Adherent Cell Type Research Articles

Generating suspension-adapted human mesenchymal stromal cells (S-hMSCs) for the scalable manufacture of extracellular vesicles

BackgroudHuman mesenchymal stromal cells (hMSCs) are a naturally adherent cell type and one of the most studied cellular agents used in cell therapy over the last 20 years. Their mechanism of action has been primarily associated with paracrine signaling, which has contributed to an increase in the number of studies focused on hMSC-related extracellular vesicles (EVs). MethodsIn this study, we demonstrate for the first time that human telomerase reverse transcriptase (hTERT) immortalized hMSCs can be adapted to suspension culture, eliminating the need for microcarriers or other matrixes to support cell growth. ResultsThis novel cell line, named suspension hMSCs (S-hMSCs), has a doubling time of approximately 55 hours, with a growth rate of 0.423/d. Regarding its immunophenotype characteristics, S-hMSCs retained close to 90% of CD73 and CD105 expression levels, with the CD90 receptor being downregulated during the adherent to suspension adaptation process. An RNA sequencing analysis showed an upregulation of the transcripts coding for CD44, CD46 and CD47 compared to the expression levels in AT-hMSCs and hTERT-hMSCs. The cell line herein established was able to generate EVs using a chemically defined medium formulation with these nanoparticles averaging 150 nm in size and displaying the markers CD63, CD81, and TSG101, while not expressing the negative marker calnexin. ConclusionThis body of evidence, combined with the visual confirmation of EV presence using transmission electron microscopy, demonstrates the EV-producing capabilities of the novel S-hMSCs. This cell line provides a platform for process development, drug discovery and translational studies in the EV field.

Integrating shear flow and trypsin treatment to assess cell adhesion strength.

Cell adhesion is of fundamental importance in cell and tissue organization and for designing cell-laden constructs for tissue engineering. Prior methods to assess cell adhesion strength for strongly adherent cells using hydrodynamic shear flow either involved the use of specialized flow devices to generate high shear stress or used simpler implementations like larger height parallel plate chambers that enable multihour cell culture but generate low wall shear stress and are, hence, more applicable for weakly adherent cells. Here, we propose a shear flow assay for adhesion strength assessment of strongly adherent cells that employs off-the-shelf parallel plate chambers for shear flow as well as simultaneous trypsin treatment to tune down the adhesion strength of cells. We implement the assay with a strongly adherent cell type and show that wall shear stress in the 0.07-7 Pa range is sufficient to dislodge the cells with simultaneous trypsin treatment. Imaging of cells over a square centimeter area allows cell morphological analysis of hundreds of cells. We show that the cell area of cells that are dislodged, on average, does not monotonically increase with wall shear stress at the higher end of wall shear stresses used and suggest that this can be explained by the likely higher resistance of high circularity cells to trypsin digestion. The adhesion strength assay proposed can be used to assess the adhesion strength of both weakly and strongly adherent cell types and has the potential to be adapted for substrate stiffness-dependent adhesion strength assessment in mechanobiology studies.

Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors.

Mesenchymal stromal cells (MSC) are a promising platform for regenerative medicine applications because of their multilineage differentiation abilities and ease of collection, isolation, and growth ex vivo. To meet the demand for clinical applications, large-scale manufacturing will be required using three-dimensional culture platforms in vessels such as stirred suspension bioreactors. As MSCs are an adherent cell type, microcarriers are added to the culture to increase the available surface area for attachment and growth. Although extensive research has been performed on efficiently culturing MSCs using microcarriers, challenges persist in downstream processing, including harvesting, filtration, and volume reduction, which all play a critical role in the translation of cell therapies to the clinic. The objective of this review is to assess the current state of downstream technologies available for microcarrier-based MSC cultures. This includes a review of current research within the three stages: harvesting, filtration, and volume reduction. Using this information, a downstream process for MSCs is proposed, which can be applied to a wide range of applications.

Performance of Cardiotropic rAAV Vectors Is Dependent on Production Method.

Gene therapy is making significant impact on a modest, yet growing, number of human diseases. Justifiably, the preferred viral vector for clinical use is that based on recombinant adeno-associated virus (rAAV). There is a need to scale up rAAV vector production with the transition from pre-clinical models to human application. Standard production methods based on the adherent cell type (HEK293) are limited in scalability and other methods, such as those based on the baculovirus and non-adherent insect cell (Sf9) system, have been pursued as an alternative to increase rAAV production. In this study, we compare these two production methods for cardiotropic rAAVs. Transduction efficiency for both production methods was assessed in primary cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and in mice following systemic delivery. We found that the rAAV produced by the traditional HEK293 method out-performed vector produced using the baculovirus/Sf9 system in vitro and in vivo. This finding provides a potential caveat for vector function when using the baculovirus/Sf9 production system and underscores the need for thorough assessment of vector performance when using diverse rAAV production methods.

