3D Cell Culture Systems Research Articles

Organoids in Haematologic Research: Advances and Future Directions

ABSTRACTOrganoid technology, as a revolutionary biomedical tool, has shown immense potential in haematological research in recent years. By using three‐dimensional (3D) cell culture systems constructed from pluripotent stem cells (PSCs) or adult stem cells (ASCs), organoids can highly mimic the characteristics of in vivo organs, thereby offering significant potential for investigating human organ development, disease processes and treatment strategies. This review introduces the development of organoids and focuses on their progress in haematological research, including haematopoietic‐related organoids, immune‐related organoids and organoids used for studying blood system diseases. It discusses the prospects, challenges and future outlook of organoids in the field of haematology. This review aims to provide the latest advancements and future directions of organoid technology in haematological research, offering references and insights into further exploration in this field.

A 3D Cell-Culture System That Uses Nano-Fibrillated Bacterial Cellulose to Prepare a Spherical Formulation of Culture Cells

A 3-dimensional (3D) cell culture is now being actively pursued to accomplish the in vivo-like cellular morphology and biological functions in cell culture. We recently obtained nano-fibrillated bacterial cellulose (NFBC). In this study, we developed a novel NFBC-based 3D cell-culture system, the OnGel method, and the Suspension method. HepG2 human liver cancer cells were cultured via these methods and formed spherical formulations in the optimized condition, 1.0% (w/v) of NFBC in the OnGel method, and 0.06-0.10% (w/v) of NFBC in the Suspension method. Non-cancerous cells such as human-induced pluripotent stem (iPS) cells and human mesenchymal stem cells (MSCs) also formed spherical formulations. It is noteworthy that both the size and cell viability of spheroids prepared via these methods were comparable to those cultured using commercially available 3D cell-culture systems. Both OnGel and Suspension methods are less complicated than the existing 3D cell-culture systems, which is an invaluable advantage for the preparation of cancer spheroids. The NFBC-based 3D cell-culture systems introduced here show great promise as a tool to prepare cultures for cell-derived spheroids for the progress of both in vitro and in vivo studies of the biological functioning of cells.

WT161, a selective HDAC6 inhibitor, decreases growth, enhances chemosensitivity, promotes apoptosis, and suppresses motility of melanoma cells.

Histone deacetylase 6 (HDAC6) plays a critical role in tumorigenesis and tumor progression, contributing to proliferation, chemoresistance, and cell motility by regulating microtubule architecture. Despite its upregulation in melanoma tissues and cell lines, the specific biological roles of HDAC6 in melanoma are not well understood. This study aims to explore the functional effects and underlying mechanisms of WT161, a selective HDAC6 inhibitor, in melanoma cell lines. Cell proliferation was assessed using both 2D and 3D cell culture systems, including MTT assays, spheroid growth analyses, and colony formation assays. The interaction between WT161 and the chemotherapeutic agents temozolomide (TMZ) or dacarbazine (DTIC) was evaluated using the Chou-Talalay method. Apoptotic cell death was analyzed through flow cytometry, while migration, adhesion, and invasion assays were conducted to evaluate the motility capacities of melanoma cells. Western blot assays quantified α-tubulin acetylation (Lys40), PARP cleavage, and protein levels of β-catenin and E-cadherin. WT161 significantly reduced cell growth in both 2D and 3D cultures, decreased clonogenic capacity, and showed synergistic interactions with TMZ and DTIC. The inhibitor also induced apoptotic cell death and enhanced TMZ-induced apoptosis. Additionally, WT161 reduced cell migration and invasion while increasing cell adhesion. These effects were linked to changes in β-catenin and E-cadherin levels, depending on the specific cell type evaluated. Our study underscores the pivotal role of HDAC6 in melanoma progression, establishing it as a promising therapeutic target. We provide the first comprehensive evidence of WT161's anti-melanoma effects, setting the stage for further research into HDAC6 inhibitors as a potential strategy for melanoma treatment.

