2D Static Culture Research Articles

Liver-on-a-Chip Integrated with Label-Free Optical Biosensors for Rapid and Continuous Monitoring of Drug-Induced Toxicity.

Drug toxicity assays using conventional 2D static cultures and animal studies have limitations preventing the translation of potential drugs to the clinic. The recent development of organs-on-a-chip platforms provides promising alternatives for drug toxicity/screening assays. However, most studies conducted with these platforms only utilize single endpoint results, which do not provide real-time/ near real-time information. Here, a versatile technology is presented that integrates a 3Dliver-on-a-chip with a label-free photonic crystal-total internal reflection (PC-TIR) biosensor for rapid and continuous monitoring of the status of cells. This technology can detect drug-induced liver toxicity by continuously monitoring the secretion rates and levels of albumin and glutathione S-transferase α (GST-α) of a 3D liver on-a-chip model treated with Doxorubicin. The PC-TIR biosensor is based on a one-step antibody functionalization with high specificity and a detection range of 21.7ngmL-1 to 7.83 x 103ngmL-1 for albumin and 2.20ngmL-1 to 7.94 x 102ngmL-1 for GST-α. This approach provides critical advantages for the early detection of drug toxicity and improved temporal resolution to capture transient drug effects. The proposed proof-of-concept study introduces a scalable and efficient plug-in solution for organ-on-a-chip technologies, advancing drug development and in vitro testing methods by enabling timely and accurate toxicity assessments.

Tracer-based metabolomics for profiling nitric oxide metabolites in a 3D microvessels-on-chip model.

Endothelial dysfunction, prevalent in cardiovascular diseases (CVDs) and linked to conditions like diabetes, hypertension, obesity, renal failure, or hypercholesterolemia, is characterized by diminished nitric oxide (NO) bioavailability-a key signaling molecule for vascular homeostasis. Current two-dimensional (2D) invitro studies on NO synthesis by endothelial cells (ECs) lack the crucial laminar shear stress, a vital factor in modulating the NO-generating enzyme, endothelial nitric oxide synthase (eNOS), under physiological conditions. Here we developed a tracer-based metabolomics approach to measure NO-specific metabolites with mass spectrometry (MS) and show the impact of fluid flow on metabolic parameters associated with NO synthesis using 2D and 3D platforms. Specifically, we tracked the conversion of stable-isotope labeled NO substrate L-Arginine to L-Citrulline and L-Ornithine to determine eNOS activity. We demonstrated clear responses in human coronary artery endothelial cells (HCAECs) cultured with 13C6, 15N4-L-Arginine, and treated with eNOS stimulator, eNOS inhibitor, and arginase inhibitor. Analysis of downstream metabolites, 13C6, 15N3 L-Citrulline and 13C5, 15N2 L-Ornithine, revealed distinct outcomes. Additionally, we evaluated the NO metabolic status in static 2D culture and 3D microvessel models with bidirectional and unidirectional fluid flow. Our 3D model exhibited significant effects, particularly in microvessels exposed to the eNOS stimulator, as indicated by the 13C6, 15N3 L-Citrulline/13C5, 15N2 L-Ornithine ratio, compared to the 2D culture. The obtained results indicate that the 2D static culture mimics an endothelial dysfunction status, while the 3D model with a unidirectional fluid flow provides a more representative physiological environment that provides a better model to study endothelial dysfunction.

Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor

Lentiviral vectors (LVVs) play a critical role in gene delivery for exvivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5×104 to 5×103 cells/cm2, delaying the transient transfection from 24 to 120h post-seeding, reducing plasmid DNA to 167ng/cm2, and adding 5mM sodium butyrate 6h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2×106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum both at small and large scale.

Serum free medium for large scale microcarrier culture system of hmsc towards clinical cell-based therapy

Background & Aim Recent advances in human mesenchymal stem cell (hMSC)-based clinical therapies for stroke, graft vs. host disease and degenerative diseases has elevated the need for the production of large numbers of functional cells in scalable, homogeneous and cost-effective processes. Current 2D static culture method can not meet the anticipated demand for cells as it is labor intensive, not scalable and not a controlled system. Bioreactor system with microcarriers as a matrix in combination with optimized xeno-free (XF) medium represent a preferred culture structure towards clinical applications. The current xeno-free culture media for hMSC are specially designed for 2D static culture and are not necessarily suitable for 3D environment. The addition of undefined animal components such as serum to the media to enable the culture of hMSC on microcarriers in suspension pose safety concerns and it can be a major obstacle for obtaining regulatory approval when required. The present study evaluated a novel GMP compliant, serum-free (SF), XF hMSC expansion medium, with auxiliary solutions for attachment and dissociation suitable for microcarrier cultures in bioreactors. Methods, Results & Conclusion Results demonstrate the feasibility of expanding hMSC from various sources (one marrow, Wharton Jelly and adipose tissue) in a scalable microcarrier-based culture system using an optimized xeno-free medium while maintaining cell characteristics. Stability studies over 1 year, validated that the media was able to support robust hMSC growth and colony formation. The microcarrier suspension culture system combined with SF media represents a significant footstep for large-scale expansion of regulatory accepted hMSC for cellular therapeutics.