Microcarrier Screening and Evaluation for Dynamic Expansion of Human Periosteum-Derived Progenitor Cells in a Xenogeneic Free Medium.

An increasing need toward a more efficient expansion of adherent progenitor cell types arises with the advancements of cell therapy. The use of a dynamic expansion instead of a static planar expansion could be one way to tackle the challenges of expanding adherent cells at a large scale. Microcarriers are often reported as a biomaterial for culturing cells in suspension. However, the type of microcarrier has an effect on the cell expansion. In order to find an efficient expansion process for a specific adherent progenitor cell type, it is important to investigate the effect of the type of microcarrier on the cell expansion. Human periosteum-derived progenitor cells are extensively used in skeletal tissue engineering for the regeneration of bone defects. Therefore, we evaluated the use of different microcarriers on human periosteum-derived progenitor cells. In order to assess the potency, identity and viability of these cells after being cultured in the spinner flasks, this study performed several in vitro and in vivo analyses. The novelty of this work lies in the combination of screening different microcarriers for human periosteum-derived progenitor cells with in vivo assessments of the cells’ potency using the microcarrier that was selected as the most promising one. The results showed that expanding human periosteum-derived progenitor cells in spinner flasks using xeno-free medium and Star-Plus microcarriers, does not affect the potency, identity or viability of the cells. The potency of the cells was assured with an in vivo evaluation, where bone formation was achieved. In summary, this expansion method has the potential to be used for large scale cell expansion with clinical relevance.

DEP-Dots for 3D cell culture: low-cost, high-repeatability, effective 3D cell culture in multiple gel systems

It is known that cells grown in 3D are more tolerant to drug treatment than those grown in dispersion, but the mechanism for this is still not clear; cells grown in 3D have opportunities to develop inter-cell communication, but are also closely packed which may impede diffusion. In this study we examine methods for dielectrophoresis-based cell aggregation of both suspension and adherent cell lines, and compare the effect of various drugs on cells grown in 3D and 2D. Comparing viability of pharmacological interventions on 3D cell clusters against both suspension cells and adherent cells grown in monolayer, as well as against a unicellular organism with no propensity for intracellular communication, we suggest that 3D aggregates of adherent cells, compared to suspension cells, show a substantially different drug response to cells grown in monolayer, which increases as the IC50 is approached. Further, a mathematical model of the system for each agent demonstrates that changes to drug response are due to inherent changes in the system of adherent cells from the 2D to 3D state. Finally, differences in the electrophysiological membrane properties of the adherent cell type suggest this parameter plays an important role in the differences found in the 3D drug response.

Developing a Microfabricated Lab-On-A-Chip Device for Patch-Clamp Applications with Internal Solution Exchange

Patch-clamp electrophysiology is a widely utilized technique in both academic and industrial laboratories to assess the function of ion channels. Despite its importance, conventional patch-clamp experiments remain inherently difficult to perform, laborious, and suffer from low data throughput. Automated patch-clamp systems have since been developed to address these issues, but have done so to the detriment of the versatility and affordability of the technique. Here, we have developed a new chip-based patch-clamp system, dubbed the SMPL system, that has been specifically microfabricated to deliver high quality patch-clamp recordings while improving upon the versatility of other chip-based patch-clamp systems. The SMPL chip is compatible with any adherent cell type, and can be used to culture and assess the electrophysiological properties of induced pluripotent stem cell (iPSC)-derived and primary cultures in addition to more conventional, stable-expression cell lines. The chip contains four independent microfluidic channels, each linked to a micropatterned patch-site, that enables for up to four sequential recordings to be taken. These microchannels also serve to allow intracellular solution to be exchanged during the course of an experiment. Since the chip has been fabricated entirely of optically transparent materials, imaging experiments can also be performed in parallel to electrophysiological assessments. The SPML chip connects to any existing patch-clamp amplifier using an affordable universal adapter and switchbox, allowing the user to decide which of the four sites is to be accessed. We are currently evaluating the capabilities of this technology applied to recording lipid bilayers and ion channels reconstituted in lipid vesicles.

