Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and t...Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.展开更多
Liver regeneration and the development of effective therapies for liver failure remain formidable challenges in modern medicine.In recent years,the utilization of 3D cell-based strategies has emerged as a promising ap...Liver regeneration and the development of effective therapies for liver failure remain formidable challenges in modern medicine.In recent years,the utilization of 3D cell-based strategies has emerged as a promising approach for addressing these urgent clinical requirements.This review provides a thorough analysis of the application of 3D cell-based approaches to liver regeneration and their potential impact on patients with end-stage liver failure.Here,we discuss various 3D culture models that incorporate hepatocytes and stem cells to restore liver function and ameliorate the consequences of liver failure.Furthermore,we explored the challenges in transitioning these innovative strategies from preclinical studies to clinical applications.The collective insights presented herein highlight the significance of 3D cell-based strategies as a transformative paradigm for liver regeneration and improved patient care.展开更多
Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible...Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.展开更多
Inhibition of mammalian target of rapamycin (m- TOR) is a potential method for cancer treatment. Effects of rapamycin (RAP) on the reversion of malignant breast epithelial cells were investigated on three-dimensional ...Inhibition of mammalian target of rapamycin (m- TOR) is a potential method for cancer treatment. Effects of rapamycin (RAP) on the reversion of malignant breast epithelial cells were investigated on three-dimensional (3D) basement membrane extract (BME) cultures. Through continuous exposure to 20 nM of RAP, cell colony size was significantly reduced in 3D BME cultures of malignant breast epithelial cells, while normal cell colony size appeared unaffected. In unfixed 3D BME cultures of normal and RAP-treated malignant breast epithelial cells, the presence of luminal cell death was confirmed by ethidium bromide and propidium iodide labeling. Increased structural organization was observed by im- munofluorescence staining of F-actin and β-catenin in RAP-treated malignant breast epithelial cells. In monolayer cultures of normal and malignant breast epithelial cells, continuous exposure to 20 nM of RAP increased caspase 3/7 activity and decreased proliferation. Reverse transcriptase polymerase ch- ain reaction (RT-PCR) array analysis indicated a fold increase in the expression of a number of proteins related to polarity, cell-cell adhesion, and cell-matrix adhesion in the presence of RAP. Our data showed that phenotypic reversion of malignancy can be ach- ieved through RAP exposure on 3D BME cultures. This 3D BME culture system will provide correct microenvironments for observing the effects of other mTOR inhibitors on phenotypic reversion of malignant breast epithelial cells.展开更多
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 syst...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.展开更多
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...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 ...展开更多
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 cel...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.展开更多
Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. ...Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.展开更多
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 discov...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.展开更多
AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow ...AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional(3-D) self-assembling scaffolds and compared with traditional two-dimentional(2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate(PEG-4-Acr) and thiolfunctionalized dextran(Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoB lue(PB) assays. Genetic expression of pluripotency markers(Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D cultureconditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining(Oct4 and Nanog) and western blot analysis(Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH(1:1 v/v) to a final concentration of 5%(w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels(P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury(mesoderm), NCAM(ectoderm), and GATA4(endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.展开更多
In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining techn...In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.展开更多
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 c...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.展开更多
Objective This study optimizes three-dimensional(3D) culture conditions of HepG2 using response surface methodology(RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues.Method HepG...Objective This study optimizes three-dimensional(3D) culture conditions of HepG2 using response surface methodology(RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues.Method HepG2 cell was 3D cultured on the VitroGel system.Cell viability was detected using Cell Counting Kit-8(CCK-8) assay of HepG2 lived cell numbers.The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test.Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit.Independent factor tests were conducted with three key factors:inoculated cell concentration,cultured time,and dilution degree of the hydrogel.The preliminary results of independent factor tests were used to determine the levels of factors for RSM.Result The selected optimal culture conditions are as follows:concentration of inoculated cells was4.44 × 10^(5)/mL,culture time was 4.86 days,and hydrogel dilution degree was 1:2.23.The result shows that under optimal conditions,the predicted optical density(OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%.Conclusion This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro.Additionally,it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.展开更多
The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a thr...The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.展开更多
Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. Methods Primary cultures of rPT cells undergoing exponential grow...Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium (2.5, 5.0 umol/L), in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate (2.5, 5.0 umol/L) induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na+, K+ -ATPase, Ca2+ -ATPase, glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants (non-enzymatic and enzymic) levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.展开更多
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-dim...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.展开更多
Objective Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related va...Objective Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.展开更多
Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positiv...Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.展开更多
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 i...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.展开更多
Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchide...Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nrnoI.L^(-1) testosterone. The effect of 1 μmol.L^(-1) flutamide was also evaluated. Results: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. Conclusion: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.展开更多
基金supported by the National Natural Science Foundation of China,No.82171380(to CD)Jiangsu Students’Platform for Innovation and Entrepreneurship Training Program,No.202110304098Y(to DJ)。
文摘Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.
