Simple Culture System Research Articles

Simple dynamic cell culture system reduces recording noise in microelectrode array recordings

Microelectrode arrays (MEAs) have applications in drug discovery, toxicology, and basic research. They measure the electrophysiological response of tissue cultures to quantify changes upon exposure to biochemical stimuli. Unfortunately, manual addition of chemicals introduces significant noise in the recordings. Here, we report a simple-to-fabricate fluidic system that addresses this issue. We show that cell cultures can be successfully established in the fluidic compartment under continuous flow conditions and that the addition of chemicals introduces minimal noise in the recordings. This dynamic cell culture system represents an improvement over traditional tissue culture wells used in MEAs, facilitating electrophysiology measurements.Graphical abstract

Reprogramming fibroblast into human iBlastoids.

The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development.

Studying Monocyte-Myofibroblast Immunomodulation in a 3D Co-culture System.

Simple and reproducible 3D cell culture systems that mimic biological interactions within physiological tissues (biomimetics) can provide unique insight for scientific inquiries compared to 2D cell cultures. Fibroblast-populated collagen lattices (FPCLs) are commonly used for mimicking physiological collagen matrices, potentiating biomechanical stresses on embedded fibroblasts. Here, we describe a novel 3D co-culture model that incorporates human Tenon's capsule fibroblasts embedded in FPCLs co-cultured with THP-1 monocytes suspended in culture media. This method can be used for the assessment of cell-cell interactions in various stages of the wound healing process and can facilitate various types of immune cells in co-culture. This system can also be used to study pharmacological agents that may eventually improve clinical outcomes in patients affected by inflammatory disorders.

The impact of cellular environment on in vitro drug screening.

There are various reasons for drug failure in the developmental stage including toxicity, adverse effects and inefficacy. This is likely due to the differences in drug behavior between a simple and controlled cell culture system to that of a more complex whole organism environment. While the use of human phenotypical cells relevant to the condition may provide more accurate screening results, they are susceptible to producing false positives as cells are continuously influenced by constant chemical and physical interaction with the surrounding microenvironment. Therefore, several microenvironmental and pharmacomechanical aspects must be factored in during tissue culture drug screening.

Ferric citrate and apo-transferrin enable erythroblast maturation with β-globin from hemogenic endothelium

Red blood cell (RBC) generation from human pluripotent stem cells (PSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with β-globin expression in culture systems. During induction of β-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we describe a simple and reproducible culture system that can be used to generate erythroblasts with β-globin expression. We prepared a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium (HE). Floating erythroblasts derived from HE cells were primarily CD45+CD71+CD235a+ cells, and their number increased remarkably upon Fe treatment. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of β-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. The rapid maturation of these erythroblasts into RBCs was observed when injected in vivo, suggesting the development of RBCs that were ready to grow. Hence, induction of β-globin expression may be explained by the effects of ferric citrate that promote cell maturation by binding with soluble transferrin and entering the cells.Taken together, upon treatment with Fe, erythroblasts showed advanced maturity with a high transcription of β-globin. These findings can help devise a stable protocol for the generation of clinically applicable RBCs.

Simple chitin-based cell culture platform for production of biopharmaceuticals.

Gene therapy using viral vectors and antibody-based therapies continue to expand within the pharmaceutical market. We evaluated whether Cellhesion® VP, a chitin-based material, can be used as 3D culture platform for cell lines used for the production of antibodies and viral vectors. The results of Cell Counting Kit-8 assay and LDH assay revealed that Cellhesion® VP had no adverse effect to Human Embryonic Kidney (HEK) 293, A549 and Chinese hamster ovary (CHO) DG44-Interferon-β (IFN) cells. Cell growth analyses showed that Cellhesion® VP supported the 3D culture of HEK293, A549 and CHO DG44- IFN-β cells with a spherical morphology. Importantly, subculture of these cell lines on Cellhesion® VP was easily performed without trypsinization because cells readily transferred to newly added scaffold. Our data also suggest that CHO DG44-IFNβ, cultured on Cellhesion® VP secreted IFNβ stably and continuously during the culture period. Cellhesion® VP provides a simple and streamlined expansion culture system for the production of biopharmaceuticals.

Efficient Generation of Chondrocytes From Bone Marrow-Derived Mesenchymal Stem Cells in a 3D Culture System: Protocol for a Practical Model for Assessing Anti-Inflammatory Therapies.

