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Matrix metalloproteinase-sensitive poly(ethylene glycol)/peptide hydrogels as an interactive platform conducive to cell proliferation during 3d cell culture

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Science China(Technological Sciences) 2021年第6期64卷 1285-1294页

作者： RONG Yan ZHANG Zhen HE ChaoLiang CHEN XueSi Key Laboratory of Polymer Ecomaterials Changchun Institute of Applied ChemistryChinese Academy of SciencesChangchun 130022China University of Chinese Academy of Sciences Beijing 100049China University of Science and Technology of China Hefei 230026China

The response of extracellular matrix(ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase(MMP)-sensitive degradable hydrogels(MSdHs) via the catalyst-free o-phthalaldehyde(OPA)/amine cross-linking reaction between o-phthalaldehyde-grafted four-arm poly(ethylene glycol)(4aPEG-OPA) and an MMP-sensitive degradable peptide. The gelation rates and storage moduli of MSdHs and the MMP-insensitive hydrogels(MIHs) based on an MMP-insensitive scramble peptide were comparable and dependent on the concentrations of precursor polymers. MSdHs were degradable while MIHs were stable in the presence of proteinase in vitro.The degradation of MSdHs was obviously faster than that of MIHs after subcutaneous injection into rats. In addition, both types of poly(ethylene glycol)/peptide hydrogels displayed excellent cytocompatibility in vitro, and showed good histocompatibility in vivo in the subcutaneous layer of rats. Furthermore, the proliferation of several MMP-expressing cell lines including MdA-MB-231 cells within MSdHs was obviously faster than that in MIHs, indicating the influence of metabolism-mediated scaffold degradation on the cell proliferation. This study provides a new biocompatible and biodegradable 3 d cell culture interactive platform for regulation of cell behavior.

关键词： hydrogels MMP-sensitive o-phthalaldehyde cross-linking cell interaction 3d cell culture

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Mechanical stretching of 3d hydrogels for neural stem cell differentiation

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Bio-design and Manufacturing 2022年第4期5卷 714-728页

作者： Quanjing Mei Ho-Yin Yuen Xin Zhao department of Biomedical Engineering The Hong Kong Polytechnic UniversityHung HomHong KongChina

While it is known that mechanical dynamics are influential in neural differentiation for critical processes like neurogenesis or neurodegeneration, studies on neural stem cell therapies usually focus on biochemical interactions rather than mechanical aspects, frequently resulting in low efficacy and unfulfilled potential. Therefore, current studies are attempting to elucidate the effect of mechanical stimulus on neural performance using conventional two-dimensional(2d) planar substrates. Yet, these2d substrates fail to capture the defining three-dimensional(3d) characteristics of the in vivo neural stem cell environment.To complete this research gap, we synthesized a series of soft and elastic 3d hydrogels to mimic the neural tissue mechanical environment for 3d cell culture, using long-chain polyethylene glycol diacrylate(PEGdA) and gelatin-methacryloyl(Gel MA).By varying the concentration of the polymer, we obtained biomimicking hydrogels with a tensile modulus as low as 10 k Pa and a compressive modulus as low as 0.8 k Pa. The in vitro results demonstrated that Gel MA-PEGdA hydrogels have the high biocompatibility required to support neural cell growth, proliferation, and differentiation, as well as neurite outgrowth. We then studied the effect of mechanical stretching on the behaviors of neural cells and observed that mechanical stretching could significantly enhance neurite extension and axon elongation. In addition, the neurites were more directionally oriented to the stretching direction. Immunocytochemistry and relative gene expression data also suggested that mechanical tension could upregulate the expression of neural differentiation protein and genes, including GFAP and βIII-Tubulin. Overall, this study shows that in addition to the specific mechanical properties of Gel MA-PEGdA that improve neural differentiation towards specific lineages, hydrogel stretching is also a potentially attractive strategy to improve the therapeutic outcomes of neural stem cell therapies.

