Adherent Cell Cultures Research Articles

A solution to prevent secondary flow in adherent cell cultures.

ABSTRACTHigh quality cell cultures require reliable laboratory practices. Today's small-scale in vitro cell culture format is dominated by circular topology vessels, with the inherent disadvantage of secondary flow induced each time the cell cultures are repositioned. The secondary flow generates uneven sedimentation and adherence that negatively impacts cell culture quality. Here we show a modification of the circular culture vessel that abrogates these disturbances. Cell culture wells were augmented with a central column to diminish secondary flow. Human carcinoma cell lines (BeWo, JEG-3), mesenchymal stem cells [human umbilical cord perivascular cells (HUCPVC)] and mouse embryonic fibroblasts (MEF) were cultured in both column-augmented and regular culture wells. Human carcinoma cell cultures showed even cell densities and significantly more viable cells in column-augmented vessels. In FTM HUCPVC cultures, cell surface MSC marker (CD90, CD105) expression and cell differentiation-related gene expression patterns were significantly more homogeneous in column-augmented vessels. MEF cells in column-augmented culture vessels showed a more consistent expression of IGF-1. Column-augmented cell culture vessels significantly improve the homogeneity of adherent cell cultures by mitigating the adverse effect of the secondary flow.This article has an associated First Person interview with the first author of the paper.

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost.Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 106 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process.Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic.The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 106 cells/mL.

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

Quantitative phase imaging of adherent mammalian cells: a comparative study.

The Quantitative phase imaging methods have several advantages when it comes to monitoring cultures of adherent mammalian cells. Because of low photo-toxicity and no need for staining, we can follow cells in a minimally invasive way over a long period of time. The ability to measure the optical path difference in a quantitative manner allows the measurement of the cell dry mass, an important metric for studying the growth kinetics of mammalian cells. Here we present and compare cell measurements obtained with three different techniques: digital holographic microscopy, lens-free microscopy and quadriwave lateral sheering interferometry. We report a linear relationship between optical volume density values measured with these different techniques and estimate the precisions of this measurement for the different individual instruments.

Efficient GMP compliant expansion of mesenchymal stromal cells (MSCs) from umbilical cord, bone marrow and adipose tissue using a closed cultivation system.

Background & Aim Human mesenchymal stem cells (MSCs) hold great promise for clinical use and cell therapy applications and can be isolated from multiple tissue, e.g. bone marrow (BM), umbilical cord (UC) or adipose tissue (AT). As there is a high number of clinical trials and applications using MSCs, quality and safety of the resulting cellular products are indispensable. The xeno-free MSC-Brew GMP Medium following the recommendations of USP on ancillary materials enables a standardized expansion and culture of MSC from different tissue sources. To increase the level of process standardization and product safety we developed the CliniMACS Prodigy® Adherent Cell Culture System for GMP compliant isolation and cultivation of adherent cells using a closed single used tubing set. The process includes the flexible combination of the following modules: - Density Gradient Centrifugation (DGC) - Surface Coating - Inoculation - Culture - Media Change - Harvest Here we show that MSCs from different tissue sources (AT, BM, UC) can be expanded and passaged using the CliniMACS Prodigy® Adherent Cell Culture System from primary tissue or single-cell suspensions combining the process modules in a flexible way. Methods, Results & Conclusion BM-MSCs were isolated via DGC and subsequent adherence to plastic using semi-automatic feeding, harvesting and reseeding procedures. AT-MSCs as well as UC-MSCs were isolated and expanded from single-cell suspensions obtained after enzymatic digestion. UC was digested using an optimized and automated procedure using the gentle MACSTM Dissociator. Subsequently, MSCs were isolated from single cell suspension using the flexible cultivation procedure of the CliniMACS Prodigy® Adherent Cell Culture System. Resulting cells displayed an MSC specific phenotype as defined by the ISCT consortium. Furthermore MSCs revealed their immunomodulatory potential as assessed by in vitro assay and multi-color flow cytometric analysis. With the CliniMACS Prodigy®, MSCs can be expanded in large scales, thus providing high numbers of cells for consecutive clinical applications.

Suspension culture of Marek’s disease virus and evaluation of its immunological effects

ABSTRACTMarek’s disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76 ± 2.64 × 106 PFU/flask in a working volume of 100 ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5 g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions.

Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)‐derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three‐dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two‐dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1‐60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin‐based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. stem cells translational medicine 2019;8:512–521

Microfluidic technology for investigation of protein function in single adherent cells.

Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency. In this chapter, we briefly review new microtechnologies for the investigation of protein function in single adherent cells, and present an overview over related recent applications of the multifunctional pipette (Biopen), a microfluidic multi-solution dispensing system that uses hydrodynamic confinement in open volume environments in order to establish a superfusion zone over selected single cells in adherent cultures.