A review of 3D bioprinting for organoids

Abstract Current two-dimensional (2D) cell models for effective drug screening suffer from significant limitations imposed by the lack of realism in the physiological environment. Three-dimensional (3D) organoids models hold immense potential in mimicking the key functions of human organs by overcoming the limitations of traditional 2D cell models. However, current techniques for preparation of 3D organoids models had limitations in reproducibility, scalability, and the ability to closely replicate the complex microenvironment found in vivo. Additionally, traditional 3D cell culture systems often involve lengthy and labor-intensive processes that hinder high-throughput applications necessary for a large-scale drug screening. Advancements in 3D bioprinting technologies offer promising solutions to these challenges by enabling precise spatial control over cell placement and material composition, thereby facilitating the creation of more physiologically relevant organoids than current techniques. This review provides a comprehensive summary of recent advances in 3D bioprinting technologies for creating organoids models, which begins with an introduction to different types of 3D bioprinting techniques (especially focus on volumetric bioprinting (VBP) technique), followed by an overview of bioinks utilized for organoids bioprinting. Moreover, we also introduce the applications of 3D bioprinting organoids in disease models, drug efficiency evaluation and regenerative medicine. Finally, the challenges and possible strategies for the development and clinical translation of 3D bioprinting organoids are concluded.

PD-L1 and IFN-γ modulate Non-Small Cell Lung Cancer (NSCLC) cell plasticity associated to immune checkpoint inhibitor (ICI)-mediated hyperprogressive disease (HPD)

BackgroundNon-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death worldwide. Although immune checkpoint inhibitors (ICIs) have shown remarkable clinical efficacy, they can also induce a paradoxical cancer acceleration, known as hyperprogressive disease (HPD), whose causative mechanisms are still unclear.MethodsThis study investigated the mechanisms of ICI resistance in an HPD-NSCLC model. Two primary cell cultures were established from samples of a NSCLC patient, before ICI initiation (“baseline”, NSCLC-B) and during HPD (“hyperprogression”, NSCLC-H). The cell lines were phenotypically and molecularly characterized through immunofluorescence, Western Blotting and RNA-Seq analysis. To assess cell plasticity and aggressiveness, cellular growth patterns were evaluated both in vitro and in vivo through 2D and 3D cell growth assays and patient-derived xenografts establishment. In vitro investigations, including the evaluation of cell sensitivity to interferon-gamma (IFN-γ) and cell response to PD-L1 modulation, were conducted to explore the influence of these factors on cell plasticity regulation.ResultsNSCLC-H exhibited increased expression of specific CD44 isoforms and a more aggressive phenotype, including organoid formation ability, compared to NSCLC-B. Plastic changes in NSCLC-H were well described by a deep transcriptome shift, that also affected IFN-γ–related genes, including PD-L1. IFN-γ–mediated cell growth inhibition was compromised in both 2D-cultured NSCLC-B and NSCLC-H cells. Further, the cytokine induced a partial activation of both type I and type II IFN-pathway mediators, together with a striking increase in NSCLC-B growth in 3D cell culture systems. Finally, low IFN-γ doses and PD-L1 modulation both promoted plastic changes in NSCLC-B, increasing CD44 expression and its ability to produce spheres.ConclusionsOur findings identified plasticity as a relevant hallmark of ICI-mediated HPD by demonstrating that ICIs can modulate the IFN-γ and PD-L1 pathways, driving tumor cell plasticity and fueling HPD development.

An agarose fluidic chip for high-throughput in toto organoid imaging.

Modern cell and developmental biology increasingly relies on 3D cell culture systems such as organoids. However, routine interrogation with microscopy is often hindered by tedious, non-standardized sample mounting, limiting throughput. To address these bottlenecks, we have developed a pipeline for imaging intact organoids in flow, utilizing a transparent agarose fluidic chip that enables efficient and consistent recordings with theoretically unlimited throughput. The chip, cast from a custom-designed 3D-printed mold, is coupled to a mechanically controlled syringe pump for fast and precise sample positioning. We benchmarked this setup on a commercial digitally scanned light sheet microscope with cleared glioblastoma spheroids. Spheroids of varying sizes were positioned in the field of view with micrometer-level stability, achieving a throughput of 40 one-minute recordings per hour. We further showed that sample positioning could be automated through online feedback microscopy. The optical quality of the agarose chip outperformed FEP tubing, glass channels and PDMS casts for the clearing agents used, as demonstrated by image contrast profiles of spheroids stained with a fluorescent nuclear counterstain and further emphasized by the resolution of fine microglial ramifications within cerebral organoids. The retention of image quality throughout 500 μm-sized spheroids enabled comprehensive spatial mapping of live and dead cells based on their nuclear morphology. Finally, imaging a batch of LMNA knockout vs. wildtype astrocytoma spheroids revealed significant differences in their DNA damage response, underscoring the system's sensitivity and throughput. Overall, the fluidic chip design provides a cost-effective, accessible, and efficient solution for high-throughput organoid imaging.

