Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting / 中华创伤杂志(英文版)

PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

In vitro co-culture of decellularized matrix of porcine colon with colon cancer hct116 cells / 中国组织工程研究

BACKGROUND: Acellular scaffolds have been widely used in the study of various tumors in recent years. The spatial arrangement, biomechanical properties and biocompatibility of the scaffolds are helpful to restore the microenvironment of tumor cell growth. OBJECTIVE: To investigate the characteristics and advantages of porcine colon acellular scaffold as an in vitro model of colon cancer. METHODS: Acellular scaffolds of porcine colon were prepared by soaking fresh porcine colon with 2% SDS, 1% TritonX-100 and 0.5% EDTA combined with repeated shaking. Human colon cancer HCT116 cells were inoculated on the mucous surface of porcine colon acellular scaffold. The growth of cells on the scaffold was observed by live-death staining. The morphology of cells on the scaffold was observed by phalloidin staining, and the longitudinal growth of cells on the scaffold was observed by hematoxylin-eosin staining and immunofluorescence staining. RESULTS AND CONCLUSION: (1) Live-death staining showed that on the first day of culture, HCT116 cells could well gather in the pores of the mucosal layer of the scaffolds, and few dead cells were found. On the third day, the cells gradually spread out of the pores, and a small number of cells grew and connected into pieces, and there were no dead cells. On the 7th day, the cell growth density further increased and grew into a sheet on the surface of the mucous layer, and there were no dead cells and exfoliated cells. (2) On the 7th day of culture, hematoxylin-eosin staining and immunofluorescence staining showed that some HCT116 cells could grow into clusters in the pores, and some cells continued to grow to the submucosa along the pores, showing the characteristics of invasive growth of intestinal cancer in situ. (3) On the 7th day of culture, the phalloidin staining showed that HCT116 cells were in close contact with the scaffold mucosa at the ultrastructural level, and had well-differentiated epithelial morphological characteristics. (4) The result shows that porcine colon acellular scaffold can be used as a carrier for three-dimensional culture of colon cancer cells.

Establishment and functional in vitro characteristics of three-dimensional collagen HepaRG microsphere / 中国组织工程研究

BACKGROUND: Some recent studies have shown that the three-dimensional (3D) model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture. OBJECTIVE: HepaRG was selected to prepare 3D collagen microspheres, and the adaptive culture and functional expression of cells in the collagen microspheres were evaluated. METHODS: Collagen hydrogel was used as the scaffold for 3D HepaRG and HepG2 microspheres. Stable cell spheres were formed. HepaRG microspheres, HepG2 microspheres, HepaRG two-dimensional culture and HepG2 two-dimensional culture were used as controls. At 1, 6, and 12 days of culture, cell survival was detected by the Live/Dead assay staining. After 1, 6, 12, and 16 days of culture, the urea synthesis and CYP3A4 secretion of the supernatant were detected in each group. After 12 days of culture, relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA was detected by qPCR. The expression levels of hepatocyte marker albumin and CYP3A4 protein were determined using western blot assay. RESULTS AND CONCLUSION: (1) In 12 days of culture, Live/Dead assay staining showed that the cell viability in the 3D collagen microsphere was well-maintained and the amount of central necrotic cells was small, with high cell viability. In the 3D collagen microsphere, especially HepaRG cells, multiple cellular clusters formed and adjacent clusters were connected closely, which created a cross-linked structure. (2) After 1, 6, and 12 days of culture, the urea content of HepaRG 3D collagen microspheres was higher than that of HepaRG two-dimensional culture (P < 0.05). After 1, 6, 12, and 16 days of culture, the urea content of HepG2 3D collagen microspheres was higher than that of HepG2 two-dimensional culture (P < 0.05). After 1, 6, and 12 days of culture, the secretion of CYP3A4 in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional culture (P < 0.05). After 6 and 12 days of culture, the secretion of CYP3A4 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (3) The relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional cells (P < 0.05), and the relative expression of CYP1A2 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (4) The expression levels of albumin and CYP3A4 protein in HepaRG 3D collagen microspheres were higher than those of HepG2 3D collagen microspheres, ordinary microspheres, and two-dimensional culture (P < 0.05). (5) These results indicated the high-level expression of hepatocyte functions in 3D collagen HepaRG microsphere, which could be taken as a reference in drug metabolism evaluation in vitro and tissue engineering application.