关键词： Mechanical property Tensile stretching Hydrogels Neural differentiation 3d cell culture

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Injectable hybrid inorganic nanoscaffold as rapid stem cell assembly template for cartilage repair

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National Science Review 2022年第4期9卷 116-127页

作者： Shenqiang Wang Letao Yang Bolei Cai Fuwei Liu Yannan Hou Hua Zheng Fang Cheng Hepeng Zhang Le Wang Xiaoyi Wang Qianxin Lv Liang Kong Ki-Bum Lee Qiuyu Zhang Key Laboratory of Special Functional and Smart Polymer Materials of Ministry of Industry and Information Technology School of Chemistry and Chemical EngineeringNorthwestern Polytechnical University Xi'an Key Laboratory of Functional Organic Porous Materials School of Chemistry and Chemical EngineeringNorthwestern Polytechnical University department of Chemistry and Chemical Biology Rutgers University department of Biomedical Engineering Columbia University State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral diseases & Shaanxi Key Laboratory of Oral diseases Department of Oral and Maxillofacial SurgerySchool of StomatologyThe Fourth Military Medical University Research & development Institute of Northwestern Polytechnical University in Shenzhen

Cartilage injuries are o?ten devastating and most cannot be cured because of the intrinsically low regenerative capacity of cartilage tissues. Although stem-cell therapy has shown enormous potential for cartilage repair, the therapeutic outcome has been restricted by low survival rates and poor chondrocyte differentiation in vivo. Here, we report an injectable hybrid inorganic(IHI) nanoscaffold that facilitates fast assembly, enhances survival and regulates chondrogenic differentiation of stem cells. IHI nanoscaffolds that strongly bind to extracellular matrix(ECM) proteins assemble stem cells through synergistic 3 d cell–cell and cell–matrix interactions, creating a favorable physical microenvironment for stem-cell survival and differentiation in vitro and in vivo. Additionally, chondrogenic factors can be loaded into nanoscaffolds with a high capacity, which allows deep, homogenous drug delivery into assembled 3 d stem-cell-derived tissues for effective control over the soluble microenvironment of stem cells. The developed IHI nanoscaffolds that assemble with stem cells are injectable. They also scavenge reactive oxygen species and timely biodegrade for proper integration into injured cartilage tissues. Implantation of stem-cell-assembled IHI nanoscaffolds into injured cartilage results in accelerated tissue regeneration and functional recovery. By establishing our IHI nanoscaffold-templated 3 d stem-cell assembly method, we provide a promising approach to better overcoming the inhibitory microenvironment associated with cartilage injuries and to advance current stem-cell-based tissue engineering.

关键词： injectable nanoscaffold 3d cell culture tissue engineering cartilage repair stem-cell therapy

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Mice 3d testicular organoid system as a novel tool to study Zika virus pathogenesis

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Virologica Sinica 2023年第1期38卷 66-74页

作者： Wei Yang Chen Zhang Yan-Hua Wu Li-Bo Liu Zi-da Zhen dong-Ying Fan Zheng-Ran Song Jia-Tong Chang Pei-Gang Wang Jing An department of Microbiology School of Basic Medical SciencesCapital Medical UniversityBeijing100069China department of Neurosurgery Capital Medical University Sanbo Brain HospitalBeijing100093China Center of Epilepsy Beijing Institute for Brain DisordersBeijing100093China

Zika virus(ZIKV)poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage.However,deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury.Organoids are relatively simple in vitro models,which could mimic the pathological changes of corresponding organs.In this study,we constructed a 3d testicular organoid model using primary testicular cells from adult BALB/c mice.Similar to the testis,this organoid system has a blood-testis barrier(BTB)-like structure and could synthesize testosterone.ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis.Therefore,our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.

关键词： Organoid 3d cell culture Testicular organoid model Zika virus(ZIKV)

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Using pipette tips to readily generate spheroids comprising single or multiple cell types

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Journal of Zhejiang University-Science A(Applied Physics & Engineering) 2023年第10期24卷 875-885页

作者： Rong PAN Xiaoyan YANG Shiming WU Yuanyuan XIE Feng CHEN Ke NING Wei SUN Ling YU Key Laboratory of Luminescence Analysis and Molecular Sensing Ministry of EducationInstitute for Clean Energy and Advanced MaterialsSchool of Materials and EnergySouthwest UniversityChongqing 400715China College of Chemistry and Chemical Engineering Hainan Normal UniversityHaikou 571158China

