Isolation and Identification of Cancer Stem Cells from Human Osteosarcom by Serum-free Three-dimensional Culture Combined with Anticancer Drugs

The cancer stem cells(CSCs)from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified.The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension,and mixed homogeneously into 1.2% alginate gel.Single-cell alginate gel was cultured with serum-free DMEM/F12 medium.Epirubicin(0.8μg/mL)was added to the medium to enrich CSCs.After cultured conventionally for 7 to 10 days,most of cells suspended in ...

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3D)culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures.In cancer and stem cell research,the natural cell characteristics and architectures are closely mimicked by the 3D cell models.Thus,the 3D cell cultures are promising and suitable systems for various proposes,ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives.This review provides a comprehensive compendium of recent advancements in culturing cells,in particular cancer and stem cells,using 3D culture techniques.The major approaches highlighted here include cell spheroids,hydrogel embedding,bioreactors,scaffolds,and bioprinting.In addition,the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed,and the prominent studies of 3D cell culture systems were discussed.

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research,the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems.Recently,researchers have been actively developing and evaluating three-dimensional(3D)cell culture-based platforms using microfluidic technologies,such as organ-on-a-chip and organoid-on-a-chip platforms,and they have achieved promising breakthroughs in stem cell engineering.In this review,we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery.In a subsequent section,we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research.In addition,some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition

Objective Adipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition. Methods ASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture. Results The 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs’ potential of cellular transformation was lower after irradiation by soft agar assay. Conclusion These findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.

3D Collagen Gels:A Promising Platform for Dendritic Cell Culture in Biomaterials Research

The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.

Three-dimensional models:from cell culture to Patient-Derived Organoid and its application to future liposarcoma research

Liposarcoma is one of the most common soft tissue sarcomas,however,its occurrence rate is still rare compared to other cancers.Due to its rarity,in vitro experiments are an essential approach to elucidate liposarcoma pathobiology.Conventional cell culture-based research(2D cell culture)is still playing a pivotal role,while several shortcomings have been recently under discussion.In vivo,mouse models are usually adopted for pre-clinical analyses with expectations to overcome the issues of 2D cell culture.However,they do not fully recapitulate human dedifferentiated liposarcoma(DDLPS)characteristics.Therefore,three-dimensional(3D)culture systems have been the recent research focus in the cell biology field with the expectation to overcome at the same time the disadvantages of 2D cell culture and in vivo animal models and fill in the gap between them.Given the liposarcoma rarity,we believe that 3D cell culture techniques,including 3D cell cultures/co-cultures,and Patient-Derived tumor Organoids(PDOs),represent a promising approach to facilitate liposarcoma investigation and elucidate its molecular mechanisms and effective therapy development.In this review,we first provide a general overview of 3D cell cultures compared to 2D cell cultures.We then focus on one of the recent 3D cell culture applications,Patient-Derived Organoids(PDOs),summarizing and discussing several PDO methodologies.Finally,we discuss the current and future applications of PDOs to sarcoma,particularly in the field of liposarcoma.

Morphological properties and proliferation analysis of olfactory ensheathing cells seeded onto three-dimensional collagen-heparan sulfate biological scaffolds

This study aimed to examine the differences in the morphological properties and proliferation of olfactory ensheathing cells in three-dimensional culture on collagen-heparan sulfate biological scaffolds and in two-dimensional culture on common flat culture plates. The proliferation rate of olfactory ensheathing cells in three-dimensional culture was higher than that in two-dimensional culture, as detected by an M-I-r assay. In addition, more than half of the olfactory ensheathing cells subcultured using the trypsinization method in three-dimensional culture displayed a spindly Schwann cell-like morphology with extremely long processes, while they showed a flat astrocyte-like morphology in two-dimensional culture. Moreover, spindle-shaped olfactory ensheathing cells tended to adopt an elongated bipolar morphology under both culture conditions. Experimental findings indicate that the morphological properties and proliferation of olfactory ensheathing cells in three-dimensional culture on collagen-heparan sulfate biological scaffolds are better than those in two-dimensional culture.