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

Drug-transporter interactions could impact renal drug clearance and should ideally be detected in early stages of drug development to avoid toxicity-related withdrawals in later stages. This requires reliable and robust assays for which current high-throughput screenings have, however, poor predictability. Kidney-on-a-chip platforms have the potential to improve predictability, but often lack compatibility with high-content detection platforms. Here, we combined conditionally immortalized proximal tubule epithelial cells overexpressing organic anion transporter 1 (ciPTEC-OAT1) with the microfluidic titer plate OrganoPlate to develop a screenings assay for renal drug-transporter interactions. In this platform, apical localization of F-actin and intracellular tight-junction protein zonula occludens-1 (ZO-1) indicated appropriate cell polarization. Gene expression levels of the drug transporters organic anion transporter 1 (OAT1; SLC22A6), organic cation transporter 2 (OCT2; SLC22A2), P-glycoprotein (P-gp; ABCB1), and multidrug resistance-associated protein 2 and 4 (MRP2/4; ABCC2/4) were similar levels to 2D static cultures. Functionality of the efflux transporters P-gp and MRP2/4 was studied as proof-of-concept for 3D assays using calcein-AM and 5-chloromethylfluorescein-diacetate (CMFDA), respectively. Confocal imaging demonstrated a 4.4 ± 0.2-fold increase in calcein accumulation upon P-gp inhibition using PSC833. For MRP2/4, a 3.0 ± 0.2-fold increased accumulation of glutathione-methylfluorescein (GS-MF) was observed upon inhibition with a combination of PSC833, MK571, and KO143. Semi-quantitative image processing methods for P-gp and MRP2/4 was demonstrated with corresponding Z′-factors of 0.1 ± 0.3 and 0.4 ± 0.1, respectively. In conclusion, we demonstrate a 3D microfluidic PTEC model valuable for screening of drug-transporter interactions that further allows multiplexing of endpoint read-outs for drug-transporter interactions and toxicity.

Comparison of growth kinetics between static and dynamic cultures of human induced pluripotent stem cells

Understanding the fundamental mechanisms that govern the growth kinetics of human induced pluripotent stem cells (hiPSCs) contributes to culture design strategies to improve large-scale production. Two hiPSC lines (Tic and 253G1) were cultured under static and dynamic suspension conditions, and growth kinetics were compared during early (24-48h), middle (48-72h), and late (72-96h) stages. In 2D static culture, similar growth profiles were observed for both hiPSC lines. However, there were significant differences in growth profile patterns and aggregate morphologies between hiPSC lines grown in 3D static and dynamic cultures. Based on immunostaining comparing the two hiPSC lines, surface distribution of collagen type I was observed in aggregates of the Tic line, but not in those of the 253G1 line. Compared to that in 3D static culture, the numbers of cells at 96h were significantly decreased in 3D dynamic culture. The apparent specific growth rate (μapp) of the Tic line was maintained continuously throughout culture, whereas that of the 253G1 line decreased gradually with culture until the late phase, at which time this parameter was reduced to μapp=(0.85±0.71)×10-2h-1. This indicates that during the growth of hiPSCs in 3D dynamic culture, cells were damaged by liquid flow, which disrupted the cell-synthesized extracellular matrix (ECM). These results demonstrate that cell-synthesized ECM is an important factor affecting cell growth and morphology, and that changes to the ECM within aggregates lead to reduced growth abilities in dynamic culture.

Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in plates and in a 3D microscale dynamic environment

We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented.

Investigating Nonalcoholic Fatty Liver Disease in a Liver-on-a-Chip Microfluidic Device.

Background and AimNonalcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide, ranging from simple steatosis to nonalcoholic steatohepatitis, which may progress to cirrhosis, eventually leading to hepatocellular carcinoma (HCC). HCC ranks as the third highest cause of cancer-related death globally, requiring an early diagnosis of NAFLD as a potential risk factor. However, the molecular mechanisms underlying NAFLD are still under investigation. So far, many in vitro studies on NAFLD have been hampered by the limitations of 2D culture systems, in which cells rapidly lose tissue-specific functions. The present liver-on-a-chip approach aims at filling the gap between conventional in vitro models, often scarcely predictive of in vivo conditions, and animal models, potentially biased by their xenogeneic nature.MethodsHepG2 cells were cultured into a microfluidically perfused device under free fatty acid (FFA) supplementation, namely palmitic and oleic acid, for 24h and 48h. The device mimicked the endothelial-parenchymal interface of a liver sinusoid, allowing the diffusion of nutrients and removal of waste products similar to the hepatic microvasculature. Assessment of intracellular lipid accumulation, cell viability/cytotoxicity and oxidative stress due to the FFA overload, was performed by high-content analysis methodologies using fluorescence-based functional probes.ResultsThe chip enables gradual and lower intracellular lipid accumulation, higher hepatic cell viability and minimal oxidative stress in microfluidic dynamic vs. 2D static cultures, thus mimicking the chronic condition of steatosis observed in vivo more closely.ConclusionsOverall, the liver-on-a-chip system provides a suitable culture microenvironment, representing a more reliable model compared to 2D cultures for investigating NAFLD pathogenesis. Hence, our system is amongst the first in vitro models of human NAFLD developed within a microfluidic device in a sinusoid-like fashion, endowing a more permissive tissue-like microenvironment for long-term culture of hepatic cells than conventional 2D static cultures.