Construction of 3D tumor in vitro models with an immune microenvironment exhibiting similar tumor properties and biomimetic physiological functionality.

Tumors pose a serious threat to people's lives and health, and the complex tumor microenvironment is the biggest obstacle to their treatment. In contrast to the basic protein matrices typically employed in 2D or 3D cell culture systems, decellularized extracellular matrix (dECM) can create complex microenvironments. In this study, a combination of physicochemical methods was established to obtain liver decellularized extracellular matrix scaffolds (dLECMs) to provide mechanical support and cell adhesion sites. By co-culturing tumor cells, tumor-associated stromal cells and immune cells, an in vitro 3D tumor model with a biomimetic immune microenvironment was constructed. By utilizing microenvironment data obtained from human liver tumor tissues and refining the double seeding modeling process, 3D in vitro liver tumor-like tissues with a tumor immune microenvironment (TIME) were obtained and designated as reconstructed human liver cancer (RHLC). These tissues demonstrated similar tumor characteristics and exhibited satisfactory physiological functionality. The results of metabolic characterisation and mouse tumorigenicity testing verified that the constructed RHLC significantly increased in vitro drug resistance while also closely mimicking in vivo tissue metabolism. This opens up new possibilities for creating effective in vitro models for screening chemotherapy drugs.

Exosomal miR-6126 as a novel therapeutic target for overcoming resistance of anti-cancer effect in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) stands as the sixth most prevalent cancer globally, presenting a substantial health challenge, particularly due to late-stage diagnoses that limit treatment effectiveness. Sorafenib, a multi-kinase inhibitor, is the primary chemotherapeutic agent for advanced HCC, but it only extends survival by 2–3 months. However, drug resistance remains a major clinical challenge, necessitating the exploration of new molecular mechanisms, including the role of microRNAs (miRNAs) in sorafenib resistance. In this study, we aimed to identify miRNAs within exosomes derived from sorafenib-resistant HCC cells to elucidate the molecular mechanisms underlying resistance.MethodsSorafenib-resistant cells were generated by culturing the human HCC cell line Huh7 in a medium containing 20 µM sorafenib for six months. Exosomes were isolated from the conditioned medium 24 h before cell harvest using exosome-depleted serum medium. miRNA sequencing and western blotting were used to analyze the expression profiles of exosomal miRNAs and proteins, respectively. pH measurement was performed to assess pH changes in response to sorafenib treatment and miRNA modulation.ResultsA total of 180 exosomal miRNAs were found to be dysregulated between sorafenib-treated control Huh7 (Huh7S) and sorafenib-resistant Huh7 (Huh7RS) cells, as well as between untreated control Huh7 and Huh7RS cells. Among these, miR-6126 was significantly downregulated in Huh7RS cells compared to Huh7S cells. Functional studies using 2-dimensional (D) and 3D cell culture systems revealed that miR-6126 overexpression reduced sorafenib resistance in Huh7RS cells, while its inhibition increased resistance in Huh7 cells. miR-6126 downregulated key proteins involved in cancer stem cell maintenance, such as CD44 and HK2. Furthermore, the pH level was elevated in cells overexpressing miR-6126 following sorafenib treatment, whereas inhibiting miR-6126 resulted in a lower pH.ConclusionsExosomal miR-6126 plays a pivotal role in sorafenib resistance and tumorigenesis, highlighting its potential as a novel therapeutic target for overcoming drug resistance in HCC.

Tumor-Infiltration Mimicking Model of Contaminated Ovarian Tissue as an Innovative Platform for Advanced Cancer Research.

