Single Cell Culture Research Articles

Lab-on-a-chip based mechanical actuators and sensors for single-cell and organoid culture studies.

All living cells constantly experience and respond to mechanical stresses. The molecular networks that activate in cells in response to mechanical stimuli are yet not well-understood. Our limited knowledge stems partially from the lack of available tools that are capable of exerting controlled mechanical stress to individual cells and at the same time observing their responses at subcellular to molecular resolution. Several tools such as rheology setups, micropipetes, and magnetic tweezers have been used in the past. While allowing to quantify short-time viscoelastic responses, these setups are not suitable for long-term observations of cells and most of them have low throughput. In this Perspective, we discuss lab-on-a-chip platforms that have the potential to overcome these limitations. Our focus is on devices that apply shear, compressive, tensile, and confinement derived stresses to single cells and organoid cultures. We compare different design strategies for these devices and highlight their advantages, drawbacks, and future potential. While the majority of these devices are used for fundamental research, some of them have potential applications in medical diagnostics and these applications are also discussed.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

COSMAS: a lightweight toolbox for cardiac optical mapping analysis

Optical mapping is widely used in experimental cardiology, as it allows visualization of cardiac membrane potential and calcium transients. However, optical mapping measurements from a single heart or cell culture can produce several gigabytes of data, warranting automated computer analysis. Here we present COSMAS, a software toolkit for automated analysis of optical mapping recordings in cardiac preparations. COSMAS generates activation and conduction velocity maps, as well as visualizations of action potential and calcium transient duration, S1-S2 protocol analysis, and alternans mapping. The software is built around our recent ‘comb’ algorithm for segmentation of action potentials and calcium transients, offering excellent performance and high resistance to noise. A core feature of our software is that it is based on scripting as opposed to relying on a graphical user interface for user input. The central role of scripts in the analysis pipeline enables batch processing and promotes reproducibility and transparency in the interpretation of large cardiac data sets. Finally, the code is designed to be easily extended, allowing researchers to add functionality if needed. COSMAS is provided in two languages, Matlab and Python, and is distributed with a user guide and sample scripts, so that accessibility to researchers is maximized.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality

AbstractWe studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial–mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Autonomous TGFβ signaling induces phenotypic variation in human acute myeloid leukemia

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell‐selective adhesion molecule (ESAM), a hematopoietic stem cell‐related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM− and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM− or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFβ signaling in these cells. AML cells secreted TGFβ1, which autonomously activated TGFβ pathway and induced their phenotypic variation. Surprisingly, TGFβ signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFβ signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

Microfluidic Single-cell Trapping and Cultivation for the Analysis of Host-viral Interactions

The isolation of single cells and their further cultivation in confined chambers are essential to the collection of statistically reliable temporal information in cell-based biological experiments. In this work, we present a hydrodynamic single-cell trapping and culturing platform that facilitates biological analysis and experimentation of virus infection into host cells. To find the optimum design of the cell trap at the microscale, we evaluated hook traps with different widths and trap intervals to obtain a high trapping efficiency of a single cell. The proposed design leverages the stochastic position of the cells as they flow into the structured microfluidic channels, where hundreds of single cells are then arrayed in nanoliter chambers for simultaneous cell-specific data collection. Optimum design is used to devise and implement a hydrodynamic cell-trapping mechanism that is minimally detrimental to the cell viability and retains a high trapping efficiency (90%), with the capability of reaching high fill factors (90%) in short loading times (10 min) in a 450-trap device. Finally, we perform an analysis of host-viral interactions under the treatment of a drug concentration gradient as a proof of concept.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Optimization of adenoviral gene transfer in human pluripotent stem cells

