Cotransplantation of marginal mass allogeneic islets with 3D culture-derived adult human skin cells improves glycemia in diabetic mice

Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.

Application of Microfluidic Liver and Kidney Chips in Toxicology Research of Chinese Medicine / 中国实验方剂学杂志

Microfluidic liver and kidney chips have become preferred model carriers in recent years for new drug development， pharmacological and toxicological research， mechanism exploration， and disease model construction. In the context of the USA. Food and Drug Administration allowing the use of in vitro model data as a substitute for animal model data in new drug applications when animal disease models are difficult to construct， microfluidic chips have received widespread attention due to their high throughput， ability to highly mimic biological characteristics of living organisms， convenient evaluation of drug toxicity in normal or pathological states with repeated dosing， real-time induction and monitoring of culture processes， and real-time data acquisition and analysis. In toxicology research， liver and kidney chips can construct in vitro models suitable for the pharmacological and toxicological detection of different substances by combining 2D monocultures and co-cultures from different species sources， 3D cultures， spheroids/organoid cells， precision-cut liver and kidney slices， immortalized cell lines， or sandwich-cultured cell lines. This model maximally simulates or retains the organ function and in vivo microenvironment of the liver and kidney， including specific physiological tissue structures， multicellular interactions/crosstalk， and multi-organ coordination/feedback， to obtain results similar to or the same as in vivo experimental data， reducing interspecies differences. At the same time， it greatly reduces the use of experimental animals and lowers costs. Microfluidic technology provides necessary shear force microenvironments for the cultivation of contents and solves problems encountered in the cultivation process of liver and kidney chips， such as insufficient tissue oxygen supply， nutrient deficiencies， and accumulation of metabolites， leading to cell apoptosis and even tissue necrosis fibrosis， which make it difficult to maintain long-term structure and function. This article reviewed the application of microfluidic technology combined with liver and kidney chips in Chinese medicine toxicology research. By summarizing the development of microfluidic technology， liver chips， kidney chips， and providing application examples of microfluidic liver and kidney chips in Chinese medicine toxicology research， combined with the characteristics of Chinese medicine administration， the article explored the advantages and future development directions of their application in the field of Chinese medicine toxicology research.

3D spheroid cell cultures and their role in bone regeneration: a systematic review / Cultivos de esferoides 3D y su papel en la regeneración ósea: una revisión sistemática

Abstract Recently, the 3D spheroid cell culture application has been extensively used in the treatment of bone defects. A wide variety of methodologies have been used, which has made the comparison of results complex. Therefore, this systematic review has two aims: (i) to perform an analysis focused on the role of 3D spheroid cell culture in bone regeneration strategies; and (ii) address the main challenges in clinical application. A search of the following keywords "3D cell culture", "spheroid", and "bone regeneration" was carried out in the PubMed, Scopus, and ScienceDirect databases and limited to the years 2010-2020. Studies were included if their primary objective was the behavior of cell aggregates to formed spheroids structures by different 3D cell culture techniques focused on the regeneration of bone tissue. To address the risk of bias for in vitro studies, the United States national toxicology program tool was applied, and descriptive statistics of the data were performed, with the SPSS V.22 program. A total of 16 studies were included, which met the established criteria corresponding to in vitro and in vitro/in vivo studies; most of these studies used stem cells for the 3D cell spheroids. The most often methods used for the 3D formation were low adherence surface and rotational methods, moreover, mesenchymal stem cells were the cell line most frequently used because of their regenerative potential in the field of bone tissue engineering. Although the advances in research on the potential use of 3D spheroids in bone regeneration have made great strides, the constant innovation in cell spheroid formation methodologies means that clinical application remains in the future as strategy for 3D tissue bioprinting.

Resumen Recientemente, la aplicación del cultivo 3D de esferoides se ha utilizado ampliamente en el tratamiento de defectos óseos. La variedad de metodologías para lograr los cultivos 3D de esferoides ha hecho compleja la comparación de resultados. Por tanto, esta revisión sistemática tiene dos objetivos: (i) realizar un análisis centrado en el papel de los cultivos 3D de esferoides en las estrategias de regeneración ósea; y (ii) abordar los principales desafíos en la aplicación clínica. Se realizó una búsqueda de las siguientes palabras clave "cultivo celular 3D", "esferoide" y "regeneración ósea" en las bases de datos PubMed, Scopus y ScienceDirect y se limitó a los años 2010-2020. Se incluyeron los estudios si su principal objetivo era el comportamiento de agregados celulares para generar las estructuras esferoidales desarrollados por diferentes técnicas de cultivo celular 3D enfocadas a la regeneración del tejido óseo. Para abordar el riesgo de sesgo de los estudios in vitro, se aplicó la herramienta del programa nacional de toxicología de Estados Unidos y se realizaron estadísticas descriptivas de los datos, con el programa SPSS V.22. Se incluyeron un total de 16 estudios, que cumplieron con los criterios establecidos correspondientes a estudios in vitro e in vitro/in vivo; la mayoría de estos estudios utilizaron células troncales para generar los esferoides celulares 3D. Los métodos más utilizados para la formación de los esferoides 3D fueron la superficie de baja adherencia y los métodos de rotación, asimismo, la línea celular de células troncales mesenquimales fueron las más utilizadas debido a su gran potencial regenerativo en el campo de la ingeniería de tejidos óseos. Aunque los avances en la investigación sobre el uso potencial de los cultivos celulares de esferoides 3D en la regeneración ósea han logrado grandes avances, la constante innovación en las metodologías de la generación de esferoides 3D deja claro que la aplicación clínica de estos permanecerá en el futuro como estrategia en la bioimpresión tisular.

Decellularized extracellular matrix mediates tissue construction and regeneration / 医学前沿

Contributing to organ formation and tissue regeneration, extracellular matrix (ECM) constituents provide tissue with three-dimensional (3D) structural integrity and cellular-function regulation. Containing the crucial traits of the cellular microenvironment, ECM substitutes mediate cell-matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo. However, these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures. Cultured cells also produce rich ECM, particularly stromal cells. Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well. Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select, produce, and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration. Overall, the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed. Moreover, current preclinical applications by which ECM components modulate the wound-healing process are reviewed.

Establishment of 3D hepatocyte genotoxicity testing assay / 中国药理学通报

Aim To establish the 3D hepatocyte model by selecting the humanized hepatocyte HepG2 cells and 3D cell culture methods, and to establish the 3D hepatocyte cytokinesis-block micronucleus cytome(CBMN-cyt)assay and 3D hepatocyte comet assay by using chemicals of different mode of action.Methods In this study, a scaffold-free culture method was used to successfully establish a 3D HepG2 hepatocyte spheroid model.The appearance of the sphere, the survival rate of cells inside the sphere, the gene expression of phase I and II metabolic enzymes, and the expression of liver-specific biomarkers were selected as the observation indicators to obtain the best culture conditions for the 3D hepatocyte model.The 3D hepatocyte model was combined with in vitro micronucleomics test and in vitro comet test to explore its applicability for genotoxicity test.Results The best culture conditions for the 3D hepatocyte model was 5×103 cells/20 μL /drop inoculation, cultivating for seven days.A 3D hepatocyte CBMN-cyt assay was established using mitomycin C(MMC), a micronucleus positive compound, and the results showed that it could successfully detect the genotoxicity and cytotoxicity of MMC.Compared with the CBMN-cyt results of 2D hepatocyte model, 3D hepatocyte model had higher sensitivity in detecting MN and Nbud.The 3D hepatocyte comet assay methods were established using the known in vivo and in vitro comet assay positive compound methyl methanesulfonate(MMS), and the results showed that MMS could significantly increase the tail DNA% of 3D hepatocytes with low cytotoxicity.The sensitivity of 3D hepatocyte model to MMS genotoxicity detection was higher than that of 2D cells.Conclusions The 3D hepatocyte model established in this study is easy to use and low in cost, and shows good sensitivity and specificity in the in vitro micronucleus test and comet test, suggesting that the 3D hepatocyte genotoxicity test method is used in early drug genotoxicity screening.It has good application prospects in additional experimental research.