Three-dimensional(3d)cell culture methods have been validated that can replicate the tumor environment in vivo to a large extent,providing an effective tool for studying tumors.In this study,we demonstrated the use of standard laboratory pipette tips as micro vessels for generating 3d cell spheroids.No microfabrication or wet-chemistry surface modifications were involved in the procedure.Spheroids consisting of single or multiple cell types were generated within 24 h just by pipetting and incubating a cell suspension in pipette tips.Scanning electron microscope and optical microscope proved that the cells grew together tightly,and suggested that while gravity force might have initiated the sedimentation of cells at the bottom of the tip,the active aggregation of cells to form tight cell-cell interactions drove the formation of spheroids.Using common laboratory micropipettes and pipette tips,the rate of spheroid generation and the generation reproducibility was characterized from five boxes each with 80 tips.The ease of transferring reagents allowed modeling of the growth of microvascular endothelial cells in tumor spheroids.Moreover,the pairing and fusion of tumor spheroids could be manipulated in the pipette tips,suggesting the potential for building and assembling heterogeneous micro-tumor tissues in vitro to mimic solid tumors in vivo.This study demonstrated that spheroids can be readily and cost-effectively generated in standard biological laboratories in a timely manner using pipette tips.

关键词： Pipette tip 3d cell culture Tumor spheroids Co-culture In-situ observation

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In vitro 3d malignant melanoma model for the evaluation of hypericin-loaded oil-in-water microemulsion in photodynamic therapy

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Bio-design and Manufacturing 2022年第4期5卷 660-673页

作者： Hui LMa Wanlu Li Mian Wang Laudemir C.Varanda Janice R.Perussi Y.Shrike Zhang Emanuel Carrilho Instituto de Química de São Carlos Universidade de São PauloUSPAv.Trabalhador São-carlenseSão CarlosSP 13566-590Brazil Instituto Nacional de Ciência e Tecnologia de Bioanalítica INCTBioCampinasSP 13083-970Brazil division of Engineering in Medicine Department of MedicineBrigham and Women’s HospitalHarvard Medical SchoolCambridgeMA 02139USA

Advances in biomimetic three-dimensional(3d) melanoma models have brought new prospects of drug screening and disease modeling, since their physiological relevancy for recapitulating in vivo tumor architectures is more accurate than traditional two-dimensional(2d) cell culture. Gelatin methacryloyl(GelMA) is widely used as a tissue-engineered scaffold hydrogel for 3d cell culture. In the present study, an in vitro 3d malignant melanoma model based on Gel MA was fabricated to evaluate the efficiency of hypericin(Hy)-loaded microemulsion(ME) in photodynamic therapy against melanoma. The ME was produced by the spontaneous emulsification method to enhance the bioavailability of Hy at tumor sites. Hy-loaded MEs were applied to a 3d malignant melanoma model made using 6% Gel MA and the co-culture of B16F10 and Balb/c 3T3 cells,followed by crosslinking using violet light(403 nm). The observation revealed excellent cell viability and the presence of F-actin cytoskeleton network. Hy-loaded MEs exhibited higher phototoxicity and cell accumulation(about threefold) than free Hy, and the cells cultured in the 3d system displayed lower susceptibility(about 2.5-fold) than those in 2d culture.These findings indicate that the developed MEs are potential delivery carriers for Hy;furthermore, Gel MA hydrogel-based modeling in polydimethylsiloxane(PdMS) molds is a user-friendly and cost-effective in vitro platform to investigate drug penetration and provide a basis for evaluating nanocarrier efficiency for skin cancer and other skin-related diseases.

关键词： Malignant melanoma 3d cell culture Hypericin Microemulsion Photodynamic therapy

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Effects of simulated zero gravity on adhesion,cell structure,proliferation,and growth behavior,in glioblastoma multiforme

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Nanotechnology and Precision Engineering 2023年第4期6卷 22-29页

作者： Saifaldeen Altaie Amera Alrawi School of Precision Instrument and Optoelectronic Engineering Tianjin UniversityTianjinChina College of Medicine Ninevah UniversityMosulIraq College of Science University of MosulMosulIraq

