TGF-ß effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions.

The TGF-ß superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-ß, TGF-ß-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-ß isoforms, with TGF-ß2 having the greater effects. Consistent with these results, treatment with TGF-ß2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-ß on adipogenesis decreased under 3D spheroid culture conditions and TGF-ß isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-ß isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.

Unexpected and Synergistical Effects of All-Trans Retinoic Acid and TGF-ß2 on Biological Aspects of 2D and 3D Cultured ARPE19 Cells.

Objectives: To study the effects of all-trans retinoic acid (ATRA) on TGF-ß2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Methods: Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of COL1, αSMA, Zo-1, HIF1α, and PGC1α (2D), and physical property analyses (3D). Results: Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-ß2 in 2D ARPE19 monolayer cells. Under a hypoxia condition, treatment with ATRA conversely increased barrier function, but the effect was masked by a marked increase in effects induced by TGF-ß2. Although ATRA alone did not affect cellular metabolism and ROS levels in 2D ARPE cells, treatment with ATRA under a hypoxia condition did not affect ROS levels but shifted cellular metabolism from mitochondrial respiration to glycolysis. The changes of cellular metabolism and ROS levels were more pronounced with treatment of both ATRA and TGF-ß2 independently of oxygen conditions. Changes in mRNA expressions of some of the above genes suggested the involvement of synergistical regulation of cellular functions by TGF-ß2 and hypoxia. In 3D ARPE spheroids, the size was decreased and the stiffness was increased by either treatment with TGF-ß2 or ATRA, but these changes were unexpectedly modulated by both ATRA and TGF-ß2 treatment regardless of oxygen conditions. Conclusions: The findings reported herein indicate that TGF-ß2 and hypoxia synergistically and differentially induce effects in 2D and 3D cultured ARPE19 cells and that their cellular properties are significantly altered by the presence of ATRA.

Cryobiological Aspects of Upscaling Cryopreservation for Encapsulated Liver Cell Therapies.

For the efficient delivery of a cell therapy a treatment must be provided rapidly, at clinical scale, contain a sufficient active cellular component (biomass), and adhere to a multitude of regulatory requirements. Cryopreservation permits many of these demands to be met more readily. Here we present the cryopreservation and recovery of large volume (2.5L) alginate encapsulated liver cell spheroids (AELS), suitable for use with a novel bioartificial liver device (HepatiCan™) for the treatment of those suffering from acute liver failure (ALF), in regulatory approved cryobags and a cryopreservation process optimised for large volumes. By first assessing the thermal profiles of large scale cryobags with a thermal mimic, the feasibility of cryopreserving a full patient dose simultaneously (3x cryobags containing 833ml biomass each) was investigated, allowing for small and subsequently large-scale testing of cellular functional recoveries. Work presented here demonstrates that optimised reproducible cooling and warming profiles could be achieved with these large volumes, leading to high biomass recoveries at full clinical scale. The recovered AELS also had high regeneration potential, achieving full pre-freeze viable cell densities within 3 days, indicating that the cell therapy could be delivered rapidly to patients with ALF. This study has presented the feasibility for rapid delivery of large volume cell therapies, whilst further research into improved speed of post-thaw recovery is warranted.

Alternative Method for Obtaining Human-Induced Pluripotent Stem Cell Lines and Three-Dimensional Growth: A Simplified, Passage-Free Approach that Minimizes Labor.

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications. Key features • Establishment of induced pluripotent stem cells in a three-dimensional environment. • Maintenance and cryopreservation of iPSCs without the need for a dissociation step.

Organ-on-chip technology: Opportunities and challenges.

Organ-on-chip (OOC) technology is an innovative approach that reproduces human organ structures and functions on microfluidic platforms, offering detailed insights into intricate physiological processes. This technology provides unique advantages over conventional in vitro and in vivo models and thus has the potential to become the new standard for biomedical research and drug screening. In this mini-review, we compare OOCs with conventional models, highlighting their differences, and present several applications of OOCs in biomedical research. Additionally, we highlight advancements in OOC technology, particularly in developing multiorgan systems, and discuss the challenges and future directions of this field.

Cancer-on-chip: a breakthrough organ-on-a-chip technology in cancer cell modeling.

