PUBLICATIONS

The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in response to toxicity and that more focus should be placed on alterations of ECM-derived protein expression as biomarkers of liver disease and chronic drug-induced hepatotoxicity, changes that can be studied in stable in vivo-like hepatic cell systems, such as the spheroids.

Three-dimensional (3D) cell culture models, such as spheroids, are a valuable
tool for mimicking the physiology of tissues and tumors in vitro. One key aspect
of spheroid physiology is their size. As spheroids grow larger, the complete
penetration of nutrients and oxygen into the core and removal of metabolites
becomes increasingly restricted, resulting in gradients and necrotic or hypoxic
zones within the interior. This is similar to what is observed in tumors in vivo.
For this reason, larger spheroids are often preferred as tumor models in vitro.
However, necrotic cores can be undesirable for other applications where high
cell viability is needed. Therefore, it is important to understand the properties
of spheroids in order to select the appropriate model for a given application.
In this study, we analyzed the presence of necrotic cores in tumor spheroids
generated from colorectal cancer cells (HCT116) using a combination of a
Live/Dead assay and high-content imaging. Our results showed that only
spheroids with a diameter larger than 700 micrometers showed a prominent
necrotic core, in line with previously published literature. Additionally, our
assay design and imaging methods are simple, cost-effective and suitable for
automation and high-throughput screening.

Treatment options for patients with NRAS-mutant melanoma lack an efficient targeted drug combination hampering overall and progression-free survival. To build a 3D melanoma model, cells were seeded at a density of 0.25 3 104 cells/well in RPMI medium in 96-well Biofloat ULA plates followed by drug treatment with Palbociclib and MEKi.

This study investigates the immune checkpoint inhibitors (ICI) targeting PD-L1/PD-1 in pancreatic ductal adenocarcinoma (PDAC), where such inhibitors have shown limited success. Macrophages in the tumor microenvironment (TME) play a crucial role in tumor progression and are examined in relation to PD-L1 expression. Immunohistochemical analysis indicates that PD-L1 is mainly expressed by stroma cells, including macrophages, and not by PDAC cells in primary PDAC tissues and liver metastases. A 3D spheroid coculture model formed using BIOFLOAT™ 96-well ultra-low attachment plates with PDAC cell lines, macrophages, and CD8+ T cells is used to assess the impact of PD-L1-expressing macrophages on PDAC cell response to PD-L1/PD-1 inhibitors. Despite strong PD-L1 expression in macrophages, the study finds that ICI treatment does not enhance the activation and cytotoxic phenotype of CD8+ T cells in PDAC.

The research discusses a novel protocol for differentiating astrocytes from human stem cell-derived ventral midbrain dopaminergic neuron (vmDAN)–generating progenitors. Astrocytes play essential roles in Parkinson’s disease, shifting the research focus from traditional dopaminergic neurons. The protocol mimics the developmental processes in the human brain, allowing the characterization of morphological, molecular, and functional features of these astrocytes. The study introduces a new calcium imaging data analysis pipeline, deCLUTTER2+, providing insights into spontaneous or cue-dependent patterns of Ca2+ transients in these astrocytes. Overall, the protocol facilitates the study of functional properties of human ventral midbrain astrocytes under physiological and disease conditions. Generated neural progenitor cells were dissociated with Accutase and then seeded in BIOFLOAT™ 96-well ultra-low-attachment plates further Imaging was done using lasers and detectors, and images were processed using Fiji software.

The study investigates the efficacy of I-CBP112, a CBP/EP300 bromodomain inhibitor, in overcoming drug resistance in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) models. I-CBP112 significantly reduces the expression of various ABC transporters associated with drug resistance, enhancing intracellular drug accumulation and cytotoxicity in both 2D and 3D cultures. The inhibitor’s impact is similar to the combined effect of specific ABC inhibitors, suggesting the need for simultaneous inhibition of multiple drug pumps for enhanced drug retention. Importantly, I-CBP112 induces proinflammatory phenotypes in human macrophages without toxicity to primary cell lines, making it a promising co-adjuvant to chemotherapy for drug-resistant cancer. Spheroids of paclitaxel-resistant A549 and doxorubicin-resistant MDA-MB-231 cell lines were treated with I-CBP112 and doxorubicin in a BIOFLOAT™ 96-well U-bottom Plate, with a repeat of the entire treatment after one week.