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

Screening ofi in vitro Cytotoxicities from methanol extract of Acehnese Murrayakoenigii Leaf

Murrayakoenigii (Linn.) Sprengis known as the miracle plant that are rich in medicinal properties. Aceh was used this plant as the local condiment that familiarize the specific food of Aceh. This research aims to screening the cytotoxic activities from methanol extract of M. Koenigii leaf from Aceh. The invitrocytotoxic activities were evaluated with using MTT assay method. Three cell lines were used as bioindicators including MCF-7, HeLa, and Raji. The result showed that the extract givesa very good activities against MCF-7 and HeLa cell lines with CD50 value of < 1 μg/ml and 2.25 μg/ml respectively, but the cytotoxicity against Raji cell lines showed as weak with CD50 value of > 1000 μg/ml. It can be concluded that the methanol extract of M. Koenigii leaf showed the potential activities against adherent cell type, but not potential to suspension cell type.

Cell-Imprint Surface Modification by Contact Photolithography-Based Approaches: Direct-Cell Photolithography and Optical Soft Lithography Using PDMS Cell Imprints.

New cell-imprint surface modification techniques based on direct-cell photolithography and optical soft lithography using poly(dimethylsiloxane) (PDMS) cell imprints are presented for enhanced cell-based studies. The core concept of engineering materials for cell-based studies is the material's ability to redesign the physicochemical characteristics of the cellular niche. There is a growing interest in direct molding from cells (cell imprinting). These negative copies of cell surface topographies have been shown to affect cell shape and direct mesenchymal stem cells' differentiation. Analyzing the results is however challenging as cells seeded on these substrates do not always end up in a cell pattern, which leads to decreased effectiveness and biased quantification. To gain control over cell seeding into the patterns and avoid unwanted cell population outside of the patterns, the cell-imprinted surface needs to be modified. From this perspective, the standard optical contact lithography process was modified and cells were introduced to the cleanroom. Direct-cell photolithography was used for a single-step PDMS cell-imprint (chondrocytes as the molding template) surface modification down to single-cell (approximately 5 μm in diameter) resolution. As cells come in a variety of shapes, sizes, and optical profiles, a complementary optical soft lithography-based photomask fabrication technique is also reported. The simplicity of the fabrication process makes this cell-imprint surface modification technique compatible with any adherent cell type and leads to efficient cell-based studies.

Mechanism of formation of soft and elastic nanoflowers; a key major guideline

Water-born flower-like nanostructures have been produced via electrospinning of an innovative bicomponent polymeric solution of cross-linkable amino-functional polysiloxane (X-AFPs) resin and polyvinyl alcohol (PVA) in a special condition, after an after-washing process. It is the first report on production of a resin-based flower-like nanostructure as well as a siliconic flower-like nanostructure. It is also a well-engineered water-based green technique. An interesting mechanism has been proposed according to the SEM outcomes as well as the principles of electrospinning. The paper gives an explanation of how some physicochemical phenomena such as specific interactions among the polymeric components as well as high-level differences in terms of electrical charge density, viscosity and elasticity can direct the formation of these amazing structures. Accordingly, this research can be considered as a key major guideline to develop other polymeric flower-like nanostructures and their associated cutting-edge applications. Developing an anti-adhesive membrane was also followed in this research. A super-hydrophobic feature was achieved on the post-treated flower-like structures with a hydrophobic X-AFPs layer, after elimination of the PVA residue. Cytocompatibility as well as anti-adhesive properties of the treated flower-like structures have been assayed and perfectly confirmed through an in vitro study on a highly adherent cell type in a high cell seeding concentration. Accordingly, these three dimensional multiple-scale flower-like nanostructures can be offered for a wide-range of biomedical applications especially for anti-adhesive requirements such as cardiovascular applications and/or controlled cell response approaches.

Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.

Using the scratch assay to study cell migration in an inquiry-based cell biology lab

Cell migration, a fundamental process in development, wound healing, and immune function, is a common topic in undergraduate cell biology courses. We developed laboratory exercises with an inquiry-based learning (IBL) approach in which cell migration could be examined with the scratch assay, adapted from the primary literature. A narrow scratch was created in a confluent monolayer of cells growing on the bottom of a cell culture dish. Migration into the resultant cell-free zone from both sides of the scratch was measured after one day using the scale bar function of a digital camera. The Chinese hamster ovary cell line was used, but any adherent cell type could be examined. Students used the scratch assay to formulate hypotheses and design experiments in which variables affecting cell migration could be investigated. For example, the effect of cytoskeletal disruption was evaluated by adding the microtubule- and microfilament-disrupting drugs, colcemid and phallacidin, respectively, to the growth medium when the scratch was made. Optimal drug concentration parameters were determined for students to reference. Low drug concentrations inhibited cell migration, while higher concentrations killed the cells. This study demonstrated that the scratch assay is an accessible IBL method for studying cell migration.