The development of advanced preclinical models is crucial for the evaluation and validation of novel therapeutic strategies in oncology. Three-dimensional (3D) microtumor models, which incorporate both cancer and stromal cells within biomimetic hydrogels, have emerged as powerful tools that more accurately replicate the complex tumor microenvironment compared to traditional two-dimensional (2D) cell culture systems. In this context, our study aims to develop 3D microtumor models by integrating cancer and stromal cells within an extracellular-matrix-mimetic hydrogel, as a physiologically accurate microtumor model that can serve as an innovative platform for advanced cancer research and drug screening. Microtumors composed of varying ratios of leukemia cells (HL-60) to healthy ovarian stromal cells (SCs) (1:1, 1:10, 1:100, or 1:1000) were encapsulated in PEGylated fibrin hydrogel and cultured for 5days. The proliferation and dynamics of cancerous and healthy cell populations were evaluated using CD43/Ki67 immunofluorescence double staining. Our findings indicate that tumor development and malignancy progression can be influenced by adjusting cell culture ratios and incubation time. Notably, the HL-60:SCs ratio of 1:100 closely replicated leukemia cell invasion in ovarian tissue, demonstrating detectable malignancy on the third and fifth days without significant changes in total cell density dynamics. This 3D leukemia microtumor model offers superior physiological relevance compared to traditional 2D in vitro assays and shows promising potential for applications in cellular analysis and drug screening.

Engineering Granular Hydrogels without Interparticle Cross-Linking to Support Multicellular Organization.

Advancing three-dimensional (3D) tissue constructs is central to creating in vitro models and engineered tissues that recapitulate biology. Materials that are permissive to cellular behaviors, including proliferation, morphogenesis of multicellular structures, and motility, will support the emergence of tissue structures. Granular hydrogels in which there is no interparticle cross-linking exhibit dynamic properties that may be permissive to such cellular behaviors. However, designing granular hydrogels that lack interparticle cross-linking but support cellular self-organization remains underexplored relative to granular systems stabilized by interparticle cross-linking. In this study, we developed a polyethylene glycol-based granular hydrogel system, with average particle diameters under 40 μm. This granular hydrogel exhibited bulk stress-relaxing behaviors and compatibility with custom microdevices to sustain cell cultures without degradation. The system was studied in conjunction with cocultures of endothelial cells and fibroblasts, known for their spontaneous network formation. Cross-linking, porosity, and cell-adhesive ligands (such as RGD) were manipulated to control system properties. Toward supporting cellular activity, increased porosity was found to enhance the formation of cellular networks, whereas RGD reduced network formation in the system studied. This research highlights the potential of un-cross-linked granular systems to support morphogenetic processes, like vasculogenesis and tissue maturation, offering insights into material design for 3D cell culture systems.

3D-environment and muscle contraction regulate the heterogeneity of myonuclei

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation.

Dynamically cultured, differentiated bovine adipose-derived stem cell spheroids as building blocks for biofabricating cultured fat

Cultured or cultivated meat, animal muscle, and fat tissue grown in vitro, could transform the global meat market, reducing animal suffering while using fewer resources than traditional meat production and no antimicrobials at all. To ensure the appeal of cultured meat to future customers, cultured fat is essential for achieving desired mouthfeel, taste, and texture, especially in beef. In this work we show the establishment of primary bovine adipose-derived stem cell spheroids in static and dynamic suspension culture. Spheroids are successfully differentiated using a single-step protocol. Differentiated spheroids from dynamic cultures maintain stability and viability during 3D bioprinting in edible gellan gum. Also, the fatty acid composition of differentiated spheroids is significantly different from control spheroids. The cells are cultured antibiotic-free to minimize the use of harmful substances. This work presents a stable and bioprintable building block for cultured fat with a high cell density in a 3D dynamic cell culture system.