Research hotspots of organoid models in recent 10 years: A search in domestic and foreign databases / 中国组织工程研究

BACKGROUND: Organoid models can partially restore the tissue and molecular characteristics of cells in the body, which is a preclinical model with good potential. Compared with precise and regenerative medicine methods, organoids with tissue organ functions can simulate body organs to the greatest extent. Moreover, organoid drug sensitivity data is more accurate than whole-genome sequencing. The technology for organoids can be combined with in vitro gene editing technology to achieve a genetic modification at organ level. OBJECTIVE: To summarize and analyze the research hotpots of organoid models in domestic and foreign databases in the past 10 years. METHODS: A computer search of WanFang, Web of Science, Chinese Clinical Trial Registry, ClinicalTrials.org and SooPAT was performed for articles regarding organoids in the past 10 years, and the research hotspots of organoid models were analyzed and concluded. RESULTS AND CONCLUSION: (1) Retrieval results of WanFang database: A total of 187 articles were included. In 2019, Chinese research on organoids showed an explosive growth. Among them, there were more studies on intestinal organoid models. The main application areas included precision medicine, tumor research and personalized medicine. (2) Retrieval results of the Web of Science Core Collection: A total of 2 450 articles were included. Twenty highly cited articles were analyzed using Histcite software. Among them, five classic original articles of organoids were screened out, and introduced intestinal organoids, pluripotent stem cell-derived three-dimensional brain organoids, prostate cancer organoids cultured from biopsy specimens and circulating tumor cells for a long time, kidney organoids containing nephrons, and three-dimensional organoid models of human stomach tissue, which are the pioneers in various fields and lay the foundation for future research on organoids. (3) Retrieval results of Chinese Clinical Trial Registry and ClinicalTrials.org: There were 13 study protocols related to organoid research in the Chinese Clinical Trial Registry and 23 in the ClinicalTrials.org. Research on clinical application of organoids in the United States is more extensive and develops earlier, but there are mostly cohort studies and single-arm trials. In recent years, China has achieved some results in the field of clinical organoid research, and two high-profile randomized controlled trials are underway. (4) Retrieval results of SooPAT Chinese patent database: There were 55 authorized patents, mainly involving the new culture method of 3D brain organoids, the research and development of high-throughput 3D cell, tissue-like and organoid dynamic culture systems. At present, researchers have successfully constructed a variety of organoid models, such as intestine, brain, kidney and various cancer tissues. However, due to insufficient evidence of clinical randomized controlled trials, the clinical applicability remains to be explored. Tumor organoids that are directly generated by tumor tissues of cancer patients in vitro can be used to analyze potential drug targets, screen anti-cancer drugs and develop new anti-tumor drugs, which will be the main research directions in the field of organoids in the future.

Advances in research and application of breast cancer organoids / 中国组织工程研究

BACKGROUND: Breast cancer is one of the most common malignant tumors in women. Its incidence rate is increasing year by year and tends to be younger. It seriously threatens women’s health. Therefore, it is particularly important to establish an ideal breast cancer model that can accurately simulate the tumor in vivo. Organoid is a new three-dimensional cultural model in vitro, which recapitulates key aspects of in vivo tissue or organ. In recent years, researches based on organoids have covered many kinds of tumors. OBJECTIVE: To review the research progress and application of breast cancer organoids, in order to provide a new research way for personalized treatment of breast cancer. METHODS: Using the key words of “organoid, breast cancer organoids, cancer organoids, mammosphere, three-dimensional culture” in English and Chinese, respectively, the first author retrieved relevant articles published from January 1980 to February 2020 in CNKI, Wanfang, and PubMed databases. The type of the article was not limited. After removal of the articles that were not related to the purpose of the study or repetitive, 66 articles were finally analyzed. RESULTS AND CONCLUSION: This review introduced organoid technology briefly and retraced the process of exploring suitable culture conditions to establish breast-cancer-origin organoids. Also, we concluded latest development of its applications and research progress. Breast cancer organoids have a wide range of application prospects in disease modeling, tumor pathogenesis, drug screening and other aspects, which provide a reliable model for breast cancer research and treatment, and in particular, open up a new perspective for personalized treatment of breast cancer.