基金This work was supported by grants fromthe Sichuan Science and Technology Program(2023NSFSC1877).
文摘Liver regeneration and the development of effective therapies for liver failure remain formidable challenges in modern medicine.In recent years,the utilization of 3D cell-based strategies has emerged as a promising approach for addressing these urgent clinical requirements.This review provides a thorough analysis of the application of 3D cell-based approaches to liver regeneration and their potential impact on patients with end-stage liver failure.Here,we discuss various 3D culture models that incorporate hepatocytes and stem cells to restore liver function and ameliorate the consequences of liver failure.Furthermore,we explored the challenges in transitioning these innovative strategies from preclinical studies to clinical applications.The collective insights presented herein highlight the significance of 3D cell-based strategies as a transformative paradigm for liver regeneration and improved patient care.
基金support from the National Key Research and Development Program of China(Grant No.2017YFA0700501),and the Innovation Fund of WNLO.
文摘Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.
文摘Inhibition of mammalian target of rapamycin (m- TOR) is a potential method for cancer treatment. Effects of rapamycin (RAP) on the reversion of malignant breast epithelial cells were investigated on three-dimensional (3D) basement membrane extract (BME) cultures. Through continuous exposure to 20 nM of RAP, cell colony size was significantly reduced in 3D BME cultures of malignant breast epithelial cells, while normal cell colony size appeared unaffected. In unfixed 3D BME cultures of normal and RAP-treated malignant breast epithelial cells, the presence of luminal cell death was confirmed by ethidium bromide and propidium iodide labeling. Increased structural organization was observed by im- munofluorescence staining of F-actin and β-catenin in RAP-treated malignant breast epithelial cells. In monolayer cultures of normal and malignant breast epithelial cells, continuous exposure to 20 nM of RAP increased caspase 3/7 activity and decreased proliferation. Reverse transcriptase polymerase ch- ain reaction (RT-PCR) array analysis indicated a fold increase in the expression of a number of proteins related to polarity, cell-cell adhesion, and cell-matrix adhesion in the presence of RAP. Our data showed that phenotypic reversion of malignancy can be ach- ieved through RAP exposure on 3D BME cultures. This 3D BME culture system will provide correct microenvironments for observing the effects of other mTOR inhibitors on phenotypic reversion of malignant breast epithelial cells.
文摘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.
文摘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 ...
文摘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.
基金supported by grants from National Natural Science Foundation of China(No.81171396)Creative Research Groups of the National Natural Science Foundation of China(No.20921062)+1 种基金National Science and Technology Major Project(No.2012ZX10002012-12)National University Students Innovation Training Project of China(No.111048673)
文摘Summary: This study aimed to establish a new in vitro three-dimensional (3D) cell culture and use quantum dots (QDs) molecular imaging to examine the invasive behaviors of hepatocellular carcinoma (HCC) cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.
基金supported by the National Research Foundation of Korea (NRF) (NRF2017R1C1B2002377, NRF-2016R1A5A1010148, and NRF2019R1A2C1003111)funded by the Ministry of Science and ICT (MSIT)partly supported by the Technology Innovation Program (No.10067787)funded by the Ministry of Trade, Industry & Energy (MOTE, Korea)
文摘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.
基金Oakland University and Oakland University-William Beaumont Institute for Stem Cell and Regenerative Medicine(OU-WB ISCRM)
文摘AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional(3-D) self-assembling scaffolds and compared with traditional two-dimentional(2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate(PEG-4-Acr) and thiolfunctionalized dextran(Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoB lue(PB) assays. Genetic expression of pluripotency markers(Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D cultureconditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining(Oct4 and Nanog) and western blot analysis(Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH(1:1 v/v) to a final concentration of 5%(w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels(P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury(mesoderm), NCAM(ectoderm), and GATA4(endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.
文摘In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.
基金supported by grants from the National Key Scientific Instrument and Equipment Development Project of China[2012YQ03014210]Major Project Specialized for Infectious Diseases of the Chinese Health and Family Planning Commission[2014ZX10004002-004-002]National Natural Science Foundation of China[31170803]to BH
文摘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.
基金funded by Toxicity Evaluation of Key Contaminants in Health Food by Cell-based Test Models and the Mechanism Analysis [2018YFC1602104]
文摘Objective This study optimizes three-dimensional(3D) culture conditions of HepG2 using response surface methodology(RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues.Method HepG2 cell was 3D cultured on the VitroGel system.Cell viability was detected using Cell Counting Kit-8(CCK-8) assay of HepG2 lived cell numbers.The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test.Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit.Independent factor tests were conducted with three key factors:inoculated cell concentration,cultured time,and dilution degree of the hydrogel.The preliminary results of independent factor tests were used to determine the levels of factors for RSM.Result The selected optimal culture conditions are as follows:concentration of inoculated cells was4.44 × 10^(5)/mL,culture time was 4.86 days,and hydrogel dilution degree was 1:2.23.The result shows that under optimal conditions,the predicted optical density(OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%.Conclusion This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro.Additionally,it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.
文摘The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.
基金supported by the National Nature Science Foundation of China (No. 31101870)Shandong Provincial Natural Science Foundation of China (No.ZR2010CQ014)
文摘Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium (2.5, 5.0 umol/L), in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate (2.5, 5.0 umol/L) induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na+, K+ -ATPase, Ca2+ -ATPase, glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants (non-enzymatic and enzymic) levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.
基金sponsored by the National Natural Science Foundation of China,No. 30570628,30770751 and 81171089
文摘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.
基金supported by grants from the Major Project Specialized for Infectious Diseases of the Chinese Health and Family Planning Commission[2014ZX10004002-004-002,2014ZX10004002-004-001]Young Talent Scholar Plan of Higher School in Hebei Province[BJ2017008]
文摘Objective Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.
基金supported by the National Natural Science Foundation of China, No. 30872646 and 30973082
文摘Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.
基金Supported by Grants-in-Aid for Scientific Research(A),No.25242040(to Tagawa YI)Grants-in-Aid for Challenging Exploratory Research,No.20K21520(to Tagawa YI)+3 种基金Grants-in-Aid for Early Career Scientists from the Japan Society for the Promotion of Science(JSPS),No.19K20655(to Tamai M)Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education,Culture,Sports,Science and Technology of Japan(MEXT),No.231190003(to Tagawa YI)Japan Agency for Medical Research and Development(AMED),No.20fk0310102(to Tagawa YI)Building of Consortia for the Development of Human Resources in Science and Technology,Ministry of Education,Culture,Sports,Science and Technology,Japan(to Tamai M)。
文摘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.
文摘Aim: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. Methods: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nrnoI.L^(-1) testosterone. The effect of 1 μmol.L^(-1) flutamide was also evaluated. Results: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. Conclusion: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.