Effect of Purmorphamine on Osteogenic Differentiation of Human Mesenchymal Stem Cells in a Three-Dimensional Dynamic Culture System

We have previously demonstrated that purmorphamine exerts an osteoinductive effect on human bone marrow mesenchymal stem cells (hBM-MSCs) in a two-dimensional (2D) static culture system. In the present study, we have evaluated the effect of purmorphamine on osteogenic differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) dynamic culture system vs. a 2D static culture condition. In this study, hMSCs were seeded in either 3D collagen scaffolds using a perfusion bioreactor or on 2D culture plates. One day later, the medium was replaced with osteogenic medium supplemented with dexamethasone, l‐ascorbic acid and β-glycerophosphate in the presence or absence of purmorphamine. Histological staining, flow cytometry, and real-time PCR were conducted for evaluation of osteogenesis in the study groups. The expressions of RUNX-2, alkaline phosphatase (ALP), osteocalcin (OC), collagen I (Col I), and bone sialoprotein (BSP) were compared between the 3D and 2D culture systems at 14 and 21 days post-induction of differentiation. Based on our results, alizarin red staining showed deposition of the mineralized matrix in the 2D static and 3D dynamic cultures. Our flow cytometric analysis indicated that the number of ALP+/OC− cells increased in the presence of purmorphamine in the 2D culture rather than the 3D system at day14. One week later, the numbers of OC+/ALP− and OC+/Stro-1− cells increased significantly in the 3D dynamic model compared with the 2D cultures. Expression of RUNX-2 was upregulated in the presence of purmorphamine in both cultures at day 14. The purmorphamine response gene, Gli-1, was upregulated during both the early and late culture periods in the 3D dynamic system, while similar up-regulation was observed only during the early culture period in 2D culture. No difference was observed in expressions of collagen type I and BSP between groups. The present study indicates that purmorphamine as agonist of Shh signaling pathway affects on osteogenic differentiation program of hMSCs in static and dynamic culture systems.

Novel mini β-TCP 3D perfusion bioreactor for proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Applications of bioreactors in combination with scaffolding materials are promising in tissue-engineering fields. To rapidly produce bone mesenchymal stem cells (MSCs) suitable for osteogenic differentiation (OSD), we developed a novel technique for β-TCP (β-tricalcium phosphate) scaffolds preparation and employed these scaffolds to build a new type of mini three dimensional (3D) perfusion bioreactor. Compared to the 2D static culture, MSCs acquired much higher amplification rate and alkaline phosphatase (ALP) activity over a 24-day culture period. Interestingly, the Specific ALP activity was independent of the growth rate under adequate osteogenic inducement, suggesting there may be an OSD bottleneck at a single-cell level. Furthermore, cells on scaffolds exhibited gradually reduced total migration rate (MR) and relatively constant local MR, both ideal for bone regeneration. Excellent adhesion of MSCs to the smooth scaffolds surface, especially the layer structures, was seen on scanning electron microscopy images, demonstrating fine compatibility between scaffold and cells. Our results indicate this simplified, integrated and potentially modularizable 3D bioreactor could enable the osteocytes producing from MSCs for expected applications.

Mechanical Stimulation Mediates Gene Expression in MC3T3 Osteoblastic Cells Differently in 2D and 3D Environments

Successful bone tissue engineering requires the understanding of cellular activity in three-dimensional (3D) architectures and how it compares to two-dimensional (2D) architecture. We developed a perfusion culture system that utilizes fluid flow to mechanically load a cell-seeded 3D scaffold. This study compared the gene expression of osteoblastic cells in 2D and 3D cultures, and the effects of mechanical loading on gene expression in 2D and 3D cultures. MC3T3-E1 osteoblastlike cells were seeded onto 2D glass slides and 3D calcium phosphate scaffolds and cultured statically or mechanically loaded with fluid flow. Gene expression of OPN and FGF-2 was upregulated at 24 h and 48 h in 3D compared with 2D static cultures, while collagen 1 gene expression was downregulated. In addition, while flow increased OPN in 2D culture at 48 h, it decreased both OPN and FGF-2 in 3D culture. In conclusion, gene expression is different between 2D and 3D osteoblast cultures under static conditions. Additionally, osteoblasts respond to shear stress differently in 2D and 3D cultures. Our results highlight the importance of 3D mechanotransduction studies for bone tissue engineering applications.

CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells.