In vitro culture of spermatogonial stem cells （SSCs） has generally been performed using two-dimensional （2D） culture systems; however, such cultures have not led to the development of complete spermatogenesis. It ...In vitro culture of spermatogonial stem cells （SSCs） has generally been performed using two-dimensional （2D） culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules （including those generated by the SSC niche） and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system （SACS） and a 3D methylcellulose culture system （MCS）. It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we （1） provide a brief overview of the differences in spermatogenesis in rodents and primates, （2） summarize data related to attempts to generate sperm in vitro, （3） report for the first time formation of colonies/clusters of cells and differentiation of meiotic （expression of CREM-1） and postmeiotic （expression of acrosin） germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and （4） indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.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
AIM： TO investigate the multicellular resistance of human hepatocellular carcinoma HepG2 cells in three-dimensional culture to delisheng, 5-fluorouracil and adriamycin, and the possible molecular mechanisms of delish...AIM： TO investigate the multicellular resistance of human hepatocellular carcinoma HepG2 cells in three-dimensional culture to delisheng, 5-fluorouracil and adriamycin, and the possible molecular mechanisms of delisheng. METHODS： Human hepatocellular carcinoma HepG2 cells were cultured with a liquid overlay technique. After the formation of multicellular spheroids, morphology was analyzed by phase contrast microscopy, scanning electron microscopy and transmission electron microscopy. Sensitivity of HepG2 cells to delisheng, 5-fluorouracil and adriamycin was investigated by Ml-I- assay in multicelluar spheroids and monolayers. Vascular endothelial growth factor （VEGF） and endostatin expression were analyzed in multicellular spheroids treated with delisheng, 5-fluorouracil, adriamycin and negative control PBS, with immunohistochemical staining. RESULTS： Multicellular spheroids exhibited structural characteristics somewhat different to those in monolayers. The cells in three-dimensional cell culture turned out to be less sensitive to delisheng, 5-fluorouracil and adriamycin than the cells cultured in monolayer. This showed that delisheng had a satisfactory cells inhibition ratio compared to 5-fluorouracil and adriamycin. Immunohistochemical staining showed that VEGF and endostatin expression was positive during growth as multicellular spheroids, and endostatin expression in spheroids with treatment of delisheng was higher than that with 5-fluorouracil, adriamycin and PBS （139.35 ± 7.83, 159.23 ± 10.34, 162.83 ± 3.47 and 148.48 ± 11.06, P 〈 0.05）.CONCLUSION： Chinese medicine compound delisheng has satisfactory anti-tumor activity in HepG2 cells in three-dimensional culture, and the effects are associated with up-regulation of endostatin.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
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
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
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
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
Nerve injury repair has been an intractable issue wor.ldwide due to the non-renew able property of neurons in vivo.Cell therapy adopting me senchymal stem cells(MSC)induced neurons stands for a promising substitute tr...Nerve injury repair has been an intractable issue wor.ldwide due to the non-renew able property of neurons in vivo.Cell therapy adopting me senchymal stem cells(MSC)induced neurons stands for a promising substitute treatment,which is hindered by the low diffe rentiation efficiency of current approaches.To improve the efficiency of MSC differentiation in vitro,we constructed a three-dimensional(3D)culture system by seeding human bone marrow-de rived MSC(hBMSCs)into collagen-nano fiber scaffolds which are a mixed hydrogel of collagen 1 and silk nanofiber solution and testified the diffe rentiation efficiency from MSC into neurons by chemical or cocktail induction methods.Results indicated MSCs cultured in 3D system showed good viability with neuron-like morphology in both groups.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
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 ...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.展开更多
BACKGROUND Fracture is one of the most pervasive injuries in the musculoskeletal system,and there is a complex interaction between macrophages and adipose tissue-derived stem cells(ADSCs)in fracture healing.However,tw...BACKGROUND Fracture is one of the most pervasive injuries in the musculoskeletal system,and there is a complex interaction between macrophages and adipose tissue-derived stem cells(ADSCs)in fracture healing.However,two-dimensional(2D)coculture of macrophages and ADSCs can not accurately mimic the in vivo cell microenvironment.AIM To establish both 2D and 3D osteogenic coculture models to investigate the interaction between macrophages and ADSCs.METHODS After obtaining ADSCs from surgery and inducing differentiation of the THP1 cell line,we established 2D and 3D osteogenic coculture models.To assess the level of osteogenic differentiation,we used alizarin red staining and measured the relative expression levels of osteogenic differentiation markers osteocalcin,Runt-related transcription factor 2,and alkaline phosphatase through polymerase chain reaction.Verification was conducted by analyzing the expression changes of N-cadherin and the activation of the Wnt/β-catenin signaling pathway using western blotting.RESULTS In this study,it was discovered that macrophages in 3D culture inhibited osteogenic differentiation of ADSCs,contrary to the effect in 2D culture.This observation confirmed the significance of intricate intercellular connections in the 3D culture environment.Additionally,the 3D culture group exhibited significantly higher N-cadherin expression and showed reducedβ-catenin and Wnt1 protein levels compared to the 2D culture group.CONCLUSION Macrophages promoted ADSC osteogenic differentiation in 2D culture conditions but inhibited it in 3D culture.The 3D culture environment might inhibit the Wnt/β-catenin signaling pathway by upregulating N-cadherin expression,ultimately hindering the osteogenic differentiation of ADSCs.By investigating the process of osteogenesis in ADSCs,this study provides novel ideas for exploring 3D osteogenesis in ADSCs,fracture repair,and other bone trauma repair.展开更多
Against the backdrop of intensified global cultural collisions and ideological competition,deeply integrating excellent traditional Chinese culture(ETCC)into university ideological and political courses(IPCs)has becom...Against the backdrop of intensified global cultural collisions and ideological competition,deeply integrating excellent traditional Chinese culture(ETCC)into university ideological and political courses(IPCs)has become an imperative of our times.Guided by General Secretary Xi Jinping’s methodology of“Two Integrations,”this paper examines the pathways for this integration from three dimensions:value,theory,and practice.The value dimension emphasizes fostering moral conviction and strengthening the spiritual foundation to meet needs such as safeguarding cultural security,preserving the spiritual lineage,and constructing a spiritual framework.The theoretical dimension reveals the mutually constitutive breakthroughs between Marxism and traditional Chinese dialectical thinking,encompassing methodological complementarity,logical coherence of values,and discursive system innovation.The practical dimension involves constructing a comprehensive educational ecosystem by localizing teaching content,modernizing traditional resources,and fostering inter-platform collaborative education,thereby internalizing the value of traditional culture.These three dimensions synergize and co-constitute each other,collectively providing methodological support and practical paradigms for cultivating cultural confidence among youth and forging a new generation capable of shouldering the mission of national rejuvenation.展开更多
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 t...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.展开更多
Background The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been dela...Background The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional （3-D） growth pattern of endometrial cells using a 3-D in vitro culture system.展开更多
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 ...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.展开更多
Through the introduction of disaster situation of Qiang Culture after Wenchuan Earthquake, the paper emphasized that carriers of Qiang Culture had been seriously damaged, the inheritance of Qiang Culture had been affe...Through the introduction of disaster situation of Qiang Culture after Wenchuan Earthquake, the paper emphasized that carriers of Qiang Culture had been seriously damaged, the inheritance of Qiang Culture had been affected, and the environment for Qiang Culture was difficult to recover. It highlighted that three-dimensional reconstruction of Qiang Culture should stress the core task and timely and effectively rescue endangered cultural heritages of Qiang Nationality from the perspectives of material and spiritual life. It had explained focuses of three-dimensional pattern construction in detail. In terms of spatial reconstruction, it should reconstruct native culture and history while material culture was constructed, and reconstruct Qiang culture highland by depending on aborigines; in terms of cluster reconstruction, it should give support to large tourism enterprises and perfect tourism chain; in terms of ecological reconstruction, it should enhance construction and demonstration of "ecological protection pilot area of Qiang culture"; in terms of development reconstruction, it should realize coordinated unity between protection and development according to classification protection, characteristic protection and key protection, so as to form the virtuous circle of post-disaster recovery protection and sustainable development.展开更多
At dawn in Wufu Village,in Dinghu District of Zhaoqing City,Guangdong Province,the newly restored Chaoyangli Cultural Retreat is already welcoming its first visitors.A visitor surnamed Chen,who has travelled from Guan...At dawn in Wufu Village,in Dinghu District of Zhaoqing City,Guangdong Province,the newly restored Chaoyangli Cultural Retreat is already welcoming its first visitors.A visitor surnamed Chen,who has travelled from Guangzhou with his child,stops in front of the Chaxi Academy to admire local intangible cultural heritage crafts.展开更多
In the year 2026,the world cultural exchanges will continue to develop,while the international public opinion environment remainsdive rseand complex.Looking back at this pivotal moment in history,the life journey of S...In the year 2026,the world cultural exchanges will continue to develop,while the international public opinion environment remainsdive rseand complex.Looking back at this pivotal moment in history,the life journey of Sunny Wu,Chairman of the Hong Kong Doyens’Club,resonates perfectly with the national development.展开更多
Under the strategic framework of rural revitalization and agricultural modernization, Xinjiashan Specialty Coffee Base, located in Zaotang Village, Lujiang Town, Longyang District, Baoshan City, has been proactively i...Under the strategic framework of rural revitalization and agricultural modernization, Xinjiashan Specialty Coffee Base, located in Zaotang Village, Lujiang Town, Longyang District, Baoshan City, has been proactively investigating innovative models for agricultural development. Through extensive communication and collaboration, this base has established close partnerships with research institutions including Kunming University of Science and Technology, Baoshan University, and Yunnan Academy of Agricultural Sciences, with a commitment to thoroughly exploring the potential for resource recycling and ecological complementarity. An innovative four-in-one three-dimensional integrated planting system incorporating "coffee, bananas, green manure, and bees" has been implemented. Concurrently, technological and digital management strategies have been comprehensively integrated to improve planting efficiency. Under this model, the proportion of specialty coffee attains 71%, and the per-unit yield is 17% greater than that of the conventional planting model. This approach not only substantially enhances economic returns but also promotes the integrated development of ecological and social benefits, offering a valuable practical example and experiential reference for the specialty and sustainable advancement of the coffee industry in comparable regions.展开更多
文摘In vitro culture of spermatogonial stem cells （SSCs） has generally been performed using two-dimensional （2D） culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules （including those generated by the SSC niche） and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system （SACS） and a 3D methylcellulose culture system （MCS）. It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we （1） provide a brief overview of the differences in spermatogenesis in rodents and primates, （2） summarize data related to attempts to generate sperm in vitro, （3） report for the first time formation of colonies/clusters of cells and differentiation of meiotic （expression of CREM-1） and postmeiotic （expression of acrosin） germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and （4） indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.
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AIM： TO investigate the multicellular resistance of human hepatocellular carcinoma HepG2 cells in three-dimensional culture to delisheng, 5-fluorouracil and adriamycin, and the possible molecular mechanisms of delisheng. METHODS： Human hepatocellular carcinoma HepG2 cells were cultured with a liquid overlay technique. After the formation of multicellular spheroids, morphology was analyzed by phase contrast microscopy, scanning electron microscopy and transmission electron microscopy. Sensitivity of HepG2 cells to delisheng, 5-fluorouracil and adriamycin was investigated by Ml-I- assay in multicelluar spheroids and monolayers. Vascular endothelial growth factor （VEGF） and endostatin expression were analyzed in multicellular spheroids treated with delisheng, 5-fluorouracil, adriamycin and negative control PBS, with immunohistochemical staining. RESULTS： Multicellular spheroids exhibited structural characteristics somewhat different to those in monolayers. The cells in three-dimensional cell culture turned out to be less sensitive to delisheng, 5-fluorouracil and adriamycin than the cells cultured in monolayer. This showed that delisheng had a satisfactory cells inhibition ratio compared to 5-fluorouracil and adriamycin. Immunohistochemical staining showed that VEGF and endostatin expression was positive during growth as multicellular spheroids, and endostatin expression in spheroids with treatment of delisheng was higher than that with 5-fluorouracil, adriamycin and PBS （139.35 ± 7.83, 159.23 ± 10.34, 162.83 ± 3.47 and 148.48 ± 11.06, P 〈 0.05）.CONCLUSION： Chinese medicine compound delisheng has satisfactory anti-tumor activity in HepG2 cells in three-dimensional culture, and the effects are associated with up-regulation of endostatin.
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.
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.
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 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.
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Nerve injury repair has been an intractable issue wor.ldwide due to the non-renew able property of neurons in vivo.Cell therapy adopting me senchymal stem cells(MSC)induced neurons stands for a promising substitute treatment,which is hindered by the low diffe rentiation efficiency of current approaches.To improve the efficiency of MSC differentiation in vitro,we constructed a three-dimensional(3D)culture system by seeding human bone marrow-de rived MSC(hBMSCs)into collagen-nano fiber scaffolds which are a mixed hydrogel of collagen 1 and silk nanofiber solution and testified the diffe rentiation efficiency from MSC into neurons by chemical or cocktail induction methods.Results indicated MSCs cultured in 3D system showed good viability with neuron-like morphology in both groups.
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.
文摘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.
文摘BACKGROUND Fracture is one of the most pervasive injuries in the musculoskeletal system,and there is a complex interaction between macrophages and adipose tissue-derived stem cells(ADSCs)in fracture healing.However,two-dimensional(2D)coculture of macrophages and ADSCs can not accurately mimic the in vivo cell microenvironment.AIM To establish both 2D and 3D osteogenic coculture models to investigate the interaction between macrophages and ADSCs.METHODS After obtaining ADSCs from surgery and inducing differentiation of the THP1 cell line,we established 2D and 3D osteogenic coculture models.To assess the level of osteogenic differentiation,we used alizarin red staining and measured the relative expression levels of osteogenic differentiation markers osteocalcin,Runt-related transcription factor 2,and alkaline phosphatase through polymerase chain reaction.Verification was conducted by analyzing the expression changes of N-cadherin and the activation of the Wnt/β-catenin signaling pathway using western blotting.RESULTS In this study,it was discovered that macrophages in 3D culture inhibited osteogenic differentiation of ADSCs,contrary to the effect in 2D culture.This observation confirmed the significance of intricate intercellular connections in the 3D culture environment.Additionally,the 3D culture group exhibited significantly higher N-cadherin expression and showed reducedβ-catenin and Wnt1 protein levels compared to the 2D culture group.CONCLUSION Macrophages promoted ADSC osteogenic differentiation in 2D culture conditions but inhibited it in 3D culture.The 3D culture environment might inhibit the Wnt/β-catenin signaling pathway by upregulating N-cadherin expression,ultimately hindering the osteogenic differentiation of ADSCs.By investigating the process of osteogenesis in ADSCs,this study provides novel ideas for exploring 3D osteogenesis in ADSCs,fracture repair,and other bone trauma repair.