Human pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have the potential to differentiate into a wide variety of cells in vitro and have applications in basic developmental biology research and regenerative medicine. To understand the process of differentiation from pluripotent stem cells to functional cells, it is necessary to efficiently and safely transfer and express exogenous genes. We attempted to optimize the efficient transfer of genes into pluripotent stem cells using adenoviral vectors. Comparative study of the activities of three representative ubiquitously active promoters revealed that only the CA promoter allowed robust transgene expression in human pluripotent stem cells. In addition, we established a protocol that allowed us to efficiently introduce target genes and ensure their expression even in small numbers of cells. Adenoviral vector infection of pluripotent stem cells in single-cell suspension culture yielded high gene transfer efficiency with low cytotoxicity, without losing the undifferentiated state of the pluripotent stem cells. This optimized system will facilitate developmental biology research and regenerative medicine using pluripotent stem cells.

Replating Protocol for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational cardiac research. Obtaining hiPSC-CM culture as a single-cell, monolayer or three-dimensional clusters for downstream applications can be challenging. Thus, it is critical to develop replating strategies for hiPSC-CMs by evaluating different enzymatic or nonenzymatic reagents for dissociation and seeding on different coating materials. To reseed hiPSC-CMs with high viability and at structures desirable for the downstream applications, here we defined optimized protocols to dissociate hiPSC-CMs by using collagenase A&B, Collagenase II, TrypLE, and EDTA and reseeding on various matrix materials including fibronectin, laminin, imatrix, Matrigel, and Geltrex. By the replating methods described here, a single cell or cluster-containing hiPSC-CM cultures can be generated effectively.

Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells Using a Single-Cell Culture Method

Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells Using a Single-Cell Culture Method

Optimizations of In Vitro Mucus and Cell Culture Models to Better Predict In Vivo Gene Transfer in Pathological Lung Respiratory Airways: Cystic Fibrosis as an Example

The respiratory epithelium can be affected by many diseases that could be treated using aerosol gene therapy. Among these, cystic fibrosis (CF) is a lethal inherited disease characterized by airways complications, which determine the life expectancy and the effectiveness of aerosolized treatments. Beside evaluations performed under in vivo settings, cell culture models mimicking in vivo pathophysiological conditions can provide complementary insights into the potential of gene transfer strategies. Such models must consider multiple parameters, following the rationale that proper gene transfer evaluations depend on whether they are performed under experimental conditions close to pathophysiological settings. In addition, the mucus layer, which covers the epithelial cells, constitutes a physical barrier for gene delivery, especially in diseases such as CF. Artificial mucus models featuring physical and biological properties similar to CF mucus allow determining the ability of gene transfer systems to effectively reach the underlying epithelium. In this review, we describe mucus and cellular models relevant for CF aerosol gene therapy, with a particular emphasis on mucus rheology. We strongly believe that combining multiple pathophysiological features in single complex cell culture models could help bridge the gaps between in vitro and in vivo settings, as well as viral and non-viral gene delivery strategies.

IP-10 and CXCR3 signaling inhibit Zika virus replication in human prostate cells.

Our previous studies have shown that Zika virus (ZIKV) replicates in human prostate cells, suggesting that the prostate may serve as a long-term reservoir for virus transmission. Here, we demonstrated that the innate immune responses generated to three distinct ZIKV strains (all isolated from human serum) were significantly different and dependent on their passage history (in mosquito, monkey, or human cells). In addition, some of these phenotypic differences were reduced by a single additional cell culture passage, suggesting that viruses that have been passaged more than 3 times from the patient sample will no longer reflect natural phenotypes. Two of the ZIKV strains analyzed induced high levels of the IP-10 chemokine and IFNγ in human prostate epithelial and stromal mesenchymal stem cells. To further understand the importance of these innate responses on ZIKV replication, we measured the effects of IP-10 and its downstream receptor, CXCR3, on RNA and virus production in prostate cells. Treatment with IP-10, CXCR3 agonist, or CXCR3 antagonist significantly altered ZIKV viral gene expression, depending on their passage in cells of relevant hosts (mosquito or human). We detected differences in gene expression of two primary CXCR3 isoforms (CXCR3-A and CXCR3-B) on the two cell types, possibly explaining differences in viral output. Lastly, we examined the effects of IP-10, agonist, or antagonist on cell death and proliferation under physiologically relevant infection rates, and detected no significant differences. Although we did not measure protein expression directly, our results indicate that CXCR3 signaling may be a target for therapeutics, to ultimately stop sexual transmission of this virus.