Stimulation of Notch Signaling in Mouse Osteoclast Precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future. This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

Lab-to-clinic application of stem cell therapy for stroke

Stem cell therapy or “cell therapy” has been demonstrated to be a potent treatment intervention in animal models of acute ischemic stroke, and recently has been introduced as an experimental therapy in early phase clinical trials. Among the many stem cells, the bone marrow adherent cell type known as mesenchymal stem cells have emerged in laboratory studies as a safe and effective therapy for ischemic stroke and other brain diseases. In particular, a unique population of adherent bone marrow-derived cells, called MultiStem cells, display immunomodulatory effects and are a promising allogeneic cell therapy in acute ischemic stroke. Here, we describe the preclinical evidence supporting the use of MultiStem in the acute setting of ischemic stroke and the translation into an early phase clinical trial in ischemic stroke.

Human pluripotent stem cell process parameter optimization in a small scale suspension bioreactor

Background As cell-based therapies begin to enter clinical trials, human pluripotent stem cells (PSCs) present a reliable and robust source of cells for differentiation into all cell types in the human body [1]. PSC may one day provide cells for new treatment options for a wide range of diseases including cardiovascular disease, neurodegenerative diseases, and diabetes. The large numbers of cells required for many of these therapies necessitates the development of scalable and cost efficient technologies for manufacturing PSCs and their derivatives [2]. PSCs are an adherent cell type that is susceptible to phenotypic change in response to environmental stress. Adherent PSCs can be grown in suspension by forming aggregates of undifferentiated cells [3,4], by encapsulating PSCs in beads or hydrogels [5,6], or by adhering PSCs to the surface of microcarriers [7,8]. Most PSC suspension expansion to date has been performed in benchtop stirred-tank bioreactors operating at volumes of 50-200mL. Little bioprocess optimization has been conducted at these scales likely because of the high cost of PSC growth media and labour requirements for bioreactor growth. This study describes the use of a small-scale bioreactor system for Design-of-Experiments based PSC expansion process development. These process developments inform strategies to bring PSC-based processes into clinical production.

AHCC Activation and Selection of Human Lymphocytes via Genotypic and Phenotypic Changes to an Adherent Cell Type: A Possible Novel Mechanism of T Cell Activation.

Active Hexose Correlated Compound (AHCC) is a fermented mushroom extract and immune supplement that has been used to treat a wide range of health conditions. It helps in augmentation of the natural immune response and affects immune cell activation and outcomes. The goal of this project was to study and understand the role and mechanisms of AHCC supplementation in the prevention of immunosuppression through T cell activation. The method described here involves “in vitro” culturing of lymphocytes, exposing them to different concentrations of AHCC (0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) at 0 hours. Interestingly, clumping and aggregation of the cells were seen between 24 and 72 hours of incubation. The cells lay down extracellular matrix, which become adherent, and phenotypical changes from small rounded lymphocytes to large macrophage-like, spindle shaped, elongated, fibroblast-like cells even beyond 360 hours were observed. These are probably translated from genotypic changes in the cells since the cells propagate for at least 3 to 6 generations (present observations). RNA isolated was subjected to gene array analysis. We hypothesize that cell adhesion is an activation and survival pathway in lymphocytes and this could be the mechanism of AHCC activation in human lymphocytes.

Transfection of Primary and iPSC‐derived Cells by Capillary Electroporation

Primary cells in culture are typically considered to be a highly relevant in vitro models. They can be cultivated and differentiated in vitro, but are typically difficult to transfect using conventional methods. In this work, we've developed a system, Cellaxess ACE, a versatile, single capillary electrode‐based electroporation system that can be used for any adherent cell type. Cellaxess electroporation offers superior transfection quality because of minimal cell processing and low voltages required for electroporation. The focused electrical field delivered by the platform minimizes electrochemical toxicity and joule heating. The platform is truly enabling, e.g. rat mouse &amp; iPSC‐derived human neurons, primary human immune cells and many more cell types with excellent viability and a completely retained morphology. It can be used in any cell culture format, and is an excellent match with high‐content and confocal microscopy readouts. The presented data shows the broad application area for the Cellaxess ACE electroporation system, including cDNA transfection and RNAi experiments on different types of primary cell cultures.