are key parameters to achieve an efficient molecular uptake. The plasma device enabled the delivery of molecules having 150 kDa-size into 4T1cells. For the first time to our knowledge, substance uptake kinetics after plasma treatment were investigated. The percentage of positive cells for propidium iodide and an anti-cancer agent, doxorubicin, was higher when the drugs were added a few minutes after treatment. The Plasma treated medium was not found to be as efficient as direct plasma treatment in 4T1 cells while allowing an efficient delivery in HeLa cells. Uptake levels as high as 39.3 ± 2.9% and 40.1 ± 9.5% for HeLa and 4 T1 cells respectively were achieved for optimized operating conditions, for which the viability of the cells was not severely affected. We also observed that plasma treatment induced the formation of actin stress fibers into cells revealing a mechanical stress.

Acoustic Affinity Cell Selection: a non-paramagnetic scalable technology for T cell selection from unprocessed apheresis products

Background & Aim Acoustic Cell Processing is a unique acousto-fluidics platform technology for minimal shear manipulation of cells using ultrasonic standing waves. The platform has broad applications in the field of cell and gene therapy, e.g., cell concentration and washing, cell culturing, microcarrier/cell separation, acoustic affinity cell selection and label-free cell selection. The acoustic radiation force exerted by the ultrasonic standing wave on the suspended cells in combination with fluid drag forces and gravitational forces is used to manipulate the cells and achieve a certain cell processing unit operation, e.g., separate, concentrate, or wash. The technology is single-use, continuous, and can be scaled up, down or out. It therefore allows for a flexible and modular approach that can be customized to process a desired cell count, cell culture volume or cell concentration within a given required process time. Utilizing its proprietary multi-dimensional standing wave platform, MilliporeSigma has been developing the Acoustic Affinity Cell Selection (AACS) system for closed and automated Cell and Gene Therapy manufacturing, e.g., CAR-T immunocellular therapies. The AACS technology is a scalable acoustic affinity cell selection method using acoustic (non-paramagnetic) affinity beads for positive or negative cell selection. A multi-dimensional acoustic standing wave is then used to separate the affinity bead-cell complexes from the unbound cells, thereby completing the process of a negative or positive cell selection. Methods, Results & Conclusion In this work the AACS system has been used to capture CD4+ and CD8+ cells from unprocessed apheresis products. The AACS column and acoustic section (Fig. 1A) allow for a continuous flow of the initial cell population (pre-labeled with biotinylated antibodies) while acoustically retaining the acoustic affinity particles in the column. The affinity particles are functionalized with Neutravidin and thus capture the target cells that are kept inside the column, while the non-target cells are washed out of the column. The small scale 5mL column has a total target cell capture capacity of 1.8B cells (60mL Leukopak volume), of which 83% are retained in the column at a 92% purity (Fig. 1B). The AACS scale up system can process a full apheresis product (300-450mL) in less than 2h.

Small-scale approach for precrystallization screening in GPCR X-ray crystallography.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor's protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

TPP Combined with DGUC as an Economic and Universal Process for Large-Scale Purification of AAV Vectors

Adeno-associated virus (AAV) vectors have been commonly purified through density gradient ultracentrifugation (DGUC) or column chromatography methods. Although the DGUC method can efficiently separate the empty from the full virus particles, its application in large-scale AAV purification is hindered due to its limitation in volume of each centrifuge tube. Alternatively, column chromatography is serotype-dependent, expensive, and complicated, which co-purifies both empty and full virus particles. In this study, we describe an economical and universal process using three-phase partitioning (TPP) combined with DGUC to purify large quantities of AAV vectors. First, TPP is used to remove up to 90% of the cellular impurities in the cell lysate and at the same time condense the AAV vectors into ∼10% of their original lysate volume. Second, two rounds of DGUC are employed to separate the empty from the full virus particles and at the same time remove the remaining cellular impurities. This combined process increases the capacity of ultracentrifugation by a factor of 5- to 10-fold depending on the yields of AAV serotypes. A variety of AAV serotypes such as AAV2, AAV5, AAV6, AAV9, and AAVDJ have been successfully purified with this process. Both in vitro and in vivo studies demonstrate that TPP has no detrimental impact on AAV infectivity. In a proof of concept, we performed several purification runs ranging from 3 to 25 L of Sf9 culture volume. We were able to purify more than 3e+15 viral genomes (vg) of AAV vectors from 3 L of cell culture volume with just two SW28 centrifuge tubes in a Beckman Coulter ultracentrifuge. Our data indicate that this TPP-DGUC process is economic, universal, and can be used to purify a large quantity of AAV vectors for clinical applications with just a few ultracentrifuges.