3D cell culture models in research: applications to lung cancer pharmacology.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, necessitating innovative research methodologies to improve treatment outcomes and develop novel strategies. The advent of three-dimensional (3D) cell cultures has marked a significant advancement in lung cancer research, offering a more physiologically relevant model compared to traditional two-dimensional (2D) cultures. This review elucidates the various types of 3D cell culture models currently used in lung cancer pharmacology, including spheroids, organoids and engineered tissue models, having pivotal roles in enhancing our understanding of lung cancer biology, facilitating drug development, and advancing precision medicine. 3D cell culture systems mimic the complex spatial architecture and microenvironment of lung tumours, providing critical insights into the cellular and molecular mechanisms of tumour progression, metastasis and drug responses. Spheroids, derived from commercialized cell lines, effectively model the tumour microenvironment (TME), including the formation of hypoxic and nutrient gradients, crucial for evaluating the penetration and efficacy of anti-cancer therapeutics. Organoids and tumouroids, derived from primary tissues, recapitulate the heterogeneity of lung cancers and are instrumental in personalized medicine approaches, supporting the simulation of in vivo pharmacological responses in a patient-specific context. Moreover, these models have been co-cultured with various cell types and biomimicry extracellular matrix (ECM) components to further recapitulate the heterotypic cell-cell and cell-ECM interactions present within the lung TME. 3D cultures have been significantly contributing to the identification of novel therapeutic targets and the understanding of resistance mechanisms against conventional therapies. Therefore, this review summarizes the latest findings in drug research involving lung cancer 3D models, together with the common laboratory-based assays used to study drug effects. Additionally, the integration of 3D cell cultures into lung cancer drug development workflows and precision medicine is discussed. This integration is pivotal in accelerating the translation of laboratory findings into clinical applications, thereby advancing the landscape of lung cancer treatment. By closely mirroring human lung tumours, these models not only enhance our understanding of the disease but also pave the way for the development of more effective and personalized therapeutic strategies.

Abstract B093: The evaluation of the gene expression profile of pancreatic ductal adenocarcinomas (PDACs) in 3D cell culture vs in vivo and development of new 3D PDACs' models for an evaluation of anti-cancer drug testing

Abstract In the ever-evolving landscape of biomedical research, 3D Cell Culture (3DCC) systems have emerged as pivotal tools for mimicking in vivo environments and fostering more physiologically relevant cellular responses. Despite promises, 3DCC based drug discovery efforts didn’t fulfil the hopes. The classic way 3DCC is done, always starts with 2D cell culture (2DCC) followed by brief 3 day 3DCC and finally drug testing. Following those observations we decided to mimic much more than just brief 3DCC state but enhance the culture by constant 3DCC to 3DCC passages. We characterized the gene expression profiles and proteomic characteristics of pancreatic tumors concluding that prolonged 3DCC is the closest to an in vivo model. The protracted cultivation periods mimic the intricacies of the in vivo environment, enabling a more accurate representation of the molecular dynamics within pancreatic tumors. In our study, we identified a panel of genes characterized by similar expression levels between prolonged 3D cell culture and in vivo conditions, while displaying significant divergence between 2D cultures and short-term 3D cultures when compared to in vivo. These genes play a pivotal role in the progression and treatment of cancer, underscoring the potential of prolonged 3DCC of LifeGel® significance in understanding tumor biology and devising therapeutic strategies. Furthermore, we also investigated the implications of prolonged 3D in vitro cultures on evaluating anti-cancer drug activities. The findings highlight the significance of 3DCC in enhancing our understanding of pancreatic tumor biology and in refining drug screening methodologies for more effective therapeutic interventions. By providing a biomimetic microenvironment, our hydrogels facilitate the formation of complex cellular structures, enabling more accurate representation of tissue architecture and cellular interactions. We delivered LifeGel® platform which presents a myriad of opportunities for researchers seeking to enhance the fidelity and translatability of their in vitro studies into pre-clinial models. Citation Format: Marcin Krzykawski, David Earnshaw, Krzysztof Kus, Artur Pirog, Sachin Kote, Mark Treherne, Justyna Kocik-Krol. The evaluation of the gene expression profile of pancreatic ductal adenocarcinomas (PDACs) in 3D cell culture vs in vivo and development of new 3D PDACs' models for an evaluation of anti-cancer drug testing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B093.

Three-dimensional Culture of Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

Background: The mammary gland is a crucial organ in dairy animals, responsible for milk production, regulated by intricate hormonal and cellular interactions. Traditional two-dimensional (2D) cell culture systems used in mammary gland research fail to replicate the complex in vivo environment, limiting the understanding of mammary gland biology. The study explores the advantages of three-dimensional (3D) culture systems for Buffalo Mammary Epithelial Cells (BuMECs), aiming to enhance morphological and functional differentiation compared to 2D cultures. Methods: BuMECs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% FBS and specific growth factors. For 2D culture, BuMECs were grown on a plastic substratum. For 3D culture, cells were cultured on top of and embedded in growth factor-reduced (GFR) Matrigel. Morphological differentiation was observed under phase-contrast microscopy. Total RNA was extracted and cDNA was synthesized to measure β-casein gene expression using quantitative real-time PCR (qPCR). Protein expression was analyzed by Western blotting, targeting total casein. Result: BuMECs grown in 3D cultures exhibited enhanced morphological differentiation, forming alveolar and ductal structures, unlike the monolayer formation observed in 2D cultures. Rapid formation of duct-like structures was noted within 24 hours in 3D cultures. Functional differentiation was significantly improved, evidenced by a five-fold increase in β-casein mRNA expression and higher protein levels of α, β and κ-casein in 3D cultured cells compared to 2D cultures. Western blot analysis confirmed the elevated casein protein expression in 3D cultures.

Deciphering the physiopathology of neurodevelopmental disorders using brain organoids.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSCs) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives an unprecedented opportunity to explore human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e. Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy syndrome, tuberous sclerosis syndrome) and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems in the realm of personalized medicine.

Protein Immobilization on Bacterial Cellulose for Biomedical Application.

New carriers for protein immobilization are objects of interest in various fields of biomedicine. Immobilization is a technique used to stabilize and provide physical support for biological micro- and macromolecules and whole cells. Special efforts have been made to develop new materials for protein immobilization that are non-toxic to both the body and the environment, inexpensive, readily available, and easy to modify. Currently, biodegradable and non-toxic polymers, including cellulose, are widely used for protein immobilization. Bacterial cellulose (BC) is a natural polymer with excellent biocompatibility, purity, high porosity, high water uptake capacity, non-immunogenicity, and ease of production and modification. BC is composed of glucose units and does not contain lignin or hemicellulose, which is an advantage allowing the avoidance of the chemical purification step before use. Recently, BC-protein composites have been developed as wound dressings, tissue engineering scaffolds, three-dimensional (3D) cell culture systems, drug delivery systems, and enzyme immobilization matrices. Proteins or peptides are often added to polymeric scaffolds to improve their biocompatibility and biological, physical-chemical, and mechanical properties. To broaden BC applications, various ex situ and in situ modifications of native BC are used to improve its properties for a specific application. In vivo studies showed that several BC-protein composites exhibited excellent biocompatibility, demonstrated prolonged treatment time, and increased the survival of animals. Today, there are several patents and commercial BC-based composites for wounds and vascular grafts. Therefore, further research on BC-protein composites has great prospects. This review focuses on the major advances in protein immobilization on BC for biomedical applications.

Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system

The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.

Establishment of 3D cell culture systems with decellularized lung-derived extracellular matrix hydrogel scaffold

Decellularized tissue hydrogels, especially that mimic the native tissue, have a high potential for tissue engineering, three-dimensional (3D) cell culture, bioprinting, and therapeutic agent encapsulation due to their excellent biocompatibility and ability to facilitate the growth of cells. It is important to note that the decellularization process significantly affects the structural integrity and properties of the extracellular matrix, which in turn shapes the characteristics of the resulting hydrogels at the macromolecular level. Therefore, our study aims to identify an effective chemical decellularization method for sheep lung tissue, using a mixing/agitation technique with a range of detergents, including commonly [Sodium dodecyl sulfate (SDS), Triton X-100, and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate] (CHAPS), and rarely used (sodium cholate hydrate, NP-40, and 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate) (ASB-14). After the effectiveness of the used detergents on decellularization was determined by histological and biochemical methods, lung derived decellularized extracellular matrix was converted into hydrogel. We investigated the interactions between lung cells and decellularized extracellular matrix using proliferation assay, scanning electron microscopy, and immunofluorescence microscopy methods on BEAS-2B cells in air-liquid interface. Notably, this study emphasizes the effectiveness of ASB-14 in the decellularization process, showcasing its crucial role in removing cellular components while preserving vital extracellular matrix biological macromolecules, including glycosaminoglycans, collagen, and elastin. The resulting hydrogels demonstrated favorable mechanical properties and are compatible with both cell-cell and cell-extracellular matrix interactions.

IPSC-derived lung and lung cancer organoid model to evaluate cisplatin encapsulated autologous iPSC-derived mesenchymal stromal cell-isolated extracellular vesicles

BackgroundLung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (iPSC)-MSC in personalized medicine approaches.MethodsFirst, we reprogrammed healthy fibroblasts into iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from iPSC and EV isolation from iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests.ResultsFibroblast-derived iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models.ConclusionWe report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.