Particokinetics and in vitro dose of high aspect ratio nanoparticles.

Computational particokinetics models become essential in the design and interpretation of in vitro nanoparticle toxicology assays involving submerged adherent cell cultures. Yet, none of the current models offers the possibility of addressing the delivered dose of high-aspect ratio nanoparticles, including nanorods, nanotubes, and nanofibers. Here we present a straightforward method that lends this ability to any of the models used currently.

Cell Proliferation High-Content Screening on Adherent Cell Cultures.

Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein immunostaining, the percentage of cells undergoing different phases of the cell cycle (G0, G1, S, G2, and M) might be established. The method can be also used to estimate the proliferation (S phase) rate of particular cell subpopulations identified through labelling with specific nuclear markers.

Investigating Age-Related Changes in Proliferation and the Cell Division Repertoire of Parenchymal Reactive Astrocytes.

Reactive gliosis is a complicated process involving all types of glial cells and is the therapeutic target of efforts to treat several types of neuropathologies. Parenchymal astrocytes continuously survey their microenvironment to identify even tiny abnormalities in the central nervous system (CNS) homeostasis and react rapidly to brain damage, such as following ischemia, trauma, or neurodegenerative diseases, to prevent propagation of tissue damage. Aging can play causal roles in certain astroglial dysfunctions, however, still little is known to what extent the heterogeneous reaction of astrocytes at the injury site might be impaired over the course of aging. Based on our experience with both in vitro and in vivo experimental paradigms, we describe here in detail the analysis of age-related changes in (1) proliferative response of parenchymal astrocytes within the posttraumatic cerebral cortex grey matter (GM), and (2) repertoire of their cell divisions in adherent cell culture prepared from the injured GM of young and old double transgenic GFAP-mRFP1/(FUCCI)-S/G2/M-mAG-hGeminin mice by single cell time-lapse imaging.

A Chemiresistor Sensor Based on Azo-Polymer and Graphene for Real-Time Monitoring of Mitochondrial Oxygen Consumption.

In the present study, a chemiresistor sensor based on a poly(Bismarck Brown Y)-reduced graphene oxide nanocomposite was developed to analyze the respiratory capacity of the constituent complexes of the electron transport chain. The sensorial platform was characterized using electrochemical impedance spectroscopy, and oxygen detection was accomplished by measuring the resistive properties of the sensor at fixed AC frequency. The impedance decreased significantly in response to small variations of the O2 concentrations tested up to saturation of the electrolyte solution with molecular oxygen. The resistive response of the sensor at 0.1 Hz was linear over the oxygen concentration range from 1.17 × 10-5 mol L-1 to 1.02 × 10-3 mol L-1, with a detection limit of 3.60 × 10-7 mol L-1. Using the new O2 sensing platform, we monitored gradients in static cultures of adherent cells exposed to graded oxygen both at rest and upon metabolic stimulation. Under high dissolved oxygen conditions, the respiration of resting cells dictated that local O2 was moderately reduced, while cell metabolic stimulation triggered a major redistribution of O2. The usefulness of the developed sensor was demonstrated by continuous monitoring of mitochondrial oxygen consumption in various biologic applications.

The Combined Effects of Co-Culture and Substrate Mechanics on 3D Tumor Spheroid Formation within Microgels Prepared via Flow-Focusing Microfluidic Fabrication.

Tumor spheroids are considered a valuable three dimensional (3D) tissue model to study various aspects of tumor physiology for biomedical applications such as tissue engineering and drug screening as well as basic scientific endeavors, as several cell types can efficiently form spheroids by themselves in both suspension and adherent cell cultures. However, it is more desirable to utilize a 3D scaffold with tunable properties to create more physiologically relevant tumor spheroids as well as optimize their formation. In this study, bioactive spherical microgels supporting 3D cell culture are fabricated by a flow-focusing microfluidic device. Uniform-sized aqueous droplets of gel precursor solution dispersed with cells generated by the microfluidic device are photocrosslinked to fabricate cell-laden microgels. Their mechanical properties are controlled by the concentration of gel-forming polymer. Using breast adenocarcinoma cells, MCF-7, the effect of mechanical properties of microgels on their proliferation and the eventual spheroid formation was explored. Furthermore, the tumor cells are co-cultured with macrophages of fibroblasts, which are known to play a prominent role in tumor physiology, within the microgels to explore their role in spheroid formation. Taken together, the results from this study provide the design strategy for creating tumor spheroids utilizing mechanically-tunable microgels as 3D cell culture platform.

Induction of epithelial-mesenchymal transition (EMT) and Gli1 expression in head and neck squamous cell carcinoma (HNSCC) spheroid cultures.