基金Center for Sinicized Marxism and Traditional Culture,Sichuan University of Science&Engineering(Project No.:ZMCY202410)。
文摘Against the backdrop of intensified global cultural collisions and ideological competition,deeply integrating excellent traditional Chinese culture(ETCC)into university ideological and political courses(IPCs)has become an imperative of our times.Guided by General Secretary Xi Jinping’s methodology of“Two Integrations,”this paper examines the pathways for this integration from three dimensions:value,theory,and practice.The value dimension emphasizes fostering moral conviction and strengthening the spiritual foundation to meet needs such as safeguarding cultural security,preserving the spiritual lineage,and constructing a spiritual framework.The theoretical dimension reveals the mutually constitutive breakthroughs between Marxism and traditional Chinese dialectical thinking,encompassing methodological complementarity,logical coherence of values,and discursive system innovation.The practical dimension involves constructing a comprehensive educational ecosystem by localizing teaching content,modernizing traditional resources,and fostering inter-platform collaborative education,thereby internalizing the value of traditional culture.These three dimensions synergize and co-constitute each other,collectively providing methodological support and practical paradigms for cultivating cultural confidence among youth and forging a new generation capable of shouldering the mission of national rejuvenation.
文摘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.
文摘Background The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional （3-D） growth pattern of endometrial cells using a 3-D in vitro culture system.
基金supported by the National Key Research andDevelopment Program of China(Grant No.2021YFC2101400).
文摘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.
文摘Through the introduction of disaster situation of Qiang Culture after Wenchuan Earthquake, the paper emphasized that carriers of Qiang Culture had been seriously damaged, the inheritance of Qiang Culture had been affected, and the environment for Qiang Culture was difficult to recover. It highlighted that three-dimensional reconstruction of Qiang Culture should stress the core task and timely and effectively rescue endangered cultural heritages of Qiang Nationality from the perspectives of material and spiritual life. It had explained focuses of three-dimensional pattern construction in detail. In terms of spatial reconstruction, it should reconstruct native culture and history while material culture was constructed, and reconstruct Qiang culture highland by depending on aborigines; in terms of cluster reconstruction, it should give support to large tourism enterprises and perfect tourism chain; in terms of ecological reconstruction, it should enhance construction and demonstration of "ecological protection pilot area of Qiang culture"; in terms of development reconstruction, it should realize coordinated unity between protection and development according to classification protection, characteristic protection and key protection, so as to form the virtuous circle of post-disaster recovery protection and sustainable development.
文摘At dawn in Wufu Village,in Dinghu District of Zhaoqing City,Guangdong Province,the newly restored Chaoyangli Cultural Retreat is already welcoming its first visitors.A visitor surnamed Chen,who has travelled from Guangzhou with his child,stops in front of the Chaxi Academy to admire local intangible cultural heritage crafts.
文摘In the year 2026,the world cultural exchanges will continue to develop,while the international public opinion environment remainsdive rseand complex.Looking back at this pivotal moment in history,the life journey of Sunny Wu,Chairman of the Hong Kong Doyens’Club,resonates perfectly with the national development.
文摘Under the strategic framework of rural revitalization and agricultural modernization, Xinjiashan Specialty Coffee Base, located in Zaotang Village, Lujiang Town, Longyang District, Baoshan City, has been proactively investigating innovative models for agricultural development. Through extensive communication and collaboration, this base has established close partnerships with research institutions including Kunming University of Science and Technology, Baoshan University, and Yunnan Academy of Agricultural Sciences, with a commitment to thoroughly exploring the potential for resource recycling and ecological complementarity. An innovative four-in-one three-dimensional integrated planting system incorporating "coffee, bananas, green manure, and bees" has been implemented. Concurrently, technological and digital management strategies have been comprehensively integrated to improve planting efficiency. Under this model, the proportion of specialty coffee attains 71%, and the per-unit yield is 17% greater than that of the conventional planting model. This approach not only substantially enhances economic returns but also promotes the integrated development of ecological and social benefits, offering a valuable practical example and experiential reference for the specialty and sustainable advancement of the coffee industry in comparable regions.