Effect of Doxycycline on vasculogenic mimicry in human pterygium fibroblasts / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effects of doxycycline(DOX)on vasculogenic mimicry(VM)in human pterygium fibroblasts(HPFs)and its molecular mechanisms.<p>METHODS: Primary cultured HPFs were identified by Vimentin and CK through immunocytochemical staining. HPFs were divided into control group and DOX group including low, medium and high concentrations(50, 100, 200mg/L). The activity and migration of HPFs were detected by cell counting kit-8(CCK-8)and wound healing assay. The density of VM was observed by three-dimensional cell culture and periodic acid schiff(PAS)staining and compared the differences of VM formation in each group. Western blot was used to analyze the expression of matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor(VEGF).<p>RESULTS:Immunocytochemical staining results showed that the cells were spindle shaped, meanwhile, they were positive for Vimentin and negative for CK, which were consistent with the characteristics of fibroblasts. Compared with the control group, the cell activity, mobility, VM density and the expression of MMP-9 and VEGF proteins in the DOX group were significantly decreased(<i>P</i><0.05). Compared among different concentrations of DOX groups, the differences were statistically significant(<i>P</i><0.05). Correlation analysis indicated that VM density formed by HPFs was significantly positively correlated with the protein expression of MMP-9 and VEGF(<i>r</i>=0.949, 0.960, all <i>P</i><0.05).<p>CONCLUSION: DOX can inhabit HPFs activity, migration, VM density by reducing the expression of MMP-9 and VEGF, suggesting that MMP-9 and VEGF may be the molecular mechanisms of VM formation in pterygium.

Three-dimensionalhanging-drop culture of mesenchymal stem cells in the treatment of tissue injury / 中国组织工程研究

BACKGROUND: In recent years, mesenchymal stem cells (MSCs) have been widely used in varieties of tissue repairs due to its easy accessibility, exceptional ability to proliferate, remarkable potential to multi-differentiate, and considerable effects on immunoregulation. However, cell pluripotency and secretory function are often severely impaired when the cells are cultured using the traditional two-dimensional method. Nevertheless, three-dimensionally cultured mesenchymal stem cells cannot only derive the advantages of mesenchymal stem cells per se, but maintain and potentiate cell capacity of proliferation, differentiation and secretion, which therefore enhance the ability to repair various tissue injuries. OBJECTIVE: To review the recent studies of three-dimensional cultured mesenchymal stem cells in the treatment of multiple tissue injuries via various aspects including its biological characteristics, underlying mechanisms of tissue repair, culture methods as well as the application in various diseases, in order to elucidate the possibility of its future application and provide theoretical support for the clinical application of three-dimensional mesenchymal stem cells in the future. METHODS: We retrieved the main databases including PubMed, CNKI, and WanFang for relevant literature published in recent 10 years. The keywords were “mesenchymal stem cells, three-dimensional culture, hanging-drop culture, tissue repair” both in Chinese and English. The type of the article was not limited. Finally, 51 articles were included for result analysis. RESULTS AND CONCLUSION: It has been widely demonstrated that compared with the two-dimensional cultured cells, three-dimensional cultured mesenchymal stem cells show stronger effects on cell proliferation, differentiation, paracrine secretion, and immunoregulation. With the rapid development of three-dimensional sphenoid culture system and stem cell transplantation, three-dimensional cultured mesenchymal stem cells will show its great potential in the treatment of multiple tissue injuries.

Effects of hydrogel three-dimensional culture on the characteristics of human amniotic mesenchymal stem cells and paracrine effect / 中国组织工程研究