The study aimed to investigate the microRNA expression profile in biphasic-sarcomatoid malignant pleural mesothelioma (MPM) cell lines and assess the impact of butein, a chemo-sensitizing compound, on these microRNAs. Nanostring-based profiling identified a set of commonly modulated microRNAs after butein treatment. Specifically, butein induced miR-186-5p-mediated modulation of TWIST1 levels, affecting various tumorigenic aspects of MPM, including 3D growth, cisplatin resistance, invasion, and bioenergetics. Analysis of TCGA data revealed an anti-correlation between miR-186-5p and TWIST1 levels in biphasic MPM specimens, suggesting a novel mechanism contributing to the chemo-sensitizing effect of butein with potential translational relevance. For generating cell spheroids, a variable number of single cells/well were seeded into BIOFLOAT TM 96-well plates.

The article discusses propranolol as a promising chemosensitizer for cancer therapy by disrupting the tumor microenvironment homeostasis. Propranolol, a non-selective beta-blocker, is explored for its potential in overcoming resistance to chemotherapy. The study analyzes the effects of the combination of propranolol and 5-fluorouracil on colorectal carcinoma cells in various models, including monolayers, spheroids, and in vivo mouse models. For the experiment, co-culture spheroids were formed with the mixture of cancer cells and MRC5 cells (1:1) were suspended in BIOFLOAT™ ultra-low attachment surface plates. Results indicate that propranolol disrupts hypoxic adaptation mechanisms, inhibits HIF1α and carbonic anhydrase IX, activates apoptosis, and slows down the growth of xenografts, suggesting its potential as a chemosensitizer in combined therapy to disrupt the tumor microenvironment homeostasis.

Various molecular mechanisms contribute to multidrug resistance in cancer, including increased drug efflux, enhanced cellular repair, and alterations in drug metabolism or targets. The focus is on ABCG2, a transporter that promotes drug efflux and induces chemotherapeutic resistance. The scientists suggest that developing selective ABCG2 inhibitors could enhance chemotherapy efficacy. The authors identify a new ABCG2 inhibitor (compound 8) and demonstrate its ability to increase mitoxantrone cytotoxicity in hepatocellular carcinoma and transfected breast cancer cell lines. Co-administration with mitoxantrone in HEK293 cells indicates the inhibitor’s efficacy in reversing multidrug resistance. Experiments on multicellular tumor spheroids and proteomic analyses support the results. Multicellular tumor spheroids (MCTS) from Hep G2 cell lines were generated by growing the cell suspensions in an ultra-low attachment BIOFLOAT™ 96-well plate by faCellitate.

The paper discusses the societal problem of thyroid hormone disruption due to chemical exposure and the traditional reliance on animal experiments for assessing environmental and health risks. However, recent biotechnological advances allow the evaluation of chemical toxicity using 3D cell cultures. The study focuses on thyroid-friendly soft (TS) microspheres and their interactive effects on thyroid cell aggregates as a potential toxicity assessment tool. Advanced characterization methods reveal that TS-microsphere-integrated thyroid cell aggregates exhibit improved thyroid function. A comparison with zebrafish embryos, commonly used for thyroid toxicity analysis, shows that the TS-microsphere-integrated aggregates are more sensitive to the thyroid inhibitor methimazole (MMI). This proof-of-concept approach suggests that controlling cellular function in the desired direction with TS-microsphere integration could provide new insights for advancing in vitro cell-based research.

The huThyrEC cell line was obtained from InSCREENeX GmbH and cultured in huThyrEC medium for 7 days. Cell aggregates were integrated with TS microspheres by seeding them in BIOFLOAT™ 96-well U-bottom plates with h7H medium, and after 7 days, the culture medium was changed to assess thyroglobulin secretion and thyroid hormone production.