Exploration of organoid in breast cancer related research / 生物工程学报

Breast cancer is the most common cancer in women. At present, the in vivo model and traditional cell culture are mainly used in breast cancer researches. However, as high as 90% clinical trials are failed for drugs explored by the above two methods, due to the inherent species differences between humans and animals, as well as the differences in the tissue structure between organs and cells. Therefore, organoid three-dimensional culture is emerging. As a new tumor research model, organoid, a three-dimensional cell complex with spatial structure, has broad application prospects, such as precision medicine, organ transplantation, establishment of refractory disease model, gene therapy and drug research and development. Therefore, organoid is considered as one of the ideal carriers for life science research in the future. Breast cancer, a heterogeneous disease with complex phenotypes, has a low survival rate. Breast cancer organoid can reproduce many key features of human breast cancer, thus, the construction of organoid biological library of breast cancer will provide a new platform for studying the occurrence, development, metastasis and drug resistance mechanism of breast cancer. In this review, we systematically introduce the culture conditions of organoids and their application in breast cancer related research, and the application prospect of organoids.

Organs-on-a-chip and engineered human tissues in drug development / 中国组织工程研究

BACKGROUND: Organs-on-a-chip (OOC) is a chip that miniatures tissues or organs in vitro to simulate human physiological or pathological activities. OBJECTIVE: To review the design considerations of single and multi-OOCs, expound its achievements, potential and application prospect in drug development and precision medicine, as well as the remaining challenges. METHODS: We searched relevant articles in PubMed and CNKI databases with the keywords of “organs-on-a-chip, liver, blood-brain barrier, tumor” in Chinese and “organs-on-a-chip, drug development, cell culture, organoids, microfluidic systems, induced pluripotent stem cells, liver” in English, respectively. Finally, 68 articles were analyzed in this review. RESULTS AND CONCLUSION: OOC is a breakthrough technology that benefits from progresses in engineered human tissue engineering, semiconductor fabrication and adult somatic cell culture, exceeding the limitations of current cell and animal models. The activities, mechanical properties and physiological reactions of the whole organ or human system can be embodied in the 3D microfluidic OOC. As it can simulate physiological or pathological states in vitro by integrating human cells with physiology-related microenvironments, OOCs are expected to supplement and reduce the pre-clinical trials of drugs, medical devices and biological materials, offering a favorable in vitro platform for screening drug-related adverse reactions.

The application and prospects of organoid technique in esophageal cancer research / 解放军医学杂志

Esophageal cancer is a common gastrointestinal tumor. The morbidity and mortality of esophageal cancer in China were 18.85/100,000 and 14.11/100,000, respectively. China is one of the regions with high incidence and high mortality of esophageal cancer in the world. Among them, esophageal squamous cell carcinoma is more common with difficult of early diagnosis. Therefore, there is a great space for research and exploration of early diagnosis and early treatment. Many factors restrict the research on the developing mechanism, drug discovery and screening as well as individualized treatment of esophageal cancer. The lack of an ideal tumor model is one of the important restrictive factors. Organoid is a novel product of 3D culture technology in vitro with the advantages of maintaining the structure, function, genome and drug sensitivity of in situ tissue, and is an excellent tumor research model with such advantages as easy to be operated, relatively low cost, and can be used in conjunction with other advanced technologies. Therefore, esophageal cancer organoid has gradually gained the attention of the academic world, and is expected to be widely used in the field of esophageal cancer research. The present review focuses on the applications and prospects of organoid in the research of esophageal cancer.