Syngeneic implantation of mouse hepatic progenitor cell-derived three-dimensional liver tissue with dense collagen fibrils

BACKGROUND Liver transplantation is a therapy for irreversible liver failure;however,at present,donor organs are in short supply.Cell transplantation therapy for liver failure is still at the developmental stage and is critically limited by a shortage of human primary hepatocytes.AIM To investigate the possibility that hepatic progenitor cells(HPCs)prepared from the portal branch-ligated hepatic lobe may be used in regenerative medicine,we attempted to enable the implantation of extracellular matrices containing organoids consisting of HPC-derived hepatocytes and non-parenchymal cells.METHODS In vitro liver organoid tissue has been generated by accumulating collagen fibrils,fibroblasts,and HPCs on a mesh of polylactic acid fabric using a bioreactor;this was subsequently implanted into syngeneic wild-type mice.RESULTS The in vitro liver organoid tissues generated transplantable tissues in the condensed collagen fibril matrix and were obtained from the mouse through partial hepatectomy.CONCLUSION Liver organoid tissue was produced from expanded HPCs using an originally designed bioreactor system.This tissue was comparable to liver lobules,and with fibroblasts embedded in the network collagen fibrils of this artificial tissue,it is useful for reconstructing the hepatic interstitial structure.

Modulation of the Nogo signaling pathway to overcome amyloid-β-mediated neurite inhibition in human pluripotent stem cell-derived neurites

Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.

Hydrogel-supported poly(L-lactic acid) and polystyrene microsphere-based three-dimensional culture systems for in vitro cell expansion

The in vitro expansion of stem cells is important for their application in different life science fields such as cellular tissue and organ repair.An objective of this paper was to achieve static cell culture in vitro through peptide hydrogel-supported microspheres(MSs).The peptides,with their gel-forming properties,microstructures,and mechanical strengths characterized,were found to have good support for the MSs and to be injectable.The internal structures of poly(L-lactic acid)microspheres(PLLA-MSs)and polystyrene microspheres(PS-MSs)made in thelaboratory were observed and statistically analyzed in terms of particle size and pore size,following which the co-cultured MSs with cells were found to have good cell adhesion.In addition,three-dimensional(3D)culturing of cells was performed on the peptide and microcarrier composite scaffolds to measure cell viability and cell proliferation.The results showed that the peptides could be stimulated by the culture medium to self-assembly form a 3D fiber network structure.Under the peptide-Ms composite scaffold-based cell culture system,further enhancement of the cell culture effect was measured.The peptide-Ms composite scaffolds have great potential for the application in 3D cell culture and in vitro cellexpansion.

Three-dimensional perfused human in vitro model of nonalcoholic fatty liver disease

AIM To develop a human in vitro model of non-alcoholic fatty liver disease(NAFLD), utilising primary hepatocytes cultured in a three-dimensional(3D) perfused platform. METHODS Fat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids(FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds. RESULTS Hepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factorbinding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantlyreduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known antisteatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium.CONCLUSION The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro

To synthesize KLD-12 peptide with sequence of AcN-KLDLKLDLKLDL-CNH2 and trigger its self-assembly in vitro, to encapsulate rabbit MSCs within peptide hydrogel for 3-D culture and to evaluate the feasibility of using it as injectable scaffold for tissue engineering of IVD. KLD-12 peptide was purified and tested with high performance liquid chromatography （HPLC） and mass spectroscopy （MS）. KLD-12 peptide solutions with concentrations of 5 g/L, 2.5 g/L and 1 g/L were triggered to self-assembly with 1 xPBS in vitro, and the self-assembled peptide hydrogel was morphologically observed. Atomic force microscope （AFM） was employed to examine the inner structure of self-assembled peptide hydrogel. Mesenchymal stem cells （MSCs） were encapsulated within peptide hydrogel for 3-D culture for 2 weeks. Calcein-AM/PI fluorescence staining was used to detect living and dead cells. Cell viability was observed to evaluate the bioactivity of MSCs in KLD-12 peptide hydrogel. The results of HPLC and MS showed that the relative molecular mass of KLD-12 peptide was 1467.83, with a purity quotient of 95.36%. KLD-12 peptide at 5 g/L could self-assemble to produce a hydrogel, which was structurally integral and homogeneous and was able to provide sufficient cohesion to retain the shape of hydrogel. AFM demonstrated that the self-assembly of KLD-12 peptide hydrogel was successful and the assembled material was composed of a kind of nano-fiber with a diameter of 3040 nm and a length of hundreds of nm. Calcein-AM/PI fluorescence staining revealed that MSCs in KLD-12 peptide hydrogel grew well. Cell activity detection exhibited that the A value increased over the culture time. It is concluded that KLD-12 peptide was synthesized successfully and was able to self-assemble to produce nano-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.