Cancer remains one of the leading causes of death worldwide. The unclear molecular mechanisms and complex in vivo microenvironment of tumors make it difficult to clarify the nature of cancer and develop effective treatments. Therefore, the development of new methods to effectively treat cancer is urgently needed and of great importance. Organ-on-a-chip (OoC) systems could be the breakthrough technology sought by the pharmaceutical industry to address ever-increasing research and development costs. The past decade has seen significant advances in the spatial modeling of cancer therapeutics related to OoC technology, improving physiological exposition criteria. This article aims to summarize the latest achievements and research results of cancer cell treatment simulated in a 3D microenvironment using OoC technology. To this end, we will first discuss the OoC system in detail and then demonstrate the latest findings of the cancer cell treatment study by Ooc and how this technique can potentially optimize better modeling of the tumor. The prospects of OoC systems in the treatment of cancer cells and their advantages and limitations are also among the other points discussed in this study.

Vitamin B6 Pathway Maintains Glioblastoma Cell Survival in 3D Spheroid Cultures.

Glioblastoma (GBM) is a deadly brain cancer. The prognosis of GBM patients has marginally improved over the last three decades. The response of GBMs to initial treatment is inevitably followed by relapse. Thus, there is an urgent need to identify and develop new therapeutics to target this cancer and improve both patient outcomes and long-term survival. Metabolic reprogramming is considered one of the hallmarks of cancers. However, cell-based studies fail to accurately recapitulate the in vivo tumour microenvironment that influences metabolic signalling and rewiring. Against this backdrop, we conducted global, untargeted metabolomics analysis of the G7 and R24 GBM 2D monolayers and 3D spheroid cultures under identical cell culture conditions. Our studies revealed that the levels of multiple metabolites associated with the vitamin B6 pathway were significantly altered in 3D spheroids compared to the 2D monolayer cultures. Importantly, we show that pharmacological intervention with hydralazine, a small molecule that reduces vitamin B6 levels, resulted in the cell death of 3D GBM spheroid cultures. Thus, our study shows that inhibition of the vitamin B6 pathway is a novel therapeutic strategy for the development of targeted therapies in GBMs.

Induced Pluripotent Stem Cell-Derived Parathyroid Organoids Resemble Parathyroid Morphology and Function.

The primary role of the parathyroid glands is to maintain calcium homeostasis through the secretion of parathyroid hormone (PTH). The limited proliferative capacity and differentiation of parathyroid cells hinder the generation of cell therapy options. In this study, parathyroid organoids are successfully generated from human-induced pluripotent stem cells (hiPSCs). At the end of the 20 days of differentiation, the parathyroid organoids exhibited distinct parathyroid morphology. Stereomicroscope, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis demonstrated the 3D arrangement of the cell layers in which intracellular structures of parathyroid cells resemble human parathyroid cellular morphology. Comprehensive molecular analyses, including RNA sequencing (RNA-Seq) and liquid chromatography/mass spectrometry (LC-MS/MS), confirmed the expression of key parathyroid-related markers. Protein expression of CasR, CxCr4, Gcm2, and PTH are observed in parathyroid organoids. Parathyroid organoids secrete PTH, demonstrate active intercellular calcium signaling, and induce osteogenic differentiation via their secretome. The tissue integration potential of parathyroid organoids is determined by transplantation into parathyroidectomized rats. The organoid transplanted animals showed significant elevations in PTH-related markers (CasR, CxCr4, Foxn1, Gcm2, and PTH). PTH secretion is detected in organoid-transplanted animals. The findings represent a significant advancement in parathyroid organoid culture and may offer a cellular therapy for treating PTH-related diseases, including hypoparathyroidism.

Innovations in 3D ovarian and follicle engineering for fertility preservation and restoration.