All life on Earth has evolved under the influence of continuous gravity,and methods have been developed to balance this influence with the biological evolution of organisms at the cellular and system levels.However,when exposed to zero gravity in space,the balance between cell structure and external forces is destroyed,resulting in changes at the cellular level(e.g.,cell morphology,adhesion,viability,apoptosis,etc.),and understanding the molecular mechanism of cell response to zero gravity will help to cope with diseases that rely on mechanical response.Therefore,biological research in space and zero gravity is a unique step in developing the best anti-cancer treatments,which is a great challenge to humanity.In this study,multicellular glioma cancer cells from a brain tumor in a 72-year-old Iraqi patient were subjected to simulated zero gravity for 24 h,and the results showed that most of the cells lost their adhesion,which is considered to be the first step toward cell apoptosis.In addition to the formation of multicellular spheroids,the results also showed that the inhibition rate for cell death was 32%in comparison to the control cells.Moreover,the cells showed a clear change in their cellular morphology and growth behavior.These results give new hope for fighting cancer distinctively,and such a treatment method has no side effects in comparison to traditional chemical and radiological ones.

关键词： 3d cell culture Space biology Glioblastoma Simulated microgravity Cancer biology

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一种高效生产细胞球的超分子水凝胶

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Science China Materials 2022年第6期65卷 1655-1661页

作者：艾思凡李惠郑浩刘金鸣高洁刘鉴峰陈佺杨志谋 Key Laboratory of Bioactive Materials Ministry of EducationState Key Laboratory of Medicinal Chemical BiologyCollege of Life SciencesCollaborative Innovation Center of Chemical Science and Engineeringand National Institute of Functional MaterialsNankai UniversityTianjin 300071China Key Laboratory of Radiopharmacokinetics for Innovative drugs Chinese Academy of Medical Sciencesand Institute of Radiation MedicineChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300192China

与传统的二维培养的细胞相比,三维培养的细胞球更能够代表天然组织和体内环境,因此细胞球在细胞生物学研究、组织工程、药物筛选方面具有巨大的潜力.然而目前开发的包括天然和合成水凝胶在内的材料,存在成分不确定和生物相容性差等缺点... 详细信息

与传统的二维培养的细胞相比,三维培养的细胞球更能够代表天然组织和体内环境,因此细胞球在细胞生物学研究、组织工程、药物筛选方面具有巨大的潜力.然而目前开发的包括天然和合成水凝胶在内的材料,存在成分不确定和生物相容性差等缺点,阻碍了它们的广泛应用.在这项研究中,我们设计了能够自组装的超分子多肽水凝胶(称为SupraGel)用于三维培养中.在多肽中引入一个d构型的氨基酸可能会减少细胞与基质之间的相互作用,从而促进自发细胞球的形成.当在SupraGel中培养两种癌细胞系MCF-7和4T1以及肠道干细胞(ISCs)时,细胞能够有效增殖并生长为细胞球.SupraGel的触变性以及自恢复行为非常适合活细胞的包埋以及细胞球的后续收集.同时,SupraGel的机械强度能够通过调整多肽的浓度轻松调节,从而使其适用于不同细胞球的三维培养.实验结果表明SupraGel能够有效生产细胞球,在细胞治疗、再生医学和药物筛选方面具有巨大的应用潜力.

关键词： cell spheroids 3d cell culture supramolecular hydrogel peptides self-assembly

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Breast Cancer MCF-7 cell Spheroid culture for drug discovery and development

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Journal of Cancer Therapy 2022年第3期13卷 117-130页

作者： Guangping Chen William Liu Bingfang Yan department of Physiological Sciences College of Veterinary Medicine Oklahoma State University Stillwater OK USA College of Pharmacy University of Cincinnati Cincinnati OH USA

In vitro 3d cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3d spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3d spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

关键词： MCF-7 cell Spheroid culture 3d cell culture Estrogen-dependent Breast Cancer Cancer drug development Personalized Cancer drug development

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Three-dimensional culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

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Biomedical and Environmental Sciences 2018年第2期31卷 136-145页

作者： CHEN Ya Xiong XIE Guang Cheng PAN dong dU Ya Rong PANG Li Li SONG Jing dong dUAN Zhao Jun HU Bu Rong Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences & Key Laboratory of Space Radiobiology of Gansu Province Institute of Modern Physics Chinese Academy of Sciences department of Pathogenic Biology Chengde Medical University department for Viral diarrhea National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention

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.

关键词： 3d cell culture Human airway epithelium （HAE） Human rhinovirus C Human bocavirus Propagation

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