BACKGROUND: Studies have shown that mesenchymal stem cells can reduce inflammation, promote wound healing, and reduce scar formation in wound healing. However, previous two-dimensional culture environment can lead to differences in gene expression, signal transduction, and morphology because of intracellular contact inhibition. OBJECTIVE: To investigate whether the ability of wound healing related factors secreted by human amniotic mesenchymal stem cells is affected by two-dimensional or three-dimensional culture environment. METHODS: The human amniotic mesenchymal stem cells cultured by traditional enzyme digestion method were inoculated in traditional cell culture flask (two-dimensional culture group) and ShakeGelTM 3D hydrogel (three-dimensional culture group) and induced to differentiate into adipocytes, osteoblasts, and chondrocytes, respectively. The direction of cell differentiation was determined by immunofluorescence staining. Human amniotic mesenchymal stem cells were fused to 70%-80% in two culture environments, and the growth characteristics and morphology of cells were observed under inverted phase contrast microscope and laser confocal microscope. After 24 hours of culture, relative mRNA expression of wound healing-related factors was detected by reverse transcription-quantitative polymerase chain reaction. After 48 hours of culture, the protein expression of wound healing-related factors was detected by the enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells cultured in two-dimensional culture group were flat and spindle-shaped, which was a typical mesenchymal stem cell-like morphology. Human amniotic mesenchymal stem cells in the three-dimensional culture group were round and evenly dispersed in each layer of the hydrogel. (2) Human amniotic mesenchymal stem cells in the three-dimensional culture group exhibited the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. (3) The mRNA expression of interleukin-6, interleukin-8, epidermal growth factor, basic fibroblast growth factor, hyaluronic acid, hepatocyte growth factor, and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture group (P 0.05). (4) The protein expression of interleukin-6, interleukin-8, interleukin-10, epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor β1 and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture (P 0.05). (5) These findings suggest that human amniotic mesenchymal stem cells cultured in three-dimensional hydrogel show better morphology and more encouraging paracrine effect of wound healing related factors than those cultured in traditional two-dimensional culture environment.

Application and progress of umbilical cord Mesenchymal stem cells in bone tissue engineering / 中国组织工程研究

BACKGROUND: As seed cells, human umbilical cord mesenchymal stem cells have many advantages, such as a broad array of sources, easy access, low immunogenicity, osteogenic differentiation potential, high proliferation and self-renewal ability. In recent years, there are more and more reports about their application for bone tissue engineering. OBJECTIVE: To summarize isolation, culture, osteogenic induction and scaffolds. METHODS: The first author searched CNKI and PubMed databases with key words of “human umbilical cord mesenchymal stem cells, isolation, culture, osteogenic differentiation, scaffold, bone tissue engineering” in both Chinese and English, so as to review the relevant literature from 2004 to 2020. Finally, 104 articles were included. RESULTS AND CONCLUSION: There are different methods of isolation and culture of umbilical cord mesenchymal stem cells. Serum-free or animal serum substitute culture system and co-culture technique have made great progress, and three-dimensional culture system will be the development direction in the future. The exact mechanisms of osteogenic differentiation of umbilical cord mesenchymal stem cells are unclear, which need further elucidation. To date, it is still the focus of researchers to develop composite scaffolds with better properties. Bio-printing technology has primarily solve the difficult problem of controlling precisely the complex inner structure of the scaffolds at the micron scale and fabricating individual scaffolds, bringing great hope for bone tissue engineering. The design and fabrication of scaffolds with multiple ideal compositions (including biocompatibility, high porosity at the micro and macro level, mechanical properties, related absorption and so on) and the less clinical side effects remain one of the key challenges in bone tissue engineering.

Study on in vitro and in vivo Genotoxicity of Emodin- 8-O-β-D-glucoside / 中国药房

ABSTRACT OBJECTIVE：To evaluate the in vitro and in vivo genotoxicity of emodin-8-O-β-D-glucoside（EG），and to compare the difference of in vitro cell test and in vivo test of rats. METHODS：2D and 3D hepatocyte models were established by in vitro two-dimensional（2D）and three-dimensional（3D）cell culture. After modeling，2D and 3D hepatocyte were divided into blank control group（0.5％ DMSO），mitomycin C group（positive control，0.1 μg/mL），EG low-dose，medium-dose and high-dose groups（10，50，200 μg/mL），respectively. The micronucleus ratio and tail DNA％ of HepaRG cells were detected. SD rats were divided into blank control group（0.5％ sodium carboxymethyl cellulose），ethyl methanesulfonate group（positive control，200 mg/kg），EG low-dose，medium-dose and high-dose groups（100，300，1 000 mg/kg），with 6 rats in each group. They were given medicine intragastrically for consecutive 15 d，once a day. 15 days later，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were measured. RESULTS：In the in vitro 2D HepaRG hepatocyte model，compared with blank control group，the micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01）. There was no statistical significance in micronucleus formation rate and tail DNA％ of HepaRG cell among EG groups（P＞0.05）. In 3D HepaRG cell model， compared with blank control group， micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01 or P＜0.001）， while tail DNA％ of HepaRG cell wasincreased significantly in EG high-dose group（P＜0.01）. In the in vivo test，compared with blank control group，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were all increased significantly in ethyl methanesulfonate group（P＜0.01）. Tail DNA％ of peripheral blood lymphocytes was increased significantly in EG high-dose group （P＜0.01）. There was no statistical significance in the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of hepatocytes among EG groups（P＞0.05）；with the increase of dose，there was an increasing trend. CONCLUSIONS：The results of this study suggest that in 2D cell model，EG not lead to chromosome breakage and DNA damage，but the long-term administration and repeated administration in vivo of 3D cell model show that EG has a certain risk of DNA damage，so the evaluation results of 3D HepaRG cell model are more similar to those of rats in vivo. KEYWORDS Emodin-8-O-β-D-glucoside；Genotoxicity；Two-dimensional culture；Three-dimensional culture；Rat；Micronucleus test