Response to Blast-like Shear Stresses Associated with Mild Blast-Induced Brain Injury

Toward the goal of understanding the pathophysiology of mild blast-induced traumatic brain injury and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a timescale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high-resolution, time-lapse optical microscopy. Using this system, we demonstrate that blast-like shear transients produced with minimal pressure transients and submillisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8 and 21 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS cells.

Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

RNA interference (RNAi) is a convenient tool to identify and characterize biological functions in organisms. Recently, it has become an alternative to chemical insecticides as a biologically based control agent. This promising technology has the potential to avoid many problems associated with conventional chemical insecticides. In order for RNAi application to be practical for field use, a major hurdle is the development of a cost-effective system of double-stranded RNA (dsRNA) production for a large quantity of dsRNA. A handful of research reports has demonstrated microbial-based dsRNA production using L4440 vector and HT115 (DE3) Escherichia coli for application to vertebrate and invertebrate systems. However, the dsRNA yield, production efficiency, and biological purity from this in vitro system is still unclear. Thus, our study detailed biochemical and molecular tools for large-scale dsRNA production using the microbial system and investigated the production efficiency and yield of crude and purified dsRNAs. An unrelated insect gene, green fluorescent protein (GFP), and an insect neuropeptide gene, pyrokinin (PK) identified from Drosophila suzukii, were used to construct the recombinant L4440 to be expressed in the HT115 (DE3) cell. A considerable amount of dsRNA, 19.5 µg/mL of liquid culture, was isolated using ultrasonic disruption followed by phenol extraction. The sonication method was further evaluated to extract crude dsRNA without the additional phenol extraction and nuclease treatments and also to reduce potential bacterial viability. The results suggest that the ultrasonic method saved time and costs to isolate crude dsRNA directly from large volumes of cell culture without E coli contamination. We investigated whether the injection of PK dsRNA into flies resulted in increased adult mortality, but it was not statistically significant at 95% confidence level. In this study, the microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.

A novel mammalian cell line development platform utilizing nanofluidics and optoelectro positioning technology

Generating a highly productive cell line is resource intensive and typically involves long timelines because of the need to screen large numbers of candidates in protein production studies. This has led to miniaturization and automation strategies to allow for reductions in resources and higher throughput. Current approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. New nanofludic technologies offer novel solutions to surpass these limits, further miniaturizing cell culture volumes (105 times smaller) by growing cells on custom nanofluidic chips. Berkeley Lights’ OptoElectro Positioning technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Using a fully integrated technology platform (Beacon), common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip. Here, we describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that commercial production Chinese hamster ovary cell lines can be isolated, cultured, screened, and exported at high efficiency. We compare this process head to head with a FACS‐enabled microtiter plate‐based workflow and demonstrate generation of comparable clonal cell lines with reduced resources. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1438–1446, 2018

Importance of dosimetry protocol for cell irradiation on a low X-rays facility and consequences for the biological response

Purpose: The main objective of radiobiology is to establish links between doses and radiation-induced biological effects. In this context, well-defined dosimetry protocols are crucial to the determination of experimental protocols. This work proposes a new dosimetry protocol for cell irradiation in a SARRP and shows the importance of the modification of some parameters defined in dosimetry protocol for physical dose and biological outcomes.Materials and methods: Once all parameters of the configuration were defined, dosimetry measurements with ionization chambers and EBT3 films were performed to evaluate the dose rate and the attenuation due to the cell culture medium. To evaluate the influence of changes in cell culture volume and/or additional filtration, 6-well plates containing EBT3 films with water were used to determine the impact on the physical dose at 80 kV. Then, experiments with the same irradiation conditions were performed by replacing EBT3 films by HUVECs. The biological response was assessed using clonogenic assay.Results: Using a 0.15 mm copper filter lead to a variation of +1% using medium thickness of 0.104 cm to −8% using a medium thickness of 0.936 cm on the physical dose compare to the reference condition (0.313 cm). For the 1 mm aluminum filter, a variation of +8 to −40% for the same medium thickness conditions has been observed. Cells irradiated in the same conditions showed significant differences in survival fraction, corroborating the effects of dosimetric changes on physical dose.Conclusions: This work shows the importance of dosimetry in radiobiology studies and the need of an accurate description of the dosimetry protocol used for irradiation.