Comparison of HepaRG and HepG2 spheroids on morphology, cytochrome P450 enzymes and albumin expressions and sensitivity to drug hepatotoxicity / 中国药理学与毒理学杂志

OBJECTIVE To establish three-dimensional (3D) spherical tissue-like models for HepaRG cells and HepG2 cells and compare Ihe difference in morphology, functional protein expressions and drug hepatotoxicity tests to provide data for the selection of drug hepatotoxicity detection models in vitro. METHODS Two in vitro 3D hepatocyte spheroid models were constructed on HepaRG and HepG2 cells in logarithmic growth phase. The two types of cells were separately seeded in ultra-low attachment surface 96-well plates at a cell density of 100 cells per well, and cultured in a conventional manner without adding any inducer. The morphology of the spheroids on the 3∗ day (D3), D7, D14, D21 after seeding was observed under the microscope, and the average diameters of spheroids were caculated. Differential expressions of cytochrome P450 enzymes and albumin at mRNA and protein levels in the two models were studied with quantitative real-time PCR, immunofluorescence staining and Western blotting. Seven drugs (thiamine, fialuridine, acetaminophen, benzbromarone, cyclophosphamide, isoniazid and nefazodone) were selected for hepatotoxicity detection. After a single dose and repeated dose experiments, the cell inhibitory rate was measured and inhibitory concentration 50 (IC∗) was calculated. RESULTS The average diameters of the two models increased with time. The average diameters of HepaRG cell spheroids on D7, D14 and D21 after seeding were 317.5, 334.3 and 397.8 pm, respectively, which were smaller than those of HepG2 spheroids (P40, 0.87, >20 , 35.74 and 2.57 mmol • L"\ respectively. Under the single administration and partial drug repeated administration, the inhibitory effect on the suivival of 2 spheroid cells did not reach half of the inhibiory level. CONCLUSION HepaRG spheroids are superior to HepG2 spheroids in morphology control and functional protein expressions. They are more sensitive in drug hepatotoxicity detection in vitro. HepaRG spheroids are a better 3D spherical tissue-like model in vitro.

Develop a 3D neurological disease model of human cortical glutamatergic neurons using micropillar-based scaffolds

Establishing an effective three-dimensional (3D) culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane (PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system, we further developed an disease-like model of traumatic brain injury (TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases.

IL-10 and TGF-ß1 evaluation in 3D human adipose-derived mesenchymal stem cells culture and hypoxic condition / Avaliação de IL-10 e TGF- ß 1 sob condições de hipóxia em cultivo celular 3D de células-tronco mesenquimais de tecido-adiposo humano

Mesenchymal stem cells (MSC) are multipotent cells derived from layer mesoderm and that have potential for self-renewal and cellular differentiation. These cells can be extracted from various tissues, being the main sources the bone marrow (BM) and adipose tissue (AT). Therefore, human Adipose-derived Mesenchymal Stem Cells (AdMSCs) are potentially able to differentiate in several cell types such as neurons, adipocytes and osteoblasts. The objective of this work was to quantify levels of the cytokines TGF-1 and IL-10 in the conditioned medium (CM) of AdMSCs c u l t i v a t e d in 2D and 3D culture after the induction of hypoxia by Cobalt chloride chemistry (CoCl2). When the AdMSCs reached 80% of confluence, the cells were transferred to t h e 24 plates wells, where they were treated with CoCl2 in 2D and 3D culture. Quantification assay was made using human TGF-1 and IL- 10 kits. The analysis was done through the sandwich ELISA assay. The IL-10 and TGF-1 production have increased when the AdMSCs were in three-dimensional culture and under hypoxic conditions, indicating that supplies of oxygen associated to the 3D culture influenced significantly the production of these cytokines. This can be a potent and low-cost strategy to improve Adipose-derived Stem Cells conditioned medium when it comes to the release of IL-10 and TGF- cytokines.

Células-tronco mesenquimais (CTMs) são células multipontes derivadas da camada mesoderma e que possuem potenciais de auto-renovação e diferenciação celular. Estas células podem ser extraídas de diversos tecidos, sendo as principais fontes a medula óssea (MO) e o tecido adiposo (TA). Assim, CTM-TA são potencialmente capazes de se diferenciarem em diversos tipos celulares como adipócitos, neurônios e osteoblastos através de ações parácrinas do microambiente de cultivo. Este trabalho teve como objetivo dosar os níveis das citocinas TGF- e IL-10 no meio condicionado de CTM-TA cultivadas em 2D e 3D após a indução de hipóxia química por Cloreto de Cobalto (CoCl2). As CTM-TA foram cultivadas até atingirem a confluência de 80% e em seguida foram transferidas para placas de 24 poços, onde foram tratadas com meio indutor de hipóxia em cultivos realizados em 2D e 3D. A quantificação foi realizada utilizando os kits TGF-1 e IL10 humanos. Nas análises foi utilizado o ensaio ELISA sanduíche. Os resultados mostraram que a produção de citocinas IL10 e TGF- aumentaram quando o cultivo celular foi tridimensional em condições de hipóxia, indicando que os fornecimentos de oxigênio associado ao cultivo em 3D influenciaram de maneira significativa na produção de tais citocinas. Esta pode ser uma estratégia potente e de baixo custo para aumentar a liberação TGF- e IL- 10 no meio condicionado de CTM-TA.