Research progress on radioresistance and sensitization of tumor cells in three-dimensional culture system / 中华放射肿瘤学杂志

Three-dimensional cell culture is a novel type of in vitro culture method.This system can mimic the microenvironment of cell growth in vivo,where cells exhibit similar state and function to that in the in vivo environment.Currently,three-dimensional culture system has been widely applied in tissue engineering and tumor cell biology fields,etc.Radiotherapy is an important treatment of cancer.Radioresistance of tumor cells is the main cause of treatment failure,tumor recurrence and metastasis.Tumor cells can exhibit the resistant characteristics in the three-dimensional culture system,similar to those of tumor cells in vivo.Therefore,three-dimensional culture system can be adopted to investigate the radioresistance and underlying mechanism of tumor cells and identify the key factors regulating the radioresistance of tumor cells,which plays a pivotal role in enhancing the radioresistance and improving the effect of radiotherapy.This article will review recent research progress in the radioresistance and sensitization of tumor cells under three-dimensional culture system,aiming to provide reference for relevant investigations.

Hypoxic three-dimensional culture microenvironment promotes proliferation of bone marrow mesenchymal stem cells through HIF-1α signaling pathway / 中国胸心血管外科临床杂志

@#Objective To investigate the effects of hypoxic three-dimensional culture microenvironment on the proliferation of bone marrow mesenchymal stem cells and its mechanism. Methods P5 generation mouse bone marrow mesenchymal stem cells and P (3HB-co-4HB) were co-cultured under normoxic three-dimensional (20%) and hypoxic three-dimensional microenvironment (4%) respectively. After 24 hours, the proliferation of the two groups was determined by CCK-8 method. The expression of HIF-1α gene was detected by real-time quantitative PCR after 12 hours. Western blotting was used to detect the expression of HIF-1α protein after 24 hours. Results After 24 hours, the CCK-8 method showed that the OD value of the hypoxia group was significantly higher than that of the normoxia group (0.455±0.027 vs. 0.352±0.090, n=12, P<0.05). After 12 hours of hypoxic culture, the expression level of HIF-1α mRNA in the hypoxia group was significantly higher than that in the normoxia group (P<0.05). Compared with the normoxia group (0.47± 0.05), the relative expression level of HIF-1α protein in the hypoxia group (0.63±0.06) significantly increased in the Western blotting after 24 hours (n=3, P<0.05). Conclusion The hypoxic three-dimensional microenvironment can promote the proliferation of bone marrow mesenchymal stem cells, which may be related to the activation of HIF-1α signaling pathway.

Decellularized matrix of human fatty liver used for three-dimensional culture of hepatocellular carcinoma cells / 南方医科大学学报

OBJECTIVE@#To construct a decellularized matrix of human fatty liver as the scaffold for three-dimensional (3D) culture of hepatocarcinoma cells.@*METHODS@#Human fatty liver decellularized matrix (hFLM) was prepared by repeated freezingthawing, perfusion with gradient SDS and 1% Triton X-100 through the portal vein and hepatic artery, and repeated agitation with Triton X-100. HepG2 cells were cultured in the prepared hFLM, and the cell survival, morphology, proliferation and cellular expressions of the adhesion molecules were detected.@*RESULTS@#The decellularization procedure shortened the time for scaffold preparation and preserved the 3D ultrastructure and the composition of the extracellular matrix. HepG2 cells cultured in hFLM scaffold maintained proliferation for up to 15 days and showed a growth pattern with a long lag phase and a slow growth rate, which was similar to the growth pattern . The cultured HepG2 exhibited a low expression of E-cadherin and a high expression of vimentin, which was consistent with the xenograft but opposite to 2D cultured cells. However, the lack of adequate nutrient transport in this hepatocarcinoma cell model led to a slowdown of cell proliferation in the later stage. The PCNA index of HepG2 cells cultured in hFLM was lowered by 29.3% on day 12 as compared with that on day 6.@*CONCLUSIONS@#We established a new protocol for preparing hFLM and confirmed the feasibility of constructing hepatocarcinoma cell models using the hFLM scaffold.