The famous versus the inconvenient-or the dawn and the rise of 3D-culture systems

One of the greatest impacts on in vitro cell biology was the introduction of three-dimensional(3D)culture systems more than six decades ago and this era may be called the dawn of 3D-tissue culture.Although the advantages were obvious,this field of research was a "sleeping beauty"until the 1970s when multicellular spheroids were discovered as ideal tumor models.With this rebirth,organotypical culture systems became valu-able tools and this trend continues to increase.While in the beginning,simple approaches,such as aggregation culture techniques,were favored due to their simplicity and convenience,now more sophisticated systems are used and are still being developed.One of the boosts in the development of new culture techniques arises from elaborate manufacturing and surface modification tech-niques,especially micro and nano system technologies that have either improved dramatically or have evolved very recently.With the help of these tools,it will soon be possible to generate even more sophisticated and more organotypic-like culture systems.Since 3D per-fused or superfused systems are much more complex to set up and maintain compared to use of petri dishes and culture flasks,the added value of 3D approaches still needs to be demonstrated.

Engineered three-dimensional rabbit oral epithelial–mesenchymal–muscular hybrid sheets

Regenerative muscles are required for swallowing and mastication, and are important for functional recovery from diseases involving oral muscular defects. Therefore, we generated three-layer hybrid sheets, similar to oral mucosal structures containing submucosal muscles, using rabbit oral mucosa epithelial, mesenchymal, and myoblastic progenitor cells, and examined the structural proteins. Each cell type was obtained from rabbit oral mucosa using enzymatic digestion. Isolated mesenchymal and myoblastic cells were multi-differentiated into osteoblasts, adipocytes, and chondrocytes or myotubes. Isolated epithelial cells were cultured on collagen gels containing isolated mesenchymal cells for 2 weeks, and these epithelial-mesenchymal cell sheets were laminated onto myoblastic cell sheets. The engineered hybrid sheets were multi-stratified in the epithelial and myoblastic layers in a time-dependent manner, expressing intermediate cytoskeletal filament proteins of epithelium and muscle. Hybrid sheets also expressed extracellular matrix basement membrane proteins. Immature cell markers for epithelial and myoblastic cells were observed continuously in hybrid sheet cultures. We established engineered three-dimensional rabbit oral mucosa hybrid sheets containing each immature cell type in vitro.

Bioengineering breakthroughs:The impact of stem cell models on advanced therapy medicinal product development

The burgeoning field of bioengineering has witnessed significant strides due to the advent of stem cell models,particularly in their application in advanced therapy medicinal products(ATMPs).In this review,we examine the multifaceted impact of these developments,emphasizing the potential of stem cell models to enhance the sophistication of ATMPs and to offer alternatives to animal testing.Stem cell-derived tissues are particularly promising because they can reshape the preclinical landscape by providing more physiologically relevant and ethically sound platforms for drug screening and disease modelling.We also discuss the critical challenges of reproducibility and accuracy in measurements to ensure the integrity and utility of stem cell models in research and application.Moreover,this review highlights the imperative of stem cell models to align with regulatory standards,ensuring using stem cells in ATMPs translates into safe and effective clinical therapies.With regulatory approval serving as a gateway to clinical adoption,the collaborative efforts between scientists and regulators are vital for the progression of stem cell applications from bench to bedside.We advocate for a balanced approach that nurtures innovation within the framework of rigorous validation and regulatory compliance,ensuring that stem cell-base solutions are maximized to promote public trust and patient health in ATMPs.