Manganese dioxide (MnO2)-based nanostructures were prepared in a one-pot reaction with Pt(IV) prodrugs to create redox-responsive theranostics for cancer treatment. The Pt(IV) complexes act as prodrugs of cisplatin (Pt(II)). The MnO2–Pt(IV) probes demonstrated effective cytotoxicity in both 2D and 3D A549 cell models, comparable to active cisplatin in 3D models. For 2D cell viability experiments, A549 cells were initially seeded in 96-well plates and treated with varying compound concentrations for 48 hours. Resazurine was added, and after a 4-hour incubation, fluorescence was measured at 590 nm using a microplate reader. In 3D cell viability studies, A549 cells were seeded in BIOFLOAT™ 96-well plates, incubated for 3 days to form spheroids, and then treated with compounds for 48 hours.

Additionally, these nanoparticles exhibited a strong off/ON magnetic resonance (MR) contrast in response to reducing agents, with a significant increase in longitudinal relaxivity (r1) upon treatment. In vivo MRI experiments demonstrated the potential of MnO2–Pt(IV) nanostructures as redox-responsive MR theranostics for cancer therapy, inducing a strong and long-lasting T1 signal enhancement upon intratumoral injection in A549 tumor-bearing mice.

This paper introduces a method to scale up the number of spheroids by factor 100 using BIOFLOAT™ 96-well plates and BIOFLOATTM FLEX coating solution to obtain low-adhesive wells. Conventional liver spheroid cultures were compared with organ liver-chip and the latter demonstrated a comparable hepatic functionality as liver spheroids, while the liver-plate did not meet the same standards. The liver chip was successfully applied to study drug safety, metabolism profiling, and drug metabolism.

Spheroid formation was achieved in a 96-well ultra-low attachment plates model to study the effects and mechanisms of pro-inflammatory cytokines on the expression of 9 different genes encoding enzymes responsible for the metabolism of > 90% of clinically used drugs

This in vivo human 3D liver spheroid model is suitable for the prediction of drug metabolism under conditions of inflammation and constitutes a versatile system for short- and long-term preclinical and mechanistic studies of cytokine-induced changes in drug metabolism.

Over the past years, research has made impressive breakthroughs towards the development and implementation of 3D cell models for a wide range of applications, such as drug development and testing, organogenesis, cancer biology, and personalized medicine. Opposed to 2D cell monolayer culture systems, advanced 3D cell models better represent the in vivo physiology. However, for these models to deliver scientific insights, appropriate investigation techniques are required. Despite the potential of fluorescence microscopy to visualize these models with high spatial resolution, sample preparation and imaging assays are not straightforward. Here, we provide different protocols of sample preparation for fluorescence imaging, for both matrix-embedded and matrix-free models ( e.g ., organoids and spheroids, respectively). Additionally, we provide detailed guidelines for imaging 3D cell models via confocal multi-photon fluorescence microscopy. We show that using these protocols, images of 3D cell culture systems can be obtained with sub-cellular resolution. Graphical abstract.

Human 3D liver microtissues/spheroids are powerful in vitro models to study drug-induced liver injury (DILI) but the small number of cells per spheroid limits the models’ usefulness to study drug metabolism. In this work, we scale up the number of spheroids on both a plate and a standardized organ-chip platform by factor 100 using a basic method which requires only limited technical expertise. We successfully generated up to 100 spheroids using polymer-coated microwells in a 96-well plate (= liver-plate) or organ-chip (= liver-chip). Liver-chips display a comparable cellular CYP3A4 activity, viability, and biomarker expression as liver spheroids for at least one week, while liver-plate cultures display an overall reduced hepatic functionality. To prove its applicability to drug discovery and development, the liver-chip was used to test selected reference compounds. The test system could discriminate toxicity of the DILI-positive compound tolcapone from its less hepatotoxic structural analogue entacapone, using biochemical and morphological readouts. Following incubation with diclofenac, the liver-chips had an increased metabolite formation compared to standard spheroid cultures. In summary, we generated a human liver-chip model using a standardized organ-chip platform which combines up to 100 spheroids and can be used for the evaluation of both drug safety and metabolism.