Research progress in organoids and applications / 中国药理学与毒理学杂志

An organoid is a novel in vitro model, formed by self-organization of stem cells or tumor cells under three-dimension culture. It recapitulates many aspects of structural organization and functionality of its in vivo organ counterparts and maintains the stability of genetic materials under long-term culture, thus holding great promise for modeling disease, drug screening and personalized medicine. So far, normal and tumor organoids from structures such as esophagus, stomach, intestine, liver, pancreas and prostate and mammary gland have been established, providing a new culture platform in vitro. This article reviews the classification and biomedical applications of organoids.

Preliminary study on the construction of three-dimensional hippocampal neural network by using microfluidic technology in vitro / 中国修复重建外科杂志

Objective: To preliminary study on the feasibility of constructing three-dimensional (3D) hippocampal neural network in vitro by using microfluidic technology. Methods: A network patterned microfluidic chip was designed and fabricated by standard wet etching process. The primary hippocampal neurons of neonatal Sprague Dawley rats were isolated and cultured, and then inoculated on microfluidic chip for culture. Immunofluorescence staining was used to observe the growth of hippocampal neurons at 3, 5, and 7 days of culture and electrophysiological detection of hippocampal neuron network at 7 days of culture. Results: The results showed that the number of hippocampal neurons increased gradually with the prolongation of culture time, and the neurite of neurons increased accordingly, and distributed uniformly and regularly in microfluidic chip channels, suggesting that the 3D hippocampal neuron network was successfully constructed in vitro. Single and multi-channel spontaneous firing signals of hippocampal neuronal networks could be detected at 7 days of culture, suggesting that neuronal networks had preliminary biological functions. Conclusion: Patterned microfluidic chips can make hippocampal neurons grow along limited paths and form 3D neuron networks with corresponding biological functions such as signal transduction, which lays a foundation for further exploring the function of neuron networks in vitro.

Effect of natural hirudin on angiogenesis of human microvascular endothelial cells / 中国修复重建外科杂志

Objective: To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods: Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results: The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Conclusion: Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.

Bioactive Glass Particles in Two-Dimensional and Three-Dimensional Osteogenic Cell Cultures

Abstract This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.

Resumo O objetivo deste estudo foi investigar a influência do modelo de cultura celular tridimensional e das partículas de vidro bioativo (BG) sobre a expressão fenotípica de culturas de células osteogênicas da calvária de ratos. As células foram mantidas em culturas sobre superfícies colágenas bi-dimensionais (2D) e em géis de colágeno tridimensional (3D) com e sem partículas de BG até 14 dias. Foram avaliadas: viabilidade celular, atividade de fosfatase alcalina (ALP), morfologia celular e imunomarcação de proteínas da matriz não-colágena do osso através de epifluorescência e microscopia confocal. As expressões de marcadores osteogênicos foram analisadas utilizando RT-PCR. A formação de nódulos mineralizados foi visualizada através de microscopia e o conteúdo de cálcio foi avaliado quantitativamente pelo Alizarina Red. As culturas experimentais produziram uma taxa crescente de viabilidade até 14 dias. Embora a atividade ALP em 7 dias tenha sido maior em culturas com BG, as células em 3D e 3D+BG apresentaram uma diminuição da atividade ALP aos 14 dias. As condições tridimensionais favoreceram a imunomarcação para OPN e BSP e a expressão de mRNAs para ALP e COL I. As partículas de BG influenciaram positivamente a expressão do mRNAs para OPN e OC e a formação de nódulos calcificados in vitro. Os resultados indicaram que as culturas em 3D e partículas BG contribuíram para a expressão do fenótipo osteoblástico e para a diferenciação e formação de matriz mineralizada.

Design and Validation of a Microfluidic Chip with Micropillar Arrays for Three-dimensional Cell Culture / 分析化学

A microfluidic chip with micropillar arrays for three-dimensional (3D) cell culture was designed and validated.The chip consisted of a polydimethylsiloxane (PDMS) channel plate and a glass cover plate.One cell culture chamber composed of two rows of micropillar arrays and two lateral channels for transporting the culture medium were integrated on the PDMS channel plate.The spacing between micropillars directly affects the chip performance, which is critical for the design of the chip.In this work, the spacing between micropillars was optimized by numerical simulation and experimental validation.With the optimized microfluidic chip, the mixture of cells and extracellular matrix mimics could be steadily injected into the cell culture chamber, the nutrients in the culture medium from the lateral channels could quickly diffuse into the chamber, and the cell metabolites could also timely diffuse out of the chamber.To test the stability of the microenvironment in the microfluidic chip, neural stem cells were three-dimensionally cultured.