Chondrocytes are the primary cells responsible for maintaining cartilage integrity and function. Their role in cartilage homeostasis and response to inflammation is crucial for understanding the progression and potential therapeutic interventions for various cartilage-related disorders. Developing an accessible and cost-effective model to generate viable chondrocytes and to assess their response to different bioactive compounds can significantly advance our knowledge of cartilage biology and contribute to the discovery of novel therapeutic approaches. We developed a novel, streamlined protocol for generating chondrocytes from bone marrow-derived mesenchymal stem cells (BMSCs) in a 3D culture system that offers significant implications for the study of cartilage biology and the discovery of potential therapeutic interventions for cartilage-related and associated disorders. We developed a streamlined protocol for generating chondrocytes from BMSCs in a 3D culture system using an "in-tube" culture approach. This simple pellet-based 3D culture system allows for cell aggregation and spheroid formation, facilitating cell-cell and cell-extracellular matrix interactions that better mimic the in vivo cellular environment compared with 2D monolayer cultures. A proinflammatory chondrocyte model was created by treating the chondrocytes with lipopolysaccharide and was subsequently used to evaluate the anti-inflammatory effects of vitamin D, curcumin, and resveratrol. The established protocol successfully generated a large quantity of viable chondrocytes, characterized by alcian blue and toluidine blue staining, and demonstrated versatility in assessing the anti-inflammatory effects of various bioactive compounds. The chondrocytes exhibited reduced inflammation, as evidenced by the decreased tumor necrosis factor-α levels, in response to vitamin D, curcumin, and resveratrol treatment. Our novel protocol offers an accessible and cost-effective approach for generating chondrocytes from BMSCs and for evaluating potential therapeutic leads in the context of inflammatory chondrocyte-related diseases. Although our approach has several advantages, further investigation is required to address its limitations, such as the potential differences between chondrocytes generated using our protocol and those derived from other established methods, and to refine the model for broader applicability and clinical translation.

Spinal Cord Organoids to Study Motor Neuron Development and Disease.

Motor neuron diseases (MNDs) are a heterogeneous group of disorders that affect the cranial and/or spinal motor neurons (spMNs), spinal sensory neurons and the muscular system. Although they have been investigated for decades, we still lack a comprehensive understanding of the underlying molecular mechanisms; and therefore, efficacious therapies are scarce. Model organisms and relatively simple two-dimensional cell culture systems have been instrumental in our current knowledge of neuromuscular disease pathology; however, in the recent years, human 3D in vitro models have transformed the disease-modeling landscape. While cerebral organoids have been pursued the most, interest in spinal cord organoids (SCOs) is now also increasing. Pluripotent stem cell (PSC)-based protocols to generate SpC-like structures, sometimes including the adjacent mesoderm and derived skeletal muscle, are constantly being refined and applied to study early human neuromuscular development and disease. In this review, we outline the evolution of human PSC-derived models for generating spMN and recapitulating SpC development. We also discuss how these models have been applied to exploring the basis of human neurodevelopmental and neurodegenerative diseases. Finally, we provide an overview of the main challenges to overcome in order to generate more physiologically relevant human SpC models and propose some exciting new perspectives.

Development of a Pneumatic-Driven Fiber-Shaped Robot Scaffold for Use as a Complex 3D Dynamic Culture System

Cells can sense and respond to different kinds of continuous mechanical strain in the human body. Mechanical stimulation needs to be included within the in vitro culture system to better mimic the existing complexity of in vivo biological systems. Existing commercial dynamic culture systems are generally two-dimensional (2D) which fail to mimic the three-dimensional (3D) native microenvironment. In this study, a pneumatically driven fiber robot has been developed as a platform for 3D dynamic cell culture. The fiber robot can generate tunable contractions upon stimulation. The surface of the fiber robot is formed by a braiding structure, which provides promising surface contact and adequate space for cell culture. An in-house dynamic stimulation using the fiber robot was set up to maintain NIH3T3 cells in a controlled environment. The biocompatibility of the developed dynamic culture systems was analyzed using LIVE/DEAD™ and alamarBlue™ assays. The results showed that the dynamic culture system was able to support cell proliferation with minimal cytotoxicity similar to static cultures. However, we observed a decrease in cell viability in the case of a high strain rate in dynamic cultures. Differences in cell arrangement and proliferation were observed between braided sleeves made of different materials (nylon and ultra-high molecular weight polyethylene). In summary, a simple and cost-effective 3D dynamic culture system has been proposed, which can be easily implemented to study complex biological phenomena in vitro.

Cell culture systems for isolation of SARS-CoV-2 clinical isolates and generation of recombinant virus

A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.

Promising Perinatal Outcome after Using a Simplified Low-Cost IVF Culture System Specifically Designed for Resource-Poor Countries.