Paclitaxel induces multidrug resistance in cancer cells therefore contributing to the high failure rates of chemotherapy and relapse of the disease. Overexpression of ABCB1 (P-glycoprotein) in lysosomal membranes is linked to drug accumulation in these organelles. The role of lysosomes in drug accumulation functional impact of drug and drug toxicity was studied using spheroids formed on Nunc™Lab-Tek™chamber slides were coated with faCellitate BIOFLOAT™ FLEX coating solution.

Cancer is a leading cause of death globally, with 18.1 million new cases and 9.6 million deaths reported in 2018, with lung cancer being the most diagnosed and lethal form. Conventional chemotherapy, combined with surgery, radiotherapy, and nanomedicines, is widely used, but challenges such as severe side effects and short drug half-life motivate research for novel drug delivery systems. Irinotecan and topotecan, derivatives of camptothecin (CPT), are used in treating various cancers, but side effects and limited delivery systems prompt exploration of alternatives. The development of nanogels containing CPT, modified with tocopherol, ergocalciferol, and testosterone, showed sustained drug release and cytotoxic activity against cancer cells. The use of silk fibroin (SF) as a carrier for CPT, due to its biomedical applications, efficient functionalization, and antiproliferative effects on cancer cells, is highlighted, along with the successful synthesis of SF aggregates for controlled drug delivery in cancer treatment. BIOFLOAT™ FLEX by faCellitate, Mannheim, Germany was used to coat non-TC treated U-bottom 96-well plate to facilitate cell aggregation and subsequent spheroid formation. The spheroids were treated with 0.1 mg/mL of the test compounds and further fluorescence imaging was performed with an Olympus IX73 inverted microscope.

EZH2, a component of Polycomb Repressive Complex 2, regulates histone modifications and is often overexpressed in lung cancers. Inhibiting EZH2 with EPZ-6438 has shown clinical promise. However, this study reveals that EZH2 suppression, while inhibiting growth in 2D cultures of A549 lung adenocarcinoma cells, stimulates growth in 3D cultures. Metabolomic analysis indicates distinct metabolic effects, including activation of Krebs cycle and purine synthesis in 3D cultures. The study emphasizes the significance of extracellular matrix and three-dimensional models in understanding the complex role of EZH2 and its inhibitors in cancer cells.

In 3D culturing without added matrix, detached cells were seeded at 300,000 cells/well in a 24-well Aggrewell™ 400 plate with microwells for spheroid formation. The plate was coated with BIOFLOATTM FLEX solution, air-dried to create a low cell-retention surface, and rinsed. Cells in lung organoid medium were added, and after gentle mixing and centrifugation, spheroids were grown at 37°C/5% CO2 with rocking. This method enables the formation of up to 1,200 isolated spheroids in the microwells.