Culture and characterization of human pseudomyxoma peritonei cells / 基础医学与临床

Objective Culture,expansion and characterization of pseudomyxoma peritonei cells to support foundation for the study of pseudomyxoma peritonei and drug screening.Methods Tumors from 5 cases of human pseudomyxoma peritonei were cultured by collagenase digestion.The cultured cells were then identified as tumor cells by chromosome karyotyping.The secretion of neutral mucopolysaccharide was detected by PAS staining.To simulate the tumor growth condition in vivo,3D culture was applied and the morphology of the cultured organoid was observed with HE staining,xenografts were used to test the tumorigenicity of PMP primary cells.Results The primary culture of pseudomyxoma peritonei cells was successful in all of the 5 cases.2D cultured cells were adherent,polygonal pebble-like arranged,and passed on up to 18 generations.Chromosome karyotype analysis showed that most of the cells were subdiploid karyotype tumor cells.Positive PAS staining suggests that these cells secrete mucus.The morphology of 3D cultured organoid was similar to that of tumor tissues.The tumor formation rate of xenografts was low,and the tumoris not similar to patient's PMP.Conclusions Pseudomyxoma peritonei tumor cells can be cultured and expanded in vitro.

Hepatogenic Differentiation Capacity of Human Wharton’s Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

BACKGROUND: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development. MATERIALS AND METHODS: HWJMSCs were extracted from human Wharton’s jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry. RESULTS: According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19. CONCLUSIONS: Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes.

3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation / 中国中药杂志

3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model.

Neuronal differentiation of PC12 and embryonic stem cells in two- and three-dimensional in vitro culture.

The quality of neuronal differentiation and reduction in apoptosis that occurred in two-dimensional (2D) and three-dimensional (3D) culture conditions is compared. PC12 and embryonic stem cells are two commonly utilized cell lines for the study of neuronal regeneration. These cells were induced to neuronally differentiate by adding NGF and retinoic acid respectively. Total neurite length and expression of neuronal markers (MAP-2 and β3-tubulin) was assessed by morphometry and immunocytochemistry. Also, TUNEL assay was used to detect apoptosis. Upon exposure to a differentiation media in the 3D fibrin gel, PC12 and embryonic stem cells stopped dividing, had increased adhesion to the substratum, extended neurite processes and expressed neuronal markers. The same results, however, were not observed with the 2D culture. Also, the apoptosis index performed by TUNEL assay demonstrated a reduction in the degree of apoptosis in the 3D culture compared to 2D culture. Fibrin matrix supports growth and neuronal differentiation of PC12 and embryonic stem cells. In addition, the 3D culture enhanced cellular resistance to apoptosis when compared to the 2D culture. It appears as if a 3D culture system may offer a better technique for future neuronal tissue engineering investigations.

Simple and Novel Three Dimensional Neuronal Cell Culture Using a Micro Mesh Scaffold

Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications.

Preparation and application of multicellular spheroid in cancer therapy / 基础医学与临床

Multicellular spheroid (MCS) can simulate many aspects of the in vivo physiological and pathological conditions in many aspects,better reflect the in-vivo behavior of cells in tumors.So it's increasingly accepted as a valuable tool for evaluating the efficacy of therapeutic intervention including chemotherapy,radiotherapy,immunotherapy and combined therapy.Various spheroid co-culture approaches have been presented to study heterologous cells interaction in solid tumors.The present review briefly introduces the methodology and applications of MCS with focus on the up-to-date information.