Tumor microenvironment provides a specialized niche in which a population of stem-like cells is enriched and contributes to cancer progression. Moreover, cancer stem cell (CSC) phenotype has been associated with epithelial-mesenchymal transition (EMT). Here we investigated the effect of tumor microenvironment on the phenotypic characteristics of head and neck cancer cells and expression of CSC markers using a three-dimensional (3D), spheroid, culture system of CAL33 cell line from human tongue squamous cell carcinoma. CAL33 cells derived from 2D monolayer cultures were grown in spheroid cultures containing serum-free medium (epidermal growth factor [EGF], fibroblast growth factor [FGF], and insulin). Adherent CAL33 cells from spheroids or standard control cultures were grown in the presence/absence of serum in combination with hypoxia/normoxia. Markers of EMT, CSC, and hypoxia were analyzed either by Western blotting, immunofluorescence, or reverse transcription quantitative PCR. Spheroid cultures showed hypoxic microenvironment (high carbonic anhydrase IX [CAIX] expression), mesenchymal-like characteristics (reduced E-cadherin and increased vimentin and N-cadherin expression, presence of larger colonies comprised of larger, spread cells with lower density), and increased expression of the CSC marker glioma-associated oncogene homolog 1 (Gli1). These effects were recapitulated in serum-free adherent CAL33 cells maintained for prolonged periods in hypoxia (1% O2) but, in contrast, were completely abolished by the presence of serum. Overall, we found that a combination of hypoxia, EGF and FGF was essential to induce the EMT in adherent CAL33 cell cultures. The addition of serum rapidly reverts the EMT of cells, affects CSC phenotype and, thus, prevents the detection of such cells in tumor cell lines.

Measuring the Nutrient Metabolism of Adherent Cells in Culture.

Metabolite extraction from cells cultured in vitro enables the comprehensive measurement of intracellular metabolites. These extracts can be analyzed using techniques such as liquid chromatography-mass spectrometry (LC-MS). This chapter describes in detail a method for metabolite extraction from cultured adherent mammalian cells to collect both polar and nonpolar intracellular metabolites. This chapter also describes experimental design considerations for performing stable isotope labeling experiments, and the use of chemical derivatization to increase the number of compounds that can be detected using one chromatography method.

Determining Compartment-Specific Metabolic Fluxes.

In this chapter, we present an experimental protocol for the targeted metabolic profiling of full cells and mitochondria in selectively permeabilized cells. Mitochondria of adherent cell cultures are made accessible by the addition of digitonin-a compound that selectively permeabilizes the cytosolic membrane without affecting mitochondrial integrity. The generated in situ mitochondria are subsequently used in a stable isotope labeling assay in which their metabolic fluxes can be analyzed without any interfering influence originating from cytosolic components. The protocol is complemented by oxygen consumption measurements of permeabilized cells on a Seahorse XF instrument. The additional data on mitochondrial respiration can be used to validate the functionality of mitochondria in the applied setup but are also a valuable add-on to the stable isotope labeling data.

An automated microfluidic perfusion device for adherent cell reprogramming, expansion and culture condition monitoring

An automated microfluidic perfusion device for adherent cell reprogramming, expansion and culture condition monitoring

Stem Cell Expansion and Fate Decision on Liquid Substrates Are Regulated by Self-Assembled Nanosheets.

The culture of adherent cells is overwhelmingly relying on the use of solid substrates to support cell adhesion. Indeed, it is typically thought that relatively strong bulk mechanical properties (bulk moduli in the range of kPa to GPa) are essential to promote cell adhesion and, in turn, regulate cell expansion and fate decision. In this report, we show that adherent stem cells such as mesenchymal stem cells and primary keratinocytes can be cultured at the surface of liquid substrates and that this phenomenon is mediated by the assembly of polymer nanosheets at the liquid-liquid interface. We use interfacial rheology to quantify this assembly and demonstrate the strong mechanical properties of such nanosheets. Importantly, we show that cell adhesion to such quasi-2D materials is mediated by the classical integrin/acto-myosin machinery, despite the absence of bulk mechanical properties of the underlying liquid substrate. Finally, we show that stem cell proliferation and fate decision are also regulated by the mechanical properties of these self-assembled protein nanosheets. Liquid substrates offer attractive features for the culture of adherent cells and stem cells, and the development of novel stem cell technologies, such as liquid-liquid systems, are particularly well-adapted to automated parallel processing and scale up.

Control Analysis of Cellular Energy Metabolism in Intact, Adherent Cell Cultures

Control Analysis of Cellular Energy Metabolism in Intact, Adherent Cell Cultures

Shaped 3D microcarriers for adherent cell culture and analysis

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting, which modulates protein expression and eliminates intercellular connections. To allow adherent culture and processing in flow, we present 3D-shaped hydrogel cell microcarriers, which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth, and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow, important for processing in flow and imaging flow cytometry. We designed a method to rapidly design, using the genetic algorithm, and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach. The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling, and is poised to enable adherent cell culture, in-flow analysis, and sorting in a single format.