Assisted reproductive techniques services are often not accessible to the majority of infertile couples in Low and Middle Income Countries (LMIC) due to high costs. Lowering IVF laboratory costs is a crucial step to make IVF affordable for a larger part of the world population. We developed a simplified culture system (SCS) which has proven to be effective, and the next step is to prove its safety. Preterm birth (PTB) and low birthweight (LBW) of 176 singletons born after using the SCS, 105 after fresh embryo transfer (fresh ET), and 71 after frozen embryo transfer (frozen ET) were compared with all IVF/ICSI singletons born in Belgium between 2013 and 2018. When comparing our 105 SCS babies born after fresh ET with all Belgian babies born after conventional IVF only, we also adjusted for 7 risk factors known to influence perinatal outcome, namelythe mother's age, day of transfer, pituitary inhibition protocol, rank of cycles, number of oocytes retrieved, number of embryos transferred, and gender of the baby. Before adjustment, we found a significantly higher PTB (10.2% vs. 3.8%, OR 2.852, 95% CI [1.042-7.803], p-value 0.0413) and LBW (9.8% vs. 2.9%, OR 3.692, 95% CI [1.163-11.721], p-value 0.0267) in the conventional IVF group versus SCS after fresh ET. After adjusting for seven risk parameters, these differences remained significant (PTB: OR 2.627, 95% CI [1.013-6.816], p-value 0.0471) and LBW: OR 3.267, 95% CI [1.118-9.549], p-value 0.0305). PTB and LBW between both groups was not significantly different for singletons born after frozen ET. Taking into account the small series, PTB and LBW rates in SCS singletons in FRET cycles are very reassuring and significantly lower compared to babies born after conventional IVF in Belgium. Being aware of its effectiveness, our results offer a good perspective for SCS to become an important tool to implement low-cost IVF in LMIC.

Establishing a simple perfusion cell culture system for light-activated liposomes

The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid flow in traditional cell models can lead to overestimation of nanoparticle uptake. In this paper, we established a perfusion cell culture platform to study light-activated liposomes and determined the effect of flow on the liposomal cell uptake. The optimal cell culturing parameters for the A549 cells under flow conditions were determined by monitoring cell viability. To determine optimal liposome treatment times, particle uptake was measured with flow cytometry. The suitability of commercial QuasiVivo flow-chambers for near-infrared light activation was assessed with a calcein release study. The chamber material did not hinder the light activation and subsequent calcein release from the liposomes. Furthermore, our results show that the standard cell culturing techniques are not directly translatable to flow cultures. For non-coated liposomes, the uptake was hindered by flow. Interestingly, hyaluronic acid coating diminished the uptake differences between the flow and static conditions. The study demonstrates that flow affects the liposomal uptake by lung cancer cell line A549. The flow also complicates the cell attachment of A549 cells. Moreover, we show that the QuasiVivo platform is suitable for light-activation studies.

Analysis of Aβ-induced neurotoxicity and microglial responses in simple two- and three-dimensional human iPSC-derived cortical culture systems

The extracellular accumulation of amyloid-β (Aβ) in plaques and associated neurodegeneration are the pathological hallmarks of Alzheimer’s disease (AD). These plaques are surrounded by microglia—the resident tissue macrophages of the brain parenchyma that originate from primitive macrophages from the embryonic yolk sac. Microglia, including a unique subpopulation called “disease-associated microglia” (DAM), are strongly implicated in AD pathology; however, their exact function and physiology remain largely unknown. Notably, simple cell and tissue culture systems that adequately recreate the brain microenvironment and can simulate critical aspects of AD pathology could fundamentally contribute to elucidating microglial function in disease development and progression. Thus, we added human-induced pluripotent stem cell (hiPSC)-induced primitive macrophages (hiMacs) to hiPSC-induced cortical neurons (cell model) and cortical organoids (tissue model). The treatment of these culture systems with the O-acyl isopeptide of Aβ1−42, which reverts to natural extracellular Aβ1−42 at neutral pH and starts self-aggregation, caused the degeneration of hiPSC-induced cortical neurons in 2D culture and within cortical organoid cultures. Notably, the hiMacs phagocytosed extracellular Aβ and exhibited a DAM-like phenotype. In both cell and tissue organoid culture systems, neurodegeneration was attenuated by the addition of hiMacs. Moreover, in cortical organoids, Aβ plaques formed more circular and fewer hotspot-like morphological structures in the vicinity of hiMacs. These findings demonstrate the utility of simple hiPSC-induced cortical cell and tissue culture systems supplemented with hiMacs for elucidating critical aspects of AD pathology, such as microglial function and physiology. Adopting such systems in routine research practice may lead to the development of novel therapeutic strategies for AD.

A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture.