Three-dimensional cell culture models for investigating human viruses

Three-dimensional(3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover,these models bridge the gap between traditional two-dimensional(2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition,3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

Assessment of pancreatic carcinoma cell chemosensitivity using a three-dimensional culture system

Background Monolayer cell culture models are the traditional culture models used for in vitro research of pancreatic carcinoma chemosensitivity. However, these models neglect the interactions between tumor cells and the impact of the tumor microenvironment. Such tumor cell monolayers poorly mimic the solid tumor microenvironment. The present study aimed to investigate the chemosensitivity characteristics of pancreatic cancer cells in a three-dimensional culture system by analyzing the differences in drug sensitivity between a scattered cell culture model and a multicellular spheroid culture model. Methods Three pancreatic cancer cell lines （SW1990, ASPC-1 and PCT-3） were cultured in three-dimensional collagen gels as well as in traditional two-dimensional monolayers. The chemosensitivities of the pancreatic carcinoma cells to 5-fluorouracil （5-FU）, gemcitabine, and oxaliplatin in vitro were detected by both the Cell Counting Kit-8 test and the collagen gel droplet-embedded culture drug-sensitivity test. Results In the two-dimensional culture model, differences in the chemosensitivities of the cloned pancreatic carcinoma cells and scattered cells existed for some concentrations of 5-FU, gemcitabine and oxaliplatin. In the three-dimensional culture model, there were significant differences in the chemosensitivities of the pancreatic cancer cells between the scattered cells and multicellular spheroids （P 〈0.05）. Conclusion Pancreatic carcinoma cells exhibit multicellular resistance in three-dimensional cultures.

Inducing human induced pluripotent stem cell differentiation through embryoid bodies:A practical and stable approach

Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research.However,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation prediction.Embryoid bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these issues.Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid establishment.EBs can potentially also be used in early prediction of iPSC differentiation capability.To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory experiences.Collectively,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.

Biomaterial-based platforms for cancer stem cell enrichment and study

Cancer stem cells(CSCs)are a relatively rare subpopulation of tumor cell with self-renewal and tumorigenesis capabilities.CSCs are associated with cancer recurrence,progression,and chemoradiotherapy resistance.Establishing a reliable platform for CSC enrichment and study is a prerequisite for understanding the characteristics of CSCs and discovering CSC-related therapeutic strategies.Certain strategies for CSC enrichment have been used in laboratory,particularly fluorescence-activated cell sorting(FACS)and mammosphere culture.However,these methods fail to recapitulate the in vivo chemical and physical conditions in tumors,thus potentially decreasing the malignancy of CSCs in culture and yielding unreliable research results.Accumulating research suggests the promise of a biomaterial-based three-dimensional(3 D)strategy for CSC enrichment and study.This strategy has an advantage over conventional methods in simulating the tumor microenvironment,thus providing a more effective and predictive model for CSC laboratory research.In this review,we first briefly discuss the conventional methods for CSC enrichment and study.We then summarize the latest advances and challenges in biomaterial-based 3 D CSC platforms.Design strategies for materials,morphology,and chemical and physical cues are highlighted to provide direction for the future construction of platforms for CSC enrichment and study.

Efficient expansion of rare human circulating hematopoietic stem/progenitor cells in steady-state blood using a polypeptide-forming 3D culture

Although widely applied in treating hematopoietic malignancies,transplantation of hematopoietic stem/progenitor cells(HSPCs)is impeded by HSPC shortage.Whether circulating HSPCs(cHSPCs)in steady-state blood could be used as an alternative source remains largely elusive.Here we develop a three-dimensional culture system(3DCS)including arginine,glycine,aspartate,and a series of factors.Fourteen-day culture of peripheral blood mononuclear cells(PBMNCs)in 3DCS led to 125-and 70-fold increase of the frequency and number of CD34+cells.Further,3DCS-expanded cHSPCs exhibited the similar reconstitution rate com-pared to CD34+HSPCs in bone marrow.Mechanistically,3DCS fabricated an immunomodulatory niche,secreting cytokines as TNF to support cHSPC survival and proliferation.Finally,3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization.Our 3DCS successfully expands rare cHSPCs,providing an alternative source for the HSPC therapy,particularly for the patients/donors who have failed in HSPC mobilization.