Secreted factors from mouse embryonic fibroblasts maintain repopulating function of single cultured hematopoietic stem cells.

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that proliferation of murine hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells.1 Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypic identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increased the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, with both two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supported self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust, alternative, serum-free source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features.

The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC–CM and single cells of hiPSC–derived EC and MC (hiPSC–EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC–EC+MC survival was promoted by the co-culture with hiPSC–CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC–EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC–CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC–CM, hiPSC–EC+MC, and extracellular matrix induced the maturation of hiPSC–CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.

Influence of a Medium’s pH on Paramecium caudatum Cells under Exposure to the Low Temperature Atmospheric Pressure Plasma

It is shown that when exposed to low-temperature atmospheric pressure plasma (LTAPP) on a single-cell culture of Paramecium caudatum as a result of changes in the external conditions (ionic composition of the solution), the nature of the movement changes and the number of viable cells decreases. The viability of Paramecium caudatum cells depends on the time of exposure to the plasma and is correlated with the changes in the pH of the medium. The kinetic features of the processes discovered in the work are analyzed: the aftereffect of exposure to the plasma and the stepwise nature of the cells’ death.

Experimental Modeling of Necrotizing Enterocolitis in Human Infant Intestinal Enteroids

Background: Experimental model systems are of paramount importance in advancing our understanding of human disease. Methods There are several limitations when using a single cell culture to recapitulate the findings in a complex organism and results often vary between species, when proxy animal models are studied. Results Human enteroids have allowed for study of human disease in complex multicellular culture systems. Here we present the novel use of human infant enteroids generated from premature infant intestine to study necrotizing enterocolitis (NEC), which is a devastating intestinal disorder that affects our most vulnerable pediatric population. Conclusions We demonstrate that NEC can be induced in premature human enteroids as supported by corresponding alterations in inflammation, apoptosis, tight junction expression, and permeability by treatment with lipopolysaccharide.

The Impact of a New Interleukin-2-Based Immunotherapy Candidate on Urothelial Cells to Support Use for Intravesical Drug Delivery.

(1) Background: The intravesical instillation of interleukin-2 (IL-2) has been shown to be very well tolerated and promising in patients with bladder malignancies. This study aims to confirm the use of a new IL-2 containing immunotherapy candidate as safe for intravesical application. IL-2, produced in mammalian cells, is glycosylated, because of its unique solubility and stability optimized for intravesical use. (2) Materials and Methods: Urothelial cells and fibroblasts were generated out of porcine bladder and cultured until they reached second passage. Afterwards, they were cultivated in renal epithelial medium (REM) and Dulbecco’s modified Eagles medium (DMEM) with the IL-2 candidate (IMS-Research) and three more types of human interleukin-2 immunotherapy products (IMS-Pure, Natural IL-2, Aldesleukin) in four different concentrations (100, 250, 500, 1000 IU/mL). Cell proliferation was analyzed by water soluble tetrazolium (WST) proliferation assay after 0, 3, and 6 days for single cell culture and co-culture. (3) Results: Proliferation assays showed that all IL-2 products induced very similar cultivation results and none of the IL-2 variants had a negative impact on the proliferation of urothelial cells and fibroblast in either concentration. (4) Conclusion: Human recombinant glycosylated IL-2 as well as human non-glycosylated IL-2 have no negative influence on the tissue cell proliferation of porcine urothelial cells and fibroblasts in vitro and represent a promising and innovative potential intravesical therapy candidate for patients in high need.