In-vitro maturation (IVM) is the process of cultivating early-stage follicles from the primordial to the antral stage and facilitating the maturation of oocytes outside the body within a supportive environment. This intricate procedure requires the careful coordination of various factors to replicate the natural ovarian conditions. Advanced techniques for IVM are designed to mimic the natural ovarian environment and enhance the development of follicles. Three-dimensional (3D) culture systems provide a more biologically relevant setting for follicle growth compared to traditional two-dimensional (2D) cultures. Traditional culture systems, often fail to support the complex process of follicle development effectively. However, modern engineered reproductive tissues and culture systems are making it possible to create increasingly physiological in-vitro models of folliculogenesis. These innovative methods are enabling researchers and clinicians to better replicate the dynamic and supportive environment of the ovary, thereby improving the outcomes of IVM offering new hope for fertility preservation and treatment. This paper focuses on the routine 3D culture, and innovative 3D culture of ovary and follicles, including a tissue engineering scaffolds, microfluidic (dynamic) culture system, organ-on-chip models, EVATAR system, from a clinical perspective to determine the most effective approach for achieving in-vitro maturation of follicles. These techniques provide critical support for ovarian function in various ovarian-associated disorders, including primary ovarian insufficiency (POI), premature ovarian failure (POF), ovarian cancer, and age-related infertility.

Mechanical confinement prevents ectopic platelet release.

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis.

Exploring Osteoarthritis Dynamics: Patient-Specific Cartilage Samples in an Organ-on-a-Chip Model.

OBJECTIVE: This study aims to tackle the existing challenges associated with the prediction and optimization of pharmaceutical interventions for osteoarthritis (OA). The primary objective is to develop an innovative tool that provides objective and patient-specific information regarding the most affected tissue in OA, articular cartilage. DESIGN: We employed an organ-on-a-chip (OoC) approach to replicate the 3D structure of cartilage in an in vitro setup. The study focused on assessing the individual drug responses of common medications using this innovative platform. Additionally, we conducted a biomarker analysis to gain insights into the variability of drug responses across patients. RESULTS: Our findings reveal that OA articular cartilage demonstrates an individualized response to pharmaceutical interventions. Despite the diverse nature of patient responses, our study indicates that Triamcinolone, a standard-of-care medication, consistently exhibits a robust anti-inflammatory response across patient tests. However, as seen in clinical studies, Triamcinolone was concurrently associated with degeneration. The biomarker analysis further underscores the importance of considering individual drug responses in developing effective treatment plans. CONCLUSION: In conclusion, this study introduces a valuable tool that not only mimics the 3D structure of cartilage but also provides crucial insights into the individualized responses of patients to various OA treatments. The application of an OoC approach may allow for a more accurate assessment of treatment efficacy. This objective biomarker analysis on patient-specific tissue offers clinicians a means to tailor treatment plans, thereby minimizing joint damage and advancing toward a more personalized approach in OA management.

Patient-derived tumor organoids mimic treatment-induced DNA damage response in glioblastoma.

Glioblastoma (GB) is the most common primary malignant brain tumor, characterized by resistance to therapy. Despite aggressive treatment options, GB remains an incurable disease. Invasiveness and heterogeneity are key GB features that cannot be studied in preclinical in vitro models. In this study, we investigated the effects of standard therapy using patient-derived GB organoids (GBOs). GBOs reflect the complexity and heterogeneity of the original tumor tissue. No significant effect on GBO viability or invasion was observed after irradiation and temozolomide treatment. E3 ubiquitin-protein ligase (MDM2), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the serine/threonine kinases ATM and ATR were upregulated at the gene and protein levels after treatment. Our results show that the p53 pathway and DNA-damage response mechanisms were triggered, suggesting that GBOs recapitulate GB therapy resistance. GBOs thus provide a highly efficient platform to assess the specific responses of GB patients to therapy and to further explore therapy resistance.

Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes.

Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechanophenotypes is not well defined. We investigated the mechanomemory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased αSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment.

Functional, patient-derived 3D tri-culture models of the uterine wall in a microfluidic array.

STUDY QUESTION: Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues? SUMMARY ANSWER: We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. WHAT IS KNOWN ALREADY: As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial-myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. STUDY DESIGN, SIZE, DURATION: We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. LIMITATIONS, REASONS FOR CAUTION: Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models. WIDER IMPLICATIONS OF THE FINDINGS: Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis. STUDY FUNDING/COMPETING INTEREST(S): C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Therapeutic Responses to Two New SN-38 Derivatives in Colorectal Cancer Patient-Derived Xenografts and Respective 3D In Vitro Cultures.