Preparation of chitosan-gelatin porous microspheres by high voltage electrostatic method combined with freeze-drying and ionic cross-linking method / 上海交通大学学报（医学版）

Objective·To prepare chitosan-gelatin porous microspheres by high voltage electrostatic method combined with freeze-drying and ionic cross-linking method and investigate the factors that influence the formation of porous medium.Methods·Porous chitosan microspheres and chitosangelatin porous microspheres were prepared using high voltage electrostatic method combined with freeze-drying and ionic cross-linking method,with sodium tripolyphosphate (STPP) as crosslinking agent.Factors that affect the porous structure and pore size of porous microspheres were compared,such as different chitosan-gelatin ratio,freezing temperature,curing time with saturated STPP in 85％ ethanol solution.The morphology,surface and internal structure,particle size of the porous chitosan microspheres and chitosan-gelatin porous microspheres were observe by using light microscope,scanning electron microscope and hematoxylin-eosin staining.Results·Microspheres prepared by freeze-drying with an electrostatic and ionic cross-linking method have open,interconnected and highly macroporous,with good spherical surface.Saturated STPP ethanol solution (85％ ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure.The order of freeze and crosslinking,cross-linking time and the second freezing temperature,can influence the pore size of porous microspheres.Gelatin and chitosan can form polyelectrolyte complexes,and can also be used as porogen in porous structure.Conclusion·The preparation of porous chitosan-gelatin microspheres via this method has a large pore size (diameter 100-200 μm),suitable for cell growth and the migration.

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine

BACKGROUND: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. METHODS: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. RESULTS: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. CONCLUSION: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

Murine enteroids culture system and Marmota Himalayana Hepatovirus replication by the enteroids / 中华实验和临床病毒学杂志

Objective@#To establish mouse intestinal tissue and its application in the proliferation of Marmota Himalayana hepatovirus (MHHAV).@*Methods@#The intestinal crypts of Balb/C mice were digested with EDTA at a high concentration, cultured in Matrigel and passaged into frozen intestinal enteroids. The monolayers were inoculated on 96-well plates and infected with MHHAV. Real-time qPCR virus concentrations at different time points were compared with those of a number of cell lines routinely used to develop hepatitis A virus.@*Results@#The mouse intestinal tissue culture system was successfully established. MHHAV did not proliferate in the cell lines commonly used for the culture of hepatitis A virus, but proliferated well in the intestinal tissues of mice. The virus multiplied about 3 700 fold in 4 days.@*Conclusions@#Mice intestinal enteroids can be used for the isolation and culture of MHHAV.

Differentiation of Rat Dermal Mesenchymal Cells and Calcification in Three-Dimensional Cultures

Three-dimensional (3D) cultures are known to promote cell differentiation. Previously, we investigated the differentiation of rat dermal fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts through transforming growth factor (TGF)-β production using a 3D culture model. Here, we investigated the phenotypic change from dermal mesenchymal cells (mostly fibroblasts) to osteoblast-like cells, being inspired by the roles of smooth muscle cells or fibroblasts during vascular calcification. Spindle-shaped cells that grew in heterologous populations out of dermal explants from 2-day-old Wistar rats were cultured within a collagen matrix. α-SMA and alkaline phosphatase (ALP) messenger RNA (mRNA) levels initially increased, followed by a rise in Runx2 and osteocalcin (OCN) mRNA levels without calcification. Calcium deposits were produced in the presence of a high concentration of inorganic phosphate (2.1 mM) or β-glycerophosphate (βGP, 10 mM) after 2 weeks of culture, and both were sensitive to an inhibitor of type III phosphate transporters. An ALP inhibitor decreased only βGP-induced calcification. Inhibition of TGF-β type-I receptors attenuated ALP mRNA levels and βGP-induced calcification, suggesting that endogenous TGF-β stimulates ALP activity and then βGP breakdown. An increase in the number of cells embedded in the collagen gel enhanced the mRNA levels of Runx2 and OCN, but not of ALP. Collectively, several factors are likely to promote the differentiation of dermal mesenchymal cells into osteoblast-like cells and ectopic calcification in a 3D collagen matrix, implying the utility of these cells as a potential autologous cell source for tissue engineering.