The design and study of efficient polymer-based drug delivery systems for the controlled release of anticancer drugs is one of the pillars of nanomedicine. The fight against metastatic and invasive cancers demands therapeutic candidates with increased and selective toxicity towards malignant cells, long-term activity and reduced side effects. In this sense, polyphosphazene nanocarriers were synthesized for the sustained release of the anticancer drugs camptothecin (CPT) and epirubicin (EPI). Linear poly(dichloro)phosphazene was modified with lipophilic tocopherol or testosterone glycinate, with antioxidant and antitumor activity, and with hydrophilic Jeffamine M1000 to obtain different polyphosphazene nanocarriers. It allowed us to encapsulate the lipophilic CPT and the more hydrophilic EPI. The encapsulation process was carried out via solvent exchange/precipitation, attaining a 9.2-13.6 wt% of CPT and 0.3-2.4 wt% of EPI. CPT-loaded polyphosphazenes formed 140-200 nm aggregates in simulated body physiological conditions (PBS, pH 7.4), resulting in an 80-100-fold increase of CPT solubility. EPI-loaded polyphosphazenes formed 250 nm aggregates in an aqueous medium. CPT and EPI release (PBS, pH 7.4, 37 °C) was monitored for 202 h, being almost linear during the first 8 h. The slow release of testosterone and tocopherol was also sustained for 150 h in PBS (pH 7.4 and 6.0) at 37 °C. The co-delivery of testosterone or tocopherol and the anticancer drugs from the nanocarriers was expected. Cells of the human breast cancer cell line MCF-7 demonstrated good uptake of anticancer-drug-loaded nanocarriers after 6 h. Similarly, MCF-7 spheroids showed good uptake of the anticancer-drug-loaded aggregates after 72 h. Almost all anticancer-drug-loaded polyphosphazenes exhibited similar or superior toxicity against MCF-7 cells and spheroids when compared to raw anticancer drugs. Additionally, cell-cycle arrest in the G2/M phase was increased in response to the drug-loaded nanocarriers. Almost no toxicity of anticancer-drug-loaded aggregates against primary human lung fibroblasts was observed. Furthermore, the aggregates displayed no hemolytic activity, which is in contrast to the parent anticancer drugs. Consequently, synthesized polyphosphazene-based nanocarriers might be potential nanomedicines for chemotherapy.

A water-soluble hydrolysate of silk fibroin (SF) (~30 kDa) was esterified with tocopherol, ergocalciferol, and testosterone to form SF aggregates for the controlled delivery of the anticancer drug camptothecin (CPT). Elemental analysis and ¹H NMR spectroscopy showed a degree of substitution (DS) on SF of 0.4 to 3.8 mol %. Yields of 58 to 71% on vitamins- and testosterone-grafted SF conjugates were achieved. CPT was efficiently incorporated into the lipophilic core of SF aggregates using a dialysis-precipitation method, achieving drug contents of 6.3-8.5 wt %. FTIR spectra and DSC thermograms showed that tocopherol- and testosterone-grafted SF conjugates predominantly adopted a β-sheet conformation. After the esterification of tyrosine residues on SF chains with the vitamin or testosterone, the hydrodynamic diameters almost doubled or tripled that of SF. The zeta potential values after esterification increased to about -30 mV, which favors the stability of aggregates in aqueous medium. Controlled and almost quantitative release of CPT was achieved after 6 days in PBS at 37 °C, with almost linear release during the first 8 h. MCF-7 cancer cells exhibited good uptake of CPT-loaded SF aggregates after 6 h, causing cell death and cell cycle arrest in the G2/M phase. Substantial uptake of the CPT-loaded aggregates into MCF-7 spheroids was shown after 3 days. Furthermore, all CPT-loaded SF aggregates demonstrated superior toxicity to MCF-7 spheroids compared with parent CPT. Blank SF aggregates induced no hemolysis at pH 6.2 and 7.4, while CPT-loaded SF aggregates provoked hemolysis at pH 6.2 but not at pH 7.4. In contrast, parent CPT caused hemolysis at both pH tested. Therefore, CPT-loaded SF aggregates are promising candidates for chemotherapy.

Neurospheres are in vitro models to study central nervous disorders but traditional methods for neurosphere cultivation are time-consuming and have low yields. This article demonstrates the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. Bioreactors were prepared prior to cell collection and BIOFLOAT™ FLEX coating solution was used to coat the reactor.

This paper introduces a method combining the microcavity array 3D culturing technique with fluorophore-based oxygen sensing via microthermoformed oxygen-sensitive polymer films. For a convenient 3D cell culture setup, spheroids can be generated directly in the microcavities. However, for this purpose, the cells must not adhere and therefore, the sensor arrays were coated with BIOFLOAT™ solution to avoid cell attachment. Using a cell-repellent coating, in this case BIOFLOAT™, allows the generation of spheroids directly within the microcavities shortening the workflow, minimizing error potentials, and enabling experiments to be carried out more reproducibly.