Culture medium exchange leads to loss of valuable auto- and paracrine factors produced by the cells. However, frequent renewal of culture medium is necessary for nutrient supply and to prevent waste product accumulation. Thus it remains the gold standard in cell culture applications. The use of dialysis as a medium refreshment method could provide a solution as low molecular weight molecules such as nutrients and waste products could easily be exchanged, while high molecular weight components such as growth factors, used in cell interactions, could be maintained in the cell culture compartment. This study investigates a dialysis culture approach for an in vitro bone remodeling model. In this model, both the differentiation of human mesenchymal stromal cells (MSCs) into osteoblasts and monocytes (MCs) into osteoclasts is studied. A custom-made simple dialysis culture system with a commercially available cellulose dialysis insert was developed. The data reported here revealed increased osteoblastic and osteoclastic activity in the dialysis groups compared to the standard nondialysis groups, mainly shown by significantly higher alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activity, respectively. This simple culture system has the potential to create a more efficient microenvironment allowing for cell interactions via secreted factors in mono- and cocultures and could be applied for many other tissues.

Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference

Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.

Multiyear outcomes using sibling oocytes demonstrates safety and efficacy of a simplified culture system consistent with use in a low-cost IVF setting

Research questionCan a novel closed simplified IVF culture system be used to achieve outcomes comparable to those obtained with intracytoplasmic sperm injection (ICSI) followed by conventional culturing? DesignThis analysis is part of a non-inferiority prospective study comparing ICSI and a simplified culture system (SCS) for gamete fertilization in a selected group of patients. According to protocol, sibling oocytes in intact cumulus–oocyte complexes were randomly distributed between ICSI and conventional insemination in the SCS. For women, selection criteria included being under 43 years of age and at least six eggs at retrieval. An inseminating motile sperm count ≥1 million was required. The primary outcome measure was ongoing pregnancy rate (>12 weeks) per cycle; secondary outcome measures included fertilization rate, miscarriage rate and implantation rate (ongoing pregnancy rate per embryo). ResultsFrom January 2016 until December 2019, 653 SCS/ICSI cycles were performed yielding a total of 7915 oocytes. The fertilization rate was 61.1% and 50.4% for SCS and ICSI (P < 0.0001), respectively. The ongoing pregnancy rate was 32.0% for SCS and 36.7% for ICSI (P = 0.27). Implantation rate was 30.6% for SCS and 34.4% for ICSI (P = 0.35). The miscarriage rate was 7.5% and 6.5% for SCS and ICSI, respectively (P = 0.75). ConclusionNo difference was found in ongoing pregnancy rate, implantation rate and the miscarriage rate between SCS and ICSI in this selected patient cohort.

3D Interfacial and Spatiotemporal Regulation of Human Neuroepithelial Organoids.

Neuroepithelial (NE) organoids with dorsal–ventral patterning provide a useful three‐dimensional (3D) in vitro model to interrogate neural tube formation during early development of the central nervous system. Understanding the fundamental processes behind the cellular self‐organization in NE organoids holds the key to the engineering of organoids with higher, more in vivo‐like complexity. However, little is known about the cellular regulation driving the NE development, especially in the presence of interfacial cues from the microenvironment. Here a simple 3D culture system that allows generation and manipulation of NE organoids from human‐induced pluripotent stem cells (hiPSCs), displaying developmental phases of hiPSC differentiation and self‐aggregation, first into NE cysts with lumen structure and then toward NE organoids with floor‐plate patterning, is established. Longitudinal inhibition reveals distinct and dynamic roles of actomyosin contractility and yes‐associated protein (YAP) signaling in governing these phases. By growing NE organoids on culture chips containing anisotropic surfaces or confining microniches, it is further demonstrated that interfacial cues can sensitively exert dimension‐dependent influence on luminal cyst and organoid morphology, successful floor‐plate patterning, as well as cytoskeletal regulation and YAP activity. This study therefore sheds new light on how organoid and tissue architecture can be steered through intracellular and extracellular means.

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

BackgroundThe liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply.MethodsWe first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects.ResultsIn our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity.ConclusionsThese results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

308 Perinatal outcome after icsi compared to a low-cost simplified ivf system in a selected population: A prospective cohort study

Aim of the study: Do babies concepted by using a low-cost simplified IVF culture system (SCS) compared with babies concepted with ICSI differ? Design: This analysis is part of a non-inferiority prospective study comparing ICSI and a simplified culture system (SCS) for gamete fertilization and subsequent embryo culture. We compared the perinatal outcome results of all babies born from January 2013 until December 2020 after using split SCS and ICSI insemination in a selected group of normoresponder women, excluding severe male infertility cases defined by an inseminating motile sperm count (IMC) of less than million after semen processing.

Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels.

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.