BACKGROUND/AIM: SN-38, an active metabolite of irinotecan, exhibits toxicity to all proliferating cells, causing dose-limiting and potentially life-threatening side effects. Newly established water-soluble derivatives of SN-38, 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin (BN-MOA) and 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (BN-NMe), exhibit a unique mechanism of spontaneous alkylation of aromatic bases in DNA and show greater in vitro activity on cancer cell lines than SN-38. The aim of this study was to compare the therapeutic responses to irinotecan, BN-MOA and BN-NMe in vivo and in vitro in 3D cultures using colorectal cancer (CRC) patient derived xenografts (PDX). MATERIALS AND METHODS: Seven established PDX tissues were subcutaneously grown on the flanks of NSG or NSG-SGM3 mice and tumor diameters were measured with a caliper. Compounds were administrated intraperitoneally at 40 mg/kg every five days. 3D PDX cultures were performed on 96-well LifeGel plates and cell viability was determined with the CellTiter Glo 3D reagent. RESULTS: Treatment with irinotecan significantly delayed or stopped the growth of 5 out of 7 PDXs, with a greater level of inhibition from BN-MOA compared to irinotecan and BN-NMe. In vitro studies exhibited the same trends in SN-38 and BN-NMe but not in BN-MOA. CONCLUSION: The new SN-38 derivatives, BN-MOA and BN-NMe, showed enhanced therapeutic effects compared to irinotecan in CRC models. BN-MOA demonstrated superior tumor inhibition in vivo, while BN-NMe had similar in vitro activity to SN-38. These findings highlight the potential of BN-MOA for greater antitumor efficacy in vivo, with BN-NMe showing comparable effectiveness to SN-38 in vitro. Future studies should optimize growth models to better predict anticancer drug responses.

Liquefied capsules containing nanogrooved microdiscs and umbilical cord-derived cells for bone tissue engineering.

Background: Surface topography has been shown to influence cell behavior and direct stromal cell differentiation into distinct lineages. Whereas this phenomenon has been verified in two-dimensional cultures, there is an urgent need for a thorough investigation of topography's role within a three-dimensional (3D) environment, as it better replicates the natural cellular environment. Methods: A co-culture of Wharton's jelly-derived mesenchymal stem/stromal cells (WJ-MSCs) and human umbilical vein endothelial cells (HUVECs) was encapsulated in a 3D system consisting of a permselective liquefied environment containing freely dispersed spherical microparticles (spheres) or nanogrooved microdiscs (microdiscs). Microdiscs presenting 358 ± 23 nm grooves and 944 ± 49 nm ridges were produced via nanoimprinting of spherical polycaprolactone microparticles between water-soluble polyvinyl alcohol counter molds of nanogrooved templates. Spheres and microdiscs were cultured in vitro with umbilical cord-derived cells in a basal or osteogenic medium within liquefied capsules for 21 days. Results: WJ-MSCs and HUVECs were successfully encapsulated within liquefied capsules containing spheres and microdiscs, ensuring high cellular viability. Results show an enhanced osteogenic differentiation in microdiscs compared to spheres, even in basal medium, evidenced by alkaline phosphatase activity and osteopontin expression. Conclusions: This work suggests that the topographical features present in microdiscs induce the osteogenic differentiation of adhered WJ-MSCs along the contact guidance, without additional differentiation factors. The developed 3D bioencapsulation system comprising topographical features might be suitable for bone tissue engineering approaches with minimum in vitro manipulation.

Production of Cartilaginous Organoids: Potential Opportunities and Challenges, A Review Article.

Damaged articular cartilage has limited self-healing potential and often leads to osteoarthritis (OA), pain, and dysfunction of the affected joint. Autologous and allogenic transplants cannot fully meet the needs of clinical treatment. Two dimensional (2D) and three-dimensional (3D) cell cultures can help to study growth modeling and physiological characteristics of the human body. Among the problems that 2D and single-layer cultures have the lack of proper and accurate tissue modeling and the lack of tissue complications similar is to the original tissue. With organoid models, cellular and tissue structural studies and functional and physiological studies of tissues have been revolutionized and more accurate. Organoids are useful for studying repair and drug efficacy. Physiological and pathological investigations by combining in vitro and in vivo methods have become more effective today. The purpose of this study is to investigate the factors involved in the formation of cartilage organoids so that we can introduce the best method of organoid production for the healing of cartilage damage by using cell types and organoid model.