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.

A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement.

Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell-cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.

A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks

We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E.coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30%. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl β-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E.coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

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.

464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

Non-replicative recombinant HIV-1 derived lentiviral vectors (LV) are increasingly used in gene therapy clinical trials for various genetic diseases, infectious diseases or cancer. Before they are used in man, preparations of LV must undergo extensive biochemical and biological quality control testing. In particular, the legislation stipulates that the absence of replication-competent lentiviruses (RCL) must be demonstrated with suitable methods, on representative fractions of batches. Current standard and widely used methods based on cell culture are challenging because high titers of vector batches achieved translate into high volumes of cell culture that have to be tested. Since vector batch titers and sizes are continuously-increasing due to the improvement of production and purification methods, it is necessary to modify the current cell culture method. Here, we propose a practical optimization of the p24-decrease-based culture assay developed by Escarpe et al. (2003) using a pairwise pooling strategy enabling the test of higher vector inoculum volumes. These modifications significantly decrease material handling, operator time, leading to a cost effective method, while maintaining optimal sensibility of the RCL testing. This optimized RCL-pooling assay ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

"RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

Design and Optimization of a Purification Process for MY32/Ls Protein Solubilizing Inclusion Bodies for a New Vaccine Against Sea Lice

Sea lice are the most problematic marine pathogens the salmon industry has to deal with, significantly affecting Europe and America. The worst offenders are genera: Pseudocaligus, Caligus, and Lepeophtheirus. Over €305 million in losses are estimated. Recent results have suggested that subolesin/akirin/myosin32 are good candidate antigens for the control of arthropod infestations such as sea lice. The aim of this study was to design and optimize the purification step of MY32/Ls protein to obtain the active pharmaceutical ingredient against sea lice. Non-chromatographic purification strategies were employed, based on published works, to establish rupture, washing, solubilization, and refolding conditions. A disruption process using a bead mill was established. Cell culture volume was flushed through the mill eight times. Three 60-minute process steps for washing inclusion bodies were performed, and the fourth washing step was a 20-minute process. The first two washes with a Tris-HCl (50 mM) buffer were performed using a non-ionic surfactant (Triton X-100). For the third wash, the surfactant was omitted. The fourth washing step was carried out at a pH of 10.5. The inclusion body solubilization process, in an alkaline condition of pH 12.5, lasted 90 minutes. For the refolding step, Tween 80 was used as a stabilizing additive. The API obtained had a better immune response (IgG) than the positive control, with higher purity.

Predictions for optimal mitigation of paracrine inhibitory signalling in haemopoietic stem cell cultures.

IntroductionRecent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output.MethodsNumerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion.ResultsThe models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered.ConclusionsThe models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to any complex cell culture scenario where cell-cell interactions and paracrine signalling networks impact upon cell fate and cell expansion.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0048-7) contains supplementary material, which is available to authorized users.

Critical factors to be considered when testing nanomaterials for genotoxicity with the comet assay.

The comet assay is widely used to test the genotoxicity of engineered nanomaterials (ENMs) but outcomes may vary when results from different laboratories, or even within one laboratory, are compared. We address some basic methodological considerations, such as the importance of carrying out physico-chemical characterisation of the ENMs in test-medium, performing uptake and cytotoxicity tests, and testing several genotoxicity-related endpoints. In this commentary, we discuss the different ways in which concentration of ENMs can be expressed, and stress the need to include appropriate controls and reference standards to monitor variation and avoid interference. Treatment conditions, including cell number, cell culture plate format and volume of treatment medium on the plate are crucial factors that may impact on results and thus should be kept constant within the study.