MTS, WST-8, and ATP viability assays in 2D and 3D cultures: Comparison of methodologically different assays in primary human chondrocytes.

BACKGROUND: Tissue engineering enables the production of three-dimensional microtissues which mimic naturally occurring conditions in special tissues. These 3D culture systems are particularly suitable for application in regenerative medicine or experimental pharmacology and toxicology. Therefore, it is important to analyse the cells in their 3D microenvironment with regard to viability and differentiation. Tetrazolium assays (WST-8 and MTS) are still the methods of choice for estimating the number of living, metabolically active cells, with WST-8 being cell-impermeable compared to MTS. In contrast to these methods, the ATP assay is an endpoint method based on the luciferase-induced reaction of ATP with luciferin after cell lysis. OBJECTIVE: We compared three methodologically different proliferation/toxicity assays (MTS, WST-8, ATP) in monolayer (2D) and 3D culture systems to improve the technically challenging determination of the number of viable cells. METHODS: Chondrocytes were isolated from human articular cartilage. Three different test systems (MTS, WST-8, ATP) were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96-well plates. The intracellular ATP concentration was determined by luciferase-induced reaction of ATP with luciferin using a luminometer. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was performed by phase contrast microscopy (toxicity), correlation of cell count and ATP concentration or absorption signal (Gompertz function) and propidium iodide (PI) staining to proof the cell lysis of all cells in spheroids. RESULTS: In 2D culture, all three assays showed a good correlation between the number of seeded cells and the ATP concentration or absorption data, whereas the MTS-assay showed the lowest specificity. In 3D culture, the spheroid sizes were directly related to the number of cells seeded. The absorption data of the WST-8 and MTS assay correlated only for certain spheroid size ranges, whereas the MTS-assay showed again the lowest specificity. Only the measured intracellular ATP content showed a linear correlation with all spheroid sizes ranging from 100-1000 µm. The WST-8 assay revealed the second-best sensitivity which allows the measurement of spheroids larger than 240 µm. Phase contrast observation of monolayer cells showed toxic effects of MTS after 6 h incubation and no signs of toxicity of WST-8. Staining with propidium iodide showed complete lysis of all cells in a spheroid in the ATP assay. CONCLUSION: Among tetrazolium-based assays, WST-8 is preferable to MTS because of its non-toxicity and better sensitivity. When determining the number of viable cells in the 2D system, caution is advised when using the ATP assay because of its two-phase slope of the correlation graph concerning cell number and intracellular ATP. In 3D systems of human chondrocytes, the ATP-assay is superior to the other two test systems, as the correlation graph between cell number and intracellular ATP is biphasic. Since differentiation processes or other metabolic events can influence the results of proliferation and toxicity assays (determination of viable cells), this should be taken into account when using these test systems.

Spontaneous Efficient Differentiation of Human Pluripotent Stem Cells (hPSC) Upon Co-culture of hPSCs with Human Neonatal Foreskin Fibroblasts in 3D.

Pluripotent stem cells (PSCs) form well-formed embryoid bodies (EBs) in 3D culture. These EBs are formed in culture media lacking leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) in mouse and human PSCs, respectively. EBs are excellent technical tools for understanding developmental biology and inducing controlled differentiation in succeeding experimental steps. Technically speaking, EBs are spontaneously differentiated PSCs in 3D and exhibit all three lineages in a time-point/sequential manner. For example, ectoderm will form first, followed by mesoderm and endoderm. We have attempted to co-culture human neonatal foreskin-derived fibroblast cells in our laboratory with the PSCs first in 2D conditions followed by the induction of EBs (PSC+fibroblasts co-cultured) in low attachment dishes. We also performed spontaneous differentiation of such EBs (co-cultured with fibroblasts). We checked the presence of markers of various lineages, namely, ectoderm, mesoderm, and endoderm in days 6, 10, and 12 day EBs. We have also compared the fibroblast co-cultured EBs, along with control EBs (derived from only PSCs). This co-culture system mimics the natural conditions of uterine implantation and the role of the endometrial fibroblasts in the induction of further embryonic development. The fibroblast co-cultured iPSC EBs had better roundness scores than the normal iPSC EBs and had a higher expression of lineage-specific markers.